Native Mass Spectrometry for the Study of PROTAC GNE-987-Containing Ternary Complexes

PRO teolysis TA rgeting C himeras (PROTACs) promote the degradation, rather than inhibition, of a drug target as a mechanism for therapeutic treatment. Bifunctional PROTAC molecules allow simultaneous binding of both the target protein and an E3-Ubiquitin ligase, bringing the two proteins into close spatial proximity to allow ubiquitinylation and degradation of the target protein via the cell's endogenous protein degradation pathway. We utilized native mass spectrometry (MS) to study the ternary complexes promoted by the previously reported PROTAC GNE-987 between Brd4 bromodomains 1 and 2, and Von Hippel Lindeau E3-Ubiquitin Ligase. Native MS at high resolution allowed us to measure ternary complex formation as a function of PROTAC concentration to provide a measure of complex affinity and stability, whilst simultaneously measuring other intermediate protein species. Native MS provides a high-throughput, low sample consumption, direct screening method to measure ternary complexes for PROTAC development.     

Short-term liveweight changes of dairy cows measured by stationary and walk-over weighing scales

Monitoring and detecting individual cows' liveweight (LW) and liveweight change (LWC) are important for estimation of nutritional requirements and health management, and could be useful to measure short-term feed intake, water consumption, defecation, and urination. Walk-over weighing (WOW) systems can facilitate measurements of LW for these purposes, providing automated LW recorded at different times of the day. We conducted a field study to (1) quantify the contribution of feed and water intake, as well as urine and feces excretions, to short-term LWC and (2) determine the feasibility of stationary and WOW scales to detect subtle changes in LW as a result of feed and water intake, urination, and defecation. In this experiment, 10 cows walked through a WOW system and then stood individually on a stationary scale collecting weights at 10 and 3.3 Hz, respectively. Cows were offered 4 kg of feed and 10 kg of water on the stationary scale. For each animal, LW before and after eating and drinking was then calculated using different approaches. Liveweight change was calculated as the difference between the initial and final LW before and after eating and drinking for each statistical measure. The weights of feed intake, water consumption, urination, and defecation were measured and used as predictors of LWC. Urine and feces were collected from individual cows while the cow was on the scale, using a container, and weighed separately. The agreement between LWC measured using either stationary or WOW scales was assessed to determine the sensitivity of the scales to detect subtle changes in LW using the coefficient of determination (R²), Lin's concordance correlation coefficient (CCC), and mean bias. The prediction model showed that most of the regression coefficients were not significantly different from +1.0 for feed and water, or ?1.0 for urine and feces. The R² and CCC values demonstrated a satisfactory agreement between calculated and stationary LWC and values ranged from 0.60 to 0.92 and 0.71 to 0.94, respectively. A moderate agreement was achieved between calculated and automated LWC with R² and Lin's CCC values of 0.45 to 0.63 and 0.60 to 0.74, respectively. Therefore, results demonstrated that new algorithms and data processing methods need to be continuously explored and improved to obtain accurate measurements of LW to measure changes in LW, especially from WOW scales.     

The impacts of cattle access points on deposited sediment levels in headwater streams in Ireland

Cattle access to streams has been linked globally with degradation of stream water quality, driven largely by bank erosion and resultant instream, fine sediment deposition. The majority of evidence on such effects is however based in arid and semiarid regions of the United States and Australia, with few studies relating to cool temperate climates such as Northwest Europe. In this study, “Quorer” resuspendable sediment samples were taken from riffle geomorphic units upstream (control) and at two points downstream (pressure and recovery) of cattle access points in headwater streams in agricultural catchments in Ireland to assess levels of deposited stream sediment. Samples were taken in April/May (2016) prior to the grazing season and in October (2016) at the end of the grazing season. Sites in good‐high ecological status catchments and less than good ecological status catchments were included in the study. Higher levels of sediment were found downstream of cattle access points in both good‐high status and less than good status catchments; however, the impacts of access points were spatially confined to, in most cases, the area immediately downstream of the point of access. There was a strong correlation between deposited sediment mass and organic matter (OM) mass, with levels of OM increasing linearly with deposited sediment mass. Levels of measured sediment were negatively correlated with riparian habitat health (measured using a qualitative habitat assessment). The results of this study highlight the need for measures to prevent cattle access to headwater streams where access points can be many in order to manage local habitat quality and downstream water quality issues.     

An Atomistic Understanding of Allosteric Inhibition of Glutamate Racemase: a Dampening of Native Activation Dynamics

Glutamate racemases (GR) are members of the family of bacterial enzymes known as cofactor-independent racemases and epimerases and catalyze the stereoinversion of glutamate. D-amino acids are universally important for the proper construction of viable bacterial cell walls, and thus have been repeatedly validated as attractive targets for novel antimicrobial drug design. Significant aspects of the mechanism of this challenging stereoinversion remain unknown. The current study employs a combination of MD and QM/MM computational approaches to show that the GR from H. pylori must proceed via a pre-activation step, which is dependent on the enzyme's flexibility. This mechanism is starkly different from previously proposed mechanisms. These findings have immediate pharmaceutical relevance, as the H. pylori GR enzyme is a very attractive allosteric drug target. The results presented in this study offer a distinctly novel understanding of how AstraZeneca's lead series of inhibitors cripple the H. pylori GR's native motions, via prevention of this critical chemical pre-activation step. Our experimental studies, using SPR, fluorescence and NMR WaterLOGSY, show that H. pylori GR is not inhibited by the uncompetitive mechanism originally put forward by Lundqvist et al.. The current study supports a deep connection between native enzyme motions and chemical reactivity, which has strong relevance to the field of allosteric drug discovery.     

Improved method for roadside barrier length of need modeling using real-world trajectories

The 2011 AASHTO Roadside Design Guide (RDG) contains perhaps the most widely used procedure for choosing an appropriate length of need (LON) for roadside barriers. However, this procedure has several limitations. The procedure uses a highly simplified model of vehicle departure, and the procedure does not allow designers to specify an explicit level of protection. A new procedure for choosing LON that addresses these limitations is presented in this paper. This new procedure is based on recent, real-world road departure trajectories and uses this departure data in a more realistic way. The new procedure also allows LON to be specified for a precisely known level of protection – a level which can be based on number of crashes, injury outcomes or even estimated crash cost – while still remaining straightforward and quick to use like the 2011 RDG procedure.     

Influence of Pluronic coating formulation on iron oxide nanoparticle transport in natural and oil-impacted sandy aquifer media

Targeted delivery of nanoparticles has the potential to enhance remediation and characterization of sites contaminated with non-aqueous phase liquids (NAPLs) by ensuring delivery of treatment or contrast agents to the NAPL/water interface. For a targeted delivery technique to be successful, nanoparticles must be capable of transporting through porous media and binding to NAPLs under relevant geological conditions. In this study, successful targeted delivery of nanoparticles to sandy aquifer material mixed with crude oil was achieved using an active targeting technique based on an amphiphilic polymer coating. It was determined that the molecular structure and concentration of the nanoparticle coating greatly influenced the recovery of nanoparticles injected into saturated columns. Coatings with longer polymer molecules and lower polymer concentrations reduced recovery, and the nanoparticle coating formulation could be adjusted to improve transport while maintaining targeted binding behaviour. This study demonstrated that nanoparticle retention in oil-impacted sand exceeded that of clean sand in flow through experiments, indicating that a nanoparticle targeted delivery strategy for soil contaminated with LNAPLs such as crude oil is possible under the experimental conditions explored.     

Comparison of sediments and organisms in identifying sources of biologically available trace metal contamination

Sediments and an indicator organism (Macoma balthica, a deposit-feeding bivalve) were used to assess the relative importance of secondary sewage, urban runoff, a landfill containing metal-enriched ash wastes and a yacht harbor in contributing to Ag, Cu and Zn enrichment in South San Francisco Bay. Spatial gradients in sediments and organisms showed Cu and Ag enrichment originated from sewage discharge, whereas Zn enrichment originated from both sewage and urban runoff. Elevated concentrations of Cu in the sediments of the yacht harbor resulted from a high abundance of fine particles. The biological availability of Cu, Ag and Zn did not coincide with metal enrichment in sediments. The availability of Cu and Ag was greatest nearest the sewage outfall and greater in winter and spring than in summer. The availability of Zn in urban runoff appeared to be lower than the availability of Zn associated with sewage.     

The characterisation of friction stir welding process effects on stiffened panel buckling performance

The introduction of advanced welding methods as an alternative joining process to riveting in the manufacture of primary aircraft structure has the potential to realise reductions in both manufacturing costs and structural weight. However, welding processes can introduce undesirable residual stresses and distortions in the final fabricated components, as well as localised loss of mechanical properties at the weld joints. The aim of this research is to determine and characterise the key process effects of friction stir welding assembly methods on stiffened panel static strength performance. Utilising experimentally validated finite element modelling methods, it is demonstrated that welding induced residual stresses have a significant influence, and that collapse behaviour is less sensitive to advanced process effects and process effect magnitudes than panel initial buckling behaviour.     

The treatment of malt whiskey distillery waste using the fungus geotrichum candidum

The imperfect fungus Geotrichum candidum, when grown in batch and in single and two-stage continuous laboratory culture, has been shown to produce reductions of up to 92% in the BOD loading of Irish malt whiskey distillery waste. Maximum COD and TOC reductions were 81% and 76% respectively. Yields of fungal biomass of over 30 g 1⁻¹, containing more than 45% “true” protein, have been obtained. The economic possibilities of employing such a process for waste treatment and perhaps single-cell protein production seem encouraging.