Direct Phenotypic Screening in Mice: Identification of Individual, Novel Antinociceptive Compounds from a Library of 734 821 Pyrrolidine Bis-piperazines

The hypothesis in the current study is that the simultaneous direct in vivo testing of thousands to millions of systematically arranged mixture-based libraries will facilitate the identification of enhanced individual compounds. Individual compounds identified from such libraries may have increased specificity and decreased side effects early in the discovery phase. Testing began by screening ten diverse scaffolds as single mixtures (ranging from 17 340 to 4 879 681 compounds) for analgesia directly in the mouse tail withdrawal model. The “all X” mixture representing the library TPI-1954 was found to produce significant antinociception and lacked respiratory depression and hyperlocomotor effects using the Comprehensive Laboratory Animal Monitoring System (CLAMS). The TPI-1954 library is a pyrrolidine bis-piperazine and totals 738 192 compounds. This library has 26 functionalities at the first three positions of diversity made up of 28 392 compounds each (26 × 26 × 42) and 42 functionalities at the fourth made up of 19 915 compounds each (26 × 26 × 26). The 120 resulting mixtures representing each of the variable four positions were screened directly in vivo in the mouse 55 °C warm-water tail-withdrawal assay (ip administration). The 120 samples were then ranked in terms of their antinociceptive activity. The synthesis of 54 individual compounds was then carried out. Nine of the individual compounds produced dose-dependent antinociception equivalent to morphine. In practical terms what this means is that one would not expect multiexponential increases in activity as we move from the all-X mixture, to the positional scanning libraries, to the individual compounds. Actually because of the systematic formatting one would typically anticipate steady increases in activity as the complexity of the mixtures is reduced. This is in fact what we see in the current study. One of the final individual compounds identified, TPI 2213-17, lacked significant respiratory depression, locomotor impairment, or sedation. Our results represent an example of this unique approach for screening large mixture-based libraries directly in vivo to rapidly identify individual compounds

Comparing Notes: Recording and Criticism

This chapter charts the ways in which recording has changed the nature of music criticism. It both provides an overview of the history of recording and music criticism, from the advent of Edison’s Phonograph to the present day, and examines the issues arising from this new technology and the consequent transformation of critical thought and practice

Wider Still and Wider: British Music Criticism since the Second World War

This chapter provides the first historical examination of music criticism in Britain since the Second World War. In the process, it also challenges the simplistic prevailing view of this being a period of decline from a golden age in music criticism

Green fluorescent protein based indicators of dynamic redox changes and reactive oxygen species

Alterations in the redox equilibrium are precipitated by changing either the glutathione/glutathione-disulfide ratio (GSH/GSSG) and/or the reduced/oxidized thioredoxin ratio. Redox-sensitive green fluorescent proteins (GFP) allow real time visualization of the oxidation state of the indicator while canceling out the amount of indicator and the absolute optical sensitivity. Because the indicator is genetically encoded, it can be targeted to specific proteins or organelles of interest and expressed in a wide variety of cells and organisms. We evaluated roGFP1 and roGFP2 with physiologically or toxicologically relevant oxidants both in vitro and in living mammalian cells. Furthermore, we investigated the response of the redox probes under physiological redox changes during superoxide bursts in macrophage cells, hyperoxic and hypoxic conditions, and in responses to H2O2-stimulating agents, e.g. epidermal growth factor and lysophosphatidic acid. Placing positive charges near cysteine residues increases sensitivity to oxidation by H2O2. We designed a series of roGFP variants with outward-facing lysine residues placed in close proximity to the key cysteine residues. The double lysine mutant RoGFP2-F223KA206K (roGFP2-FA) was used in three distinct applications for roGFPs; The protein was expressed in primary neurons; the redox indicator was evaluated for sensitivity to environmental toxicants; and finally the potential for clamping roGFP2-FA in a particular redox state following equilibration using formaldehyde was investigated. Sensitivity of selenocysteine to oxidation was harnessed to further optimize roGFPs. By employing the 3'UTR of gluthathione peroxidase and an opal codon at each of the cysteine residues of roGFP we were able to create seleno- GFPs, which have a greater sensitivity to oxidation. A rational approach was taken for the discovery of the green fluorescent protein based singlet oxygen sensors (SOS- GFP). There was a strong response to singlet oxygen in particularly for mutations expressing histidines at positions 147 and 204. These double histidine mutants were evaluated for specificity and sensitivity to singlet oxygen. The most reactive/sensitive mutations were then used in biological settings to sense singlet oxygen generated from a nearby sourc

Green fluorescent protein based indicators of dynamic redox changes and reactive oxygen species

Alterations in the redox equilibrium are precipitated by changing either the glutathione/glutathione-disulfide ratio (GSH/GSSG) and/or the reduced/oxidized thioredoxin ratio. Redox-sensitive green fluorescent proteins (GFP) allow real time visualization of the oxidation state of the indicator while canceling out the amount of indicator and the absolute optical sensitivity. Because the indicator is genetically encoded, it can be targeted to specific proteins or organelles of interest and expressed in a wide variety of cells and organisms. We evaluated roGFP1 and roGFP2 with physiologically or toxicologically relevant oxidants both in vitro and in living mammalian cells. Furthermore, we investigated the response of the redox probes under physiological redox changes during superoxide bursts in macrophage cells, hyperoxic and hypoxic conditions, and in responses to H2O2-stimulating agents, e.g. epidermal growth factor and lysophosphatidic acid. Placing positive charges near cysteine residues increases sensitivity to oxidation by H2O2. We designed a series of roGFP variants with outward-facing lysine residues placed in close proximity to the key cysteine residues. The double lysine mutant RoGFP2-F223KA206K (roGFP2-FA) was used in three distinct applications for roGFPs; The protein was expressed in primary neurons; the redox indicator was evaluated for sensitivity to environmental toxicants; and finally the potential for clamping roGFP2-FA in a particular redox state following equilibration using formaldehyde was investigated. Sensitivity of selenocysteine to oxidation was harnessed to further optimize roGFPs. By employing the 3'UTR of gluthathione peroxidase and an opal codon at each of the cysteine residues of roGFP we were able to create seleno- GFPs, which have a greater sensitivity to oxidation. A rational approach was taken for the discovery of the green fluorescent protein based singlet oxygen sensors (SOS- GFP). There was a strong response to singlet oxygen in particularly for mutations expressing histidines at positions 147 and 204. These double histidine mutants were evaluated for specificity and sensitivity to singlet oxygen. The most reactive/sensitive mutations were then used in biological settings to sense singlet oxygen generated from a nearby sourc

Communicating with students: what works, what doesn’t and how to get your message across

Across the university, communicating with students is key.   However, disseminating important messages, getting students to engage with course information, events, or feedback surveys can often be difficult. Messages often get lost or confused, and students have fed back that they struggle with the sheer volume of emails and ‘important’ messages coming their way.   This workshop, delivered by Corporate Communications, will explore the best ways of communicating with students, when to do it, when not to do it, and how to make best use of the platforms available from central comms.  It will cover social media, Canvas announcements, emails and digital signage.   The workshop will also consider how individuals and teams can best utilise student-friendly language to increase engagement using some key case studies of what has worked well over the past 12 months.   At the end of the workshop, delegates should have insight in to how they can improve communication with students, as well as a few practical tips and techniques on how to manage different approaches, dependent on the channel.   Any potential delegates who currently run LJMU-related social media channels, or communicate with students regularly will find this session particularly useful. Please note this session will not explicitly address one-to-one communication, although many of the principles may still apply

Bridging immunogenetics and immunoproteomics: Model positional scanning library analysis for Major Histocompatibility Complex class II DQ in Tursiops truncatus.

The Major Histocompatibility Complex (MHC) is a critical element in mounting an effective immune response in vertebrates against invading pathogens. Studies of MHC in wildlife populations have typically focused on assessing diversity within the peptide binding regions (PBR) of the MHC class II (MHC II) family, especially the DQ receptor genes. Such metrics of diversity, however, are of limited use to health risk assessment since functional analyses (where changes in the PBR are correlated to recognition/pathologies of known pathogen proteins), are difficult to conduct in wildlife species. Here we describe a means to predict the binding preferences of MHC proteins: We have developed a model positional scanning library analysis (MPSLA) by harnessing the power of mixture based combinatorial libraries to probe the peptide landscapes of distinct MHC II DQ proteins. The algorithm provided by NNAlign was employed to predict the binding affinities of sets of peptides generated for DQ proteins. These binding affinities were then used to retroactively construct a model Positional Scanning Library screen. To test the utility of the approach, a model screen was compared to physical combinatorial screens for human MHC II DP. Model library screens were generated for DQ proteins derived from sequence data from bottlenose dolphins from the Indian River Lagoon (IRL) and the Atlantic coast of Florida, and compared to screens of DQ proteins from Genbank for dolphin and three other cetaceans. To explore the peptide binding landscape for DQ proteins from the IRL, combinations of the amino acids identified as active were compiled into peptide sequence lists that were used to mine databases for representation in known proteins. The frequency of which peptide sequences predicted to bind the MHC protein are found in proteins from pathogens associated with marine mammals was found to be significant (p values <0.0001). Through this analysis, genetic variation in MHC (classes I and II) can now be associated with the binding repertoires of the expressed MHC proteins and subsequently used to identify target pathogens. This approach may be eventually applied to evaluate individual population and species risk for outbreaks of emerging diseases

Use and Implications of the Harmonic Mean Model on Mixtures for Basic Research and Drug Discovery

The use of the harmonic mean model for predicting the activities of a given mixture and its constituents has not previously been explored in the context of combinatorial libraries and drug discovery. Herein, the analyses of historical data confirm the harmonic mean as an accurate predictor of mixture activity. The implications of these results are discussed

Use and Implications of the Harmonic Mean Model on Mixtures for Basic Research and Drug Discovery

The use of the harmonic mean model for predicting the activities of a given mixture and its constituents has not previously been explored in the context of combinatorial libraries and drug discovery. Herein, the analyses of historical data confirm the harmonic mean as an accurate predictor of mixture activity. The implications of these results are discussed