Impact of a Biological Chelator, Lanmodulin, on Minor Actinide Aqueous Speciation and Transport in the Environment.

Minor actinides are major contributors to the long-term radiotoxicity of nuclear fuels and other radioactive wastes. In this context, understanding their interactions with natural chelators and minerals is key to evaluating their transport behavior in the environment. The lanmodulin family of metalloproteins is produced by ubiquitous bacteria and Methylorubrum extorquens lanmodulin (LanM) was recently identified as one of nature's most selective chelators for trivalent f-elements. Herein, we investigated the behavior of neptunium, americium, and curium in the presence of LanM, carbonate ions, and common minerals (calcite, montmorillonite, quartz, and kaolinite). We show that LanM's aqueous complexes with Am(III) and Cm(III) remain stable in carbonate-bicarbonate solutions. Furthermore, the sorption of Am(III) to these minerals is strongly impacted by LanM, while Np(V) sorption is not. With calcite, even a submicromolar concentration of LanM leads to a significant reduction in the Am(III) distribution coefficient (Kd, from >104 to ∼102 mL/g at pH 8.5), rendering it even more mobile than Np(V). Thus, LanM-type chelators can potentially increase the mobility of trivalent actinides and lanthanide fission products under environmentally relevant conditions. Monitoring biological chelators, including metalloproteins, and their biogenerators should therefore be considered during the evaluation of radioactive waste repository sites and the risk assessment of contaminated sites.

Label-Free Multiphoton Imaging of Microbes in Root, Mineral, and Soil Matrices with Time-Gated Coherent Raman and Fluorescence Lifetime Imaging.

Imaging biogeochemical interactions in complex microbial systems─such as those at the soil-root interface─is crucial to studies of climate, agriculture, and environmental health but complicated by the three-dimensional (3D) juxtaposition of materials with a wide range of optical properties. We developed a label-free multiphoton nonlinear imaging approach to provide contrast and chemical information for soil microorganisms in roots and minerals with epi-illumination by simultaneously imaging two-photon excitation fluorescence (TPEF), coherent anti-Stokes Raman scattering (CARS), second-harmonic generation (SHG), and sum-frequency mixing (SFM). We used fluorescence lifetime imaging (FLIM) and time gating to correct CARS for the autofluorescence background native to soil particles and fungal hyphae (TG-CARS) using time-correlated single-photon counting (TCSPC). We combined TPEF, TG-CARS, and FLIM to maximize image contrast for live fungi and bacteria in roots and soil matrices without fluorescence labeling. Using this instrument, we imaged symbiotic arbuscular mycorrhizal fungi (AMF) structures within unstained plant roots in 3D to 60 µm depth. High-quality imaging was possible at up to 30 µm depth in a clay particle matrix and at 15 µm in complex soil preparation. TG-CARS allowed us to identify previously unknown lipid droplets in the symbiotic fungus, Serendipita bescii. We also visualized unstained putative bacteria associated with the roots of Brachypodium distachyon in a soil microcosm. Our results show that this multimodal approach holds significant promise for rhizosphere and soil science research.

Wavefront shaping with a Hadamard basis for scattering soil imaging.

Soil is a scattering medium that inhibits imaging of plant-microbial-mineral interactions that are essential to plant health and soil carbon sequestration. However, optical imaging in the complex medium of soil has been stymied by the seemingly intractable problems of scattering and contrast. Here, we develop a wavefront shaping method based on adaptive stochastic parallel gradient descent optimization with a Hadamard basis to focus light through soil mineral samples. Our approach allows a sparse representation of the wavefront with reduced dimensionality for the optimization. We further divide the used Hadamard basis set into subsets and optimize a certain subset at once. Simulation and experimental optimization results demonstrate our method has an approximately seven times higher convergence rate and overall better performance compared to that with optimizing all pixels at once. The proposed method can benefit other high-dimensional optimization problems in adaptive optics and wavefront shaping.

Influence of Uranium Concentration and pH on U-Phosphate Biomineralization by Caulobacter OR37.

Uranium contamination of soils and groundwater in the United States represents a significant health risk and will require multiple remediation approaches. Microbial phosphatase activity coupled to the addition of an organic P source has recently been studied as a remediation strategy that provides an extended release of inorganic P (Pi) into U-contaminated sites, resulting in the precipitation of meta-autunite minerals. Previous laboratory- and field-based biomineralization studies have investigated environments with relatively high U concentrations (>20 µM). However, most contaminated sites have much lower U concentrations (<2 µM). The Environmental Protection Agency (EPA) limit for U in drinking water is 0.126 µM. Reaching this regulatory limit becomes challenging as U concentrations approach autunite solubility. We studied the precipitation of U(VI)-phosphate minerals by an environmental isolate of Caulobacter sp. (strain OR37) from an Oak Ridge, Tennessee, U-contaminated site. Abiotic U(VI) solubility experiments reveal that U(VI)-phosphate minerals do not form in the presence of excess Pi (500 µM) when U(VI) concentrations are <1 µM and pH is <5. When OR37 cells are reacted under the same conditions with Pi or glycerol-2-phosphate, U(VI)-phosphate mineral formation was observed, along with the formation of intracellular polyphosphate granules. These results show that bacteria provide supersaturated microenvironments needed for U(VI)-phosphate mineralization while hydrolyzing organic P sources. This provides a pathway to lower U concentrations to below EPA limits for drinking water.

Selective Prostacyclin Receptor Agonist Selexipag, in Contrast to Prostacyclin Analogs, Does Not Evoke Paradoxical Vasoconstriction of the Rat Femoral Artery.

Selexipag [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide] is a selective nonprostanoid prostacyclin (PGI2) receptor (IP receptor) agonist that is approved for the treatment of pulmonary arterial hypertension (PAH). In contrast to selexipag, PGI2 analogs used in the clinic are nonselective agonists at prostanoid receptors and can also activate contractile prostaglandin E receptor 3 (EP3) receptors. Leg pain is a common side effect in patients receiving treatment with PGI2 analogs and peripheral vasoconstriction can be responsible for side effects related to muscular ischemia. This study tested the hypothesis that PGI2 analogs could cause paradoxical vasoconstriction of the femoral artery via EP3 receptor activation but that only vasorelaxation would be observed in response to selexipag and its active metabolite ACT-333679 [{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}acetic acid]. Selexipag and ACT-333679 relaxed rings of the isolated rat femoral artery contracted with either prostaglandin F2α (PGF2α ) or the α1 adrenoceptor (α1AR) agonist phenylephrine. ACT-333679 also inhibited contraction of the femoral artery to sympathetic nerve stimulation. In contrast, PGI2 analogs (iloprost, beraprost, and treprostinil) caused additional contraction of arterial rings precontracted with phenylephrine, which was reverted to relaxation by antagonism of EP3 receptors. Treprostinil augmented contraction of the femoral artery to sympathetic nerve stimulation in an EP3 receptor-dependent manner. Mechanistically, concomitant EP3 and α1AR receptor activation synergistically constricted femoral arteries. It is concluded that selexipag and ACT-333679 are vasorelaxants of the rat femoral artery and, unlike PGI2 analogs, do not cause paradoxical vasoconstriction via activation of EP3 receptors. EP3 receptor-mediated vasoconstriction may contribute to the well documented peripheral muscle pain reported in patients with PAH receiving PGI2 analogs. Leg pain may be less in patients treated with selexipag.

Reduction of Plutonium(VI) to (V) by Hydroxamate Compounds at Environmentally Relevant pH.

Natural organic matter is known to influence the mobility of plutonium (Pu) in the environment via complexation and reduction mechanisms. Hydroxamate siderophores have been specifically implicated due to their strong association with Pu. Hydroxamate siderophores can also break down into di and monohydroxamates and may influence the Pu oxidation state, and thereby its mobility. In this study we explored the reactions of Pu(VI) and Pu(V) with a monohydroxamate compound (acetohydroxamic acid, AHA) and a trihydroxamate siderophore desferrioxamine B (DFOB) at an environmentally relevant pH (5.5-8.2). Pu(VI) was instantaneously reduced to Pu(V) upon reaction with AHA. The presence of hydroxylamine was not observed at these pHs; however, AHA was consumed during the reaction. This suggests that the reduction of Pu(VI) to Pu(V) by AHA is facilitated by a direct one electron transfer. Importantly, further reduction to Pu(IV) or Pu(III) was not observed, even with excess AHA. We believe that further reduction of Pu(V) did not occur because Pu(V) does not form a strong complex with hydroxamate compounds at a circum-neutral pH. Experiments performed using desferrioxamine B (DFOB) yielded similar results. Broadly, this suggests that Pu(V) reduction to Pu(IV) in the presence of natural organic matter is not facilitated by hydroxamate functional groups and that other natural organic matter moieties likely play a more prominent role.

Selexipag Active Metabolite ACT-333679 Displays Strong Anticontractile and Antiremodeling Effects but Low ß-Arrestin Recruitment and Desensitization Potential.

Prostacyclin (PGI2) receptor (IP receptor) agonists, which are indicated for the treatment of pulmonary arterial hypertension (PAH), increase cytosolic cAMP levels and thereby inhibit pulmonary vasoconstriction, pulmonary arterial smooth muscle cell (PASMC) proliferation, and extracellular matrix synthesis. Selexipag (Uptravi, 2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide) is the first nonprostanoid IP receptor agonist, it is available orally and was recently approved for the treatment of PAH. In this study we show that the active metabolite of selexipag and the main contributor to clinical efficacy ACT-333679 (previously known as MRE-269) behaved as a full agonist in multiple PAH-relevant receptor-distal-or downstream-cellular assays with a maximal efficacy (Emax) comparable to that of the prototypic PGI2 analog iloprost. In PASMC, ACT-333679 potently induced cellular relaxation (EC50 4.3 nM) and inhibited cell proliferation (IC50 4.0 nM) as well as extracellular matrix synthesis (IC50 8.3 nM). In contrast, ACT-333679 displayed partial agonism in receptor-proximal-or upstream-cAMP accumulation assays (Emax 56%) when compared with iloprost and the PGI2 analogs beraprost and treprostinil (Emax ∼100%). Partial agonism of ACT-333679 also resulted in limited ß-arrestin recruitment (Emax 40%) and lack of sustained IP receptor internalization, whereas all tested PGI2 analogs behaved as full agonists in these desensitization-related assays. In line with these in vitro findings, selexipag, but not treprostinil, displayed sustained efficacy in rat models of pulmonary and systemic hypertension. Thus, the partial agonism of ACT-333679 allows for full efficacy in amplified receptor-distal PAH-relevant readouts while causing limited activity in desensitization-related receptor-proximal readouts.

Importance of Real-Time Assays To Distinguish Multidrug Efflux Pump-Inhibiting and Outer Membrane-Destabilizing Activities in Escherichia coli.

UNLABELLED: The constitutively expressed AcrAB multidrug efflux system of Escherichia coli shows a high degree of homology with the normally silent AcrEF system. Exposure of a strain with acrAB deleted to antibiotic selection pressure frequently leads to the insertion sequence-mediated activation of the homologous AcrEF system. In this study, we used strains constitutively expressing either AcrAB or AcrEF from their normal chromosomal locations to resolve a controversy about whether phenylalanylarginine ß-naphthylamide (PAßN) inhibits the activities of AcrAB and AcrEF and/or acts synergistically with antibiotics by destabilizing the outer membrane permeability barrier. Real-time efflux assays allowed a clear distinction between the efflux pump-inhibiting activity of PAßN and the outer membrane-destabilizing action of polymyxin B nonapeptide (PMXBN). When added in equal amounts, PAßN, but not PMXBN, strongly inhibited the efflux activities of both AcrAB and AcrEF pumps. In contrast, when outer membrane destabilization was assessed by the nitrocefin hydrolysis assay, PMXBN exerted a much greater damaging effect than PAßN. Strong action of PAßN in inhibiting efflux activity compared to its weak action in destabilizing the outer membrane permeability barrier suggests that PAßN acts mainly by inhibiting efflux pumps. We concluded that at low concentrations, PAßN acts specifically as an inhibitor of both AcrAB and AcrEF efflux pumps; however, at high concentrations, PAßN in the efflux-proficient background not only inhibits efflux pump activity but also destabilizes the membrane. The effects of PAßN on membrane integrity are compounded in cells unable to extrude PAßN. IMPORTANCE: The increase in multidrug-resistant bacterial pathogens at an alarming rate has accelerated the need for implementation of better antimicrobial stewardship, discovery of new antibiotics, and deeper understanding of the mechanism of drug resistance. The work carried out in this study highlights the importance of employing real-time fluorescence-based assays in differentiating multidrug efflux-inhibitory and outer membrane-destabilizing activities of antibacterial compounds.

Reversal of the Drug Binding Pocket Defects of the AcrB Multidrug Efflux Pump Protein of Escherichia coli.

UNLABELLED: The AcrB protein of Escherichia coli, together with TolC and AcrA, forms a contiguous envelope conduit for the capture and extrusion of diverse antibiotics and cellular metabolites. In this study, we sought to expand our knowledge of AcrB by conducting genetic and functional analyses. We began with an AcrB mutant bearing an F610A substitution in the drug binding pocket and obtained second-site substitutions that overcame the antibiotic hypersusceptibility phenotype conferred by the F610A mutation. Five of the seven unique single amino acid substitutions--Y49S, V127A, V127G, D153E, and G288C--mapped in the periplasmic porter domain of AcrB, with the D153E and G288C mutations mapping near and at the distal drug binding pocket, respectively. The other two substitutions--F453C and L486W--were mapped to transmembrane (TM) helices 5 and 6, respectively. The nitrocefin efflux kinetics data suggested that all periplasmic suppressors significantly restored nitrocefin binding affinity impaired by the F610A mutation. Surprisingly, despite increasing MICs of tested antibiotics and the efflux of N-phenyl-1-naphthylamine, the TM suppressors did not improve the nitrocefin efflux kinetics. These data suggest that the periplasmic substitutions act by influencing drug binding affinities for the distal binding pocket, whereas the TM substitutions may indirectly affect the conformational dynamics of the drug binding domain. IMPORTANCE: The AcrB protein and its homologues confer multidrug resistance in many important human bacterial pathogens. A greater understanding of how these efflux pump proteins function will lead to the development of effective inhibitors against them. The research presented in this paper investigates drug binding pocket mutants of AcrB through the isolation and characterization of intragenic suppressor mutations that overcome the drug susceptibility phenotype of mutations affecting the drug binding pocket. The data reveal a remarkable structure-function plasticity of the AcrB protein pertaining to its drug efflux activity.

Mineralogical variables that control the antibacterial effectiveness of a natural clay deposit.

As antibiotic-resistant bacterial strains emerge and pose increased global health risks, new antibacterial agents are needed as alternatives to conventional antimicrobials. Naturally occurring antibacterial clays have been identified which are effective in killing antibiotic-resistant bacteria. This study examines a hydrothermally formed antibacterial clay deposit near Crater Lake, OR (USA). Our hypothesis is that antibacterial clays buffer pH and Eh conditions to dissolve unstable mineral phases containing transition metals (primarily Fe(2+)), while smectite interlayers serve as reservoirs for time release of bactericidal components. Model pathogens (Escherichia coli ATCC 25922 and Staphylococcus epidermidis ATCC 14990) were incubated with clays from different alteration zones of the hydrothermal deposit. In vitro antibacterial susceptibility testing showed that reduced mineral zones were bactericidal, while more oxidized zones had variable antibacterial effect. TEM images showed no indication of cell lysis. Cytoplasmic condensation and cell wall accumulations of <100 nm particles were seen within both bacterial populations. Electron energy loss analysis indicates precipitation of intracellular Fe(3+)-oxide nanoparticles (<10 nm) in E. coli after 24 h. Clay minerals and pyrite buffer aqueous solutions to pH 2.5-3.1, Eh > 630 mV and contain elevated level (mM) of soluble Fe (Fe(2+) and Fe(3+)) and Al(3+). Our interpretation is that rapid uptake of Fe(2+) impairs bacterial metabolism by flooding the cell with excess Fe(2+) and overwhelming iron storage proteins. As the intracellular Fe(2+) oxidizes, it produces reactive oxygen species that damage biomolecules and precipitates Fe-oxides. The ability of antibacterial clays to buffer pH and Eh in chronic non-healing wounds to conditions of healthy skin appears key to their healing potential and viability as an alternative to conventional antibiotics.

The application of synthetic antibacterial minerals to combat topical infections: exploring a mouse model of MRSA infection.

The development of new antibiotics has stalled, and novel strategies are needed as we enter the age of antibiotic resistance. Certain naturally occurring clays have been shown to be effective in killing antibiotic resistant bacteria. However, these natural clays are too variable to be used in clinical settings. Our study shows that synthetic antibacterial minerals exhibit potent antibacterial activity against topical MRSA infections and increase the rate of wound closure relative to controls. The antibacterial minerals maintain a redox cycle between Fe2+/Fe3+ and the surfaces of pyrite minerals, which act as a semiconductor and produce reactive oxygen species (ROS), while smectite minerals act as a cation exchange reservoir. Acidic conditions are maintained throughout the application of the hydrated minerals and can mitigate the alkaline pH conditions observed in chronic non-healing wounds. These results provide evidence for the strategy of 'iron overload' to combat antibiotic resistant infections through the maintained release of Fe2+ and generation of ROS via distinct geochemical reactions that can break the chronic wound damage cycle.

Discovery of a Novel Orally Active, Selective LPA Receptor Type 1 Antagonist, 4-(4-(2-Isopropylphenyl)-4-((2-methoxy-4-methylphenyl)carbamoyl)piperidin-1-yl)-4-oxobutanoic Acid, with a Distinct Molecular Scaffold.

Lysophosphatidic acid receptor 1 (LPAR1) antagonists show promise as potentially novel antifibrotic treatments. In a human LPAR1 ß-arrestin recruitment-based high-throughput screening campaign, we identified urea 19 as a hit with a LPAR1 IC50 value of 5.0 µM. Hit-to-lead activities revealed that one of the urea nitrogen atoms can be replaced by carbon and establish the corresponding phenylacetic amide as a lead structure for further optimization. Medicinal chemistry efforts led to the discovery of piperidine 18 as a potent and selective LPAR1 antagonist with oral activity in a mouse model of LPA-induced skin vascular leakage. The molecular scaffold of 18 shares no obvious structural similarity with any other LPAR1 antagonist disclosed so far.

Discovery of the Novel, Orally Active, and Selective LPA1 Receptor Antagonist ACT-1016-0707 as a Preclinical Candidate for the Treatment of Fibrotic Diseases.

Piperidine 3 is a potent and selective lysophosphatidic acid receptor subtype 1 receptor (LPAR1) antagonist that has shown efficacy in a skin vascular leakage target engagement model in mice. However, compound 3 has very high human plasma protein binding and high clearance in rats, which could significantly hamper its clinical development. Continued lead optimization led to the potent, less protein bound, metabolically stable, and orally active azetidine 17. Rat pharmacokinetics (PK) studies revealed that 17 accumulated in the liver. In vitro studies indicated that 17 is an organic anion co-transporting polypeptide 1B1 (OATP1B1) substrate. Although analogue 24 was no longer a substrate of OATP1B1, PK studies suggested that the compound undergoes enterohepatic recirculation. Replacing the carboxylic acidic side chain by a non-acidic sulfamide moiety and further fine-tuning of the scaffold yielded the potent, orally active LPAR1 antagonist 49, which was selected for preclinical development for the treatment of fibrotic diseases.

Potential of APSIS-InSAR for measuring surface oscillations of tropical peatlands.

Tropical peatland across Southeast Asia is drained extensively for production of pulpwood, palm oil and other food crops. Associated increases in peat decomposition have led to widespread subsidence, deterioration of peat condition and CO2 emissions. However, quantification of subsidence and peat condition from these processes is challenging due to the scale and inaccessibility of dense tropical peat swamp forests. The development of satellite interferometric synthetic aperture radar (InSAR) has the potential to solve this problem. The Advanced Pixel System using Intermittent Baseline Subset (APSIS, formerly ISBAS) modelling technique provides improved coverage across almost all land surfaces irrespective of ground cover, enabling derivation of a time series of tropical peatland surface oscillations across whole catchments. This study aimed to establish the extent to which APSIS-InSAR can monitor seasonal patterns of tropical peat surface oscillations at North Selangor Peat Swamp Forest, Peninsular Malaysia. Results showed that C-band SAR could penetrate the forest canopy over tropical peat swamp forests intermittently and was applicable to a range of land covers. Therefore the APSIS technique has the potential for monitoring peat surface oscillations under tropical forest canopy using regularly acquired C-band Sentinel-1 InSAR data, enabling continuous monitoring of tropical peatland surface motion at a spatial resolution of 20 m.

New thermal decomposition pathway for TATB.

Understanding the thermal decomposition behavior of TATB (1,3,5-triamino-2,4,6-trinitrobenzene) is a major focus in energetic materials research because of safety issues. Previous research and modelling efforts have suggested benzo-monofurazan condensation producing H2O is the initiating decomposition step. However, early evolving CO2 (m/z 44) along with H2O (m/z 18) evolution have been observed by mass spectrometric monitoring of head-space gases in both constant heating rate and isothermal decomposition studies. The source of the CO2 has not been explained, until now. With the recent successful synthesis of 13C6-TATB (13C incorporated into the benzene ring), the same experiments have been used to show the source of the CO2 is the early breakdown of the TATB ring, not adventitious C from impurities and/or adsorbed CO2. A shift in mass m/z 44 (CO2) to m/z 45 is observed throughout the decomposition process indicating the isotopically labeled 13C ring breakdown occurs at the onset of thermal decomposition along with furazan formation. Partially labeled (N18O2)3-TATB confirms at least some of the oxygen comes from the nitro-groups. This finding has a significant bearing on decomposition computational models for prediction of energy release and deflagration to detonation transitions, with respect to conditions which currently do not recognize this oxidation step.

Differential effects of Selexipag [corrected] and prostacyclin analogs in rat pulmonary artery.

{4-[(5,6-Diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}acetic acid (ACT-333679) is the main metabolite of the selective prostacyclin (PGI(2)) receptor (IP receptor) agonist selexipag. The goal of this study was to determine the influence of IP receptor selectivity on the vasorelaxant efficacy of ACT-333679 and the PGI(2) analog treprostinil in pulmonary artery under conditions associated with pulmonary arterial hypertension (PAH). Selexipag and ACT-333679 evoked full relaxation of pulmonary artery from control and monocrotaline (MCT)-PAH rats, and ACT-333679 relaxed normal pulmonary artery contracted with either endothelin-1 (ET-1) or phenylephrine. In contrast, treprostinil evoked weaker relaxation than ACT-333679 of control pulmonary artery and failed to induce relaxation of pulmonary artery from MCT-PAH rats. Treprostinil did not evoke relaxation of normal pulmonary artery contracted with either ET-1 or phenylephrine. Expression of prostaglandin E(3) (EP(3)) receptor mRNA was increased in pulmonary artery from MCT-PAH rats. In contraction experiments, the selective EP(3) receptor agonist sulprostone evoked significantly greater contraction of pulmonary artery from MCT-PAH rats compared with control rats. The presence of a threshold concentration of ET-1 significantly augmented the contractile response to sulprostone in normal pulmonary artery. ACT-333679 did not evoke direct contraction of rat pulmonary artery, whereas treprostinil evoked concentration-dependent contraction that was inhibited by the EP(3) receptor antagonist (2E)-3-(3',4'-dichlorobiphenyl-2-yl)-N-(2-thienylsulfonyl)acrylamide. Antagonism of EP(3) receptors also revealed a relaxant response to treprostinil in normal pulmonary artery contracted with ET-1. These data demonstrate that the relaxant efficacy of the selective IP receptor agonist selexipag and its metabolite ACT-333679 is not modified under conditions associated with PAH, whereas relaxation to treprostinil may be limited in the presence of mediators of disease.

Synthetic antibacterial minerals: harnessing a natural geochemical reaction to combat antibiotic resistance.

The overuse of antibiotics in clinical and livestock settings is accelerating the selection of multidrug resistant bacterial pathogens. Antibiotic resistant bacteria result in increased mortality and financial strain on the health care and livestock industry. The development of new antibiotics has stalled, and novel strategies are needed as we enter the age of antibiotic resistance. Certain naturally occurring clays have been shown to have antimicrobial properties and kill antibiotic resistant bacteria. Harnessing the activity of compounds within these clays that harbor antibiotic properties offers new therapeutic opportunities for fighting the potentially devastating effects of the post antibiotic era. However, natural samples are highly heterogenous and exhibit variable antibacterial effectiveness, therefore synthesizing minerals of high purity with reproducible antibacterial activity is needed. Here we describe for the first time synthetic smectite clay minerals and Fe-sulfide microspheres that reproduce the geochemical antibacterial properties observed in natural occurring clays. We show that these mineral formulations are effective at killing the ESKAPE pathogens (Enterococcus sp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter sp., Pseudomonas aeruginosa and Enterobacter sp.) by maintaining Fe2+ solubility and reactive oxygen species (ROS) production while buffering solution pH, unlike the application of metals alone. Our results represent the first step in utilizing a geochemical process to treat antibiotic resistant topical or gastrointestinal infections in the age of antibiotic resistance.

Selexipag: a selective prostacyclin receptor agonist that does not affect rat gastric function.

Selexipag [2-{4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl)acetamide] is an orally available prostacyclin (PGI(2)) receptor (IP receptor) agonist that is chemically distinct from PGI(2) and is in clinical development for the treatment of pulmonary arterial hypertension. Selexipag is highly selective for the human IP receptor in vitro, whereas analogs of PGI(2) can activate prostanoid receptors other than the IP receptor. The goal of this study was to determine the impact of selectivity for the IP receptor on gastric function by measuring 1) contraction of rat gastric fundus ex vivo and 2) the rates of gastric emptying and intestinal transport in response to selexipag in comparison with other PGI(2) analogs. The rat gastric fundus expresses mRNA encoding multiple prostanoid receptors to different levels: prostaglandin E receptor 1 (EP(1)) > prostaglandin E receptor 3 (EP(3)), IP receptor > prostaglandin D(2) receptor 1, thromboxane receptor. Selexipag and metabolite {4-[(5,6-diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}acetic acid (ACT-333679) did not contract gastric fundus at concentrations up to 10(-3) M. In contrast, the PGI(2) analogs iloprost and beraprost evoked concentration-dependent contraction of gastric fundus. Contraction to treprostinil was observed at high concentration (10(-4) M). Contraction to all PGI(2) analogs was mediated via activation of EP(3) receptors, although EP(1) receptors also contributed to the contraction of gastric fundus to iloprost and beraprost. Antagonism of IP receptors did not affect responses. Oral selexipag did not significantly alter gastric function in vivo, as measured by rates of stomach emptying and intestinal transport, whereas beraprost slowed gastrointestinal transport. The high functional selectivity of selexipag and ACT-333679 for the IP receptor precludes a stimulatory action on gastric smooth muscle and may help minimize gastric side effects such as nausea and vomiting.

Pharmacology of macitentan, an orally active tissue-targeting dual endothelin receptor antagonist.

Macitentan, also called Actelion-1 or ACT-064992 [N-[5-(4-bromophenyl)-6-(2-(5-bromopyrimidin-2-yloxy)ethoxy)-pyrimidin-4-yl]-N'-propylaminosulfonamide], is a new dual ET(A)/ET(B) endothelin (ET) receptor antagonist designed for tissue targeting. Selection of macitentan was based on inhibitory potency on both ET receptors and optimization of physicochemical properties to achieve high affinity for lipophilic milieu. In vivo, macitentan is metabolized into a major and pharmacologically active metabolite, ACT-132577. Macitentan and its metabolite antagonized the specific binding of ET-1 on membranes of cells overexpressing ET(A) and ET(B) receptors and blunted ET-1-induced calcium mobilization in various natural cell lines, with inhibitory constants within the nanomolar range. In functional assays, macitentan and ACT-132577 inhibited ET-1-induced contractions in isolated endothelium-denuded rat aorta (ET(A) receptors) and sarafotoxin S6c-induced contractions in isolated rat trachea (ET(B) receptors). In rats with pulmonary hypertension, macitentan prevented both the increase of pulmonary pressure and the right ventricle hypertrophy, and it markedly improved survival. In diabetic rats, chronic administration of macitentan decreased blood pressure and proteinuria and prevented end-organ damage (renal vascular hypertrophy and structural injury). In conclusion, macitentan, by its tissue-targeting properties and dual antagonism of ET receptors, protects against end-organ damage in diabetes and improves survival in pulmonary hypertensive rats. This profile makes macitentan a new agent to treat cardiovascular disorders associated with chronic tissue ET system activation.