SLIDES: Paying the Price for Power: When L.A. Turns on the Lights, Northwestern New Mexico Feels It

Presenter: Jonathan Thompson, Editor, High Country News 23 slide

SLIDES: Paying the Price for Power: When L.A. Turns on the Lights, Northwestern New Mexico Feels It

Presenter: Jonathan Thompson, Editor, High Country News 23 slide

Administrative Law\u27s Extraordinary Cases

The Supreme Court\u27s major questions doctrine is grounded in the Chevron framework. Reconstituting it as a major rules exception to Chevron or as a non-delegation principle are misguided and create greater uncertainty

Iron uptake and homeostasis in the veterinary pathogen Rhodococcus equi: an integrated omics approach

Rhodococcus equi, a veterinary pathogen that causes pyogranulomatous pneumonia, can secrete low molecular weight chelators called siderophores to scavenge iron when its bioavailability is limited. When iron is plentiful, synthesis of siderophores and ferri–siderophore transport systems are repressed. Current literature on bacterial iron regulation and homeostasis indicates two distinct protein families of global iron-dependent transcriptional repressor: Fur and DtxR. Gram-negative bacteria produce Fur to regulate iron uptake genes and the biosynthesis of siderophores in response to the iron level in the cell. However, the Gram-positive Corynebacteriaceae produce DtxR-like proteins to regulate analogous genes. Much remains undefined with respect to rhodococcal siderophore biosynthesis and uptake. Detailed analysis of the R. equi 103S genome for genes related to iron homeostasis identified two potential metal regulatory genes each from the Fur and DtxR families: iron dependent regulatory protein (IdeR), Diphtheria toxin repressor (DtxR), Ferric uptake regulator A (FurA) and Ferric uptake regulator B (FurB). Bioinformatic analysis confirmed that this complement of genes was conserved throughout Rhodococcus and the Corynebacteriaceae in general. To investigate their individual roles in metal homeostasis, molecular cloning and gene expression was performed, to facilitate analysis of regulator-metal specificities. Each gene was cloned but over-expression for functional analysis could only be achieved for ideR; thus, a thorough systematic analysis could not be achieved. In order to address their individual roles, homology-based protein modelling was used, and comparisons made with characterised homologues from M. tuberculosis. The geometrical conservation of key ligand amino acid residues strongly suggests R. equi utilises ideR as an iron regulator; furB as a zinc regulator, dtxR as a manganese regulator and furA as an oxidative stress response protein. Most bacteria generate an exaggerated response to iron limitation in vitro, however R. equi produces very small siderophore yields s, which has complicated their characterisation. In-frame deletion of the putative metal regulator genes ideR, dtxR, furA and furB was attempted in order to address the hypothesis that de-repression might generate greater yields. All genes were deleted individually; a marked phenotypic difference was noted only for R. equi-ΔfurA, which significantly upregulated the catalase encoded by the neighbouring gene and was coincidentally hyper-resistant to hydrogen peroxide. Surprisingly, analysis of siderophore production in the mutants indicated no increase in yield. The thesis discusses the relevance of this observation to microbial ecology. The availability of these mutants in combination with their predicted metal specificities facilitates the design of experiments to define their individual roles in metal homeostasis beyond the scope of this thesis. The combination of ‘omic’ analyses was attempted here to initiate the ultimate definition of the complex molecular network associated with iron uptake. The genomic investigation informed hypothesis building for the other omic analyses. It suggested R. equi is capable of synthesising two siderophores, rhequibactin and rhequichelin; up to three had previously been postulated in the literature. Culture optimisation was required to deliver a robust experimental design to impose iron limitation in isolation from other stresses. Once medium composition and biomarker-indicated harvesting criteria were established, biomass and associated secretomes were produced en masse for integrated omics analysis. A comparative untargeted metabolomics study demonstrated an adapted iron-starved metabolome; strong siderophore candidates were then investigated using a targeted strategy. A strong candidate metabolite was identified by mass that appeared to be responsible for a heterobactin-like chromophore, however further biochemical characterisation has been elusive. Interestingly, the metabolite readily precipitates on complexation with iron, an observation also made for heterobactins. Secondly, a transcriptomic study was attempted to study the global gene expression under iron starvation, and the impact of the loss of the IdeR in the deletion mutant generated in this work. However, the RNA extraction proved particularly challenging likely due to difficulties arising from lysis of the mycolic acid-containing cell wall. In the absence of a high-quality transcriptome sample, the study did not advance further and other aspects of the study were prioritised. Finally, a comparative proteomic analysis into iron regulatory mechanisms associated with the rhodococcal cell wall was performed. Current literature deliberates how R. equi uses a range of strategies to overcome iron limitation through proposed uptake mechanisms associated with translocation across the cytoplasmic membrane via ABC transport systems, while no consideration has yet been made with regards to transport across the mycolic acid-containing cell wall structure. In this study no obvious candidate proteins for ferri-sideophore transport across the mycolate region were identified, therefore it is possible that R. equi utilises facilitated diffusion via a porin for entry of ferri-siderophore complexes into the pseudoperiplasm, where a substrate-binding lipoprotein may act as the primary receptor to facilitate cytosolic transfer through an ABC transport system

Galvanic corrosion of aluminium–copper model alloys

Galvanic coupling between different α and θ phase-containing model Al–Cu alloys, deposited by magnetron sputtering, has revealed that the anodic α phase did not suffer corrosion and remained in the passive state in sulphate solution. Conversely, sulphate ions induced pitting of the cathodic θ phase. Pitting susceptibility of the cathode increased when the difference between the copper content of the anode and cathode increased. Similar observations were made for all the galvanic couples; further, the higher the copper content of a phase, then the greater its susceptibility to pitting