On emotionally intelligent time travel: Individual differences in affective forecasting ability

available options would make them feel. Yet, recent research suggests that such predictions, or affective forecasts, often are inaccurate. There are, after all, a number of obstacles to successful emotional time travel-people are likely to err in affective forecasting if they inaccurately envision the future event itself, misremember how they felt in response to similar past events, or fail to adequately take into account the host of potential differences between their physical and psychological state at the time of forecasting and the time of experiencin

The mechanism of catalysis by type-II NADH : quinone oxidoreductases

Type II NADH:quinone oxidoreductase (NDH-2) is central to the respiratory chains of many organisms. It is not present in mammals so may be exploited as an antimicrobial drug target or used as a substitute for dysfunctional respiratory complex I in neuromuscular disorders. NDH-2 is a single-subunit monotopic membrane protein with just a flavin cofactor, yet no consensus exists on its mechanism. Here, we use steady-state and pre-steady-state kinetics combined with mutagenesis and structural studies to determine the mechanism of NDH-2 from Caldalkalibacillus thermarum. We show that the two substrate reactions occur independently, at different sites, and regardless of the occupancy of the partner site. We conclude that the reaction pathway is determined stochastically, by the substrate/product concentrations and dissociation constants, and can follow either a ping-pong or ternary mechanism. This mechanistic versatility provides a unified explanation for all extant data and a new foundation for the development of therapeutic strategies

Reaction hijacking inhibition of Plasmodium falciparum asparagine tRNA synthetase

Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl-tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure-activity relationship and the selectivity mechanism.</p

Reaction hijacking inhibition of Plasmodium falciparum asparagine tRNA synthetase

Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl-tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure-activity relationship and the selectivity mechanism

Reaction hijacking inhibition of Plasmodium falciparum asparagine tRNA synthetase

Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl-tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure-activity relationship and the selectivity mechanism

Characterization of Alkyltriphenylphosphonium Cations and Their Interaction with Bacterial Cells

Tuberculosis (TB) is a difficult to treat disease caused by the bacterium Mycobacterium tuberculosis. M. tuberculosis is able to shut down its metabolism in response to diverse environmental cues and enter a stage of non-replicating persistence that makes it resistant to many frontline TB drugs. This is further compounded by the “walling off” of M. tuberculosis in granulomatous lesions during infection. New drugs and strategies are in desperate need to combat TB, which currently kills two million people a year. The goal of this thesis was to explore the chemotherapeutic potential of alkyltriphenylphosphonium (alkylTPP) cations; lipophilic positively charged molecules known to accumulate at biological membranes in response to the membrane potential. To address this goal, a structure-function analysis of alkylTPP cations was carried out against several clinically important microorganisms: Mycobacterium tuberculosis, Staphylococcus aureus, Enterococcus faecalis and Escherichia coli; and the non-pathogenic Mycobacterium smegmatis. In addition, we determined if these compounds were toxic to murine RAW macrophages. A series of alkylTPP cations ranging in lipophilicity were characterized, where their toxicity against each cell type was used as a measure of effective accumulation. AlkylTPP cations were shown to be highly toxic to bacteria and mammalian macrophages at concentrations of as low as 1 – 2 μg/mL, where this toxicity increased with respect to lipophilicity. This was deemed an important structure- function relationship for their efficacy. The alkylTPP cation Aa10 was shown to be an effective inhibitor of all bacterial strains used in this study, where it elicited bactericidal killing in M. smegmatis and collapsed the membrane potential. On the basis of these data it is proposed that Aa10 inhibits bacterial growth in a bactericidal manner by dissipating the membrane potential. At toxic concentrations this is due to the accumulation of positively charged alkylTPP cations in the cytoplasmic membrane, where the specifics of this mechanism are yet to be defined. This is validated by the ability of Aa10 to effectively inhibit the anaerobic growth of E. faecalis JH2-2, implying that the action of Aa10 is not dependent on an electron transport chain. Future work will focus on investigating other structure- function relationships that attribute to effective alkylTPP cation toxicity. This includes the addition of different substituents around the central phosphonium ion and variations of the central cation (such as ammonium). Defining these relationships is key in developing alkylTPP cations for a therapeutic application

Characterisation of Bacterial Type II NADH Dehydrogenase (NDH-2)

Compounds that target energy generation in many bacterial pathogens are effective inhibitors of growth, however their precise molecular targets and interactions are poorly understood. Type II (non-proton-translocating) NADH dehydrogenase is a monotopic membrane protein (40 – 70 kDa) that catalyses electron transfer from NADH to quinone, via a flavin cofactor. The absence of mammalian homologs and its essentiality in many pathogenic microorganisms such as Mycobacterium tuberculosis makes NDH-2 an attractive target for drug development. Despite this potential, no potent nanomolar inhibitors of NDH-2 are in the development pipeline and our mechanistic and biochemical understanding of the bacterial enzyme is limited. The aim of the current study was to improve our fundamental understanding of bacterial NDH-2, namely its biochemical features, catalytic mechanism and substrate binding interactions, and apply this information to the development of potent inhibitory molecules. A combinational approach using the recent crystal structure of Caldalkalibacillus thermarum NDH-2 and kinetic analysis revealed that this enzyme conducts catalysis by a two-site ping-pong mechanism. The active sites of NDH-2 were further explored, where the nucleotide-binding site was shown to have a strong (nanomolar) affinity for NADH and residues involved in NADH/NADPH substrate specificity were identified. Structural and sequence analysis revealed a conserved quinone-binding motif (316 –AQXAXQ-321). The enzymatic environment of NDH-2 for inhibitor screening was investigated by comparing detergent-solubilized and lipid-reconstituted NDH-2 systems. The detergent-solubilized system was shown to detect more drug-like compounds and inhibitor scaffolds than the lipid-reconstituted system, however lipid-reconstitution significantly reduced the number insoluble (i.e. highly lipophilic) compounds detected. Conjugation of the alkyltriphenylphosphonium cation delivery functionality to a previously reported NDH-2 inhibitor class (the phenothiazines) caused a 130-fold increase in potency against growing cultures of M. tuberculosis. The conjugate was also shown to target NDH-2 linked process, such as NADH oxidation and oxygen consumption. Overall, the findings of this study provide valuable insight into the biochemistry and catalysis of bacterial NDH-2, where this information is utilized to develop methods for the identification of potent inhibitors of this enzyme

Characterisation of Bacterial Type II NADH Dehydrogenase (NDH-2)

Compounds that target energy generation in many bacterial pathogens are effective inhibitors of growth, however their precise molecular targets and interactions are poorly understood. Type II (non-proton-translocating) NADH dehydrogenase is a monotopic membrane protein (40 – 70 kDa) that catalyses electron transfer from NADH to quinone, via a flavin cofactor. The absence of mammalian homologs and its essentiality in many pathogenic microorganisms such as Mycobacterium tuberculosis makes NDH-2 an attractive target for drug development. Despite this potential, no potent nanomolar inhibitors of NDH-2 are in the development pipeline and our mechanistic and biochemical understanding of the bacterial enzyme is limited. The aim of the current study was to improve our fundamental understanding of bacterial NDH-2, namely its biochemical features, catalytic mechanism and substrate binding interactions, and apply this information to the development of potent inhibitory molecules. A combinational approach using the recent crystal structure of Caldalkalibacillus thermarum NDH-2 and kinetic analysis revealed that this enzyme conducts catalysis by a two-site ping-pong mechanism. The active sites of NDH-2 were further explored, where the nucleotide-binding site was shown to have a strong (nanomolar) affinity for NADH and residues involved in NADH/NADPH substrate specificity were identified. Structural and sequence analysis revealed a conserved quinone-binding motif (316 –AQXAXQ-321). The enzymatic environment of NDH-2 for inhibitor screening was investigated by comparing detergent-solubilized and lipid-reconstituted NDH-2 systems. The detergent-solubilized system was shown to detect more drug-like compounds and inhibitor scaffolds than the lipid-reconstituted system, however lipid-reconstitution significantly reduced the number insoluble (i.e. highly lipophilic) compounds detected. Conjugation of the alkyltriphenylphosphonium cation delivery functionality to a previously reported NDH-2 inhibitor class (the phenothiazines) caused a 130-fold increase in potency against growing cultures of M. tuberculosis. The conjugate was also shown to target NDH-2 linked process, such as NADH oxidation and oxygen consumption. Overall, the findings of this study provide valuable insight into the biochemistry and catalysis of bacterial NDH-2, where this information is utilized to develop methods for the identification of potent inhibitors of this enzyme

The cooking and eating experiences of Australian families with children, living in private, inner-city, high-rise apartments

More Australian families are choosing to live in private, inner-city, high-rise apartments. To date, the cooking and eating experiences of such families, and how their practices are shaped by the design of these apartments, have not been investigated. As children’s health status can benefit from participating in family meals, this study aimed to explore the cooking and eating experiences of apartment-dwelling families in two Australian capital cities. Participants were recruited using purposive sampling and data was collected using photo-elicitation interviews. The experiences of seven mothers aged between 31 and 44 years were explored via two data sources: photographs and semi-structured interviews. Thematic analysis revealed three themes: family values related to cooking and eating; adapting to the space and design constraints of small apartments; and compromising values due to these constraints. While open plan kitchen and dining layouts were found to facilitate children’s involvement in cooking, a lack of space and other design constraints of apartment living forced families to compromise their cooking and eating values. The findings substantiate calls for family-friendly apartment design guidelines, and for these to specifically consider what is needed to support families with the health-promoting practices of home cooking and eating.</p

Annotated compound data for modulators of detergent-solubilised or lipid-reconstituted respiratory type II NADH dehydrogenase activity obtained by compound library screening

The energy-generating membrane protein NADH dehydrogenase (NDH-2), a proposed antibacterial drug target (see “Inhibitors of type II NADH:menaquinone oxidoreductase represent a class of antitubercular drugs” Weinstein et al. 2005 [1]), was screened for modulators of activity in either detergent-solublised or lipid reconstituted (proteolipsome) form. Here we present an annotated list of compounds identified in a small-scale screen against NDH-2. The dataset contains information regarding the libraries screened, the identities of hit compounds and the physicochemical properties governing solubility and permeability. The implications of these data for future antibiotic discovery are discussed in our associated report, “Comparison of lipid and detergent enzyme environments for identifying inhibitors of membrane-bound energy-transducing proteins” [2]