Search

The over-enrichment of nitrogen (N) in the environment has contributed to severe and recurring harmful cyanobacterial blooms, especially by the non-N(2)-fixing Microcystis spp. N chemical speciation influences cyanobacterial growth, persistence and the production of the hepatotoxin microcystin, but the physiological mechanisms to explain these observations remain unresolved. Stable-labelled isotopes and metabolomics were employed to address the influence of nitrate, ammonium, and urea on cellular physiology and production of microcystins in Microcystis aeruginosa NIES-843. Global metabolic changes were driven by both N speciation and diel cycling. Tracing (15)N-labelled nitrate, ammonium, and urea through the metabolome revealed N uptake, regardless of species, was linked to C assimilation. The production of amino acids, like arginine, and other N-rich compounds corresponded with greater turnover of microcystins in cells grown on urea compared to nitrate and ammonium. However, (15)N was incorporated into microcystins from all N sources. The differences in N flux were attributed to the energetic efficiency of growth on each N source. While N in general plays an important role in sustaining biomass, these data show that N-speciation induces physiological changes that culminate in differences in global metabolism, cellular microcystin quotas and congener composition.

Bacterial membranes are complex mixtures of lipids and proteins, the combination of which confers biophysical properties that allows cells to respond to environmental conditions. Carotenoids are sterol analogs that are important for regulating membrane dynamics. The membrane of Pantoea sp. YR343 is characterized by the presence of the carotenoid zeaxanthin, and a carotenoid-deficient mutant, ΔcrtB, displays defects in root colonization, reduced secretion of indole-3-acetic acid, and defects in biofilm formation. Here we demonstrate that the loss of carotenoids results in changes to the membrane lipid composition in Pantoea sp. YR343, including increased amounts of unsaturated fatty acids in the ΔcrtB mutant membranes. These mutant cells displayed less fluid membranes in comparison to wild type cells as measured by fluorescence anisotropy of whole cells. Studies with artificial systems, however, have shown that carotenoids impart membrane rigidifying properties. Thus, we examined membrane fluidity using spheroplasts and vesicles composed of lipids extracted from either wild type or mutant cells. Interestingly, with the removal of the cell wall and membrane proteins, ΔcrtB vesicles were more fluid than vesicles made from lipids extracted from wild type cells. In addition, carotenoids appeared to stabilize membrane fluidity during rapidly changing temperatures. Taken together, these results suggest that Pantoea sp. YR343 compensates for the loss of carotenoids by changing lipid composition, which together with membrane proteins, results in reduced membrane fluidity. These changes may influence the abundance or function of membrane proteins that are responsible for the physiological changes observed in the ΔcrtB mutant cells.

Nonequilibrium quasiparticles are ubiquitous in superconducting electronics. These quasiparticles can trap in the internal Andreev bound states of a phase-biased Josephson junction, providing a mechanism for studying their presence and behavior. We characterize a quasiparticle trapping detector device based on a two junction aluminum nanobridge superconducting quantum interference device incorporated into a transmission line resonator. When the device is flux-biased, distinct resonant frequencies develop depending on the trapped quasiparticle number. We demonstrate continuous detection of up to 3 trapped quasiparticles, with detection of a trapped quasiparticle with a signal-to-noise ratio of 27 in 5 μs. We describe initial measurements of quasiparticle behavior and discuss the possible optimization and application of such detector devices.

Metallic fuel relocation experiments were conducted in which molten uranium was dropped into the cladding of a single fuel pin where eutectic interaction between fuel and cladding led to the release of eutectic fuel mixture into a narrow sodium channel. The thermal hydraulic relocation behavior of the molten uranium was analyzed in a previous study. Here, the extent of the eutectic formation was evaluated by scanning electron microscopy and energy-dispersive spectroscopy (SEM/EDS). The SEM/EDS analysis confirmed that cladding failure was caused by eutectic penetration. The eutectic penetration initiated around the boundary between cladding and uranium fuel, progressed into the cladding, and caused the cladding failure. Near the cladding failure region, the original cladding shape was unrecognizable, and many cracks, breakthroughs, and partial losses of the cladding were observed. The main components of the cladding failure region were UFe2 and U6Fe, which are the key components in the eutectic penetration.

The objectives were to determine the optimal feeding amount of choline in a ruminally protected form to reduce the triacylglycerol (TAG) concentration in liver and to increase TAG in blood plasma of dairy cows. Pregnant, nonlactating multiparous Holstein cows (n = 77) were blocked by body condition score (3.59 ± 0.33) and assigned to treatment at 64 ± 10 d before calculated calving date. Dietary treatments were top-dressing of 0, 30, 60, 90, or 120 g/d of ruminally protected choline (RPC; Balchem Corp., New Hampton, NY) ions to supply the equivalent of 0, 6.5, 12.9, 19.4, and 25.8 g/d of choline ions. Diets were formulated to exceed nutrient requirements for maintenance and pregnancy and fed in ad libitum amounts for the first 5 d. From d 6 to 15, cows were restricted to consume approximately 31% of their net energy requirements to simulate early lactating cows in negative energy balance. Methionine intake was maintained throughout each 15-d period. Liver was biopsied at 5 and 14 d and analyzed for TAG and glycogen. Blood was sampled on d 5 and 14 and plasma analyzed for glucose, insulin, cholesterol, β-hydroxybutyrate, long-chain fatty acids, and haptoglobin. On d 14, a mixture of saturated long-chain fatty acids, ground corn, and dried molasses (50:37:13) was offered (908 g, as-is basis) 10 h after the single daily feeding. Blood samples were collected for 19 h and plasma analyzed for TAG and cholesterol to assess apparent absorption of dietary fat. Mean dry matter intake and energy balance decreased from means of 9.5 to 3.3 kg/d and from 0.6 to −9.2 Mcal of net energy for lactation/d during the ad libitum and restricted feeding periods, respectively. Plasma concentrations of the lipid-soluble choline biomolecules, namely total phosphatidylcholines, total lysophosphatidylcholines, and sphingomyelin, increased with choline supplementation. Feed restriction increased plasma concentrations of β-hydroxybutyrate and free long-chain fatty acids, whereas those of glucose, insulin, and total cholesterol decreased. During feed restriction, concentration of hepatic TAG and plasma haptoglobin decreased linearly, whereas concentration of hepatic glycogen tended to increase quadratically with increasing intake of RPC. After fat supplementation, mean plasma concentration of TAG increased by an average of 21% with intake of RPC ions, peaking at intakes of ≥6.5 g/d of RPC ion. In summary, feeding RPC ions to cows in negative energy balance had increasing lipotropic effects on the liver when consumed up to 25.8 g/d, whereas feeding only 6.5 g/d increased concentrations of hepatic glycogen and TAG in the blood.

Cobamides such as vitamin B12 are structurally conserved, cobalt-containing tetrapyrrole biomolecules that have essential biochemical functions in all domains of life. In organohalide respiration, a vital biological process for the global cycling of natural and anthropogenic organohalogens, cobamides are the requisite prosthetic groups for carbon-halogen bond-cleaving reductive dehalogenases. This study reports the biosynthesis of a new cobamide with unsubstituted purine as the lower base and assigns unsubstituted purine a biological function by demonstrating that Coα-purinyl-cobamide (purinyl-Cba) is the native prosthetic group in catalytically active tetrachloroethene reductive dehalogenases of Desulfitobacterium hafniense. Cobamides featuring different lower bases are not functionally equivalent, and purinyl-Cba elicits different physiological responses in corrinoid-auxotrophic, organohalide-respiring bacteria. Given that cobamide-dependent enzymes catalyze key steps in essential metabolic pathways, the discovery of a novel cobamide structure and the realization that lower bases can effectively modulate enzyme activities generate opportunities to manipulate functionalities of microbiomes.

Understanding the relocation behavior of metallic fuel in severe accident scenarios is one of the most important factors in the safety assessment of sodium-cooled fast reactors (SFRs). In the present study, the relocation behavior of uranium metallic fuel was investigated by conducting metallic fuel relocation experiments using 19-pin bundle test sections in the Argonne’s Metallic Uranium Safety Experiment (MUSE) facility. Post-test radiographic images were taken to characterize the uranium fuel relocation within the pin bundle test section. In addition, the pressure drop in the test sections was measured after the relocation experiments to evaluate the blockage of the sodium coolant channel. The pressure drop in the pin bundle test sections was also analytically calculated by varying the porosity and the pore size of the relocated fuel. The porosity and pore size of the relocated fuel in the pin bundle test sections were estimated by comparing those calculated pressure drop values with the measured pressure drop values from the pressure drop tests. The experimental results showed that the more formation of the eutectics could lead to more fuel dispersion and less blockage due to the lower freezing point of the eutectics.

Paenibacillus polymyxa is a Gram-positive bacterium commonly found associated with plant roots. P. polymyxa can exhibit two forms of flagellar motility: swimming in liquid culture and swarming on a surface. Here, swimming cells were compared to swarming cells using an integrated proteomic and lipidomic approach, yielding information about how lipid modifications and protein/enzyme pathways are tailored for these specific phenotypes. Observed differences in both phospholipid composition and metabolism between the two conditions suggest membrane remodeling in response to the surrounding environment. Key enzymes involved in glycerophospholipid metabolism were abundant in swimming bacteria, while enzymes associated with glycerol-3-phosphate metabolism were more abundant in swarming bacteria. Several glycoside hydrolases were either unique to or more abundant during swarming. This likely reflects the degradation of their own exopolysaccharides to both enhance swarming and supply the necessary chemical energy to compensate for increased flagellar synthesis. The observed upregulation of biosynthetic gene clusters (polyketides, lantibiotics, and surfactin) in swarming bacteria suggest the importance of signaling, antimicrobial activity, and surfactin production during this mode of motility – the latter of which is confirmed via RT-PCR.

The use of urea as a nitrogenous fertilizer has increased over the past two decades, with urea itself being readily detected at high concentrations in many lakes. Urea has been linked to cyanobacterial blooms as it is a readily assimilated nitrogen (N) - source for cyanobacteria that possess the enzyme urease. We tested the hypothesis that urea may also act as a carbon (C) source to supplemental growth requirements during the alkaline conditions created by dense cyanobacterial blooms, when concentrations of dissolved CO2 are vanishingly low. High rates of photosynthesis markedly reduce dissolved CO2 concentrations and drive up pH. This was observed in Lake Erie during the largest bloom on record (2015) over long periods (months) and short periods (days) of time, suggesting blooms experience periods of CO2-limitation on a seasonal and daily basis. We used 13C-urea to demonstrate that axenic cultures of the model toxic cyanobacterium, Microcystis aeruginosa NIES843, assimilated C at varying environmentally relevant pH conditions directly into a spectrum of metabolic pools during urea hydrolysis. Primarily, 13C from urea was assimilated into central C metabolism and amino acid biosynthesis pathways, including those important for the production of the hepatotoxin, microcystin, and incorporation into these pathways was at a higher percentage during growth at higher pH. This corresponded to increased growth rates on urea as the sole N source with increasing pH. We propose this ability to incorporate C from urea represents yet another competitive advantage for this cyanobacterium during dense algal blooms.

INTRODUCTION: Lipidomics can reveal global alterations in a broad class of molecules whose functions are innately linked to physiology. Monitoring changes in the phospholipid composition of biological membranes in response to stressors can aid the development of targeted therapies. However, exact quantitation of cardiolipins is not a straightforward task due to low ionization efficiencies and poor chromatographic separation of these compounds. OBJECTIVE: The aim of this study was to develop a quantitative method for the detection of cardiolipins and other phospholipids using both a targeted and untargeted analyses with a Q-Exactive. METHODS: HILIC chromatography and high-resolution mass spectrometry with parallel reaction monitoring was used to measure changes in lipid concentration. Internal standards and fragmentation techniques allowed for the reliable quantitation of lipid species including: lysyl-phosphatidylglycerol, phosphatidylglycerol, and cardiolipin. RESULTS: The untargeted analysis was capable to detecting 6 different phospholipid classes as well as free fatty acids. The targeted analysis quantified up to 23 cardiolipins, 10 phosphatidylglycerols and 10 lysyl-phosphatidylglycerols with detection limits as low as 50 nM. Biological validation with Enterococcus faecalis demonstrates sensitivity in monitoring the incorporation of exogenously supplied free fats into membrane phospholipids. When supplemented with oleic acid, the amount of free oleic acid in the membrane was 100 times greater and the concentration of polyunsaturated cardiolipin increased to over 3.5 μM compared to controls. CONCLUSIONS: This lipidomics method is capable of targeted quantitation for challenging biologically relevant cardiolipins as well as broad, untargeted lipid profiling.

Amyloidosis is a malignant pathology associated with the formation of proteinaceous amyloid fibrils that deposit in organs and tissues, leading to dysfunction and severe morbidity. More than 25 proteins have been identified as components of amyloid, but the most common form of systemic amyloidosis is associated with the deposition of amyloid composed of Ig light chains (AL). Clinical management of amyloidosis focuses on reducing synthesis of the amyloid precursor protein. However, recently, passive immunotherapy using amyloid fibril-reactive antibodies, such as 11-1F4, to remove amyloid from organs has been shown to be effective at restoring organ function in patients with AL amyloidosis. However, 11-1F4 does not bind amyloid in all AL patients, as evidenced by PET/CT imaging, nor does it efficiently bind the many other forms of amyloid. To enhance the reactivity and expand the utility of the 11-1F4 mAb as an amyloid immunotherapeutic, we have developed a pretargeting "peptope" comprising a multiamyloid-reactive peptide, p5+14, fused to a high-affinity peptide epitope recognized by 11-1F4. The peptope, known as p66, bound the 11-1F4 mAb in vitro with subnanomolar efficiency, exhibited multiamyloid reactivity in vitro and, using tissue biodistribution and SPECT imaging, colocalized with amyloid deposits in a mouse model of systemic serum amyloid A amyloidosis. Pretreatment with the peptope induced 11-1F4 mAb accumulation in serum amyloid A deposits in vivo and enhanced 11-1F4-mediated dissolution of a human AL amyloid extract implanted in mice.

This study offers insight into the roles anodic and cathodic processes play in electrochemically activated persulfate (EAP) and screens EAP as a viable technique for ciprofloxacin degradation in wastewater. Sulfate radical formation at a boron-doped diamond (BDD) anode and persulfate activation at a graphite cathode were experimentally elucidated using different electrolytes and electrochemical setups. Rapid ciprofloxacin transformation occurred via pseudo-first-order mechanisms with respect to ciprofloxacin in persulfate electrolyte, reaching 84% removal in 120 min using EAP. Transformation pathways were compared to those in nitrate and sulfate electrolytes. Ciprofloxacin removal rates in the electrochemical system were 88% and 33% faster in persulfate than nitrate and sulfate electrolytes, respectively. Total organic carbon removal rates were 93% and 48% faster in persulfate than nitrate and sulfate, respectively. Use of sulfate electrolyte resulted in removal rates 6-7 times faster than those in nitrate solution. Accelerated removal in sulfate was attributed to anodic sulfate radical formation, while enhanced removal in persulfate was associated with cathodic persulfate activation and nonradical persulfate activation at the BDD anode. Quenching experiments indicated both sulfate radicals and hydroxyl radicals contributed to degradation. Comparisons between platinum and graphite cathodes showed similar cathodic persulfate activation and ciprofloxacin degradation.

Ultra low-background radiation measurements are essential to several large-scale physics investigations. Assay of solid polymer materials for extremely low levels of radioactive elements, such as uranium, presents challenges. This paper describes an initial investigation into the use of laser ablation with inductively coupled plasma mass spectrometry for screening a solid plastic, polyethylene, for gross uranium levels.

Corrinoid auxotrophic organohalide-respiring Dehalococcoides mccartyi (Dhc) strains are keystone bacteria for reductive dechlorination of toxic and carcinogenic chloroorganic contaminants. We demonstrate that the lower base attached to the essential corrinoid cofactor of reductive dehalogenase (RDase) enzyme systems modulates dechlorination activity and affects the vinyl chloride (VC) RDases BvcA and VcrA differently. Amendment of 5,6-dimethylbenzimidazolyl-cobamide (DMB-Cba) to Dhc strain BAV1 and strain GT cultures supported cis-1,2-dichloroethene-to-ethene reductive dechlorination at rates of 107.0 (±12.0) μM and 67.4 (±1.4) μM Cl(-) released per day, respectively. Strain BAV1, expressing the BvcA RDase, reductively dechlorinated VC to ethene, although at up to fivefold lower rates in cultures amended with cobamides carrying 5-methylbenzimidazole (5-MeBza), 5-methoxybenzimidazole (5-OMeBza) or benzimidazole (Bza) as the lower base. In contrast, strain GT harboring the VcrA RDase failed to grow and dechlorinate VC to ethene in medium amended with 5-OMeBza-Cba or Bza-Cba. The amendment with DMB to inactive strain GT cultures restored the VC-to-ethene-dechlorinating phenotype and intracellular DMB-Cba was produced, demonstrating cobamide uptake and remodeling. The distinct responses of Dhc strains with BvcA versus VcrA RDases to different cobamides implicate that the lower base exerts control over Dhc reductive dechlorination rates and extents (that is, detoxification), and therefore the dynamics of Dhc strains with discrete reductive dechlorination capabilities. These findings emphasize that the role of the corrinoid/lower base synthesizing community must be understood to predict strain-specific Dhc activity and achieve efficacious contaminated site cleanup.

A new approach to isotopic purification has been developed and applied to the production of a small quantity of 244Pu with an isotopic purity >99.996 %, as compared against the standard 244Pu available that displays an isotopic purity of 97.87 %. The presence of Pu isotopes 239Pu, 240Pu, 241Pu and 242Pu have been greatly reduced, allowing for higher spiking levels of the isotopically purified 244Pu tracer to be used in environmental level sample analysis. Details of the isotopic purification process will be described along with the effect this improved Pu tracer could have on analytical Pu mass spectrometry measurements.

The use of naturally occurring antimicrobial peptides provides a promising route to selectively target pathogenic agents and to shape microbiome structure. Lantibiotics, such as duramycin, are one class of bacterially produced peptidic natural products that can selectively inhibit the growth of other bacteria. However, despite longstanding characterization efforts, the microbial selectivity and mode of action of duramycin are still obscure. We describe here a suite of biological, chemical and physical characterizations that shed new light on the selective and mechanistic aspects of duramycin activity. Bacterial screening assays have been performed using duramycin and Populus-derived bacterial isolates to determine species selectivity. Lipidomic profiles of selected resistant and sensitive strains show that the sensitivity of Gram-positive bacteria depends on the presence of phosphatidylethanolamine (PE) in the cell membrane. Further the surface and interface morphology were studied by high resolution Atomic Force Microscopy (AFM) and showed a progression of cellular changes in the cell envelope after treatment with duramycin for the susceptible bacterial strains. Together, these molecular and cellular level analyses provide insight into duramycin’s mode of action and a better understanding of its selectivity.

We report the development and testing of a collector arrangement for quadrupole inductively coupled plasma mass spectrometry (ICP-MS) that for the first time has been used to isolate small quantities of highly enriched (>99.99%) single isotopes, with deposition rates >10 ng h −1 . The collector assembly replaces the standard instrument detector allowing for implantation with simultaneous monitoring of the incident ion current. Even under zero bias implant conditions, low energy ( 151 Eu ion currents of 0.1–0.5 nA were collected on a simple, planar foil without resorting to any type of cup configuration. Recovery of the enriched isotope from such foils is much simpler than from a more complex cup configuration. High rejection of adjacent mass isotopes was demonstrated by selectively implanting 167 Er, then using laser ablation ICP-MS imaging to confirm the absence of any co-implanted 166 Er or 168 Er. The important analytical possibilities of this new approach to isotope ratio measurement, tracer purification, and radiation measurements are discussed.

Maternal intake of eicosapentaenoic acid (EPA; 20:5 n-3) and docosahexaenoic acid (22:6 n-3) has been associated with reduced adiposity in children, suggesting the possibility to program adipose development through dietary fatty acids before birth. This study determined if enriching the maternal diet in fish oil, the primary source of EPA and DHA, affected adipose development in offspring. Broiler chickens were used because they are obesity-prone, and because fatty acids provided to the embryo can be manipulated through the hen diet. Hens were fed diets supplemented (2.8% wt:wt) with corn oil (CO; n-6) or fish oil (FO; n-3) for 28 d. Chicks from both maternal diet groups were fed the same diet after hatch. Maternal FO consumption enriched chick adipose tissue in EPA and DHA and reduced adiposity by promoting more, but smaller, adipocytes. This adipocyte profile was paralleled by lower expression of the adipogenic regulator PPARG and its co-activator PPARGC1B, and elevated expression of LPL. Proteomics identified 95 differentially abundant proteins between FO and CO adipose tissue, including components of glucose metabolism, lipid droplet trafficking, and cytoskeletal organization. These results demonstrate that the maternal dietary fatty acid profile programs offspring adipose development.

Ultra-pure construction materials are required for the next generation of neutrino physics, dark matter and environmental science applications. These materials are also important for use in high-purity germanium spectrometers used in screening materials for radiopurity. The next-generation science applications require materials with radiopurity levels at or below 1 μBq/kg 232Th and 238U. Yet radiometric analysis lacks sensitivity below ~10 μBq/kg for the U and Th decay chains. This limits both the selection of clean materials and the validation of purification processes. Copper is an important high-purity material for low-background experiments due to the ease with which it can be purified by electrochemical methods. Electroplating for purification into near-final shapes, known as electroforming, is one such method. Continued refinement of the copper electroforming process is underway, for the first time guided by an ICP-MS based assay method that can measure 232Th and 238U near the desired purity levels. An assay of electroformed copper at a μBq/kg level has been achieved and is described. The implications of electroformed copper at or better than this purity on next-generation low-background experiments are discussed.

This work was supported by the National Institutes of Health NIH R01AL105690. Phosphatidylserine (PS) synthase (Cho1p) and the PS decarboxylase enzymes (Psd1p and Psd2p), which synthesize PS and phosphatidylethanolamine (PE), respectively, are crucial for Candida albicans virulence. Mutations that disrupt these enzymes compromise virulence. These enzymes are part of the cytidine diphosphate-diacylglycerol pathway (i.e. de novo pathway) for phospholipid synthesis. Understanding how losses of PS and/or PE synthesis pathways affect the phospholipidome of Candida is important for fully understanding how these enzymes impact virulence. The cho1Δ/Δ and psd1Δ/Δ psd2Δ/Δ mutations cause similar changes in levels of phosphatidic acid, phosphatidylglycerol, phosphatidylinositol and PS. However, only slight changes were seen in PE and phosphatidylcholine (PC). This finding suggests that the alternative mechanism for making PE and PC, the Kennedy pathway, can compensate for loss of the de novo synthesis pathway. Candida albicans Cho1p, the lipid biosynthetic enzyme with the most potential as a drug target, has been biochemically characterized, and analysis of its substrate specificity and kinetics reveal that these are similar to those previously published for Saccharomyces cerevisiae Cho1p. Postprint

Astrocytes show a complex structural and physiological interplay with neurons and respond to neuronal activation in vitro and in vivo with intracellular calcium elevations. These calcium changes enable astrocytes to modulate synaptic transmission and plasticity through various mechanisms. However, the response pattern of astrocytes to single neuronal depolarization events still remains unresolved. This information is critical for fully understanding the coordinated network of neuron–glial signaling in the brain. To address this, we developed a system to map astrocyte calcium responses along apical dendrites of CA1 pyramidal neurons in hippocampal slices using single-neuron stimulation with channelrhodopsin-2. This technique allowed selective neuronal depolarization without invasive manipulations known to alter calcium levels in astrocytes. Light-evoked neuronal depolarization was elicited and calcium events in surrounding astrocytes were monitored using the calcium-sensitive dye Calcium Orange. Stimulation of single neurons caused calcium responses in populations of astrocytes along the apical axis of CA1 cell dendrites. Calcium responses included single events that were synchronized with neuronal stimulation and poststimulus changes in calcium event frequency, both of which were modulated by glutamatergic and purinergic signaling. Individual astrocytes near CA1 cells showed low ability to respond to repeated neuronal depolarization events. However, the response of the surrounding astrocyte population was remarkably accurate. Interestingly, the reliability of responses was graded with respect to astrocyte location along the CA1 cell dendrite, with astrocytes residing in the primary dendrite subregion being most responsive. This study provides a new perspective on the dynamic response property of astrocyte ensembles to neuronal activity.

Lanthanides are common fission products and the ability to separate and quantify these elements is critical to rapid radiochemistry applications. Published lanthanide separations using Eichrom Ln Spec resin utilize an HCl gradient. Here it is shown that the efficacy and resolution of the separation is improved when a nitric acid gradient is used instead. The described method allows parallel processing of many samples in 1.5 h followed by 60 min counting for quantification of 9 radioisotopes of 7 lanthanide elements.

Discovery and optimization of potency and selectivity of a non-Zn-chelating MMP-13 inhibitor with the aid of protein co-crystal structural information is reported. This inhibitor was observed to have a binding mode distinct from previously published MMP-13 inhibitors. Potency and selectivity were improved by extending the hit structure out from the active site into the S1' pocket.

A series of inhibitors of Pim-2 kinase identified by high-throughput screening is described. Details of the hit validation and lead generation process and structure-activity relationship (SAR) studies are presented. Disclosure of an unconventional binding mode for 1, as revealed by X-ray crystallography using the highly homologous Pim-1 protein, is also presented, and observed binding features are shown to correlate with the Pim-2 SAR. While highly selective within the kinase family, the series shows similar potency for both Pim-1 and Pim-2, which was expected on the basis of homology, but unusual in light of reports in the literature documenting a bias for Pim-1. A rationale for these observations based on Pim-1 and Pim-2 K(M(ATP)) values is suggested. Some interesting cross reactivity with casein kinase-2 was also identified, and structural features which may contribute to the association are discussed.

The goals of the Global Nuclear Energy Partnership (GNEP) are to expand the use of nuclear energy to meet increasing global energy demand in an environmentally sustainable manner, to address nuclear waste management issues without making separated plutonium, and to address nonproliferation concerns. The advanced burner reactor (ABR) is a fast reactor concept which supports the GNEP fuel cycle system. Since the integral fast reactor (IFR) and advanced liquid-metal reactor (ALMR) projects were terminated in 1994, there has been no major development on sodium-cooled fast reactors in the United States. Therefore, in support of the GNEP fast reactor program, the history of sodium-cooled reactor development was reviewed to support the initiation of this technology within the United States and to gain an understanding of the technology gaps that may still remain for sodium fast reactor technology. The fuel-handling system is a key element of any fast reactor design. The major functions of this system are to receive, test, store, and then load fresh fuel into the core; unload from the core; then clean, test, store, and ship spent fuel. Major requirements are that the system must be reliable and relatively easy to maintain. In addition, the system should be designed more » so that it does not adversely impact plant economics from the viewpoints of capital investment or plant operations. In this gap analysis, information on fuel-handling operating experiences in the following reactor plants was carefully reviewed: EBR-I, SRE, HNPF, Fermi, SEFOR, FFTF, CRBR, EBR-II, DFR, PFR, Rapsodie, Phenix, Superphenix, KNK, SNR-300, Joyo, and Monju. The results of this evaluation indicate that a standardized fuel-handling system for a commercial fast reactor is yet to be established. However, in the past sodium-cooled reactor plants, most major fuel-handling components-such as the rotatable plug, in-vessel fuel-handling machine, ex-vessel fuel transportation cask, ex-vessel sodium-cooled storage, and cleaning stations-have accumulated satisfactory construction and operation experiences. In addition, two special issues for future development are described in this report: large capacity interim storage and transuranic-bearing fuel handling. « less

During World War II, the Hanford Site in Washington became the location for U.S. plutonium production. In 2004, a bottle containing a sample of plutonium was recovered from a Hanford waste trench. Here, state-of-the-art instrumental analyses, reactor model simulations, and investigative science techniques were used to provide insights as to the origin of this unknown sample, a process collectively termed as nuclear archeology. Isotopic age dating conducted on the sample in 2007 indicated the sample was separated from the spent fuel 61.6 +/- 4.5 years earlier. The isotope (22)Na, a detectable product of a secondary nuclear reaction, proved useful as a powerful tool for nuclear forensic analysis as (1) an easily detectable signifier of the presence of alpha emitting actinides, (2) an indicator of sample splitting, and (3) a measure of the time since sample splitting. Analytical results of minor actinide isotopes and reactor model simulations confirmed the material originated from the X-10 reactor in Oak Ridge, TN. Corroborated by historical documents, we concluded this sample was part of the first batch of Pu separated at T-Plant, Hanford, the world's first industrial-scale reprocessing facility, on December 9, 1944. This sample represents the oldest known collection of man-made (239)Pu in the world.

CFE88 is a conserved essential gene product from Streptococcus pneumoniae. This 227-residue protein has minimal sequence similarity to proteins of known 3D structure. Sequence alignment models and computational protein threading studies suggest that CFE88 is a methyltransferase. Characterization of the conformation and function of CFE88 has been performed by using several techniques. Backbone atom and limited side-chain atom NMR resonance assignments have been obtained. The data indicate that CFE88 has two domains: an N-terminal domain with 163 residues and a C-terminal domain with 64 residues. The C-terminal domain is primarily helical, while the N-terminal domain has a mixed helical/extended (Rossmann) fold. By aligning the experimentally observed elements of secondary structure, an initial unrefined model of CFE88 has been constructed based on the X-ray structure of ErmC' methyltransferase (Protein Data Bank entry 1QAN). NMR and biophysical studies demonstrate binding of S-adenosyl-L-homocysteine (SAH) to CFE88; these interactions have been localized by NMR to the predicted active site in the N-terminal domain. Mutants that target this predicted active site (H26W, E46R, and E46W) have been constructed and characterized. Overall, our results both indicate that CFE88 is a methyltransferase and further suggest that the methyltransferase activity is essential for bacterial survival.

The goals of the Global Nuclear Energy Partnership (GNEP) are to expand the use of nuclear energy to meet increasing global energy demand in an environmentally sustainable manner, to address nuclea...

Spectrometers for the lowest-level radiometric measurements require materials of extreme radiopurity. Measurements of rare nuclear decays, e.g., neutrinoless double-beta decay, can require construction and shielding materials with bulk radiopurity reaching one micro-Becquerel per kilogram or less. When such extreme material purity is achieved, surface contamination, particularly solid daughters in the natural radon decay chains, can become the limiting background. High-purity copper is an important material for ultra-low-background spectrometers and thus is the focus of this work. A method for removing surface contamination at very low levels without attacking the bulk material is described. An assay method using a low-background proportional counter made of the material under examination is employed, and the preliminary result of achievable surface contamination levels is presented.

A project has been undertaken at Pacific Northwest National Laboratory (PNNL) to tailor a series of efficient chemical separations to allow the rapid quantification of gamma-ray emitting isotopes in mixed fission product (MFP) samples. In support of that goal, modeling of singles and coincident gamma-ray spectra that would result from various chemical separation strategies has been performed. These simulated spectra have identified likely instances of spectral interference and have provided an estimate of the time window available for the detection of radionuclides following various chemical separation schemes. A description of results to date is presented here, demonstrating the utility of this approach for improved processing and analysis of fission product samples.

A method for the separation and determination of total and isotopic uranium and plutonium by ICP/MS was developed for IAEA samples on cellulose-based media. Preparation of the IAEA samples involved a series of redox chemistries and separations using TRU® resin (Eichrom). The sample introduction system, an APEX nebulizer (Elemental Scientific, Inc.), provided enhanced nebulization for a several-fold increase in sensitivity and reduction in background. Application of mass bias (ALPHA) correction factors greatly improved the precision of the data. By combining the enhancements of chemical separation, instrumentation and data processing, detection levels for uranium and plutonium approached high attogram levels.

The search for neutrinoless double beta decay in 76Ge has driven the need for ultra-low background Ge detectors shielded by electroformed copper of ultra-high radiopurity (

The subsurface distribution of a nuclear waste tank leak on the U.S. Department of Energy’s Hanford Site was sampled by slant drilling techniques in order to characterize the chemical and radiological characteristics of the leaked material and assess geochemical transport properties of hazardous constituents. Sediment core samples recovered from the borehole were subjected to distilled water and acid leaching procedures with the resulting leachates analyzed for isotopic and chemical signatures. High-sensitivity inductively coupled plasma/mass spectrometry (ICP/MS) techniques were used for determination of isotopic ratios for Cs, I, Mo. Analysis of the isotopic patterns of I and Mo combined with associated chemical data showed evidence for at least two separate intrusions of nuclear waste into the subsurface. Isotopic data for Cs was inconclusive with respect to a source attribution signature.

Experimental results are provided for the sample analyses for technetium in charcoal samples placed in-line with a Savannah River Site (SRS) processing stack effluent stream as a part of an environmental surveillance program. The method for Tc removal from charcoal was based on that originally developed with high purity charcoal. Presented is the process that allowed for the quantitative analysis of 99Tc in SRS charcoal stack samples with and without 97Tc as a tracer. The results obtained with the method using the 97Tc tracer quantitatively confirm the results obtained with no tracer added. All samples contain 99Tc at the pg·g−1 level.

(241)Am has been deposited using a novel technique that employs a commercial inductively coupled plasma mass spectrometer. This work presents results of high-resolution alpha spectrometry on the (241)Am samples using a small area passivated implanted planar silicon detector. We have also investigated the mass-based separation capability by developing a (238)Pu sample, present as a minor constituent in a (244)Pu standard, and performed subsequent radiometric counting. With this new sample development method, the (241)Am samples achieved the intrinsic energy resolution of the detector used for these measurements. There was no detectable trace of any other isotopes contained in the (238)Pu implant demonstrating the mass-based separation (or enhancement) attainable with this technique.

The NMR structure is presented for compound 1 (BMS-480404) (Ki = 33 (+/-2) nM) bound to keratinocyte fatty acid-binding protein. This article describes interactions between a high affinity drug-like compound and a member of the fatty acid-binding protein family. A benzyl group ortho to the mandelic acid in 1 occupies an area of the protein that fatty acids do not normally contact. Similar to that in the kFABP-palmitic acid structure, the acid moiety in 1 is proximal to R129 and Y131. Computational modeling indicates that the acid moiety in 1 interacts indirectly via a modeled water molecule to R109.

RadICalc was developed to address the need for a computer program that could calculate the composition, activity, and measurable radiation of arbitrary radionuclide mixtures over time without significant effort from end-users. It provides an interface to perform decay calculations and can search and display the resulting data in graphical or tabular form. RadICalc can also determine radiation expected at specific masses with user-defined molecules in addition to atomic species for use in mass-based isotope separations for radiometric counting applications, a novel method under development at Pacific Northwest National Laboratory.

Measurements of volatile organic compounds (VOCs) were made at three rural sites in the southeast U.S. national parks: Mammoth Cave National Park, Kentucky; Cove Mountain, Great Smoky Mountains National Park, Tennessee; and Big Meadows, Shenandoah National Park, Virginia. In 1995 the three locations were sampling sites for the Southern Oxidants Study (SOS) Nashville Intensive, and the measurements of VOCs for Shenandoah were also made under contract with the National Park Service. Starting in 1996, the National Park Service added the other two parks to the monitoring contract. Hydrocarbon measurements made during June through September for the years 1995, 1996, and 1997 were analyzed in this study. Source classification techniques based on correlation coefficient, chemical reactivity, and ratioing were developed and applied to these data. The results show that anthropogenic VOCs from automobile exhaust appeared to be dominant at Mammoth Cave National Park, and at Cove Mountain, Great Smoky Mountains National Park, but other sources were also important at Big Meadows, Shenandoah National Park. Correlation and ratio analysis based on chemical reactivity provides a basis for source-receptor relationship. The most abundant ambient VOCs varied both in concentration and order depending on park and year, but the following VOCs appeared on the top 10 list for all three sites: isoprene (6.3 to 18.4 ppbv), propane (2.1 to 12.9 ppbv), isopentane (1.3 to 5.7 ppbv), and toluene (1.0 to 7.2 ppbv). Isoprene is naturally emitted by vegetation, and the others are produced mainly by fossil fuel combustion and industrial processes. Propylene-equivalent concentrations were calculated to account for differences in reaction rates between the hydroxyl radical and individual hydrocarbons, and to thereby estimate their relative contributions to ozone formation.

A sequential injection (SI) separation system has been developed for separation and analysis of Am, Pu, and Np isotopes using on-line inductively coupled plasma mass spectrometry (ICP MS) detection. On-line actinide separations were carried out using an actinide-specific extraction chromatographic material (TRU-resin, Eichrom Industries, Inc. USA). Separations are demonstrated and characterized that are compatible with on-line ICP MS detection and address isobaric (241Am/241Pu, 244Pu/244Cm, 238U/238Pu), molecular (238UH/239Pu), and spectral (238U/237Np) interferences encountered in the analysis of Am, Pu and Np isotopic composition using ICP MS. The effects of reductive sample treatment on the extent of U matrix removal were investigated. Uranium separation factors were limited by the formation of U(IV) species, which exhibited separation behaviors similar to that of Pu(IV) and Np(IV). Nevertheless, separation factors as high as 3.0 × 105 can be achieved at the 0.1 mg ml−1 U concentration levels using Fe(II) sulfamate as a reductant. The sequential injection (SI) ICP MS technique has been applied towards analysis of Am, Pu, and Np isotopes in a dissolved vitrified nuclear waste sample. Results obtained using the automated separation technique with ICP MS detection were in satisfactory agreement with results obtained using standard analytical methodologies.

Previous evidence suggests there is an association between cardiovascular disease and depression. Tricyclic antidepressants (TCAs) are contraindicated in patients with a recent history of cardiovascular disease. 5678 patients between the ages of 50 and 79 years and with a new acute cardiac event were selected from a computerised general practice database and followed up for one year. ‘New episodes’ of diagnosed depression were identified and treatment with antidepressants was investigated. 183 656 patients aged 50–79 years without a cardiac event were used for comparison. A significant association was found between cardiovascular disease and a new diagnosis of depression (adjusted OR=2.23, 95 per cent CI 1.90–2.62). Patients who had suffered a recent cardiac event were less likely to be prescribed a TCA than an SSRI (adjusted OR=0.59, 95 per cent CI 0.41–0.85). However, 35 per cent of patients with a recent cardiac event were initially prescribed a TCA. The findings indicate that general practitioners recognise the dangers of prescribing TCAs to patients with a recent history of cardiovascular disease. However, a substantial minority of cardiac patients were prescribed TCAs where they may not have been the optimal choice. Copyright © 1999 John Wiley & Sons, Ltd.

Several trimethylenemethane-type (TMM-type) dinitroxide biradicals have been prepared that differ in tert-butylaminoxylphenyl ring torsion angles by virtue of different steric demands of their “spin-protecting groups”. In addition, the synthesis of a TMM-type dinitroxide having a planar π-system is reported. The synthetic route employed for the planar TMM-type biradical is general and should therefore expand the utility of TMM-type biradicals that are susceptible to bond torsions that attenuate exchange coupling. The compounds presented are designed to be effective to map out J-coupling/conformation space in TMM-type biradicals. EPR spectroscopic parameters (hfcc and D-values) were compared to determine if their values reflected differences in conformation in the series of biradicals. Interestingly, neither N- nor H-hfcc varied within our series of biradicals/monoradicals in a regular fashion, indicating that there is no apparent relationship between N- or H-hfcc and conformation (as judged by molecular m...

The syntheses of three new bis(semiquinone)s (2••2-, 3••2-, and 5••2-) linked through carbon−carbon double bonds in a geminal fashion (2••2- and 3••2-), and through an sp3 carbon (5••2-), are presented, as well as the results of variable-temperature EPR (VT-EPR) spectroscopy on these biradicals and two previously reported bis(semiquinone)s, 1••2- and 4••2-. We suggest that the potential difference in redox couples associated with a biradical is useful for qualitatively assessing changes in the exchange parameter within an isostructural series. The zero-field-splitting parameters for 1••2-−5••2- are consistent with their electronic structures: biradicals 1••2-−3••2- which have conjugating groups attached to the semiquinone rings have D-values less than 5••2-, a bis(semiquinone) that lacks such a conjugating group. Also, the D-value of 3••2- is significantly less than those of 1••2- and 2••2-, in agreement with larger interelectron separation in 3••2-, a biradical with quinone-methide π-system delocalizati...

The synthesis and characterization of an anion biradical, 1(-)(*)(*), composed of semiquinone and nitroxide functionalities is described. The biradical was prepared by reduction of the nitroxide-orthoquinone precursor using both electrochemical and chemical methods. The zero-field-splitting parameter, |D/hc|, for 1(-)(*)(*) is consistent with the proposed electronic structure, based on a comparison to |D/hc| of related biradicals. Finally, the results of variable-temperature EPR spectroscopy are in agreement with ferromagnetic (high-spin) coupling of the two unpaired electrons through the m-phenylene group.

The detection of129I by Inductively Coupled Plasma Mass Spectrometry (ICP/MS) in environmental samples can be used to determine anthropogenic release of this long-lived radionuclide, which is a definitive indicator of certain nuclear activities. By using selective precipitation techniques with on-line ICP/MS detection, low levels of129I can be detected. The major interference for the ICP/MS detection of129I is due the presence of natural129Xe found in water samples at a concentration of about 1 ng/ml. This work will demonstrate a instrument detection of less than 50 fg129I from environmental air samples and shows promise for a rugged ICP/MS technique to monitor129I levels in ambient air for nonproliferation monitoring purposes.

Adipocyte lipid-binding protein (A-LBP) and muscle fatty acid-binding protein (M-FABP) are members of a family of small ( approximately 15 kDa) cytosolic proteins that are involved in the metabolism of fatty acids and other lipid-soluble molecules. Although highly homologous (65%) and structurally very similar, A-LBP and M-FABP display distinct ligand binding characteristics. Since ligand binding may be influenced by intrinsic protein dynamical properties, we have characterized the backbone and side chain dynamics of uncomplexed (apo) human A-LBP and M-FABP. Backbone dynamics were characterized by measurements of 15N T1 and T2 values and ¿1H¿-15N NOEs. These data were analyzed using model-free spectral density functions and reduced spectral density mapping. The dynamics of methyl-containing side chains were charaterized by measurements of 2H T1 and T1rho relaxation times of 13C1H22H groups. The 2H relaxation data were analyzed using the model-free approach. For A-LBP, 15N relaxation data were obtained for 111 residues and 2H relaxation data were obtained for 42 methyl groups. For M-FABP, 15N relaxation data were obtained for 111 residues and 2H relaxation data were obtained for 53 methyl groups. The intrinsic flexibilities of these two proteins are compared, with particular emphasis placed on binding pocket residues. There are a number of distinct dynamical differences among corresponding residues between the two proteins. In particular, many residues display greater backbone picosecond to nanosecond and/or microsecond to millisecond time scale mobility in A-LBP relative to M-FABP, including F57, K58, and most residues in alpha-helix 2 (residues 28-35). Variations in the dynamics of this region may play a role in ligand selectivity. The side chains lining the fatty acid binding pocket display a wide range of motional restriction in both proteins. Side chains showing distinct dynamical differences between the two proteins include those of residues 20, 29, and 51. This information provides a necessary benchmark for determining dynamical changes induced by ligand binding and may ultimately lead to an enhanced understanding of ligand affinity and selectivity among fatty acid-binding proteins.

We hypothesize that paramagnetic, multidentate, chelating ligands can be used to create molecular magnetic materials with high ordering temperatures. The ligands proposed for such materials are di-, tri-, and oligosemiquinone molecules. This paper will discuss the synthesis and characterization of these semiquinone ligands.

Anomalous crosspeaks and additional resonances in the indirectly detected dimension have been previously observed in a number of 2D NMR experiments applied to samples having at least one concentrated species. These unexpected peaks exhibit all the characteristics of intermolecular multiple-quantum coherences. Since these coherences are possible within the concentrated species alone, their creation and subsequent detection may be one of the causes for poor water suppression in a variety of biomolecular NMR experiments, e.g., the conventional MQ-filtered- (MQF) and MQ-COSY experiments applied to proteins/peptides in 90% water. In this report, we experimentally characterize the creation/observation of intermolecular water−water MQ coherences using variable-angle pulsed field gradients. We show that the existing theoretical picture is consistent with all of our experimental observations, thereby validating the predictive power of the intuition gained from this picture. We also provide an increased understandi...

Volatile hydrocarbons play an important role in the photochemical formation of tropospheric ozone, especially in urban atmospheres. In order to assess the significance and relative impact of biogenic and anthropogenic hydrocarbon emissions as precursors of ozone formation, a program to monitor a suite of 56 common nonmethane hydrocarbons (NMHCs), the 1992 Atlanta Intensive, was implemented as part of the Southern Oxidants Study (SOS). During the last decade, Atlanta has averaged approximately 12 days of ozone nonattainment per year, typically occurring in summer during periods of air stagnation. Under current EPA guidelines, this classifies the Atlanta metropolitan area as having a serious ozone nonattainment problem. This study constituted a significant example of an intensive data collection effort for NMHCs. Three independent laboratory groups using a common standard operating procedure were involved in the analysis of both time integrated (hourly) and discrete canister samples collected over ...