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The New Zealand glowworm, Arachnocampa luminosa, is well-known for displays of blue-green bioluminescence, but details of its bioluminescent chemistry have been elusive. The glowworm is evolutionarily distant from other bioluminescent creatures studied in detail, including the firefly. We have isolated and characterised the molecular components of the glowworm luciferase-luciferin system using chromatography, mass spectrometry and 1 H NMR spectroscopy. The purified luciferase enzyme is in the same protein family as firefly luciferase (31% sequence identity). However, the luciferin substrate of this enzyme is produced from xanthurenic acid and tyrosine, and is entirely different to that of the firefly and known luciferins of other glowing creatures. A candidate luciferin structure is proposed, which needs to be confirmed by chemical synthesis and bioluminescence assays. These findings show that luciferases can evolve independently from the same family of enzymes to produce light using structurally different luciferins.

Background: The purpose of this study was to determine whether mechanically ventilated trauma patients with a positive urine drug screen (UDS) for cocaine and/or amphetamines have different opioid analgesic and sedative requirements compared with similar patients with a negative drug screen for these stimulants., Methods: This retrospective, single-center cohort study at a tertiary care, academic medical and level 1 trauma center in the United States included patients ≥16 years of age who were admitted to an adult intensive care unit with a diagnosis of trauma between 2009 and 2013 with a UDS documented within 24 hours of admission, and were mechanically ventilated for >24 hours. The primary end point was the daily dose of opioid received during mechanical ventilation, expressed as morphine equivalents, for patients presenting with a positive UDS for cocaine and/or amphetamines compared with patients with a negative UDS for these stimulants. Secondary end points included the daily benzodiazepine dose and median infusion rates of propofol and dexmedetomidine received during mechanical ventilation, duration of mechanical ventilation, intensive care unit and hospital length of stay, and in-hospital mortality. Analgesic and sedative goals were similar for the duration of the study period, and both intermittent and continuous infusions of opioids and sedatives were administered to achieve these targets, although a standardized approach was not used. A multivariate logistic regression analysis and a propensity-adjusted model evaluated patient characteristics predictive of a higher median opioid requirement., Results: A total of 150 patients were included in the final analysis. In a univariate analysis, opioid and sedative requirements were similar for patients presenting with a positive UDS for cocaine and/or amphetamines compared with patients with a negative UDS for these stimulants. In the multivariate regression analysis, increasing age and Abbreviated Injury Scale (head and neck) were associated with decreased daily opioid requirements (odds ratio [OR], .95, 95% confidence interval [CI], .93-.97 and OR, .71, 95% CI, .65-.77, respectively), whereas preinjury stimulant use was not predictive of opioid requirements (OR, .88, 95% CI, .40-1.90). In a propensity score--adjusted model, preinjury stimulant use was similarly not predictive of opioid requirements during mechanical ventilation (OR, .97, 95% CI, .44-2.11)., Conclusions: For trauma patients presenting with acute, preinjury use of cocaine and/or amphetamines, analgesic and sedative requirements are variables and may not be greater than those patients presenting with a stimulant-negative UDS to achieve desirable pain control and depth of sedation, although this observation should be interpreted cautiously in light of the wide CI observed in the propensity score--adjusted model. Although unexpected, these findings indicate that empirically increasing analgesic and sedative doses based on positive UDS results for these stimulants may not be necessary.

The Japanese firefly squid Hotaru-ika (Watasenia scintillans) produces intense blue light from photophores at the tips of two arms. These photophores are densely packed with protein microcrystals that catalyse the bioluminescent reaction using ATP and the substrate coelenterazine disulfate. The squid is the only organism known to produce light using protein crystals. We extracted microcrystals from arm tip photophores and identified the constituent proteins using mass spectrometry and transcriptome libraries prepared from arm tip tissue. The crystals contain three proteins, wsluc1-3, all members of the ANL superfamily of adenylating enzymes. They share 19 to 21% sequence identity with firefly luciferases, which produce light using ATP and the unrelated firefly luciferin substrate. We propose that wsluc1-3 form a complex that crystallises inside the squid photophores, and that in the crystal one or more of the proteins catalyses the production of light using coelenterazine disulfate and ATP. These results suggest that ANL superfamily enzymes have independently evolved in distant species to produce light using unrelated substrates.

Background: The New Zealand glowworm is the larva of a carnivorous fungus gnat that produces bioluminescence to attract prey. The bioluminescent system of the glowworm is evolutionarily distinct from other well-characterised systems, especially that of the fireflies, and the molecules involved have not yet been identified. We have used high throughput sequencing technology to produce a transcriptome for the glowworm and identify transcripts encoding proteins that are likely to be involved in glowworm bioluminescence., Results: Here we report the sequencing and annotation of the first transcriptome of the glowworm, and a differential analysis of expression from the glowworm light organ compared with non-light organ tissue. The analysis identified six transcripts encoding proteins that are potentially involved in glowworm bioluminescence. Three of these proteins are members of the ANL superfamily of adenylating enzymes, with similar amino acid sequences to that of the luciferase enzyme found in fireflies (31 to 37 % identical), and are candidate luciferases for the glowworm bioluminescent system. The remaining three transcripts encode putative aminoacylase, phosphatidylethanolamine-binding and glutathione S-transferase proteins., Conclusions: This research provides a basis for further biochemical studies into how the glowworm produces light, and a source of genetic information to aid future ecological and evolutionary studies of the glowworm.

Dendritic cells (DCs) competent to express the regulatory enzyme IDO in mice are a small but distinctive subset of DCs. Previously, we reported that a high-dose systemic CpG treatment to ligate TLR9 in vivo induced functional IDO exclusively in splenic CD19(+) DCs, which stimulated resting Foxp3-lineage regulatory T cells (Tregs) to rapidly acquire potent suppressor activity. In this paper, we show that IDO was induced in spleen and peripheral lymph nodes after CpG treatment in a dose-dependent manner. Induced IDO suppressed local T cell responses to exogenous Ags and inhibited proinflammatory cytokine expression in response to TLR9 ligation. IDO induction did not occur in T cell-deficient mice or in mice with defective B7 or programmed death (PD)-1 costimulatory pathways. Consistent with these findings, CTLA4 or PD-1/PD-ligand costimulatory blockade abrogated IDO induction and prevented Treg activation via IDO following high-dose CpG treatment. Consequently, CD4(+)CD25(+) T cells uniformly expressed IL-17 shortly after TLR9 ligation. These data support the hypothesis that constitutive interactions from activated T cells or Tregs and IDO-competent DCs via concomitant CTLA4→B7 and PD-1→PD-ligand signals maintain the default potential to regulate T cell responsiveness via IDO. Acute disruption of these nonredundant interactions abrogated regulation via IDO, providing novel perspectives on the proinflammatory effects of costimulatory blockade therapies. Moreover, interactions between IDO-competent DCs and activated T cells in lymphoid tissues may attenuate proinflammatory responses to adjuvants such as TLR ligands.

Background: Streptococcus pneumoniae is a globally important pathogen. The Gram-positive diplococcus is a leading cause of pneumonia, otitis media, bacteremia, and meningitis, and antibiotic resistant strains have become increasingly common over recent years. Alanine racemase is a ubiquitous enzyme among bacteria and provides the essential cell wall precursor, D-alanine. Since it is absent in humans, this enzyme is an attractive target for the development of drugs against S. pneumoniae and other bacterial pathogens., Results: Here we report the crystal structure of alanine racemase from S. pneumoniae (AlrSP). Crystals diffracted to a resolution of 2.0 Å and belong to the space group P3121 with the unit cell parameters a = b = 119.97 Å, c = 118.10 Å, α = β = 90° and γ = 120°. Structural comparisons show that AlrSP shares both an overall fold and key active site residues with other bacterial alanine racemases. The active site cavity is similar to other Gram positive alanine racemases, featuring a restricted but conserved entryway., Conclusions: We have solved the structure of AlrSP, an essential step towards the development of an accurate pharmacophore model of the enzyme, and an important contribution towards our on-going alanine racemase structure-based drug design project. We have identified three regions on the enzyme that could be targeted for inhibitor design, the active site, the dimer interface, and the active site entryway.

Candida albicans exo-β-1,3-glucanase (Exg; EC 3.2.1.58) is implicated in cell wall β-D-glucan remodelling through its glucosyl hydrolase and/or transglucosylase activities. A pair of antiparallel phenylalanyl residues (F144 and F258) flank the entrance to the active site pocket. Various Exg mutants were studied using steady-state kinetics and crystallography aiming to understand the roles played by these residues in positioning the β-1,3-D-glucan substrate. Mutations at the Phe-Phe entranceway demonstrated the requirement for double-sided CH/π interactions at the +1 subsite, and the necessity for phenylalanine rather than tyrosine or tryptophan. The Tyr-Tyr double mutations introduced ordered water molecules into the entranceway. A third Phe residue (F229) nearby was evaluated as a possible +2 subsite. The inactive double mutant E292S/F229A complexed with laminaritriose has provided the first picture of substrate binding to Exg and demonstrated how the Phe-Phe arrangement acts as a clamp at the +1 subsite. The terminal sugar at the -1 site showed displacement from the position of a monosaccharide analogue with interchange of water molecules and sugar hydroxyls. An unexpected additional glucose binding site, well removed from the active site, was revealed. This site may enable Exg to associate with the branched glucan structure of the C. albicans cell wall., (© 2010 The Authors Journal compilation © 2010 FEBS.)

Polycomb group proteins have an essential role in the epigenetic maintenance of repressive chromatin states. The gene-silencing activity of the Polycomb repressive complex 2 (PRC2) depends on its ability to trimethylate lysine 27 of histone H3 (H3K27) by the catalytic SET domain of the EZH2 subunit, and at least two other subunits of the complex: SUZ12 and EED. Here we show that the carboxy-terminal domain of EED specifically binds to histone tails carrying trimethyl-lysine residues associated with repressive chromatin marks, and that this leads to the allosteric activation of the methyltransferase activity of PRC2. Mutations in EED that prevent it from recognizing repressive trimethyl-lysine marks abolish the activation of PRC2 in vitro and, in Drosophila, reduce global methylation and disrupt development. These findings suggest a model for the propagation of the H3K27me3 mark that accounts for the maintenance of repressive chromatin domains and for the transmission of a histone modification from mother to daughter cells.

Mycobacterium tuberculosis adapts to cellular stresses such as decreased oxygen concentration, at least in part, by upregulation of the dormancy survival regulon, which is thought to be important for the bacterium's ability to enter a persistent state in its human host. We have determined the structure of hypoxic response protein 1, a protein encoded by one of the most strongly upregulated genes in the dormancy survival regulon. Hypoxic response protein 1 is an example of a 'cystathionine-beta-synthase-domain-only' protein; however, unlike other cystathionine-beta-synthase domains, it does not appear to bind AMP. The protein is proteolytically sensitive at its C-terminus and contains two unexpected disulfide bonds, one of which appears resistant to reducing agents in solution and is, therefore, most likely buried in the protein and is not solvent-accessible. We show that the protein is secreted from the bacterium in hypoxic in vitro culture and does not accumulate in the bacterial cell wall. The biological function of the protein remains unclear, but we suggest that it may contribute to the modulation of the host immune response. The work reported advances our understanding of the chemistry and cell biology of this intriguing and potentially important protein, and establishes a structural framework for future functional and immunological studies.

Hypoxic response protein I (HRPI) is a protein of unknown biochemical function whose expression is very strongly upregulated in response to oxygen depletion in Mycobacterium tuberculosis. Crystals have been grown from a solution of full-length HRPI by the unusual method of dehydration without the use of precipitants. The crystals produced diffract to a maximum resolution of 2.1 A and belong to space group P4(1)2(1)2 (or P4(3)2(1)2), with unit-cell parameters a = b = 79.18, c = 37.34 A.