The structural components of the Azotobacter vinelandii iron-only nitrogenase, AnfDKG, form a protein complex within the plant mitochondrial matrix.

A long-held goal of synthetic biology has been the transfer of a bacterial nitrogen-fixation pathway into plants to reduce the use of chemical fertiliser on crops such as rice, wheat and maize. There are three classes of bacterial nitrogenase, named after their metal requirements, containing either a MoFe-, VFe- or FeFe-cofactor, that converts N2 gas to ammonia. Relative to the Mo-nitrogenase the Fe-nitrogenase is not as efficient for catalysis but has less complex genetic and metallocluster requirements, features that may be preferable for engineering into crops. Here we report the successful targeting of bacterial Fe-nitrogenase proteins, AnfD, AnfK, AnfG and AnfH, to plant mitochondria. When expressed as a single protein AnfD was mostly insoluble in plant mitochondria, but coexpression of AnfD with AnfK improved its solubility. Using affinity-based purification of mitochondrially expressed AnfK or AnfG we were able to demonstrate a strong interaction of AnfD with AnfK and a weaker interaction of AnfG with AnfDK. This work establishes that the structural components of the Fe-nitrogenase can be engineered into plant mitochondria and form a complex, which will be a requirement for function. This report outlines the first use of Fe-nitrogenase proteins within a plant as a preliminary step towards engineering an alternative nitrogenase into crops.

Role of toll-like receptor 7 (TLR7) in voluntary alcohol consumption.

Overactivation of neuroimmune signaling has been linked to excessive ethanol consumption. Toll-like receptors (TLRs) are a major component of innate immune signaling and initiate anti- and pro-inflammatory responses via intracellular signal transduction cascades. TLR7 is upregulated in post-mortem brain tissue from humans with alcohol use disorder (AUD) and animals with prior exposure to ethanol. Despite this evidence, the role of TLR7 in the regulation of voluntary ethanol consumption has not been studied. We test the hypothesis that TLR7 activation regulates voluntary ethanol drinking behavior by administering a TLR7 agonist (R848) during an intermittent access drinking procedure in mice. Acute activation of TLR7 reduced ethanol intake, preference, and total fluid intake due, at least in part, to an acute sickness response. However, chronic pre-treatment with R848 resulted in tolerance to the adverse effects of the drug and a subsequent increase in ethanol consumption. To determine the molecular machinery that mediates these behavioral changes, we evaluated gene expression after acute and chronic TLR7 activation. We found that acute TLR7 activation produces brain region specific changes in expression of immune pathway genes, whereas chronic TLR7 activation causes downregulation of TLRs and blunted cytokine induction, suggesting molecular tolerance. Our results demonstrate a novel role for TLR7 signaling in regulating voluntary ethanol consumption. Taken together, our findings suggest TLR7 may be a viable target for development of therapies to treat AUD.

Massive in Silico Study of Noble Gas Binding to the Structural Proteome.

Noble gases are chemically inert, and it was therefore thought they would have little effect on biology. Paradoxically, it was found that they do exhibit a wide range of biological effects, many of which are target-specific and potentially useful and some of which have been demonstrated in vivo. The underlying mechanisms by which useful pharmacology, such as tissue and neuroprotection, anti-addiction effects, and analgesia, is elicited are relatively unexplored. Experiments to probe the interactions of noble gases with specific proteins are more difficult with gases than those with other chemicals. It is clearly impractical to conduct the large number of gas-protein experiments required to gain a complete picture of noble gas biology. Given the simplicity of atoms as ligands, in silico methods provide an opportunity to gain insight into which noble gas-protein interactions are worthy of further experimental or advanced computational investigation. Our previous validation studies showed that in silico methods can accurately predict experimentally determined noble gas binding sites in X-ray structures of proteins. Here, we summarize the largest reported in silico reverse docking study involving 127 854 protein structures and the five nonradioactive noble gases. We describe how these computational screening methods are implemented, summarize the main types of interactions that occur between noble gases and target proteins, describe how the massive data set that this study generated can be analyzed (freely available at group18.csiro.au), and provide the NDMA receptor as an example of how these data can be used to understand the molecular pharmacology underlying the biology of the noble gases. We encourage chemical biologists to access the data and use them to expand the knowledge base of noble gas pharmacology, and to use this information, together with more efficient delivery systems, to develop "atomic drugs" that can fully exploit their considerable and relatively unexplored potential in medicine.

Impact of psychiatric medication co-exposure on Neonatal Abstinence Syndrome severity.

BACKGROUND: Among opioid-exposed infants, psychiatric medication co-exposure is common. Our objective was to compare Neonatal Abstinence Syndrome (NAS) outcomes based on individual psychiatric medication co-exposures. METHODS: A retrospective study of 744 opioid-exposed mother-infant dyads from a single institution was performed. Mothers on pharmacotherapy with methadone or buprenorphine at delivery were included. Data were collected on maternal demographics, psychiatric medication use, and NAS outcomes, including any medication treatment, adjunctive medication treatment, length of hospital stay (LOS), and opioid treatment days. The extent to which individual psychiatric medication and polypharmacy exposure were associated with NAS outcomes was assessed using multivariable regression. RESULTS: Fifty-four percent of the mothers were on ≥1 psychiatric medication, with 32% on ≥2 or psychiatric medications (polypharmacy group). In adjusted models, polypharmacy exposure was associated with longer LOS (ß = 4.31 days, 95% CI 2.55-6.06) and opioid treatment days (ß = 3.98 days, 95% CI 2.24-5.72) and more treatment with adjunctive medication for NAS (aOR = 2.49, 95% CI 1.57-3.95). Benzodiazepines were associated with longer LOS (ß = 4.94, 95% CI 2.86-7.03) and opioid treatment days (ß = 4.86, 95% CI 2.61-6.75), and more adjunctive medication treatment (aOR = 2.57, 95% CI 1.49-4.42). Gabapentin was associated with longer LOS (ß = 2.79, 95% CI 0.54-5.03), more NAS medication treatment (aOR = 2.96, 95% CI 1.18-7.42) including more adjunctive medications (aOR = 1.92, 95% CI 1.05-3.53). CONCLUSION: For infants of mothers with OUD who are also on concurrent psychiatric medications, polypharmacy was associated with worse NAS severity. When medically indicated, limiting use of multiple psychiatric medications, particularly benzodiazepines and gabapentin, during pregnancy should be considered to improve NAS outcomes.

The structure of the hexameric atrazine chlorohydrolase AtzA.

Atrazine chlorohydrolase (AtzA) was discovered and purified in the early 1990s from soil that had been exposed to the widely used herbicide atrazine. It was subsequently found that this enzyme catalyzes the first and necessary step in the breakdown of atrazine by the soil organism Pseudomonas sp. strain ADP. Although it has taken 20 years, a crystal structure of the full hexameric form of AtzA has now been obtained. AtzA is less well adapted to its physiological role (i.e. atrazine dechlorination) than the alternative metal-dependent atrazine chlorohydrolase (TrzN), with a substrate-binding pocket that is under considerable strain and for which the substrate is a poor fit.

Synthesis of novel derivatives of 1,4,7-triazacyclononane.

[structure: see text]. The coordination environment of 1,4,7-triazacyclononane can be adapted, through sequential functionalization of two secondary amines, to generate ligands applicable in biomimetic studies. Two "amino acids" and an amino derivative have been prepared from 1,4,7-triazatricyclo[5.2.1.0(4,10)]decane. This synthon allows efficient attachment of one functional group to the macrocyclic ring, forming a monoamidinium salt. Hydrolysis generates a formyl derivative, which was further functionalized at the secondary amine and hydrolyzed in strong acid to generate ligands 1-3.

Hospital mortality in relation to staff workload: a 4-year study in an adult intensive-care unit.

BACKGROUND: Few studies have examined mortality rates in relation to the workload of hospital staff. We investigated this issue in one adult intensive-care unit (ICU) in the UK. METHODS: We measured ICU workload per shift during each patient's stay for all admissions between 1992 and 1995 that met criteria for adjustment of mortality risk by the APACHE II equation (n=1050). APACHE II data were validated by one observer. Measures of workload in each patient's stay included occupancy, total ICU nursing requirement as defined by the UK Intensive Care Society, and the ratio of occupied to appropriately staffed beds. Over the period, staffing was appropriate for between 4.1 and 5.3 occupied beds (1.3 nurses per patient). FINDINGS: There were 337 deaths, 49 more (95% CI 34-65) than predicted by the APACHE II equation. Median occupancy was 5.8 beds, and median nursing requirement was 1.6 per patient. On multiple logistic regression analysis, adjusted mortality was more than two times higher (odds ratio 3.1 [1.9-5.0]) in patients exposed to high than in those exposed to low ICU workload, defined by average nursing requirement per occupied bed and peak occupancy; the unadjusted odds ratio for this comparison was 4.0 (2.6-6.2). After exclusion of measures of nursing requirement, adjusted mortality increased with the ratio of occupied to appropriately staffed beds during each patient's stay. All logistic regression models fitted the data satisfactorily. INTERPRETATION: Variations in mortality may be partly explained by excess ICU workload. This methodology may have implications for planning and clinical governance.

Circadian rhythms of self-selected lighting in golden hamsters: relation to gonadal condition.

Golden hamster (20 males, 8 females) were maintained in isolation boxes for 4-7 weeks. The animals had access to wheels and selected their own lighting by pressing one bar to turn light-on and another bar to turn the light-off. All hamsters maintained circadian rhythms of wheel-running activity. Seventeen of 28 hamsters selected lighting with a circadian periodicty. For 9 hamsters, there was a significant positive correlation between wheel-running activity and self-selected darkness, while this correlation was significantly negative for 10 hamsters. Four hamsters had regressed testes at the end of the experiment. These 4 had significant positive correlations between activity and self-selected darkness, while none of the hamsters with significant negative correlations between activity and self-selected darkness had regressed testes. Light in phase with activity seems to be more important to the prevention of testicular regression than is the total daily amount of light.