The nitrate to ammonia and ceramic (NAC) process for the denitration and immobilization of low-level radioactive liquid waste (LLW)

Hazardous radioactive liquid waste is the legacy of more than 50 years of plutonium production associated with the United States' nuclear weapons program. It is estimated that more than 245,000 tons of nitrate wastes are stored at facilities such as the single-shell tanks (SST) at the Hanford Site in the state of Washington, and the Melton Valley storage tanks at Oak Ridge National Laboratory (ORNL) in Tennessee. In order to develop an innovative, new technology for the destruction and immobilization of nitrate-based radioactive liquid waste, the United State Department of Energy (DOE) initiated the research project which resulted in the technology known as the Nitrate to Ammonia and Ceramic (NAC) process. However, inasmuch as the nitrate anion is highly mobile and difficult to immobilize, especially in relatively porous cement-based grout which has been used to date as a method for the immobilization of liquid waste, it presents a major obstacle to environmental clean-up initiatives. Thus, in an effort to contribute to the existing body of knowledge and enhance the efficacy of the NAC process, this research involved the experimental measurement of the rheological and heat transfer behaviors of the NAC product slurry and the determination of the optimal operating parameters for the continuous NAC chemical reaction process. Test results indicate that the NAC product slurry exhibits a typical non-Newtonian flow behavior. Correlation equations for the slurry's rheological properties and heat transfer rate in a pipe flow have been developed; these should prove valuable in the design of a full-scale NAC processing plant. The 20-percent slurry exhibited a typical dilatant (shear thickening) behavior and was in the turbulent flow regime due to its lower viscosity. The 40-percent slurry exhibited a typical pseudoplastic (shear thinning) behavior and remained in the laminar flow regime throughout its experimental range. The reactions were found to be more efficient in the lower temperature range investigated. With respect to leachability, the experimental final NAC ceramic waste form is comparable to the final product of vitrification, the technology chosen by DOE to treat these wastes. As the NAC process has the potential of reducing the volume of nitrate-based radioactive liquid waste by as much as 70 percent, it not only promises to enhance environmental remediation efforts but also effect substantial cost savings. ^

Covalent protein adduction by drugs of abuse

Recreational abuse of the drugs cocaine, methamphetamine, and morphine continues to be prevalent in the United States of America and around the world. While numerous methods of detection exist for each drug, they are generally limited by the lifetime of the parent drug and its metabolites in the body. However, the covalent modification of endogenous proteins by these drugs of abuse may act as biomarkers of exposure and allow for extension of detection windows for these drugs beyond the lifetime of parent molecules or metabolites in the free fraction. Additionally, existence of covalently bound molecules arising from drug ingestion can offer insight into downstream toxicities associated with each of these drugs. This research investigated the metabolism of cocaine, methamphetamine, and morphine in common in vitro assay systems, specifically focusing on the generation of reactive intermediates and metabolites that have the potential to form covalent protein adducts. Results demonstrated the formation of covalent adduction products between biological cysteine thiols and reactive moieties on cocaine and morphine metabolites. Rigorous mass spectrometric analysis in conjunction with in vitro metabolic activation, pharmacogenetic reaction phenotyping, and computational modeling were utilized to characterize structures and mechanisms of formation for each resultant thiol adduction product. For cocaine, data collected demonstrated the formation of adduction products from a reactive arene epoxide intermediate, designating a novel metabolic pathway for cocaine. In the case of morphine, data expanded on known adduct-forming pathways using sensitive and selective analysis techniques, following the known reactive metabolite, morphinone, and a proposed novel metabolite, morphine quinone methide. Data collected in this study describe novel metabolic events for multiple important drugs of abuse, culminating in detection methods and mechanistic descriptors useful to both medical and forensic investigators when examining the toxicology associated with cocaine, methamphetamine, and morphine.

Covalent protein adduction of nitrogen mustards and related compounds

Chemical warfare agents continue to pose a global threat despite the efforts of the international community to prohibit their use in warfare. For this reason, improvement in the detection of these compounds remains of forensic interest. Protein adducts formed by the covalent modification of an electrophilic xenobiotic and a nucleophilic amino acid may provide a biomarker of exposure that is stable and specific to compounds of interest (such as chemical warfare agents), and have the capability to extend the window of detection further than the parent compound or circulating metabolites. This research investigated the formation of protein adducts of the nitrogen mustard chemical warfare agents mechlorethamine (HN-2) and tris(2-chloroethyl)amine (HN-3) to lysine and histidine residues found on the blood proteins hemoglobin and human serum albumin. Identified adducts were assessed for reproducibility and stability both in model peptide and whole protein assays. Specificity of these identified adducts was assessed using in vitro assays to metabolize common therapeutic drugs containing nitrogen mustard moieties. Results of the model peptide assays demonstrated that HN-2 and HN-3 were able to form stable adducts with lysine and histidine residues under physiological conditions. Results for whole protein assays identified three histidine adducts on hemoglobin, and three adducts (two lysine residues and one histidine residue) on human serum albumin that were previously unknown. These protein adducts were determined to be reproducible and stable at physiological conditions over a three-week analysis period. Results from the in vitro metabolic assays revealed that adducts formed by HN-2 and HN-3 are specific to these agents, as metabolized therapeutic drugs (chlorambucil, cyclophosphamide, and melphalan) did not form the same adducts on lysine or histidine residues as the previously identified adducts formed by HN-2 and HN-3. Results obtained from the model peptide and full protein work were enhanced by comparing experimental data to theoretical calculations for adduct formation, providing further confirmatory data. This project was successful in identifying and characterizing biomarkers of exposure to HN-2 and HN-3 that are specific and stable and which have the potential to be used for the forensic determination of exposure to these dangerous agents.

Covalent Protein Adduction by Drugs of Abuse

Recreational abuse of the drugs cocaine, methamphetamine, and morphine continues to be prevalent in the United States of America and around the world. While numerous methods of detection exist for each drug, they are generally limited by the lifetime of the parent drug and its metabolites in the body. However, the covalent modification of endogenous proteins by these drugs of abuse may act as biomarkers of exposure and allow for extension of detection windows for these drugs beyond the lifetime of parent molecules or metabolites in the free fraction. Additionally, existence of covalently bound molecules arising from drug ingestion can offer insight into downstream toxicities associated with each of these drugs. This research investigated the metabolism of cocaine, methamphetamine, and morphine in common in vitro assay systems, specifically focusing on the generation of reactive intermediates and metabolites that have the potential to form covalent protein adducts. Results demonstrated the formation of covalent adduction products between biological cysteine thiols and reactive moieties on cocaine and morphine metabolites. Rigorous mass spectrometric analysis in conjunction with in vitro metabolic activation, pharmacogenetic reaction phenotyping, and computational modeling were utilized to characterize structures and mechanisms of formation for each resultant thiol adduction product. For cocaine, data collected demonstrated the formation of adduction products from a reactive arene epoxide intermediate, designating a novel metabolic pathway for cocaine. In the case of morphine, data expanded on known adduct-forming pathways using sensitive and selective analysis techniques, following the known reactive metabolite, morphinone, and a proposed novel metabolite, morphine quinone methide. Data collected in this study describe novel metabolic events for multiple important drugs of abuse, culminating in detection methods and mechanistic descriptors useful to both medical and forensic investigators when examining the toxicology associated with cocaine, methamphetamine, and morphine.

Importance of water source in controlling leaf leaching losses in a dwarf red mangrove (Rhizophora mangle L.) wetland

The southern Everglades mangrove ecotone is characterized by extensive dwarf Rhizophora mangle L. shrub forests with a seasonally variable water source (Everglades – NE Florida Bay) and residence times ranging from short to long. We conducted a leaf leaching experiment to understand the influence that water source and its corresponding water quality have on (1) the early decay of R. mangle leaves and (2) the early exchange of total organic carbon (TOC) and total phosphorus (TP) between leaves and the water column. Newly senesced leaves collected from lower Taylor River (FL) were incubated in bottles containing water from one of three sources (Everglades, ambient mangrove, and Florida Bay) that spanned a range of salinity from 0 to 32‰, [TOC] from 710 to 1400 μM, and [TP] from 0.17 to 0.33 μM. We poisoned half the bottles in order to quantify abiotic processes (i.e., leaching) and assumed that non-poisoned bottles represented both biotic (i.e., microbial) and abiotic processes. We sacrificed bottles after 1,2, 5, 10, and 21 days of incubation and quantified changes in leaf mass and changes in water column [TOC] and [TP]. We saw 10–20% loss of leaf mass after 24 h—independent of water treatment—that leveled off by Day 21. After 3 weeks, non-poisoned leaves lost more mass than poisoned leaves, and there was only an effect of salinity on mass loss in poisoned incubations—with greatest leaching-associated losses in Everglades freshwater. Normalized concentrations of TOC in the water column increased by more than two orders of magnitude after 21 days with no effect of salinity and no difference between poisoned and non-poisoned treatments. However, normalized [TP] was lower in non-poisoned incubations as a result of immobilization by epiphytic microbes. This immobilization was greatest in Everglades freshwater and reflects the high P demand in this ecosystem. Immobilization of leached P in mangrove water and Florida Bay water was delayed by several days and may indicate an initial microbial limitation by labile C during the dry season.

Proton nuclear magnetic resonance investigation of the native and modified active site structure of heme proteins

Hemoproteins are a very important class of enzymes in nature sharing the essentially same prosthetic group, heme, and are good models for exploring the relationship between protein structure and function. Three important hemoproteins, chloroperoxidase (CPO), horseradish peroxidase (HRP), and cytochrome P450cam (P450cam), have been extensively studied as archetypes for the relationship between structure and function. In this study, a series of 1D and 2D NMR experiments were successfully conducted to contribute to the structural studies of these hemoproteins. ^ During the epoxidation of allylbenzene, CPO is converted to an inactive green species with the prosthetic heme modified by addition of the alkene plus an oxygen atom forming a five-membered chelate ring. Complete assignment of the NMR resonances of the modified porphyrin extracted and demetallated from green CPO unambiguously established the structure of this porphyrin as an NIII-alkylated product. A novel substrate binding motif of CPO was proposed from this concluded regiospecific N-alkylation structure. ^ Soybean peroxidase (SBP) is considered as a more stable, more abundant and less expensive substitute of HRP for industrial applications. A NMR study of SBP using 1D and 2D NOE methods successfully established the active site structure of SBP and consequently fills in the blank of the SBP NMR study. All of the hyperfine shifts of the SBP-CN- complex are unambiguously assigned together with most of the prosthetic heme and all proximal His170 resonances identified. The active site structure of SBP revealed by this NMR study is in complete agreement with the recombinant SBP crystal structure and is highly similar to that of the HRP with minor differences. ^ The NMR study of paramagnetic P450cam had been greatly restricted for a long time. A combination of 2D NMR methods was used in this study for P450cam-CN - complexes with and without camphor bound. The results lead to the first unequivocal assignments of all heme hyperfine-shifted signals, together with certain correlated diamagnetic resonances. The observed alternation of the assigned novel proximal cysteine β-CH2 resonances induced by camphor binding indicated a conformational change near the proximal side.^

Nano-grids of Yeast Cytochrome C

One innovative thought in biomolecular electronics is the exploitation of electron transfer proteins. Using nature's self assembly techniques, proteins can build highly organized edifices with retained functional activity, and they can serve as platforms for biosensors. In this research work, Yeast Cytochrome C (YCC) is immobilized with a help of a linker molecule, 3-Mercaptopropyltrimethoxysilane (3-MPTS) on a hydroxylated surface of a silicon substrate. Atomic Force Microscopy (AFM) is used for characterization. AFM data shows immobilization of one YCC molecule in between eight grids that are formed by the linker molecules. 3-MPTS monolayers are organized in grids that are 1.2 nm apart. Immobilization of 3-MPTS was optimized using a concentration of 5 mM in a completely dehydrated state for 30 minutes. The functionally active grids of YCC can now be incorporated with Cytochrome C oxidase on a Platinum electrode surface for transfer of electrons in development of biosensors, such as nitrate sensor, that are small in size, cheaper, and easier to manufacture than the top-down approach of fabrication of molecular biodevices

Design and development of an enzyme-linked biosensor for detection and quantification of phosphate species

The objective of this study is to design and development of an enzyme-linked biosensor for detection and quantification of phosphate species. Various concentrations of phosphate species were tested and completed for this study. Phosphate is one of the vital nutrients for all living organisms. Phosphate compounds can be found in nature (e.g., water sediments), and they often exist in aninorganic form. The amount of phosphates in the environment strongly influences the operations of living organisms. Excess amount of phosphate in the environment causes eutrophication which in turn causes oxygen deficit for the other living organisms. Fish die and degradation of habitat in the water occurs as a result of eutrophication. In contrast, low phosphate concentration causes death of vegetation since plants utilize the inorganic phosphate for photosynthesis, respiration, and regulation of enzymes. Therefore, the phosphate quantity in lakes and rivers must be monitored. Result demonstrated that phosphate species could be detected in various organisms via enzyme-linked biosensor in this research.