Characterization of Neutral Lipids of the Oleaginous Alga Micractinum inermum.

An oleaginous microalga Micractinum inermum isolated from Mariana Lake, AB, Canada was cultured in a 1000 L photobioreactor with an f/2 medium to study its lipid content and neutral lipid profile. Algal biomass was collected at the stationary phase contained a significant amount of lipids (44.2%), as determined by Folch's method. The lipid was fractionated into neutral lipid, glycolipid and phospholipid fractions. The neutral lipid constitutes almost 77.3% of the total lipid species and is mainly composed of triacylglycerols (TAGs) determined by a proton NMR study. UHPLC-HRMS analysis allows us for the first time to identify 81 TAGs in the neutral lipid fraction of M. inermum. The fatty acid acyl side chains were identified based on fragment ions observed in MSMS analysis. TAGs with fatty acid acyl chains 18:1/18:1/18:1, 18:1/18:1/16:0, 18:2/18:1/16:0, and 18:2/18:2/18:0 were the major ones among the identified TAGs. Fatty acid analysis further supports the fact that oleic acid was the major fatty acid present in the neutral lipid fraction of M. inermum constituting 41.7%, followed by linoleic acid at 21.5%, and palmitic acid at 21.2%. The saturated and monounsaturated fatty acids were 67.8% or higher in the lipid fraction. Long-chain fatty acids were only present in a minor quantity. The results clearly demonstrate that M. inermum is an excellent source for TAGs.

Plant Growth-Promoting Rhizobacteria (PGPR) with Microbial Growth Broth Improve Biomass and Secondary Metabolite Accumulation of Cannabis sativa L.

Plant growth-promoting rhizobacteria (PGPR) are a sustainable crop production input; some show positive effects under laboratory conditions but poorly colonize host field-grown plants. Inoculating with PGPR in microbial growth medium (e.g., King's B) could overcome this. We evaluated cannabis plant (cv. CBD Kush) growth promotion by inoculating three PGPR (Bacillus sp., Mucilaginibacter sp., and Pseudomonas sp.) in King's B at vegetative and flower stages. At the vegetative stage, Mucilaginibacter sp. inoculation increased flower dry weight (24%), total CBD (11.1%), and THC (11.6%); Pseudomonas sp. increased stem (28%) dry matter, total CBD (7.2%), and THC (5.9%); and Bacillus sp. increased total THC by 4.8%. Inoculation with Mucilaginibacter sp. and Pseudomonas sp. at the flowering stage led to 23 and 18% increases in total terpene accumulation, respectively. Overall, vegetative inoculation with PGPR enhanced cannabis yield attributes and chemical profiles. Further research into PGPR inoculation onto cannabis and the subsequent level of colonization could provide key insights regarding PGPR-host interactions.

Evaluation of the Uptake, Metabolism, and Secretion of Toxicants by Zebrafish Larvae.

Zebrafish larvae have classically been used as a high-throughput model with which to test both the bioactivity and toxicity of known and novel compounds, making them a promising whole organism New Approach Method in the context of the international momentum to eliminate animal testing. Larvae are generally exposed to the chemicals being tested in a static environment and the concentration-response patterns are calculated based on the initial bath concentrations of the compounds. This approach rarely takes into account the absorption, distribution, metabolism, and excretion of the compounds being tested, which can have a significant effect on the toxicokinetic profiles of the compounds and thus impact the predictive ability of the model. In this study, we have evaluated the toxicokinetic profile of 5 known toxicants, 3 phenolic compounds, along with thiabendazole and 3,4-dicholoronalanine, at 6, 8, 24, 72, and 120 h postfertilization in order to match the exposure timelines of a standard in vitro fish embryo toxicity test. It was revealed that in addition to bioaccumulation effects, the compounds were all actively metabolized and excreted by the larvae. Importantly, comparisons between the toxicants revealed that the patterns of uptake and metabolism were varied and could often partially explain the differences in their concentration-response patterns. The findings of this study are significant as they highlight the requirement for an assessment of the stability and toxicokinetic profile of chemicals tested using standard zebrafish larval toxicity assays in order to better understand and compare their toxicity profiles.

Analysis of Polar Lipids in Hemp (Cannabis sativa L.) By-Products by Ultra-High Performance Liquid Chromatography and High-Resolution Mass Spectrometry.

Polar lipids were extracted from residual biomass of hemp (Cannabis sativa L.) by-products with EtOH and partitioned into aqueous and chloroform fractions. The chloroform fractions were studied for their lipid composition using solid-phase extraction (SPE) followed by UHPLC/HRMS and NMR analyses. The 1H NMR and gravimetric yield of SPE indicated triacylglycerols covered ≥ 51.3% of the chloroform fraction of hemp seed hulls and hemp cake. UHPLC/HRMS analyses of remaining polar lipids led to the identification of nine diacylglycerols (DAGs), six lysophosphatidylcholines (LPCs), five lysophosphatidylethanolamines (LPEs), eight phosphatidylethanolamines (PEs), and thirteen phosphatidylcholines (PCs) for the first time from hemp seed hulls. The regiospecificity of fatty acyl substitutes in glycerol backbone of individual phospholipids were assigned by analyzing the diagnostic fragment ions and their intensities. The heat-map analysis suggested that DAG 18:2/18:2, 1-LPC 18:2, 1-LPE 18:2, PE 18:2/18:2, and PC 18:2/18:2 were the predominant molecules within their classes, supported by the fact that linoleic acid was the major fatty acid covering > 41.1% of the total fatty acids determined by GC-FID analysis. The 31P NMR analysis confirmed the identification of phospholipids and suggested PC covers ≥ 37.9% of the total phospholipid present in hemp by-products. HPLC purification led to the isolation of 1,2-dilinoleoylphosphatidylcholine and 1-palmitoyl-2-linoleoylphosphatidylcholine. These two major PCs further confirmed the UHPLC/HRMS finding.

Production of quorum sensing-related metabolites and phytoalexins during Pseudomonas aeruginosa-Brassica napus interaction.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that has been shown to interact with many organisms throughout the domains of life, including plants. How this broad-host-range bacterium interacts with each of its diverse hosts, especially the metabolites that mediate these interactions, is not completely known. In this work, we used a liquid culture root infection system to collect plant and bacterial metabolites on days 1, 3 and 5 post-P. aeruginosa (strain PA14) infection of the oilseed plant, canola (Brassica napus). Using MS-based metabolomics approaches, we identified the overproduction of quorum sensing (QS)-related (both signalling molecules and regulated products) metabolites by P. aeruginosa while interacting with canola plants. However, the P. aeruginosa infection induced the production of several phytoalexins, which is a part of the hallmark plant defence response to microbes. The QS system of PA14 appears to only mediate part of the canola-P. aeruginosa metabolomic interactions, as the use of isogenic mutant strains of each of the three QS signalling branches did not significantly affect the induction of the phytoalexin brassilexin, while induction of spirobrassinin was significantly decreased. Interestingly, a treatment of purified QS molecules in the absence of bacteria was not able to induce any phytoalexin production, suggesting that active bacterial colonization is required for eliciting phytoalexin production. Furthermore, we identified that brassilexin, the only commercially available phytoalexin that was detected in this study, demonstrated a MIC of 400 µg ml-1 against P. aeruginosa PA14. The production of phytoalexins can be an effective component of canola innate immunity to keep potential infections by the opportunistic pathogen P. aeruginosa at bay.

Triacylglycerols and Other Lipids Profiling of Hemp By-Products.

Hemp seed by-products, namely hemp cake (hemp meal) and hemp hulls were studied for their lipid content and composition. Total lipid content of hemp cake and hemp hulls was 13.1% and 17.5%, respectively. Oil extraction yields using hexane, on the other hand, were much lower in hemp cake (7.4%) and hemp hulls (12.1%). Oil derived from both hemp seeds and by-products were primarily composed of neutral lipids (>97.1%), mainly triacylglycerols (TAGs), determined by SPE and confirmed by NMR study. Linoleic acid was the major fatty acid present in oils derived from hemp by-products, covering almost 55%, followed by α-linolenic acid, covering around 18% of the total fatty acids. For the first time, 47 intact TAGs were identified in the hemp oils using UPLC-HRMS. Among them, TAGs with fatty acid acyl chain 18:3/18:2/18:2 and 18:3/18:2/18:1 were the major ones, followed by TAGs with fatty acid acyl chain of 18:3/18:3/18:2, 18:2/18:2/16:0, 18:2/18:2/18:1, 18:3/18:2.18:0, 18:2/18:2/18:0, 18:2/18:1/18:1 and 18:3/18:2:16:0. Besides TAGs, low levels of terpenes, carotenoids and cannabidiolic acid were also detected in the oils. Moreover, the oils extracted from hemp by-products possessed a dose-dependent DPPH radical scavenging property and their potencies were in a similar range compared to other vegetable oils.

9,10-Dihydro-5-hydroxy-2,3,6-trimethoxyphenanthrene-1,4-dione: a new dihydrophenanthrene from commercial cannabis and its effect on zebrafish larval behaviour.

A new dihydrophenanthrene derivative namely 9,10-dihydro-5-hydroxy-2, 3,6-trimethoxyphenanthrene-1,4-dione (1) was isolated from commercial cannabis product together with 4,5-dihydroxy-2,3,6-trimethoxy-9,10-dihydrophenanthrene (2), 4-hydroxy-2,3,6,7-tetramethoxy-9,10-dihydrophenanthrene (3), combretastatin B-2 (4) and isocannbispiradienone (5). Structure elucidation of the isolated compounds were done based on the interpretation of the mass spectrometry (MS) and nuclear magnetic resonance (NMR) data. New dihydrophenanthrene derivative (1) was tested for its effect on zebrafish larval behaviour. Preliminary results suggested that the new dihydrophenanthrene derivative (1) exhibits similar effect on zebrafish larval behaviour as cannabidiol (CBD), a biologically active component of Cannabis.

Low Mannitol Concentrations in Arabidopsis thaliana Expressing Ectocarpus Genes Improve Salt Tolerance.

Mannitol is abundant in a wide range of organisms, playing important roles in biotic and abiotic stress responses. Nonetheless, mannitol is not produced by a vast majority of plants, including many important crop plants. Mannitol-producing transgenic plants displayed improved tolerance to salt stresses though mannitol production was rather low, in the µM range, compared to mM range found in plants that innately produce mannitol. Little is known about the molecular mechanisms underlying salt tolerance triggered by low concentrations of mannitol. Reported here is the production of mannitol in Arabidopsis thaliana, by expressing two mannitol biosynthesis genes from the brown alga Ectocarpus sp. strain Ec32. To date, no brown algal genes have been successfully expressed in land plants. Expression of mannitol-1-phosphate dehydrogenase and mannitol-1-phosphatase genes was associated with the production of 42.3-52.7 nmol g-1 fresh weight of mannitol, which was sufficient to impart salinity and temperature stress tolerance. Transcriptomics revealed significant differences in the expression of numerous genes, in standard and salinity stress conditions, including genes involved in K+ homeostasis, ROS signaling, plant development, photosynthesis, ABA signaling and secondary metabolism. These results suggest that the improved tolerance to salinity stress observed in transgenic plants producing mannitol in µM range is achieved by the activation of a significant number of genes, many of which are involved in priming and modulating the expression of genes involved in a variety of functions including hormone signaling, osmotic and oxidative stress, and ion homeostasis.

Insights into the κ/ι-carrageenan metabolism pathway of some marine Pseudoalteromonas species.

Pseudoalteromonas is a globally distributed marine-associated genus that can be found in a broad range of aquatic environments, including in association with macroalgal surfaces where they may take advantage of these rich sources of polysaccharides. The metabolic systems that confer the ability to metabolize this abundant form of photosynthetically fixed carbon, however, are not yet fully understood. Through genomics, transcriptomics, microbiology, and specific structure-function studies of pathway components we address the capacity of newly isolated marine pseudoalteromonads to metabolize the red algal galactan carrageenan. The results reveal that the κ/ι-carrageenan specific polysaccharide utilization locus (CarPUL) enables isolates possessing this locus the ability to grow on this substrate. Biochemical and structural analysis of the enzymatic components of the CarPUL promoted the development of a detailed model of the κ/ι-carrageenan metabolic pathway deployed by pseudoalteromonads, thus furthering our understanding of how these microbes have adapted to a unique environmental niche.

Analysis of the Uptake, Metabolism, and Behavioral Effects of Cannabinoids on Zebrafish Larvae.

The Cannabis sativa plant contains numerous phytocannabinoids and terpenes with known or potential biological activity. For decades, plant breeders have been breeding the Cannabis plant to control for a desired ratio of the major cannabinoids. A high-throughput in vivo model to understand the relationship between the chemical composition of different strains and their therapeutic potential then becomes of value. Measuring changes in the behavioral patterns of zebrafish larvae is an established model with which to test the biological activity of neuroactive compounds. However, there is currently little information regarding the uptake kinetics and metabolism of compounds by larvae. In this study, we chose to compare the uptake kinetics and metabolism of Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) alone or in combination with their effects on larval behavior. We have shown that both compounds have distinct behavioral patterns and concentration response profiles. Additionally, the uptake kinetics observed for each compound appears to correlate with the change in behavior observed in the behavioral assays. When combinations of THC and CBD were tested there were shifts in both the behavioral activity and the uptake kinetics of each compound compared with when they were tested alone. Finally, the THC/CBD-derived metabolites detected in the larvae are similar to those found in mammalian systems. This study thus provides a model for further testing of additional cannabinoids and potentially plant extracts.

Characterization of Shrimp Oil from Pandalus borealis by High Performance Liquid Chromatography and High Resolution Mass Spectrometry.

Northern shrimp (Pandalus borealis) oil, which is rich in omega-3 fatty acids, was recovered from the cooking water of shrimp processing facilities. The oil contains significant amounts of omega-3 fatty acids in triglyceride form, along with substantial long-chain monounsaturated fatty acids (MUFAs). It also features natural isomeric forms of astaxanthin, a nutritional carotenoid, which gives the oil a brilliant red color. As part of our efforts in developing value added products from waste streams of the seafood processing industry, we present in this paper a comprehensive characterization of the triacylglycerols (TAGs) and astaxanthin esters that predominate in the shrimp oil by using HPLC-HRMS and MS/MS, as well as 13C-NMR. This approach, in combination with FAME analysis, offers direct characterization of fatty acid molecules in their intact forms, including the distribution of regioisomers in TAGs. The information is important for the standardization and quality control, as well as for differentiation of composition features of shrimp oil, which could be sold as an ingredient in health supplements and functional foods.

Strategic identification of in vitro metabolites of 13-desmethyl spirolide C using liquid chromatography/high-resolution mass spectrometry.

A strategy to identify metabolites of a marine biotoxin, 13-desmethyl spirolide C, has been developed using liquid chromatography coupled to high-resolution mass spectrometry (LC/HRMS). Metabolites were generated in vitro through incubation with human liver microsomes. A list of metabolites was established by selecting precursor ions of a common fragment ion characteristic of the spirolide toxin which was known to contain a cyclic imine ring. Accurate mass measurements were subsequently used to confirm the molecular formula of each biotransformation product. Using this approach, a total of nine phase I metabolites was successfully identified with deviations of mass accuracy less than 2 ppm. The biotransformations observed included hydroxylation, dihydroxylation, oxidation of a quaternary methyl group to hydroxymethyl or carboxylic acid groups, dehydrogenation and hydroxylation, as well as demethylation and dihydroxylation reactions. In a second step, tandem mass spectrometry (MS/MS) was performed to elucidate structures of the metabolites. Using the unique fragment ions in the spectra, the structures of the three major metabolites, 13,19-didesmethyl-19-carboxy spirolide C, 13,19-didesmethyl-19-hydroxymethyl spirolide C and 13-desmethyl-17-hydroxy spirolide C, were assigned. Levels of 13-desmethyl spirolide C and its metabolites were monitored at selected time points over a 32-h incubation period with human liver microsomes. It was determined that 13,19-didesmethyl-19-carboxy spirolide C became the predominant metabolite after 2 h of incubation. The stability plot of 13-desmethyl spirolide C showed first-order kinetics for its metabolism and the intrinsic clearance was calculated to be 41 µL/min/mg, suggesting first-pass metabolism may contribute to limiting oral toxicity of 13-desmethyl spirolide C.

Molecular characterization of zebrafish embryogenesis via DNA microarrays and multiplatform time course metabolomics studies.

One of the greatest strengths of "-omics" technologies is their ability to capture a molecular snapshot of multiple cellular processes simultaneously. Transcriptomics, proteomics, and metabolomics have, individually, been used in wide-ranging studies involving cell lines, tissues, model organisms, and human subjects. Nonetheless, despite the fact that their power lies in the global acquisition of parallel data streams, these methods continue to be employed separately. We highlight work done to merge transcriptomics and metabolomics technologies to study zebrafish (Danio rerio) embryogenesis. We combine information from three bioanalytical platforms, that is, DNA microarrays, (1)H nuclear magnetic resonance ((1)H NMR), and mass spectrometry (MS)-based metabolomics, to identify and provide insights into the organism's developmental regulators. We apply a customized approach to the analysis of such time-ordered measurements to provide temporal profiles that depict the modulation of metabolites and gene transcription. Initially, the three data sets were analyzed individually but later they were fused to highlight the advantages gained through such an integrated approach. Unique challenges posed by fusion of such data are discussed given differences in the measurement error structures, the wide dynamic range for the molecular species, and the analytical platforms used to measure them (i.e., fluorescence ratios, NMR, and MS intensities). Our data analysis reveals that changes in transcript levels at specific developmental stages correlate with previously published data with over 90% accuracy. In addition, transcript profiles exhibited trends that were similar to the accumulation of metabolites over time. Profiles for metabolites such as choline-like compounds (Trimethylamine-N-oxide, phosphocholine, betaine), creatinine/creatine, and other metabolites involved in energy metabolism exhibited a steady increase from 15 hours post fertilization (hpf) to 48 hpf. Other metabolite and transcript profiles were transiently rising and then falling back to baseline. The "house keeping" metabolites such as branched chain amino acids exhibited a steady presence throughout embryogenesis. Although the transcript profiling corresponds to only 16 384 genes, a subset of the total number of genes in the zebrafish genome, we identified examples where gene transcript and metabolite profiles correlate with one another, reflective of a relationship between gene and metabolite regulation over the course of embryogenesis.

Screening for multiple classes of marine biotoxins by liquid chromatography-high-resolution mass spectrometry.

Marine biotoxins pose a significant food safety risk when bioaccumulated in shellfish, and adequate testing for biotoxins in shellfish is required to ensure public safety and long-term viability of commercial shellfish markets. This report describes the use of a benchtop Orbitrap system for liquid chromatography-mass spectrometry (LC-MS) screening of multiple classes of biotoxins commonly found in shellfish. Lipophilic toxins such as dinophysistoxins, pectenotoxins, and azaspiracids were separated by reversed phase LC in less than 7 min prior to MS data acquisition at 2 Hz with alternating positive and negative scans. This approach resulted in mass accuracy for analytes detected in positive mode (gymnodimine, 13-desmethyl spirolide C, pectenotoxin-2, and azaspiracid-1, -2, and -3) of less than 1 ppm, while those analytes detected in negative mode (yessotoxin, okadaic acid, and dinophysistoxin-1 and -2) exhibited mass errors between 2 and 4 ppm. Hydrophilic toxins such as domoic acid, saxitoxin, and gonyautoxins were separated by hydrophilic interaction LC (HILIC) in less than 4 min, and MS data was collected at 1 Hz in positive mode, yielding mass accuracy of less than 1 ppm error at a resolving power of 100,000 for the analytes studied (m/z 300-500). Data were processed by extracting 5 ppm mass windows centered around the calculated masses of the analytes. Limits of detection (LOD) for the lipophilic toxins ranged from 0.041 to 0.10 µg/L (parts per billion) for the positive ions, 1.6-5.1 µg/L for those detected in negative mode, while the domoic acid and paralytic shellfish toxins yielded LODs ranging from 3.4 to 14 µg/L. Toxins were detected in mussel tissue extracts free of interference in all cases.

Metabolomics in glycomics.

Metabolomics is essentially the study of all low molecular weight molecules in a biological system under defined conditions. In glycomics, there is much potential to gain insight into the biosynthesis of novel glycoconjugate structures by probing the metabolome for substrates that are suspected, or known, to be involved in the biosynthetic processes. Recently, we employed the use of hydrophilic interaction liquid chromatography-mass spectrometry (HILIC-MS) in a focused metabolomic study of sugar-nucleotides relevant to the biosynthesis of highly novel carbohydrate modifications on the flagellin of Campylobacter sp. We exploited the unique selectivity of the HILIC-MS method for discriminating between closely related sugar-nucleotide intermediates and allowed their subsequent structural identification using a combination of high-resolution mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. In addition, the HILIC-MS method permitted screening of selected isogenic mutants for sugar-nucleotide intermediates to determine a role for the corresponding genes on the flagellin glycosylation locus in the biosynthesis of the novel carbohydrate modifications.

Campylobacter jejuni glycosylation island important in cell charge, legionaminic acid biosynthesis, and colonization of chickens.

Previously, we identified five genes (Cj1321 to Cj1326, of which Cj1325 and Cj1326 are a single gene) in the O-linked flagellin glycosylation island that are highly prevalent in Campylobacter jejuni isolates from chickens. We report mutagenesis, functional, and structural data to confirm that this locus, and Cj1324 in particular, has a significant contributory role in the colonization of chickens by C. jejuni. A motile DeltaCj1324 mutant with intact flagella was considerably less hydrophobic and less able to autoagglutinate and form biofilms than the parent strain, 11168H, suggesting that the surface charge of flagella of Cj1324-deficient strains was altered. The physical and functional attributes of the parent were restored upon complementation. Structural analysis of flagellin protein purified from the DeltaCj1324 mutant revealed the absence of two legionaminic acid glycan modifications that were present in the parent strain, 11168H. These glycoform modifications were shown to be prevalent in chicken isolates and confirm that differences in the highly variable flagellin glycosylation locus can relate to the strain source. The discovery of molecular mechanisms influencing the persistence of C. jejuni in poultry aids the rational design of approaches to control this problematic pathogen in the food chain.

Identification of novel carbohydrate modifications on Campylobacter jejuni 11168 flagellin using metabolomics-based approaches.

It is well known that the flagellin of Campylobacter jejuni is extensively glycosylated by pseudaminic acid and the related acetamindino derivative, in addition to flagellin glycosylation being essential for motility and colonization of host cells. Recently, the use of metabolomics permitted the unequivocal characterization of unique flagellin modifications in Campylobacter, including novel legionaminic acid sugars in Campylobacter coli, which had been impossible to ascertain in earlier studies using proteomics-based approaches. To date, the precise identities of the flagellin glycosylation modifications have only been elucidated for C. jejuni 81-176 and C. coli VC167 and those present in the first genome-sequenced strain C. jejuni 11168 remain elusive due to lability and respective levels of individual glycan modifications. We report the characterization of the carbohydrate modifications on C. jejuni 11168 flagellin using metabolomics-based approaches. Detected as their corresponding CMP-linked precursors, structural information on the flagellin modifications was obtained using a combination of MS and NMR spectroscopy. In addition to the pseudaminic acid and legionaminic acid sugars known to be present on Campylobacter flagellin, two unusual 2,3-di-O-methylglyceric acid modifications of a nonulosonate sugar were identified. By performing a metabolomic analysis of selected isogenic mutants of genes from the flagellin glycosylation locus of this pathogen, these novel CMP-linked precursors were confirmed to be di-O-methylglyceric acid derivatives of pseudaminic acid and the related acetamidino sugar. This is the first comprehensive analysis of the flagellar modifications in C. jejuni 11168 and structural elucidation of di-O-methylglyceric acid derivatives of pseudaminic acid on Campylobacter flagellin.

Estrogenic compounds in seawater and sediment from Halifax Harbour, Nova Scotia, Canada.

Abstract-Samples of seawater and surface sediment were collected from seven locations around Halifax Harbour, Nova Scotia, Canada, and analyzed for the presence of the organic estrogenic contaminants, bisphenol A (BPA), 17beta-estradiol (E2), and 17alpha-ethinylestradiol (EE2). Samples were extracted using solid phase extraction (seawater) or sonication (sediments), followed by fractionation on a two-layer alumina/silica gel column prior to analysis by liquid chromatography-tandem mass spectrometry (LCMS/MS) with negative-ion electrospray ionization. Levels of the three compounds consistently ranked as BPA > E2 > EE2. The least potent compound and plasticizer BPA reached levels of up to 2.6 ng/L in seawater and 9.5 ng/g in sediments; the natural product E2 was detected at concentrations up to 0.57 ng/L and 0.86 ng/g; while the synthetic estrogen EE2 was in most cases below the method detection limit (0.14 ng/L and 0.28 ng/g). The highest levels were observed in the influent of a secondary treatment plant that discharges into the harbor, with concentrations of 32.4 ng/L for BPA and 5.3 ng/L for E2. Overall, the results indicate that these compounds readily associate with suspended particles rather than remaining in the soluble phase. Measurement of the octanol-water partition coefficient (log K(OW)) confirmed these results, with values of 3.41, 3.89, and 4.16 for BPA, E2, and EE2, respectively. Partitioning experiments using spiked field samples further confirmed these findings, with sorption directly related to sediment total organic content and following the order EE2 > E2 > BPA.

Lipophilic sugar nucleotide synthesis by structure-based design of nucleotidylyltransferase substrates.

Structure-based design of alkyl sugar-1-phosphates provides an efficient nucleotidylyltransferase-catalyzed synthesis of a series of new lipophilic sugar nucleotides possessing long or branched alkyl chains, thereby demonstrating the utility of nucleotidylyltransferases to catalyze the synthesis of sugar nucleotides with potential applications in lipopolysaccharide and lipoglycopeptide biosynthesis.

Enzyme-catalyzed synthesis of furanosyl nucleotides.

A bacterial alpha-d-glucopyranosyl-1-phosphate thymidylyltransferase was found to couple four hexofuranosyl-1-phosphates, as well as a pentofuranosyl-1-phosphate, with deoxythymidine 5'-triphosphate, providing access to furanosyl nucleotides. The enzymatic reaction mixtures were analyzed by electrospray ionization mass spectrometry and NMR spectroscopy to determine the anomeric stereochemistry of furanosyl nucleotide products. This is the first demonstration of a nucleotidylyltransferase discriminating between diastereomeric mixtures of sugar-1-phosphates to produce stereopure, biologically relevant furanosyl nucleotides.