Evaluation of muscle lipid extraction and non-lethal fin tissue use for carbon, nitrogen, and sulfur isotope analyses in adult salmonids.

RATIONALE: Chemical lipid extraction or using alternative tissues such as fish fin as opposed to muscle may alter isotopic ratios and influence interpretations of δ13 C, δ15 N, and previously unassessed δ34 S values in stable isotope analyses (SIA). Our objectives were to determine if lipid extraction alters these isotope ratios in muscle, if lipid normalization models can be used for lipid-rich salmonids, and if fin isotope ratios are comparable with those of muscle in adult salmonids. METHODS: In six adult salmonid species (n = 106) collected from Lake Ontario, we compared three isotope ratios in lipid-extracted (LE) muscle with bulk muscle, and LE muscle with fin tissue, with paired t-tests and linear regressions. We compared differences between δ13 C values in LE and bulk muscle with predicted values from lipid normalization models and the log-linear model of best fit and determined model efficiency. RESULTS: The δ15 N values in LE muscle increased (<1‰) relative to bulk muscle for most salmonids, with relationships nearing 1:1. There were either no differences or strong 1:1 relationships in δ34 S values between species-specific bulk and LE muscle. One lipid normalization model had greater model efficiency (97%) than the model of best fit (94%). Fin had higher δ13 C values than LE muscle while δ15 N trends varied (<1‰); however, both isotope ratios had either no or weak linear relationships with fin and LE muscle within species. The δ34 S values in fin were similar to those in LE muscle and had strong 1:1 relationships across species. CONCLUSIONS: We recommend using the lipid normalization model to adjust for δ13 C values in lipid-rich muscle (C:N >3.4). LE muscle could be used without δ15 N or δ34 S adjustments, but the minimal increase in δ15 N values may affect SIA interpretation. With high unexplained variability among adult species in fin-muscle δ13 C and δ15 N relationships, species-specific fin-muscle adjustments are warranted. No fin-muscle tissue adjustment would be required for δ34 S values.

Fractionation of carbon isotopes of dissolved organic matter adsorbed to goethite in the presence of arsenic to study the origin of DOM in groundwater.

Natural dissolved organic matter (DOM) in groundwater plays a crucial role in mobilizing arsenic (As). The complex contribution of DOM sources makes it hard to predict how the variation of environmental conditions would affect the distribution of As concentrations. Identifying the carbon isotope fractionation of DOM is the key to quantify DOM sources based on stable carbon isotopes. To understand the magnitude and variability in the carbon isotopic fractionation of DOM in competitive adsorption with As(V), this study investigated the δ13C values of fulvic acid (FA) and DOM during adsorption to goethite in the presence of As(V), at a specific pH and temperature. The carbon isotopic enrichment factor (ε) of FA in the adsorption to goethite was 0.65 ± 2.11‰ at pH 4.1, 25 °C, suggesting that FA molecules containing 13C were more easily adsorbed to goethite. An increasing temperature increased εFA from 0.32 ± 1.17‰ to 0.82 ± 5.39‰ at 15-35 °C. For dissolved sediment organic matter (DSOM) cases, molecules containing 13C were more easily adsorbed to goethite. However, enrichment factors were not detected due to a reduction in DSOM adsorption and the diversity of natural humic substances or groups. The findings provide basic data for accurately ascertaining DOM sources through carbon isotopes, which is significant for predicting As fluctuation in aquifers affected by monsoon climate and/or human activities.

Growth-related trophic changes of Thunnus thynnus as evidenced by stable nitrogen isotopic values in the first dorsal spine.

The bluefin tuna, Thunnus thynnus, is a highly migratory and long-living fish at the top of the pelagic food web. As top predator, it plays a key role in the stability of marine food webs by exerting top-down control on its prey. The diet composition of bluefin tuna varies in relation to its growth, seasons and migratory patterns, making it difficult to evaluate spatial and temporal effects. This latter aspect is further complicated to be determined during the first months of life, when T. thynnus specimens have a rapid growth rate leading to changes in the trophic status. In this study, the potential collagen-related effects on δ15N and δ13C values were evaluated on the whole spine of adult tuna specimens collected in the central Mediterranean Sea. Obtained results showed non-significant differences between extracted and non-extracted collagen samples for δ15N in whole spine, allowing adopting the isotopic analysis both for annuli in the spine section of adults and for younger specimens, whose spine size does not permit the collagen extraction. Specifically, isotopic analysis of whole spine of the young of the year specimens, showed a rapid change in δ15N values with length, following an exponential model. For older specimens, δ15N values were higher and varied around a plateau, likely due to a higher specificity in the choice of prey and/or to change in the geographical location. Such variability was also mirrored in annuli of spines sections of adult tunas. As far as δ13C values are concerned, a strong collagen-related effect was evidenced, likely highlighting the influence of lipids. Consequently, δ13C analysis may be used only on adult specimens where collagen extraction is possible. This research also showed how isotopic analysis of both whole sample and sequence of annuli in the cross-section of dorsal spine might produce isotopic profiles useful to detect specific trophic dynamics along the bluefin tuna growth.

High Accuracy of Convolutional Neural Network for Evaluation of Helicobacter pylori Infection Based on Endoscopic Images: Preliminary Experience.

OBJECTIVES: Application of artificial intelligence in gastrointestinal endoscopy is increasing. The aim of the study was to examine the accuracy of convolutional neural network (CNN) using endoscopic images for evaluating Helicobacter pylori (H. pylori) infection. METHODS: Patients who received upper endoscopy and gastric biopsies at Sir Run Run Shaw Hospital (January 2015-June 2015) were retrospectively searched. A novel Computer-Aided Decision Support System that incorporates CNN model (ResNet-50) based on endoscopic gastric images was developed to evaluate for H. pylori infection. Diagnostic accuracy was evaluated in an independent validation cohort. H. pylori infection was defined by the presence of H. pylori on immunohistochemistry testing on gastric biopsies and/or a positive 13C-urea breath test. RESULTS: Of 1,959 patients, 1,507 (77%) including 847 (56%) with H. pylori infection (11,729 gastric images) were assigned to the derivation cohort, and 452 (23%) including 310 (69%) with H. pylori infection (3,755 images) were assigned to the validation cohort. The area under the curve for a single gastric image was 0.93 (95% confidence interval [CI] 0.92-0.94) with sensitivity, specificity, and accuracy of 81.4% (95% CI 79.8%-82.9%), 90.1% (95% CI 88.4%-91.7%), and 84.5% (95% CI 83.3%-85.7%), respectively, using an optimal cutoff value of 0.3. Area under the curve for multiple gastric images (8.3 ± 3.3) per patient was 0.97 (95% CI 0.96-0.99) with sensitivity, specificity, and accuracy of 91.6% (95% CI 88.0%-94.4%), 98.6% (95% CI 95.0%-99.8%), and 93.8% (95% CI 91.2%-95.8%), respectively, using an optimal cutoff value of 0.4. DISCUSSION: In this pilot study, CNN using multiple archived gastric images achieved high diagnostic accuracy for the evaluation of H. pylori infection.

Labeling Cancer Stem Cells with 13C6 Glucose and 13C5 Glutamine for Metabolic Flux Analysis.

Cancer stem cells (CSCs) or tumor-initiating cells (TICs) are a population of cells present within tumor that have increased self-renewal, chemoresistance, and aggressiveness, thereby contributing to tumor relapse. Literature shows that CSCs or TICs typically originate within the hypoxic niches of the tumor, making hypoxia one of the driving factors for generation of this population. Hypoxic stress promotes adaptation to low oxygen tension in the tissues by altering metabolic properties of the CSCs. This leads to a number of altered enzymatic activities in the CSC population that further contribute to the survival of the CSCs leading to resistance to standard therapy. Hence, understanding this altered metabolic pathways as well as targeting key nodes in these may pave the way for cancer management.Glucose and glutamine are the major substrates utilized by cancer cells and feed into multiple biosynthetic pathways. Hence, labeling and tracking these compounds may reveal some novel metabolic pathways exploited by cancer stem cells to acquire survival advantage. In these current book chapters, we elaborately summarized the basic steps required for isolation, characterization, and metabolic labeling (13C6 glucose and 13C5 glutamine) of CSC for flux analysis.

Rapid NMR-scale purification of 15N,13C isotope-labeled recombinant human STIM1 coiled coil fragments.

We report a new NMR-scale purification procedure for two recombinant wild type fragments of the stromal interaction molecule 1 (STIM1). This protein acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol accumulating at ER - plasma membrane (PM) junctions upon calcium store depletion ultimately leading to activation of the Orai/CRAC channel. The functionally relevant cytosolic part of STIM1 consists of three coiled coil domains, which are mainly involved in intra- and inter-molecular homomeric interactions as well as coupling to and gating of CRAC channels. The optimized one-step rapid purification procedure for two 15N,13C isotope-labeled cytosolic coiled coil fragments, which avoids the problems of previous approaches. The high yields of soluble well folded 15N,13C isotope-labeled cytosolic coiled coil fragments followed by detergent screening provide for initial NMR characterization of these domains. The longer 30.5 kDa fragment represents the largest STIM1 wild type fragment that has been recombinantly prepared and characterized in solution without need for mutation or refolding.

Optimisation of extraction methods and quantification of benzo[a]pyrene and benz[a]anthracene in yerba maté tea by isotope dilution mass spectrometry.

A gas chromatography-isotope dilution mass spectrometry (GC-IDMS) technique was developed for the quantification of two heavy polyaromatic hydrocarbons (PAHs), benz[a]anthracene and benzo[a]pyrene, in yerba maté tea (maté). The optimisation of two extraction methods, namely liquid-liquid extraction and accelerated solvent extraction, was carried out. Both optimised methods were validated using a certified reference material of fine dust and the results were within the expanded uncertainties at 95% confidence level. Recoveries of 99.2-106.7% with RSD of measurements of 1.1-2.3% were achieved for benz[a]anthracene. Recoveries of 95.7-101.9% with RSD of measurements of 0.4-1.4% were achieved for benzo[a]pyrene. The validated methods were applied for the extraction of benz[a]anthracene and benzo[a]pyrene in maté powder from NIST. A metrological approach was undertaken to ensure the traceability of measurement results. The uncertainties associated with the results were rigorously evaluated and also reported herein. Graphical abstract Quantification of benz[a]anthracene and benzo[a]pyrene using IDMS.

Synthesis of stable isotope-labeled epothilone D using a degradation-reconstruction approach.

The stabilization of microtubules using epothilones represents a novel mechanism of action to treat Alzheimer's disease. Epothilone D is one such microtubule-stabilizing drug that has been investigated by Bristol-Myers Squibb. An important step in the development process was the synthesis of a stable isotope-labeled analog for use in bioanalytical assays to accurately quantify the concentration of the drug in biological samples. A novel synthetic route to stable isotope-labeled epothilone D is described. The synthetic route was based on a strategy to degrade epothilone B and then use that key intermediate to reconstruct stable isotope-labeled epothilone D. Epothilone B was treated with potassium osmate and sodium periodate. The thiazole moiety in epothilone B was efficiently cleaved to give (1S,3S,7S,10R,11S,12S,16R)-3-acetyl-7,11-dihydroxy-8,8,10,12,16-pentamethyl-4,17-dioxabicyclo[14.1.0]heptadecane-5,9-dione. The epoxide in the macrocyclic ring of that intermediate was cleanly removed by treatment with tungsten hexachloride and n-butyllithium to give the corresponding olefin (4S,7R,8S,9S,16S,Z)-16-acetyl-4,8-dihydroxy-5,5,7,9,13-pentamethyloxacyclohexadec-13-ene-2,6-dione. Bis(triethylsilyl) protection produced (4S,7R,8S,9S,16S,Z)-16-acetyl-5,5,7,9,13-pentamethyl-4,8-bis(triethylsilyloxy)-oxacyclohexadec-13-ene-2,6-dione. This intermediate was coupled to a stable isotope-labeled thiazole using a Wittig reaction as the key step to provide (13)C5, (15)N-labeled epothilone D. In summary, the synthesis was completed in nine total steps, only six of which involved isotopically labeled reagents. A total of 168 mg of (13)C5, (15)N-labeled epothilone D was prepared in an 8% overall yield from (13)C2, (15)N-labeled thioacetamide and (13)C3-labeled ethyl bromopyruvate.

Variability in the carbon isotope fractionation of trichloroethene on its reductive dechlorination by vitamin B12.

Stable carbon isotope fractionation through the reductive dechlorination of trichloroethylene by vitamin B12 was determined to assess the possibility of using stable carbon isotope analysis to determine the efficacy of remediation of trichloroethylene using vitamin B12. We elucidated the effects of environmental conditions, including the pH, reaction temperature, and vitamin B12 concentration, on the carbon isotope enrichment factor (ε). The ε values were relatively insensitive to the reaction temperature and vitamin B12 concentration, ranging from -15.7‰ to -16.2‰, with a mean of -15.9 ± 0.2‰, at different temperatures and vitamin B12 concentrations. Such a reproducible ε value could be particularly useful for estimating the extent of degradation in reactions in which a mass balance is difficult to achieve. However, changing the initial solution pH from 6.5 to 9.0 caused a notable change in the ε values, from -14.0‰ to -18.0‰. Reactions were investigated by calculating the apparent kinetic isotope effects for carbon, which, at 1.029-1.037, were smaller than the kinetic isotope effect values previously found for C-Cl bond cleavage. This indicates that a reaction other than the elimination of chloride may be a competitive degradation pathway. The dominant degradation pathway may be different for different initial solution pH values, and this will clearly influence carbon isotope fractionation. Therefore, if the ε value varies with reaction conditions, such as the solution pH, the calculations should take into account the actual environmental conditions that affect the rate limiting pathways.

Synthesis of isotopically labeled 1,3-dithiane.

The 1,3-dithiane is a protected formaldehyde anion equivalent that could serve as a useful labeled synthon. We report a facile synthesis of 1,3-[2-(13)C]- and 1,3-[2-(13)C, 2-(2)H2]dithiane in two steps from [(13)C]- or [(13) C, (2)H3 ]methyl phenyl sulfoxide. We have previously reported the high yield synthesis of [(13)C]methyl phenyl sulfide from [(13)C]MEOH and the oxidation of [(13)C]methyl phenyl sulfide to [(13)C]methyl phenyl sulfoxide. Here, we describe the facile exchange of deuterium from (2) H2 O into [(13)C]methyl phenyl sulfoxide to yield [(13)C, (2)H3]methyl phenyl sulfoxide. Thus, from [(13)C]MEOH and (2)H2O, all possible C2 stable isotopomers of 1,3-dithiane are available. Our synthetic route is also amenable to preparation of radiolabeled 1,3-dithianes.

Carbon and chlorine isotope fractionation during Fenton-like degradation of trichloroethene.

Dual isotope approach has been proposed as a viable tool for characterizing and assessing in situ contaminant transformation, however, little data is currently available on its applicability to chlorinated ethenes. This study determined carbon and chlorine isotope fractionation during Fenton-like degradation of trichloroethene (TCE). Carbon and chlorine isotope enrichment factors were εC=-2.9 ± 0.3‰ and εCl=-0.9 ± 0.1‰, respectively. An observed small secondary chlorine isotope effect (AKIECl=1.001) was consistent with an initial transformation by adding hydroxyl radicals (OH) to CC bonds without cleavage of CCl bonds. The relative change in carbon and chlorine isotope ratios (Δ=Δδ(13)C/Δδ(37)Cl) was calculated to be 3.1 ± 0.2, approximately equal to the ratio of chlorine and carbon isotope enrichment factors (εC/εCl=3.2). The similarity of the Δ (or εC/εCl) values between Fenton-like degradation and microbial reductive dechlorination of TCE was observed, indicating that application of solely dual isotope approach may be limited in distinguishing the two transformation pathways.

Synthesis of three alpha 7 agonists in labeled form.

In support of a program to develop an alpha 7 agonist as a treatment for Alzheimer's disease, three drug candidates, 1, 2, and 3, were prepared in labeled forms. Compound 1 was prepared in C-14 labeled form by lithiation of [2,6-(14)C2]2-chloropyridine and subsequent coupling with spirooxirane-2,3'-quinuclidine. When this same coupling was attempted using [3,4,5,6-(2)H4]2-chloropyridine, alcohol [(2)H6]-6 was the major product indicating that the primary isotope effect for the lithiation step was significant enough to shift the reaction pathway. Therefore, an alternate site of labeling was used to prepare [(2)H4]-1. [(13)C5]-2 was prepared in five steps from [(13)C5 ]2-furoic acid, but the C-14 labeled compound used [(14)C2]-1 as the starting material instead. [(14)C2]-3 was prepared in two steps from [carbonyl-(14)C]nicotinic acid.

Production of [11C]CO2 with gas target at low proton energies.

Nowadays the demand and the installation of self-shielded low-energy cyclotrons is growing, allowing the use of (11)C in many more centers. The aim of this study was the design of a new target and the evaluation of the production of (11)C as [(11)C]CO2 at low proton energies. The target was coupled to an IBA Cyclone-18/9 and the energy was decreased to 4-16 MeV. The newly designed target allowed the production of [(11)C]CO2 at different proton energies, and the results suggest that the cyclotron energy of Cyclone-18/9 is slightly higher than the nominal 18 MeV.

Use of Cl and C isotopic fractionation to identify degradation and sources of polychlorinated phenols: mechanistic study and field application.

The widespread use of chlorinated phenols (CPs) as a wood preservative has led to numerous contaminated sawmill sites. However, it remains challenging to assess the extent of in situ degradation of CPs. We evaluated the use of compound-specific chlorine and carbon isotope analysis (Cl- and C-CSIA) to assess CP biotransformation. In a laboratory system, we measured isotopic fractionation during oxidative 2,4,6-trichlorophenol dechlorination by representative soil enzymes (C. fumago chloroperoxidase, horseradish peroxidase, and laccase from T. versicolor). Using a mathematical model, the validity of the Rayleigh approach to evaluate apparent kinetic isotope effects (AKIE) was confirmed. A small but significant Cl-AKIE of 1.0022 ± 0.0006 was observed for all three enzymes, consistent with a reaction pathway via a cationic radical species. For carbon, a slight inverse isotope effect was observed (C-AKIE = 0.9945 ± 0.0019). This fractionation behavior is clearly distinguishable from reported reductive dechlorination mechanisms. Based on these results we then assessed degradation and apportioned different types of technical CP mixtures used at two former sawmill sites. To our knowledge, this is the first study that makes use of two-element CSIA to study sources and transformation of CPs in the environment.

Validation of adsorbents for sample preconcentration in compound-specific isotope analysis of common vapor intrusion pollutants.

Isotope ratios of volatile organic compounds (VOCs) in the environment are often of interest in contaminant fate studies. Adsorbent preconcentration-thermal desorption of VOCs can be used to collect environmental vapor samples for compound-specific isotope analysis (CSIA). While active adsorbent samplers offer logistic benefits in handling large volumes of air, their performance in preserving VOCs isotope ratios was not previously tested under sampling conditions corresponding to typical indoor air sampling conditions. In this study, the performance of selected adsorbents was tested for preconcentration of TCE (for determination of C and Cl isotope ratios), PCE (C and Cl) and benzene (C and H). The key objective of the study was to identify the adsorbent(s) permitting preconcentration of the target VOCs present in air at low µg/m(3) concentrations, without significant alteration of their isotope ratios. Carboxen 1016 was found to perform well for the full range of tested parameters. Carboxen 1016 can be recommended for sampling of TCE, PCE and benzene, for CSIA, from air volumes up to 100 L. Variable extent of isotope ratio alteration was observed in the preconcentration of the target VOCs on Carbopack B and Carbopack X, resulting from partial analyte loss via adsorbent bed breakthrough and (possibly) via incomplete desorption. The results from testing the Carbopack B and Carbopack X highlight the need of adsorbent performance validation at conditions fully representative of actual sample collection conditions, and caution against extrapolation of performance data toward more challenging sampling conditions.

HPLC purification of higher plant-dervied lignin phenols for compound specific radiocarbon analysis.

The ability to measure the radiocarbon content of compounds isolated from complex mixtures has begun to revolutionize our understanding of carbon transformations on earth. Because samples are often small, each new compound isolation method must be tested for background carbon contamination (C(ex)). Here, we present a new method for compound-specific radiocarbon analysis (CSRA) of higher plant-derived lignin phenols. To test for C(ex), we compared the Δ(14)C values of unprocessed lignin phenol containing standard materials (woods, leaves, natural vanillin, and synthetic vanillin) with those of lignin phenols liberated by CuO oxidation and purified by two-dimensional high-pressure liquid chromatography (HPLC) coupled to mass spectrometry (MS) and UV detection. We assessed C(ex) associated with (1) microwave assisted CuO oxidation of bulk samples to lignin phenol monomers, (2) HPLC purification, and (3) accelerator mass spectrometry (AMS) sample preparation. The Δ(14)C of purified compounds (corrected for C(ex)) agreed, within error, with those of bulk materials for samples that were >10 µg C. This method will allow routine analysis of the Δ(14)C of lignin phenols isolated from terrestrial, aquatic, and marine settings, revealing the time scale for the processing of one of the single largest components of active organic carbon reservoirs on earth.

Carbon and hydrogen isotope fractionation during anaerobic quinoline degradation.

Quinoline is a N-heterocyclic compound often found at tar oil contaminated field sites. To provide information whether stable isotope analysis can help to characterize the fate of quinoline within contaminated aquifers, carbon and hydrogen isotope fractionation of quinoline were investigated during biodegradation under sulfate-reducing conditions. No significant carbon isotope effect was observed, however, substantial hydrogen isotope fractionation was detected. Thus, hydrogen isotope fractionation may be used as an indicator for in situ biodegradation of quinoline. The bulk hydrogen isotope enrichment factor was εH(bulk)=-33±12‰. During the biodegradation of quinoline the primary intermediate 2-hydroxyquinoline was detected indicating hydroxylation at the C2-position. According to this reaction mechanism, the reactive position specific hydrogen enrichment factor (εH(reactive position)) and apparent kinetic hydrogen isotope effect (AKIE(H)) were calculated and gave values of εH(reactive position)=-205±75‰ and AKIE(H)=1.26±0.12, respectively. The missing carbon isotope effect may be explained by strong masking or an enzymatic direct side-on insertion of oxygen from the MoOH(H) group of the molybdenum center across the CH bond at the C2-position of quinoline with concomitant hydride transfer. The later assumption is supported by recent studies showing that initial step of hydroxylation of N-heteroaromatic compounds proceeds via a similar reaction mechanism.

Extracting metabolite ions out of a matrix background by combined mass defect, neutral loss and isotope filtration.

Mass defect, neutral loss and isotope filtration techniques were applied to electrospray ionization mass spectrometry (ESI-MS) data obtained for in vivo and in vitro samples of drug metabolism studies. A combination of these post-acquisition processing techniques was shown to be more powerful than the use of one of these tools alone for the detection in complex matrices of metabolites of candidate drugs with a characteristic isotope pattern (e.g. containing bromine, chlorine, or a high proportion of radiolabeled drug ((12)C/(14)C)) or characteristic neutral losses. In combination with 'all-in-one' data acquisition this methodology is able to perform software-driven constant neutral loss scanning for an unlimited number of mass differences at any time after analysis. Highly selective MS chromatograms were obtained with excellent correlation with their corresponding radiochromatograms.

Producing over 100 ml of highly concentrated hyperpolarized solution by means of dissolution DNP.

New low-temperature inserts compatible with an existing hyperpolarizer were developed to dynamically polarize nuclei in large samples. The performance of the system was tested on 8 ml glassy frozen solutions containing 13C-labeled molecules and doped with nitroxyl free radicals. The obtained 13C low-temperature polarization was comparable to the one measured on 20 times smaller sample volume with only 3-4 times higher microwave power. By using a dissolution insert that fits to the new design, it was possible to obtain about 120 ml of room-temperature hyperpolarized solution. The polarization as well as the molecule concentration was comparable to the values obtained in standard size hyperpolarized samples. Such large samples are interesting for future studies on larger animals and possibly for potential clinical applications.