iTRAQ-based quantitative proteomics analysis of the effect of ACT001 on non-alcoholic steatohepatitis in mice.

ACT001 is a novel sesquiterpene lactone derivative that has been shown to have significant antitumor and anti-inflammatory effects. However, the effect of ACT001 on nonalcoholic steatohepatitis (NASH) is unknown. Methionine and choline deficient (MCD) diet induced NASH model in C57BL/6J mice. Steatosis, inflammation and fibrosis-related indices of serum and liver tissues were detected by fully automated biochemical analyzer, enzyme-linked immunosorbent assay (ELISA) kit, flow cytometry, hematoxylin and eosin (H&E), Masson and immunohistochemical staining. The results showed that ACT001 reduced serum lipid and inflammatory factor levels, attenuated hepatic steatosis, inflammation and fibrosis, and inhibited hepatic oxidative stress and activation of NOD-like receptor protein 3 (NLRP3) inflammatory vesicles in NASH mice. In addition, 381 differentially expressed proteins (DEPs), including 162 up-regulated and 219 down-regulated proteins, were identified in the MCD group and ACT001 high-dose group using isotope labeling relative and absolute quantification (iTRAQ) technique analysis. Among these DEPs, five proteins associated with NAFLD were selected for real-time fluorescence quantitative PCR (RT-qPCR) validation, and the results were consistent with proteomics. In conclusion, ACT001 has a therapeutic effect on NASH, and the results of proteomic analysis will provide new ideas for the mechanism study of ACT001 for NASH treatment.

Stable isotope labeling as a promising tool for rapid drug susceptibility testing in Neisseria gonorrhoeae.

The world is heading towards an era of intractable and impending untreatable N. gonorrhoeae, thereby underlining the significance of rapid and accurate prediction of drug resistance as an indispensable need of the hour. In the present study, we optimized and evaluated a stable isotope labeling-based approach using the MALDI-TOF MS (Matrix-Assisted Laser Desorption/Ionization-Time of Flight Mass Spectrometry) for rapid and reliable detection of ciprofloxacin and azithromycin resistance in N. gonorrhoeae. All the isolates were cultured under three varied condition setups viz. medium supplemented with normal lysine, heavy lysine (isotope), and heavy lysine along with the antibiotics (ciprofloxacin/azithromycin), respectively. After incubation, spectra were acquired using the MALDI-TOF MS which were further screened for unique patterns (media-specific spectra) to differentiate drug-susceptible and resistant isolates. The results of the stable isotope labeling assay were comparable to the results of phenotypic methods used for susceptibility testing.

Isotope-coded derivatization with designed Girard-type reagent as charged isobaric mass tags for non-targeted profiling and discovery of natural aldehydes by liquid chromatography-tandem mass spectrometry.

Aldehyde-containing metabolites are reactive electrophiles that have attracted extensive attention due to their widespread occurrence in organisms and natural foods. Herein we described a newly-designed Girard's reagent, 1-(4-hydrazinyl-4-oxobutyl)pyridin-1-ium bromide (HBP), as charged tandem mass (MS/MS) tags to facilitate selective capture, sensitive detection and semi-targeted discovery of aldehyde metabolites via hydrazone formation. After HBP labeling, the detection signals of the test aldehydes were increased by 21-2856 times, with the limits of detection were 2.5-7 nM. Upon isotope-coded derivatization with a pair of labeling reagents, HBP-d0 and its deuterium-labeled counterpart HBP-d5, the aldehyde analytes were converted to hydrazone derivatives, which generated characteristic neutral fragments of 79 Da and 84 Da, respectively. The isobaric HBP-d0/HBP-d5 labeling based LC-MS/MS method was validated by relative quantification of human urinary aldehydes (slope=0.999, R2 > 0.99, RSDs ≤ 8.5%) and discrimination analysis between diabetic and control samples. The unique isotopic doubles (Δm/z = 5 Da) by dual neutral loss scanning (dNLS) provided a generic reactivity-based screening strategy that allowed non-targeted profiling and identification of endogenous aldehydes even amidst noisy data. The LC-dNLS-MS/MS screening of cinnamon extracts led to finding 61 possible natural aldehydes and guided discovery of 10 previously undetected congeners in this medicinal plant.

Profiling of Secreted Proteins in Serum-Containing Media Using BONCAT and Pulsed SILAC.

Secreted proteins play pivotal roles in signal transduction and cell-to-cell communication. Despite increasing interest in secretome analysis over the past decade, most studies on this topic have utilized serum-free medium (SFM). However, fetal bovine serum (FBS) is the most widely used serum supplement for cell culture, and secretome analysis using serum-containing medium (SCM) is important to identify proteins secreted under realistic conditions and to understand their physiological roles. In this chapter, we describe a simple and robust protocol based on bioorthogonal non-canonical amino acid tagging (BONCAT) and pulsed stable isotope labeling by amino acids in cell culture (pSILAC), for identification and quantitation of the cell secretome in SCM. In this protocol, the secretome of SFM is compared with that of SCM to confirm the effect of FBS. Additionally, for mass spectrometric data processing, we provide parameters that increase true positives and decrease both false positives and false negatives.

Multi-element stable isotope Raman microspectroscopy of bacterial carotenoids unravels rare signal shift patterns and single-cell phenotypic heterogeneity.

The combination of single-cell Raman microspectroscopy (SCRM) and stable isotope probing (SIP) enables in situ tracking of carbon or hydrogen fluxes into microorganisms at the single-cell level. Therefore, it has high potential for the analysis of metabolic processes and biogeochemical cycles. However, especially for high throughput applications such as imaging or cell sorting, it is hampered by low Raman scattering intensities (and therefore long acquisition times). In order to overcome these limitations, this study brings forward a systematic investigation of Resonance Raman (RR) enhanced SCRM for SIP of bacterial carotenoids. Dynamic carbon uptake from 13C-glucose was successfully monitored and quantified utilizing 13C stable isotope-induced red-shifts of RR signals. High single-cell phenotypic heterogeneity was revealed in terms of carbon uptake and, unlike in previous studies, clear evidence for de novo synthesis of carotenoids was found. For the first time, hydrogen uptake into carotenoids was systematically investigated by deuterium labeling (providing a direct probe for metabolic activity of single cells). In carotenoid single-cell Resonance Raman (SCRR) spectra, a unique pattern of signal red-shifts and apparent blue-shifts was observed and quantitatively evaluated. Finally, a novel combined approach for simultaneous monitoring of carbon and hydrogen uptake revealed complementary effects in carotenoid SCRR spectra that can be analyzed in parallel. Overall, it was shown that the high RR intensity, simplicity of spectral features and straightforward signal processing make microbial carotenoids an ideal target for quantitative multi-element SIP, with great potential for high throughput applications.

Tag you're it: Application of stable isotope labeling and LC-MS to identify the precursors of specialized metabolites in plants.

Untargeted liquid chromatography/mass spectrometry (LC-MS) can contribute a comprehensive and unbiased picture of the metabolic space of plants. These data can be used to quantify natural metabolite variation for genome wide association studies, to compare global metabolic responses from environmental or genetic perturbations, and to identify previously undescribed metabolites in Nature. A major limitation with untargeted metabolomics is the classification and identification of the thousands of metabolite features that can be detected in a single analytical run. Isotopic labeling improves the informational value of these datasets by categorizing metabolites as being derived from specific upstream precursors and/or to known metabolic pathways. When a 13C-labeled precursor is fed to either a plant or tissue, the downstream metabolites produced from it have a higher m/z value than the molecules in the pre-existing pool, generating an m/z peak pair that can be specifically identified within the MS data. This paper outlines methods and principles to consider when supplementing untargeted MS data with isotopic labeling, including how to choose the appropriate isotopic label, grow and feed plant tissues to maximize label uptake and incorporation into derivatives, optimize LC-MS methods, and interpret the resulting labeling data. Although the focus here is on annotation of amino acid-derived metabolites using LC-MS, we anticipate that the methods are generally adaptable to other precursors, plant species, and chromatographic approaches.

Generation of 13C-Labeled Inositol and Inositol Phosphates by Stable Isotope Labeling Cell Culture for Quantitative Metabolomics.

Inositol and inositol phosphates (IPx) are central metabolites. Their accurate quantitative analysis in complex biological samples is challenging due to lengthy sample preparation procedures, sample losses by strong adsorption to surfaces, and unpredictable matrix effects. Currently, U13C-inositol and U13C-IPx are not available from commercial sources. In this study, we developed a method that is capable of generating U13C-inositol and U13C-IPx. An inositol-independent cell line L929S was cultured in inositol-free medium supplemented with U13C-glucose. Inositol contamination in FBS was observed as the critical parameter for labeling efficiency (LE). A balance between cell growth and LE was achieved by adopting a two-step labeling strategy. In the first step, a LE of 90% could be obtained by normal cell growth in the long-term. Cells were then cultured in a second step in ultra-labeling medium for improved LE for a short duration before harvesting. The generated U13Canalogs were of high isotopic purity (>99%). Utilized as internal standards spiked before sample preparation in biological applications, U13Canalogs can effectively compensate sample loss during sample preparation as well as the matrix effect during electrospray ionization. An exemplary pharmacological study was conducted with phospholipase C inhibitor and activator to document the great utility of the prepared stable isotope-labeled internal standards in elucidating the PLC-dependent IP code. U13CIPx are used as internal standards to generate quantitative profiles of IPx in HeLa cell samples after treatment with PLC inhibitor and activator. This established method generating U13Canalogs is cost-effective, robust, and reproducible, which can facilitate quantitative studies of inositol and IPx in biological scenarios.

Tracing the Transformation and Allocation of the Newly Applied-P in Calcareous Soil Using an Enriched Oxygen Isotope Labeling Technique.

Different phosphorus (P) fertilizations significantly impact the transformation of the applied-P in soils. However, knowledge about how different P fertilization regimes influence the allocation of the amended-P in soil remains incomplete. Herein, we carried out a pot experiment to explore the fate of applied-P in calcareous soil using an oxygen isotope labeling technique (18O-P18O43-). Treatments included check (CK), single, and repeated applications. The phosphorus mass balance result showed that more than 48.5% of the applied-P was held in labile and moderately labile fractions with the repeated treatment, while approximately 27.4% of the added-P was recovered in nonlabile forms in the single application treatment. The isotopic tracer (18O-P18O43-) result demonstrated that the δ18OP values of NaHCO3-P and NaOH-P in the repeated P application were significantly higher than those in the single P application. Ultimately, better agronomic performances of the crops and higher PUE were achieved in the repeated treatment. Our findings highlighted that repeated P fertilization can improve P availability by reducing P fixation. These results pronounced that the enriched oxygen isotope technique can be considered an effective approach for tracing applied-P in soils.

ProMetheusDB: An In-Depth Analysis of the High-Quality Human Methyl-proteome.

Protein arginine (R) methylation is a post-translational modification involved in various biological processes, such as RNA splicing, DNA repair, immune response, signal transduction, and tumor development. Although several advancements were made in the study of this modification by mass spectrometry, researchers still face the problem of a high false discovery rate. We present a dataset of high-quality methylations obtained from several different heavy methyl stable isotope labeling with amino acids in cell culture experiments analyzed with a machine learning-based tool and show that this model allows for improved high-confidence identification of real methyl-peptides. Overall, our results are consistent with the notion that protein R methylation modulates protein-RNA interactions and suggest a role in rewiring protein-protein interactions, for which we provide experimental evidence for a representative case (i.e., NONO [non-POU domain-containing octamer-binding protein]-paraspeckle component 1 [PSPC1]). Upon intersecting our R-methyl-sites dataset with the PhosphoSitePlus phosphorylation dataset, we observed that R methylation correlates differently with S/T-Y phosphorylation in response to various stimuli. Finally, we explored the application of heavy methyl stable isotope labeling with amino acids in cell culture to identify unconventional methylated residues and successfully identified novel histone methylation marks on serine 28 and threonine 32 of H3. The database generated, named ProMetheusDB, is freely accessible at https://bioserver.ieo.it/shiny/app/prometheusdb.

Site-Specific Conjugation Quantitation of a Cysteine-Conjugated Antibody-Drug Conjugate Using Stable Isotope Labeling Peptide Mapping LC-MS/MS Analysis.

Drug-load (DL) characterization of antibody-drug conjugates (ADCs) is an important analytical task due to its designation as a critical quality attribute (CQA) affecting potency and stability. Intact and subunit liquid chromatography-mass spectrometry (LC-MS) analyses can determine global drug-to-antibody ratios (DARs) that correlate well with other orthogonal analytical methods; however, peptide mapping liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis has struggled to provide complementary site-specific quantitation of drug conjugation sites. The peptide mapping method described herein utilizes stable isotope labeling to accurately quantitate the site-specific conjugation levels of a cysteine-conjugated ADC to provide "bottom-up" DAR characterization in parallel with protein sequence and post-translational modification (PTM) characterization in one multi-attribute analytical method (MAM).

A Synergistic Consortium Involved in rac-Dichlorprop Degradation as Revealed by DNA Stable Isotope Probing and Metagenomic Analysis.

rac-Dichlorprop, a commonly used phenoxyalkanoic acid herbicide, is frequently detected in environments and poses threats to environmental safety and human health. Microbial consortia are thought to play key roles in rac-dichlorprop degradation. However, the compositions of the microbial consortia involved in rac-dichlorprop degradation remain largely unknown. In this study, DNA stable isotope probing (SIP) and metagenomic analysis were integrated to reveal the key microbial consortium responsible for rac-dichlorprop degradation in a rac-dichlorprop-degrading enrichment. OTU340 (Sphingobium sp.) and OTU348 (Sphingopyxis sp.) were significantly enriched in the rac-[13C]dichlorprop-labeled heavy DNA fractions. A rac-dichlorprop degrader, Sphingobium sp. strain L3, was isolated from the enrichment by a traditional enrichment method but with additional supplementation of the antibiotic ciprofloxacin, which was instructed by metagenomic analysis of the associations between rac-dichlorprop degraders and antibiotic resistance genes. As revealed by functional profiling of the metagenomes of the heavy DNA, the genes rdpA and sdpA, involved in the initial degradation of the (R)- and (S)-enantiomers of dichlorprop, respectively, were mostly taxonomically assigned to Sphingobium species, indicating that Sphingopyxis species might harbor novel dichlorprop-degrading genes. In addition, taxonomically diverse bacterial genera such as Dyella, Sphingomonas, Pseudomonas, and Achromobacter were presumed to synergistically cooperate with the key degraders Sphingobium/Sphingopyxis for enhanced degradation of rac-dichlorprop. IMPORTANCE Understanding of the key microbial consortium involved in the degradation of the phenoxyalkanoic acid herbicide rac-dichlorprop is pivotal for design of synergistic consortia used for enhanced bioremediation of herbicide-contaminated sites. However, the composition of the microbial consortium and the interactions between community members during the biodegradation of rac-dichlorprop are unclear. In this study, DNA-SIP and metagenomic analysis were integrated to reveal that the metabolite 2,4-dichlorophenol degraders Dyella, Sphingomonas, Pseudomonas, and Achromobacter synergistically cooperated with the key degraders Sphingobium/Sphingopyxis for enhanced degradation of rac-dichlorprop. Our study provides new insights into the synergistic degradation of rac-dichlorprop at the community level and implies the existence of novel degrading genes for rac-dichlorprop in nature.

Tracing the fate of phosphorus fertilizer derived cadmium in soil-fertilizer-wheat systems using enriched stable isotope labeling.

Applying mineral phosphorus (P) fertilizers introduces a considerable input of the toxic heavy metal cadmium (Cd) into arable soils. This study investigates the fate of P fertilizer derived Cd (Cddff) in soil-wheat systems using a novel combination of enriched stable Cd isotope mass balances, sequential extractions, and Bayesian isotope mixing models. We applied an enriched 111Cd labeled mineral P fertilizer to arable soils from two long-term field trials with distinct soil properties (a strongly acidic pH and a neutral pH) and distinct past mineral P fertilizer application rates. We then cultivated wheat in a pot trial on these two soils. In the neutral soil, Cd concentrations in the soil and the wheat increased with increasing past mineral P fertilizer application rates. This was not the case in the strongly acidic soil. Less than 2.3% of freshly applied Cddff was taken up by the whole wheat plant. Most of the Cddff remained in the soil and was predominantly (>95% of freshly applied Cddff) partitioned into the easily mobilizable acetic acid soluble fraction (F1) and the potentially mobile reducible fraction (F2). Soil pH was the determining factor for the partitioning of Cddff into F1, as revealed through a recovery of about 40% of freshly applied Cddff in F1 in the neutral pH soil compared with about 60% in the strongly acidic soil. Isotope mixing models showed that F1 was the predominant source of Cd for wheat on both soils and that it contributed to over 80% of the Cd that was taken up by wheat. By tracing the fate of Cddff in entire soil-plant systems using different isotope source tracing approaches, we show that the majority of Cddff remains mobilizable and is potentially plant available in the subsequent crop cycle.

Quantitative analysis of n-3 polyunsaturated fatty acids and their metabolites by chemical isotope labeling coupled with liquid chromatography - mass spectrometry.

n-3 polyunsaturated fatty acids (PUFAs) and their metabolites play the crucial role in a wide range of physiologic and pathologic processes, including cardiovascular, neurodegenerative diseases, and inflammation-associated disorders. However, the quantitative analysis of n-3 PUFAs and their metabolites, oxylipins, is obstructed by high structural similarity, poor ionization efficiency and low abundance. In this study, a sensitive method was developed to quantify 28 n-3 PUFAs/oxylipins using chemical isotope labeling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Standards labeled with cholamine-d9 were used as one-to-one internal standards to achieve accurate quantification. The cholamine-d0-derivatized biological samples were mixed with cholamine d9-labeled standards for LC-MS/MS with multiple reaction monitoring. After cholamine derivatization, both MS sensitivity and chromatographic performance of n-3 PUFAs/oxylipins were substantially improved. Furthermore, the relationship between retention time and substituent position of regioisomers, and their fragmentation patterns were investigated, which may facilitate the identification of unknown oxylipins. Additionally, the developed method was applied to quantify the target n-3 PUFAs/oxylipins in serum and brain tissue from fish oil-supplemented mice, which exhibited its great potential and practicability. Collectively, this sensitive and reliable method may facilitate the elucidation of the roles of n-3 PUFAs/oxylipins in the physiological and pathological processes.

Discovery of Cyanobacterial Natural Products Containing Fatty Acid Residues*.

In recent years, extensive sequencing and annotation of bacterial genomes has revealed an unexpectedly large number of secondary metabolite biosynthetic gene clusters whose products are yet to be discovered. For example, cyanobacterial genomes contain a variety of gene clusters that likely incorporate fatty acid derived moieties, but for most cases we lack the knowledge and tools to effectively predict or detect the encoded natural products. Here, we exploit the apparent absence of a functional ß-oxidation pathway in cyanobacteria to achieve efficient stable-isotope-labeling of their fatty acid derived lipidome. We show that supplementation of cyanobacterial cultures with deuterated fatty acids can be used to easily detect natural product signatures in individual strains. The utility of this strategy is demonstrated in two cultured cyanobacteria by uncovering analogues of the multidrug-resistance reverting hapalosin, and novel, cytotoxic, lactylate-nocuolin A hybrids-the nocuolactylates.

Neonatal Piglets Can Synthesize Adequate Creatine, but Only with Sufficient Dietary Arginine and Methionine, or with Guanidinoacetate and Excess Methionine.

BACKGROUND: Suckling piglets synthesize most of their creatine requirement, which consumes substantial amounts of arginine in order to synthesize guanidinoacetic acid (GAA) and methionine in order to transmethylate GAA to creatine. OBJECTIVES: To determine whether supplemental GAA or creatine spare arginine and/or methionine for protein synthesis and, if GAA is supplemented, whether excess methionine is needed for conversion to creatine. METHODS: Yucatan miniature piglets (9-11 days old; both sexes) were fed 1 of 5 elemental diets for 5 days: 1) low arginine (0.3 g·kg-1·d-1) and low methionine (0.20 g·kg-1·d-1; Base); 2) Base plus GAA (0.093 g·kg-1·d-1; +GAA); 3) Base plus GAA plus excess methionine (0.5 g·kg-1·d-1; +GAA/Met); 4) Base plus creatine (0.12 g·kg-1·d-1; +Cre); or 5) excess arginine (1.8 g·kg-1·d-1) and excess methionine (+Arg/Met). Isotope tracers were infused to determine whole-body GAA, creatine, and protein synthesis; tissues were analyzed for creatine synthesis enzymes and metabolite concentrations. Data were analyzed by 1-way ANOVA. RESULTS: : GAA and creatine syntheses were 115% and 32% higher, respectively, with the +Arg/Met diet (P < 0.0001), in spite of 33% lower renal L-arginine: glycine amidinotransferase activity (P < 0.0001) compared to Base, suggesting substrate availability dictates synthesis rather than enzyme capacity. GAA or creatine supplementation reduced arginine conversion to creatine by 46% and 43%, respectively (P < 0.01), but did not spare amino acids for whole-body protein synthesis, suggesting that limited amino acids were diverted to protein at the expense of creatine synthesis. The +GAA/Met diet led to higher creatine concentrations in the kidney (2.6-fold) and liver (7.6-fold) than the +GAA diet (P < 0.01), suggesting excess methionine is needed for GAA conversion to creatine. CONCLUSIONS: Piglets are capable of synthesizing sufficient creatine from the precursor amino acids arginine and methionine, or from GAA plus methionine.

18O Isotope Labeling Combined with 31P Nuclear Magnetic Resonance Spectroscopy for Accurate Quantification of Hydrolyzable Phosphorus Species in Environmental Samples.

31P nuclear magnetic resonance (NMR) spectra can be biased due to the hydrolysis of labile P species during sample treatment and NMR analysis. This paper offers an approach to circumvent this problem by performing sample preparation and analysis in 18O-enriched medium. Heavy 18O isotope atoms were introduced into the resulting artificial hydrolysis products. The NMR signal of 18O-labeled P was shifted upfield relative to the unlabeled P nuclei in natural metabolites. This isotope shift enabled an immediate differentiation of artificial hydrolysis products from natural metabolites. Moreover, the hydrolysis products could be accurately quantified. Our data suggest that the extent to which artificial hydrolysis alters NMR spectra varies among different types of environmental samples. For instance, 72-84% of the detected monoesters in the organic soils of this study were actually artificially hydrolyzed diesters. By contrast, artificial hydrolysis products in the mineral soils used for this study accounted for less than 6% of the total monoesters. Polyphosphate was also hydrolyzed to yield 18O-labeled products in algal biomass.

Exploring the metabolic fate of medium-chain triglycerides in healthy individuals using a stable isotope tracer.

BACKGROUND & AIMS: Medium chain triglyceride (MCT) supplementation is often recommended as treatment for patients with long-chain fatty acid ß-oxidation (lcFAO) disorders, since they can be utilized as an energy source without the use of the defective enzyme. However, studies in mice and preterm infants suggest that not all medium-chain fatty acids (MCFA) are oxidized and may undergo elongation to long-chain fatty acids (LCFA). In this single blinded study, we explored the metabolic fates of MCT in healthy individuals using a 13C-labeled MCT tracer. METHOD: Three healthy males in rest received on two test days a primed continuous infusion of glyceryl tri[1,2,3,4-13C4]-octanoate with either an isocaloric supplementation of 1) exclusively MCT (MCT-only) or 2) a mixture of MCT, proteins and carbohydrates (MCT-mix). Gas chromatography - combustion - isotope ratio mass spectrometry (GC-C-IRMS) was used to determine 13C-enrichment of long-chain fatty acids in plasma and of 13CO2 in exhaled air. RESULTS: When provided as single energy source, an estimated 42% of administered MCT was converted to CO2. In combination with carbohydrates and proteins in the diet, oxidation of MCT was higher (62%). In both diets <1% of 13C-label was incorporated in LCFA in plasma, indicating that administered MCT underwent chain-elongation to LCT. CONCLUSIONS: Although the relative MCT oxidation rate was higher when combined with carbohydrates and protein, quantitatively more MCT was oxidized when given an isocaloric meal with solely MCT. As these results were obtained in the resting state opposed to during exercise, it is too early to give a recommendation concerning the use of MCT in lcFAO disorders. The data show that in resting healthy individuals only a very small part of the MCT is traced back as LCFA in plasma, suggesting that MCT treatment does not result in a large LCFA burden, however further research on storage of MCT in tissues is warranted. REGISTRATION: The study was registered in the Nederlands Trialregister. Protocol ID: Trial NL7417 (NTR7650).

Studies of ApoD-/- and ApoD-/-ApoE-/- mice uncover the APOD significance for retinal metabolism, function, and status of chorioretinal blood vessels.

Apolipoprotein D (APOD) is an atypical apolipoprotein with unknown significance for retinal structure and function. Conversely, apolipoprotein E (APOE) is a typical apolipoprotein with established roles in retinal cholesterol transport. Herein, we immunolocalized APOD to the photoreceptor inner segments and conducted ophthalmic characterizations of ApoD-/- and ApoD-/-ApoE-/- mice. ApoD-/- mice had normal levels of retinal sterols but changes in the chorioretinal blood vessels and impaired retinal function. The whole-body glucose disposal was impaired in this genotype but the retinal glucose metabolism was unchanged. ApoD-/-ApoE-/- mice had altered sterol profile in the retina but apparently normal chorioretinal vasculature and function. The whole-body glucose disposal and retinal glucose utilization were enhanced in this genotype. OB-Rb, both leptin and APOD receptor, was found to be expressed in the photoreceptor inner segments and was at increased abundance in the ApoD-/- and ApoD-/-ApoE-/- retinas. Retinal levels of Glut4 and Cd36, the glucose transporter and scavenger receptor, respectively, were increased as well, thus linking APOD to retinal glucose and fatty acid metabolism and suggesting the APOD-OB-Rb-GLUT4/CD36 axis. In vivo isotopic labeling, transmission electron microscopy, and retinal proteomics provided additional insights into the mechanism underlying the retinal phenotypes of ApoD-/- and ApoD-/-ApoE-/- mice. Collectively, our data suggest that the APOD roles in the retina are context specific and could determine retinal glucose fluxes into different pathways. APOD and APOE do not play redundant, complementary or opposing roles in the retina, rather their interplay is more complex and reflects retinal responses elicited by lack of these apolipoproteins.

Quantification of phenolic compounds in vegetable oils by mixed-mode solid-phase extraction isotope chemical labeling coupled with UHPLC-MS/MS.

In the present work, a rapid, accurate and cost-effective method has been developed for the simultaneous quantification of phenolic compounds in oil using mixed-mode solid-phase extraction (SPE) coupled with chemical labeling UHPLC-MS/MS. Mix-mode SPE weak cation cartridges were selected to enrich and purify phenolic compounds in oil, and hydroxyl moiety was dansylation as stable-isotope internal standard. The major parameters that affected the extraction and chemical labeling efficiency were investigated, and the method was fully validated. The limit of quantifications and the limit of detections were 0.002 µg kg-1 ~ 0.10 µg kg-1 and 0.006 µg kg-1 ~ 0.30 µg kg-1, respectively. The recoveries were 61.2% ~ 129.3% with intra-day and inter-day precision less than 12%. The results for 38 rapeseed oils revealed that 14 phenolic compounds, including canolol, phenolic acids, phenolic alcohols, tyrosol and vanillin from trace levels to relatively high content.

Specialized cattle farming in the Neolithic Rhine-Meuse Delta: Results from zooarchaeological and stable isotope (δ18O, δ13C, δ15N) analyses.

Schipluiden (3630-3380 cal BC), the earliest known year-round settlement in the Rhine-Meuse Delta in the Netherlands, is a key site for addressing the nature of Neolithic subsistence in the wetlands of northwestern Europe. A preliminary zooarchaeological study suggested that cattle husbandry was a major activity at Schipluiden. In contrast, stable carbon and nitrogen isotope analyses of human remains from the site indicated a marine-oriented diet, implying that the Mesolithic-Neolithic dietary transition continued well into the mid-4th Millennium BC in this region. Here, we re-investigate the role and nature of cattle husbandry at Neolithic Schipluiden using mortality profiles and stable isotope analysis (δ18O, δ13C, δ15N) of animal bone collagen and tooth enamel. The age-at-death analysis suggests that cattle were managed for both meat and milk production. The δ18O and δ13C analysis of tooth enamel provide evidence that calving spread over five-and-a-half-months, which would have led to a longer availability of milk throughout the year. Cattle were grazing in open, marshy environments near the site and winter foddering was practiced occasionally. The faunal isotopic data also reveal that the high 15N in human bone collagen is more likely to signal the consumption of products from cattle that grazed on 15N-enriched salt marsh plants around the site, rather than a marine-oriented diet. This undermines the previous interpretation of the dietary practices at Schipluiden by showing that human diet in mid-4th millennium BC Rhine-Meuse area was fully "Neolithic", based primarily on products from domesticates, especially cattle, with some input from wild terrestrial and aquatic resources available in their surroundings, contrary to what has been proposed before. Collating these results demonstrates a high level of investment in cattle husbandry, highlighting the social and economic importance of cattle at the lower Rhine-Meuse Delta during the 4th millennium BC.