Inhibition of oncogenic transcription factor REL by the natural product derivative calafianin monomer 101 induces proliferation arrest and apoptosis in human B-lymphoma cell lines

Increased activity of transcription factor NF-κB has been implicated in many B-cell lymphomas. We investigated effects of synthetic compound calafianin monomer (CM101) on biochemical and biological properties of NF-κB. In human 293 cells, CM101 selectively inhibited DNA binding by overexpressed NF-κB subunits REL (human c-Rel) and p65 as compared to NF-κB p50, and inhibition of REL and p65 DNA binding by CM101 required a conserved cysteine residue. CM101 also inhibited DNA binding by REL in human B-lymphoma cell lines, and the sensitivity of several B-lymphoma cell lines to CM101-induced proliferation arrest and apoptosis correlated with levels of cellular and nuclear REL. CM101 treatment induced both phosphorylation and decreased expression of anti-apoptotic protein Bcl-XL, a REL target gene product, in sensitive B-lymphoma cell lines. Ectopic expression of Bcl-XL protected SUDHL-2 B-lymphoma cells against CM101-induced apoptosis, and overexpression of a transforming mutant of REL decreased the sensitivity of BJAB B-lymphoma cells to CM101-induced apoptosis. Lipopolysaccharide-induced activation of NF-κB signaling upstream components occurred in RAW264.7 macrophages at CM101 concentrations that blocked NF-κB DNA binding. Direct inhibitors of REL may be useful for treating B-cell lymphomas in which REL is active, and may inhibit B-lymphoma cell growth at doses that do not affect some immune-related responses in normal cells.R01 GM094551 - NIGMS NIH HHS; P50 GM067041 - NIGMS NIH HHS; GM094551 - NIGMS NIH HHS; R24 GM111625 - NIGMS NIH HHS; GM067041 - NIGMS NIH HH

CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein

The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter A) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support canonical NF-κB signaling in response to treatment with tumor necrosis factor (TNF). Nevertheless, non-canonical NF-κB signaling is intact in these NEMO-deficient cells. Expression of ectopic NEMO in the edited cells restores downstream NF-κB signaling in response to TNF. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter A) in the human SNU-423 liver cancer cell line. We have also used homology directed repair (HDR) to fix the promoter B element in a 293T cell clone. Overall, we have created a strategy for selectively eliminating cell type-specific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-κB signaling are discussed.GM117350 - National Institutes of Health; CA077474 - National Institutes of HealthPublished versio

Multi-chord fiber-coupled interferometer with a long coherence length laser

This paper describes a 561 nm laser heterodyne interferometer that provides time-resolved measurements of line-integrated plasma electron density within the range of 10^15-10^18 cm^(-2). Such plasmas are produced by railguns on the Plasma Liner Experiment (PLX), which aims to produce \mu s-, cm-, and Mbar-scale plasmas through the merging of thirty plasma jets in a spherically convergent geometry. A long coherence length, 320 mW laser allows for a strong, sub-fringe phase-shift signal without the need for closely-matched probe and reference path lengths. Thus only one reference path is required for all eight probe paths, and an individual probe chord can be altered without altering the reference or other probe path lengths. Fiber-optic decoupling of the probe chord optics on the vacuum chamber from the rest of the system allows the probe paths to be easily altered to focus on different spatial regions of the plasma. We demonstrate that sub-fringe resolution capability allows the interferometer to operate down to line-integrated densities of order 10^15 cm^(-2).Comment: submitted to Rev. Sci. Instrum. (2011

The European dilemma:evaluating the implications of UEFA Europa League participation on English Premier League clubs

For clubs qualifying for the UEFA Europa League, participation generates a series of tensions. UEFA has provided financial rewards for those clubs, but with qualification comes additional pressures on playing squads which are often less able to deal with the performative demands of an extra competition, as they generally possess fewer financial resources compared with teams gaining Champions League qualification. This paper evaluates these conflicting tensions. The study takes a detailed quantitative analysis of the costs and benefits for four Premier League clubs that have repeatedly participated in the Europa League over eight seasons. It demonstrates a positive financial benefit of participation, but this is only significant if the club progresses to the later competition stages and has a robust playing squad. This research fills a gap in a lack of analysis based on reliable empirical data as to the impact of participation in this competition on clubs

Developing Water and Methane Potentials for MD Simulations of Methane Clathrate Hydrate

PhDThe aim of this thesis is to develop a set of intermolecular potentials that enable the study of nucleation or decomposition of methane hydrates. The potentials are developed for water, methane, and the water-methane pair by fitting to SAPT(DFT) reference energies. The first set of potentials developed differ from recent polarisable models in that they have rank 4 ISA multipoles, rank 3 anisotropic polarisabilities, rank 3 isotropic dispersion, and anisotropic exchange-repulsion terms. These potentials are validated based on the structures and energies of small clusters and second virial coefficients. The potentials are then significantly simplified for use in MD simulations with DL_POLY4. Simplifying the methane potential makes it difficult to fit simultaneously the global minimum dimer and a set of randomly generated dimers used as reference energies. Several methods are tested to account for polarisation in water within the limitations of DL_POLY and it is found that for MD simulations good results can be attained by increasing the charge values to match the multipole moments of a water molecule in a dielectric. Simulations are carried out for liquid water, ice Ih, and methane gas to validate the new models. The models developed are compared in MD simulations with TIP4P/Ice and the United Atom Methane (UAM) model in simulations of sI methane clathrate; both under stable conditions and while undergoing decomposition at different temperatures. It is found that the melting behaviour differs according the methane- and water-methane interactions; the behaviour of methane under clathrate decomposition using either methane model is discussed.Queen Mary University of Londo

Coefficientwise total positivity of some matrices defined by linear recurrences

We exhibit a lower-triangular matrix of polynomials $T(a,c,d,e,f,g)$ in six indeterminates that appears empirically to be coefficientwise totally positive, and which includes as a special case the Eulerian triangle. We prove the coefficientwise total positivity of $T(a,c,0,e,0,0)$, which includes the reversed Stirling subset triangle.Comment: LaTeX2e, 12 pages with 2 figures: extended abstract submitted to FPSAC 202

CRISPR/Cas9-based editing of a sensitive transcriptional regulatory element to achieve cell type-specific knockdown of the NEMO scaffold protein

The use of alternative promoters for the cell type-specific expression of a given mRNA/protein is a common cell strategy. NEMO is a scaffold protein required for canonical NF-κB signaling. Transcription of the NEMO gene is primarily controlled by two promoters: one (promoter B) drives NEMO transcription in most cell types and the second (promoter D) is largely responsible for NEMO transcription in liver cells. Herein, we have used a CRISPR/Cas9-based approach to disrupt a core sequence element of promoter B, and this genetic editing essentially eliminates expression of NEMO mRNA and protein in 293T human kidney cells. By cell subcloning, we have isolated targeted 293T cell lines that express no detectable NEMO protein, have defined genomic alterations at promoter B, and do not support activation of canonical NF-κB signaling in response to treatment with tumor necrosis factor. Nevertheless, noncanonical NF-κB signaling is intact in these NEMO-deficient cells. Expression of ectopic wildtype NEMO, but not certain human NEMO disease mutants, in the edited cells restores downstream NF-κB signaling in response to tumor necrosis factor. Targeting of the promoter B element does not substantially reduce NEMO expression (from promoter D) in the human SNU423 liver cancer cell line. Thus, we have created a strategy for selectively eliminating cell typespecific expression from an alternative promoter and have generated 293T cell lines with a functional knockout of NEMO. The implications of these findings for further studies and for therapeutic approaches to target canonical NF-κB signaling are discussed.Published versio

The impact of dark matter cusps and cores on the satellite galaxy population around spiral galaxies

We use N-body simulations to study the effects that a divergent (i.e. ‘cuspy') dark matter profile introduces on the tidal evolution of dwarf spheroidal galaxies (dSphs). Our models assume cosmologically motivated initial conditions where dSphs are dark-matter-dominated systems on eccentric orbits about a host galaxy composed of a dark halo and a baryonic disc. We find that the resilience of dSphs to tidal stripping is extremely sensitive to the cuspiness of the inner halo profile; whereas dwarfs with a cored profile can be easily destroyed by the disc component, those with cusps always retain a bound remnant, even after losing more than 99.99 per cent of the original mass. For a given halo profile, the evolution of the structural parameters as driven by tides is controlled solely by the total amount of mass lost. This information is used to construct a semi-analytic code that follows the tidal evolution of individual satellites as they fall into a more massive host, which allows us to simulate the hierarchical build-up of spiral galaxies assuming different halo profiles and disc masses. We find that tidal encounters with discs tend to decrease the average mass of satellite galaxies at all galactocentric radii. Of all satellites, those accreted before re-ionization (z≳ 6), which may be singled out by anomalous metallicity patterns, provide the strongest constraints on the inner profile of dark haloes. These galaxies move on orbits that penetrate the disc repeatedly and survive to the present day only if haloes have an inner density cusp. We show that the size-mass relationship established from Milky Way (MW) dwarfs strongly supports the presence of cusps in the majority of these systems, as cored models systematically underestimate the masses of the known ultra-faint dSphs. Our models also indicate that a massive M31 disc may explain why many of its dSphs with suitable kinematic data fall below the size-mass relationship derived from MW dSphs. We also examine whether our modelling can constrain the mass threshold below which star formation is suppressed in dark matter haloes. We find that luminous satellites must be accreted with masses above 108-109 M⊙ in order to explain the size-mass relation observed in MW dwarf