The ethics of ethnographic methods in conflict zones.

This article examines the ethics of using ethnographic methods in contemporary conflict zones. Ethnographic research is an embodied research practice of immersion within a field site whereby researchers use ethnographic sensibility to study how people make sense of their world. Feminist, conflict and peacebuilding scholars who research vulnerable populations and local dynamics especially value ethnographic approaches for their emphasis on contextual understanding, human agency, egalitarian research relationships and researcher empathy. While immersion leads to knowledge that can hardly be replaced by using more formal approaches, it also elicits ethical dilemmas. These arise not only from the specific research context but also from who the researcher is and how they may navigate violent and often misogynous settings. I argue that many dilemmas may and perhaps should not be overcome by researcher skill and perseverance. Instead, ethical challenges may lead researchers to adopt limited and/or uneven immersion in their field site, not as failed or flawed ethnography but as an ethical research strategy that incorporates ethnographic sensibility to a varying extent. Examining why researchers may opt for limited and uneven immersion is important because in conflict research, stereotypes of the intrepid (male) researcher with a neutral gaze still tend to mute open discussions of how gender, race, ethnicity, nationality, class and other background factors inevitably shape immersion. This article seeks to contribute to creating discursive space for these conversations, which are vital for researchers to analyse, reflect and write from the position of a 'vulnerable observer' and incorporate greater transparency in the discussion of research findings.

Discovery of GSK251: A Highly Potent, Highly Selective, Orally Bioavailable Inhibitor of PI3Kδ with a Novel Binding Mode.

Optimization of a previously reported lead series of PI3Kδ inhibitors with a novel binding mode led to the identification of a clinical candidate compound 31 (GSK251). Removal of an embedded Ames-positive heteroaromatic amine by reversing a sulfonamide followed by locating an interaction with Trp760 led to a highly selective compound 9. Further optimization to avoid glutathione trapping, to enhance potency and selectivity, and to optimize an oral pharmacokinetic profile led to the discovery of compound 31 (GSK215) that had a low predicted daily dose (45 mg, b.i.d) and a rat toxicity profile suitable for further development.

Identifying drug targets in tissues and whole blood with thermal-shift profiling.

Monitoring drug-target interactions with methods such as the cellular thermal-shift assay (CETSA) is well established for simple cell systems but remains challenging in vivo. Here we introduce tissue thermal proteome profiling (tissue-TPP), which measures binding of small-molecule drugs to proteins in tissue samples from drug-treated animals by detecting changes in protein thermal stability using quantitative mass spectrometry. We report organ-specific, proteome-wide thermal stability maps and derive target profiles of the non-covalent histone deacetylase inhibitor panobinostat in rat liver, lung, kidney and spleen and of the B-Raf inhibitor vemurafenib in mouse testis. In addition, we devised blood-CETSA and blood-TPP and applied it to measure target and off-target engagement of panobinostat and the BET family inhibitor JQ1 directly in whole blood. Blood-TPP analysis of panobinostat confirmed its binding to known targets and also revealed thermal stabilization of the zinc-finger transcription factor ZNF512. These methods will help to elucidate the mechanisms of drug action in vivo.

Puberty in female cavies (Cavia aperea) is affected by photoperiod and social conditions.

In many environments, photoperiod is a reliable predictor of ecological conditions. Such predictability generally declines towards low latitudes as the yearly cycle becomes less marked. It has been claimed that photoperiodic effects are small in cavies and guinea pigs that reproduce throughout the year. We here investigated photoperiodic influences on the onset of puberty in female cavies (Cavia aperea) and show that photoperiod exerts a major influence. Female pups kept in groups of two matured at about 47 days when born into lengthening (from 10:14 to 12.5:11.5 L:D) and 79 days when born into shortening day length (from 14.5:9.5 to 12:12 L:D) and kept under identical short day conditions after weaning on day 20 of life (12.25:11.75 L:D). As shown previously, social conditions, especially the presence of an adult male, proved important modifiers of the onset of maturity in females. Differential stress cannot be responsible for the social effects on puberty as social conditions did not affect cortisol levels in young females. We conclude that photoperiod plays an important role in gearing the onset of cavy reproduction to the seasons and that specific male stimuli rather than unspecific effects of stressors accelerate female maturation.

Discovery of a Highly Selective Tankyrase Inhibitor Displaying Growth Inhibition Effects against a Diverse Range of Tumor Derived Cell Lines.

The availability of high quality probes for specific protein targets is fundamental to the investigation of their function and their validation as therapeutic targets. We report the utilization of a dedicated chemoproteomic assay platform combining affinity enrichment technology with high-resolution protein mass spectrometry to the discovery of a novel nicotinamide isoster, the tetrazoloquinoxaline 41, a highly potent and selective tankyrase inhibitor. We also describe the use of 41 to investigate the biology of tankyrase, revealing the compound induced growth inhibition of a number of tumor derived cell lines, demonstrating the potential of tankyrase inhibitors in oncology.

A Modular Probe Strategy for Drug Localization, Target Identification and Target Occupancy Measurement on Single Cell Level.

Late stage failures of candidate drug molecules are frequently caused by off-target effects or inefficient target engagement in vivo. In order to address these fundamental challenges in drug discovery, we developed a modular probe strategy based on bioorthogonal chemistry that enables the attachment of multiple reporters to the same probe in cell extracts and live cells. In a systematic evaluation, we identified the inverse electron demand Diels-Alder reaction between trans-cyclooctene labeled probe molecules and tetrazine-tagged reporters to be the most efficient bioorthogonal reaction for this strategy. Bioorthogonal biotinylation of the probe allows the identification of drug targets in a chemoproteomics competition binding assay using quantitative mass spectrometry. Attachment of a fluorescent reporter enables monitoring of spatial localization of probes as well as drug-target colocalization studies. Finally, direct target occupancy of unlabeled drugs can be determined at single cell resolution by competitive binding with fluorescently labeled probe molecules. The feasibility of the modular probe strategy is demonstrated with noncovalent PARP inhibitors.

Type III secretion chaperones ShcS1 and ShcO1 from Pseudomonas syringae pv. tomato DC3000 bind more than one effector.

The hrp-type III secretion (TTS) system is a key pathogenicity factor of the plant pathogen Pseudomonas syringae pv. tomato DC3000 that translocates effector proteins into the cytosol of the eukaryotic host cell. The translocation of a subset of effectors is dependent on specific chaperones. In this study an operon encoding a TTS chaperone (ShcS1) and the truncated effector HopS1' was characterized. Yeast two-hybrid analysis and pull-down assays demonstrated that these proteins interact. Using protein fusions to AvrRpt2 it was shown that ShcS1 facilitates the translocation of HopS1', suggesting that ShcS1 is a TTS chaperone for HopS1' and that amino acids 1 to 118 of HopS1' are required for translocation. P. syringae pv. tomato DC3000 carries two shcS1 homologues, shcO1 and shcS2, which are located in different operons, and both operons include additional putative effector genes. Transcomplementation experiments showed that ShcS1 and ShcO1, but not ShcS2, can facilitate the translocation of HopS1' :: AvrRpt2. To characterize the specificities of the putative chaperones, yeast two-hybrid interaction studies were performed between the three chaperones and putative target effectors. These experiments showed that both ShcS1 and ShcO1 bind to two different effectors, HopS1' and HopO1-1, that share only 16% amino acid sequence identity. Using gel filtration it was shown that ShcS1 forms homodimers, and this was confirmed by yeast two-hybrid experiments. In addition, ShcS1 is also able to form heterodimers with ShcO1. These data demonstrate that ShcS1 and ShcO1 are exceptional class IA TTS chaperones because they can bind more than one target effector.