19F-labeling of the adenine H2-site to study large RNAs by NMR spectroscopy

In comparison to proteins and protein complexes, the size of RNA amenable to NMR studies is limited despite the development of new isotopic labeling strategies including deuteration and ligation of differentially labeled RNAs. Due to the restricted chemical shift dispersion in only four different nucleotides spectral resolution remains limited in larger RNAs. Labeling RNAs with the NMR-active nucleus [superscript 19]F has previously been introduced for small RNAs up to 40 nucleotides (nt). In the presented work, we study the natural occurring RNA aptamer domain of the guanine-sensing riboswitch comprising 73 nucleotides from Bacillus subtilis. The work includes protocols for improved in vitro transcription of 2-fluoroadenosine-5′-triphosphat (2F-ATP) using the mutant P266L of the T7 RNA polymerase. Our NMR analysis shows that the secondary and tertiary structure of the riboswitch is fully maintained and that the specific binding of the cognate ligand hypoxanthine is not impaired by the introduction of the [superscript 19]F isotope. The thermal stability of the [superscript 19]F-labeled riboswitch is not altered compared to the unmodified sequence, but local base pair stabilities, as measured by hydrogen exchange experiments, are modulated. The characteristic change in the chemical shift of the imino resonances detected in a 1H,15N-HSQC allow the identification of Watson–Crick base paired uridine signals and the [superscript 19]F resonances can be used as reporters for tertiary and secondary structure transitions, confirming the potential of [superscript 19]F-labeling even for sizeable RNAs in the range of 70 nucleotides.Deutsche Forschungsgemeinschaft (collaborative research center: SFB902

Integration of genome-level data to allow identification of subtype-specific vulnerability genes as novel therapeutic targets

The identification of cancer-specific vulnerability genes is one of the most promising approaches for developing more effective and less toxic cancer treatments. Cancer genomes exhibit thousands of changes in DNA methylation and gene expression, with the vast majority likely to be passenger changes. We hypothesised that, through integration of genome-wide DNA methylation/expression data, we could exploit this inherent variability to identify cancer subtype-specific vulnerability genes that would represent novel therapeutic targets that could allow cancer-specific cell killing. We developed a bioinformatics pipeline integrating genome-wide DNA methylation/gene expression data to identify candidate subtype-specific vulnerability partner genes for the genetic drivers of individual genetic/molecular subtypes. Using acute lymphoblastic leukaemia as an initial model, 21 candidate subtype-specific vulnerability genes were identified across the five common genetic subtypes, with at least one per subtype. To confirm the approach was applicable across cancer types, we also assessed medulloblastoma, identifying 15 candidate subtype-specific vulnerability genes across three of four established subtypes. Almost all identified genes had not previously been implicated in these diseases. Functional analysis of seven candidate subtype-specific vulnerability genes across the two tumour types confirmed that siRNA-mediated knockdown induced significant inhibition of proliferation/induction of apoptosis, which was specific to the cancer subtype in which the gene was predicted to be specifically lethal. Thus, we present a novel approach that integrates genome-wide DNA methylation/expression data to identify cancer subtype-specific vulnerability genes as novel therapeutic targets. We demonstrate this approach is applicable to multiple cancer types and identifies true functional subtype-specific vulnerability genes with high efficiency

NMR quality control of fragment libraries for screening

Fragment-based screening has evolved as a remarkable approach within the drug discovery process both in the industry and academia. Fragment screening has become a more structure-based approach to inhibitor development, but also towards development of pathway-specific clinical probes. However, it is often witnessed that the availability, immediate and long-term, of a high quality fragment-screening library is still beyond the reach of most academic laboratories. Within iNEXT (Infrastructure for NMR, EM and X-rays for Translational research), a EU-funded Horizon 2020 program, a collection of 782 fragments were assembled utilizing the concept of "poised fragments" with the aim to facilitate downstream synthesis of ligands with high affinity by fragment ligation. Herein, we describe the analytical procedure to assess the quality of this purchased and assembled fragment library by NMR spectroscopy. This quality assessment requires buffer solubility screening, comparison with LC/MS quality control and is supported by state-of-the-art software for high throughput data acquisition and on-the-fly data analysis. Results from the analysis of the library are presented as a prototype of fragment progression through the quality control process

Researching retired ex-servicemen: reflections on ethnographic encounters

The opportunities and challenges that younger, female, civilian researchers can encounter when undertaking ethnographic research with predominantly male military veterans are relatively underexplored sociologically. This is despite a growing literature on reflexivity in military studies over the past decade. To address this gap, we draw on symbolic interactionist insights to examine the reflective account of a British, female researcher in her mid-20s, who conducted qualitative research with 20 ‘older’ (aged 60+) retired servicemen from the Royal British Legion, a United Kingdom charity providing support for military veterans and their families. The study explored ex-servicemen’s embodied experiences of physical activity. The findings presented here cohere around four salient themes identified in the ethnographic reflections: (1) researcher positionality as a young, female, civilian researcher in a traditionally masculine militarised world; (2) managing distressing topics and interactional discomfort; (3) maintaining an ‘ethic of care’; and (4) dilemmas regarding representational issues and ex-servicemen’s embodied experiences

Equilibria-based Probabilistic Model Checking for Concurrent Stochastic Games

Probabilistic model checking for stochastic games enables formal verification of systems that comprise competing or collaborating entities operating in a stochastic environment. Despite good progress in the area, existing approaches focus on zero-sum goals and cannot reason about scenarios where entities are endowed with different objectives. In this paper, we propose probabilistic model checking techniques for concurrent stochastic games based on Nash equilibria. We extend the temporal logic rPATL (probabilistic alternating-time temporal logic with rewards) to allow reasoning about players with distinct quantitative goals, which capture either the probability of an event occurring or a reward measure. We present algorithms to synthesise strategies that are subgame perfect social welfare optimal Nash equilibria, i.e., where there is no incentive for any players to unilaterally change their strategy in any state of the game, whilst the combined probabilities or rewards are maximised. We implement our techniques in the PRISM-games tool and apply them to several case studies, including network protocols and robot navigation, showing the benefits compared to existing approaches

Boron-Based Inhibitors of the NLRP3 Inflammasome.

NLRP3 is a receptor important for host responses to infection, yet is also known to contribute to devastating diseases such as Alzheimer's disease, diabetes, atherosclerosis, and others, making inhibitors for NLRP3 sought after. One of the inhibitors currently in use is 2-aminoethoxy diphenylborinate (2APB). Unfortunately, in addition to inhibiting NLRP3, 2APB also displays non-selective effects on cellular Ca2+ homeostasis. Here, we use 2APB as a chemical scaffold to build a series of inhibitors, the NBC series, which inhibit the NLRP3 inflammasome in vitro and in vivo without affecting Ca2+ homeostasis. The core chemical insight of this work is that the oxazaborine ring is a critical feature of the NBC series, and the main biological insight the use of NBC inhibitors led to was that NLRP3 inflammasome activation was independent of Ca2+. The NBC compounds represent useful tools to dissect NLRP3 function, and may lead to oxazaborine ring-containing therapeutics

Quasi Harmonic Lattice Dynamics and Molecular Dynamics calculations for the Lennard-Jones solids

We present Molecular Dynamics (MD), Quasi Harmonic Lattice Dynamics (QHLD) and Energy Minimization (EM) calculations for the crystal structure of Ne, Ar, Kr and Xe as a function of pressure and temperature. New Lennard-Jones (LJ) parameters are obtained for Ne, Kr and Xe to reproduce the experimental pressure dependence of the density. We employ a simple method which combines results of QHLD and MD calculations to achieve densities in good agreement with experiment from 0 K to melting. Melting is discussed in connection with intrinsic instability of the solid as given by the QHLD approximation. (See http://www.fci.unibo.it/~valle for related papers)Comment: 7 pages, 5 figures, REVte