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The 18O/16O ratio of a-cellulose in land plants has proved of interest for climate, environmental, physiological and metabolic studies. Reliable application of such ratio may be compromised by the presence of hemicellulose impurities in the a-cellulose product obtainable with current extraction methods, as the impurities are known to be isotopically different from that of the a-cellulose. We firstly compared the quality of hydrolysates of “a-cellulose products” obtained with four repre-sentative extraction methods (JAYME and WISE; BRENDEL; ZHOU; LOADER) and quantified the hemicellulose-derived non-glucose sugars in the “a-cellulose products” from 40 land grass species using GC/MS. Secondly, we performed com-pound-specific oxygen isotope analysis for the hydrolysates using GC/Pyrolysis/IRMS. These results were then compared with the bulk isotope analysis using EA/Pyrolysis/IRMS of the “a-cellulose products”. We found that the ZHOU method afforded overall the highest purity a-cellulose as judged by the minimal presence of lignin, and the second lowest presence of non-glucose sugars in the investigated grass species. Isotopic analysis then showed that the O-2~O-6 of the a-cellulose glucosyl units were all depleted in 18O by 0.0-4.3 mUr (with an average of 1.9 mUr) in a species-dependent manner relative to the a-cellulose products. The positive isotopic bias of using a-cellulose product instead of the glucosyl units stems mainly from the fact that the pentoses that dominate hemicellulose contamination in the a-cellulose product are relatively enriched in 18O (compared to hexoses) as they inherit only the relatively 18O-enriched O-2~O-5 moiety of sucrose, the common bio-chemical precursor of pentoses and hexoses in cellulose, and are further enriched in 18O by the (incomplete) hydrolysis. Failure to prepare a-cellulose with the highest possible purity, free from lignin and hemicellulose contamination, can bias predictions based on mechanistic models linking a-cellulose oxygen isotope composition to plant growth conditions.

Water data is a crucial asset for sustainable water resource management. However, the availability of China’s water datasets lags far behind modern expectations for open geoscientific data. This dataset is a part of the China Water Data Archive (CWDA), an upcoming national collection of water-related data covering all aspects of water data for boosting data sharing in China. The CWDA aims at providing free, clean, non-sensitive, coherent, and reliable water data within China for global researchers to support the national and global water resources management and the United Nations-Water Integrated Monitoring Initiative for Sustainable Development Goals 6 and 14. In this paper, we used Python and R language to collect, tidy, reorganize, and curate the publicly available inland and coastal/ocean surface water quality data in China, following a series of data quality dimensions (integrity, completeness, consistency, and accuracy). As the most comprehensive, publicly available, handy, and clean water quality dataset in China so far, it included water quality data for daily, weekly, and monthly in the period of 1980–2022, with 17 indicators for over 330,000 observations at 2384 sites from inland to coastal/ocean areas. This dataset will greatly support works relevant to the assessment, modelling, and projection of water quality, ocean biomass, and biodiversity in China.

Temperature is a critical environmental factor that affects the cell growth of dinoflagellates and bloom formation. To date, the molecular mechanisms underlying the physiological responses to temperature variations are poorly understood. Here, we applied quantitative proteomic and untargeted metabolomic approaches to investigate protein and metabolite expression profiles of a bloom-forming dinoflagellate Prorocentrum shikokuense at different temperatures. Of the four temperatures (19, 22, 25, and 28°C) investigated, P. shikokuense at 25°C exhibited the maximal cell growth rate and maximum quantum efficiency of photosystem II (Fv/Fm) value. The levels of particulate organic carbon (POC) and nitrogen (PON) decreased with increasing temperature, while the POC/PON ratio increased and peaked at 25°C. Proteomic analysis showed proteins related to photoreaction, light harvesting, and protein homeostasis were highly expressed at 28°C when cells were under moderate heat stress. Metabolomic analysis further confirmed reallocated amino acids and soluble sugars at this temperature. Both omic analyses showed glutathione metabolism that scavenges the excess reactive oxygen species, and transcription and lipid biosynthesis that compensate for the low translation efficiency and plasma membrane fluidity were largely upregulated at suboptimal temperature. Higher accumulations of glutathione, glutarate semialdehyde, and 5-KETE at 19°C implied their important roles in low-temperature acclimation. The strikingly active nitrate reduction and nitrogen flux into asparagine, glutamine, and aspartic acid at 19°C indicated these three amino acids may serve as nitrogen storage pools and help cells cope with low temperature. Our study provides insights into the effects of temperature on dinoflagellate resource allocation and advances our knowledge of dinoflagellate bloom formation in marine environments. IMPORTANCE Marine phytoplankton is one of the most important nodes in global biogeochemical cycle. Deciphering temperature-associated marine phytoplankton cell stoichiometric changes and the underlying molecular mechanisms are therefore of great ecological concerns. However, knowledge of how phytoplankton adjust the cell stoichiometry to sustain growth under temperature changes is still lacking. This study investigates the variations of protein and metabolite profiles in a marine dinoflagellate across temperatures at which the field blooms usually occur and highlights the temperature-dependent molecular traits and key metabolites that may be associated with rapid cell growth and temperature stress acclimation.

Dinoflagellates are important primary producers and major causative agents of harmful algal blooms in the global ocean. Despite the great ecological significance, the photosynthetic carbon acquisition by dinoflagellates is still poorly understood. The pathways of photosynthetic carbon assimilation in a marine dinoflagellate Prorocentrum donghaiense under both in situ and laboratory-simulated bloom conditions were investigated using a combination of metaproteomics, qPCR, stable carbon isotope and targeted metabolomics approaches. A rapid consumption of dissolved CO2 to generate high biomass was observed as the bloom proceeded. The carbon assimilation genes and proteins including intracellular carbonic anhydrase 2, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase and RubisCO as well as their enzyme activities were all highly expressed at the low CO2 level, indicating that C4 photosynthetic pathway functioned in the blooming P. donghaiense cells. Furthermore, δ13 C values and content of C4 compound (malate) significantly increased with the decreasing CO2 concentration. The transition from C3 to C4 pathway minimizes the internal CO2 leakage and guarantees efficient carbon fixation at the low CO2 level. This study demonstrates the existence of C4 photosynthetic pathway in a marine dinoflagellate and reveals its important complementary role to assist carbon assimilation for cell proliferation during the bloom period.

The hydrogen isotope ratio of water cryogenically extracted from plant stem samples (δ(2)H(stem_CVD)) is routinely used to aid isotope applications that span hydrological, ecological, and paleoclimatological research. However, an increasing number of studies have shown that a key assumption of these applications—that δ(2)H(stem_CVD) is equal to the δ(2)H of plant source water (δ(2)H(source))—is not necessarily met in plants from various habitats. To examine this assumption, we purposedly designed an experimental system to allow independent measurements of δ(2)H(stem_CVD), δ(2)H(source), and δ(2)H of water transported in xylem conduits (δ(2)H(xylem)) under controlled conditions. Our measurements performed on nine woody plant species from diverse habitats revealed a consistent and significant depletion in δ(2)H(stem_CVD) compared with both δ(2)H(source) and δ(2)H(xylem). Meanwhile, no significant discrepancy was observed between δ(2)H(source) and δ(2)H(xylem) in any of the plants investigated. These results cast significant doubt on the long-standing view that deuterium fractionation occurs during root water uptake and, alternatively, suggest that measurement bias inherent in the cryogenic extraction method is the root cause of δ(2)H(stem_CVD) depletion. We used a rehydration experiment to show that the stem water cryogenic extraction error could originate from a dynamic exchange between organically bound deuterium and liquid water during water extraction. In light of our finding, we suggest caution when partitioning plant water sources and reconstructing past climates using hydrogen isotopes, and carefully propose that the paradigm-shifting phenomenon of ecohydrological separation (“two water worlds”) is underpinned by an extraction artifact.

The 18O/16O (and 15N/14N) ratio of natural nitrate (NO3-) and nitrite (NO2-) can be used to extract valuable information about their source and fate as environmental contaminants, their metabolism as macronutrients in plants and animals, and their behavior in the N biogeochemical cycle. We developed an accurate, precise, sensitive (minimum sample size: 0.2 μg NO3--equivalent), and reliable (minimal oxygen exchange, loss, or gain) method to selectively isolate and purify nitrate and nitrite from natural water, soil, air, and plant materials by strong anion exchange (SAX) for low- to normal-salinity samples or strong cation exchange (SCX) for high-salinity samples, followed by quantitative conversion to their respective benzyl esters, which can be separated and individually analyzed for δ18O (and potentially δ15N) by gas chromatography (GC)/pyrolysis/GC/isotope-ratio mass spectrometry (IRMS). The method compares favorably with the currently popular bacterial denitrification and chemical reduction methods, in terms of sensitivity and reliability, and has the potential to simultaneously measure δ15N and δ18O of nitrate and nitrite from natural samples of various origins.

Information about plant photosynthetic carbon assimilation, physiology, and biochemistry is locked in the 18O/16O ratios of the individual positions of higher plants carbohydrates but is under-utilized, because of the difficulty of making these determinations. We report the extension of the wet chemistry approach we used to access the 18O/16O ratio of O-3 of glucose with a novel GC/Pyrolysis/IRMS-based method, to determine the 18O/16O ratios of O-4, O-5, and O-6. The O atoms (OH groups) at positions 1, 2, 5, and 6 of glucose were protected by acetonation (converting to 1,2;5,6-di-O-isopropylidene-glucofuranose, DAGF). The DAGF was then converted to 6-bromo-6-deoxy-1,2;3,5-di-O-isopropylidene-glucofuranose (6-bromoDAGF) with the simultaneous removal of O-6 with N-bromosuccinimide and triphenylphosphine. The DAGF was also methylated at O-3 with CH3I under the catalysis of NaH to 3-methylDAGF, which was then deacetonated to 1,2-O-isopropylidene-3-O-methyl-glucofuranose (3-methylMAGF). O-5 and O-6 were then removed as a whole from 3-methylMAGF by I2 oxidization under the catalysis of Ph3P and imidazole. Isotope mass balance was then applied to calculate the 18O/16O of O-5 and O-6 as a whole and O-6, respectively. Sampling at different stages of substrate conversion to product and applying a Rayleigh-type fractionation model were employed, when quantitative conversion of substrate was unachievable to calculate the δ18O of the converted substrate. Quantitative conversion of glucose with phenylhydrazine to phenylglucosazone also allowed for the calculation of δ18O2 by applying isotope mass balance between the two. A C4 starch-derived glucose intramolecular δ18O profile is now determined: O-3 is relatively enriched (by 12.16 mUr), O-4 is relatively depleted (by 20.40-31.11 mUr), and O-2 is marginally enriched (by 2.40 mUr) against the molecular average.

SCD1 is a key enzyme controlling lipid metabolism and a link between its activity and NAFLD has been proposed. Lipophagy is a novel regulatory approach to lipid metabolism regulation, which is involved in the development of NAFLD. However, the possible functional connection between SCD1 and lipophagy in NAFLD remains unknown. To investigate the molecular mechanisms through which SCD1 regulates lipophagy in hepatic steatosis, the model of hepatic steatosis was established by inducing mouse primary hepatocytes with sodium palmitate and feeding C57BL/6 mice with HFD. Our results indicated that sodium palmitate-treated hepatocytes exhibited increased SCD1 expression, AMPK inactivation and defective lipophagy. Inhibition of SCD1 expression in hepatocytes resulted in enhanced AMPK activity and lipophagy, and reduced lipid deposition. Although SCD1 overexpression led to decreased AMPK activity and lipophagy, lipid deposition was increased in hepatocytes. SCD1 regulated lipophagy through AMPK to affect lipid metabolism in mouse primary hepatocytes. Additionally, compared to HFD-fed mice, CAY10566(an SCD1-specific inhibitor)-treated mice exhibited significantly decreased hepatic steatosis and hepatic lipid droplet accumulation, as well as enhanced AMPK activity and lipophagy. This study elucidated that SCD1 inhibition ameliorates hepatic steatosis by inducing AMPK-mediated lipophagy, suggesting that the SCD1-AMPK-lipophagy pathway is a potential therapeutic target for NAFLD.

Source-specific highly branched isoprenoids (HBIs) have been recently served as a binary or semi-quantitative biomarker to indicate the sea ice extent in the past. Since the light intensity controlled by overlying snow cover and sea ice thickness has a significant impact on the productivity of photoautotrophic organisms and environmental water is the sole source of the hydrogen for the biosynthesis of these organisms, the hydrogen isotope ratio (2H/1H) of HBIs holds the potential to reveal more characteristics of sea ice. In this study, based on the observation of natural settings underneath sea ice, diatom Pleurosigma intermedium were grown at irradiances from 20 to 300 μmol m-2 s-1 in laboratory conditions and harvested from exponential phase and stationary phase respectively to investigate the effect of light and growth phase on hydrogen isotope fractionation in HBIs. Gas chromatography-mass spectrometry (GC-MS) screening showed that a triene (C25:3) and a tetraene (C25:4) C25 HBI alkene were detected in all samples from varying irradiances. A remarkable decline of the ratio of C25:3/C25:4 from higher to lower irradiances was observed. However, there was no significant change in the concentration of C14 (myristic), C16:1 (palmitoleic) and C16 (palmitic) fatty acids with varying light intensity. In addition, terpenoids such as phytol, squalene and range of sterols were also be identified. Published studies on phytol, fatty acid and sterol from Thalassiosira pseudonana and alkenones from Emiliania huxleyi have shown dramatic changes in hydrogen isotope fractionation and concluded that the source of nicotinamide adenine dinucleotide phosphate (NADPH) and the operation of acetogenic pathway, plastidic methylerythritol phosphate (MEP) and/or cytosolic mevalonic acid (MVA) of lipids are the key factors controlling 2H/1H fractionation. The integration of molecular distribution of HBIs, fatty acids and terpenoids in Pleurosigma intermedium together with our ongoing work on their 2H/1H and 13C/12C compositions will lead to a better understanding of diatom metabolism and biochemistry under different light conditions. This knowledge will be instrumental to a more robust interpretation of stable isotope data from environmental samples and thus will contribute to further developing HBI biomarkers as a tool for estimating not only the absence/presence of sea ice but also the ice type, thickness, and snow cover.

Carbon-13 position-specific isotope analysis of fatty acids from vegetable oils was performed using Nuclear Magnetic Resonance in the present study. The measured 13C patterns are not totally in accordance with the conventional view of the relative 13C-depletion of acetogenic lipids and their alternation of 13C-enriched and 13C-depleted carbon positions. The results presented here provide a new evaluation of the isotopic fractionation associated with fatty acids biosynthesis. Whereas it is commonly acknowledged that the pyruvate dehydrogenase (PDH) is responsible for the 13C distribution within fatty acids, data from the present work demonstrate that the conversion of acetyl-CoA to malonyl-CoA catalyzed by acetyl-CoA carboxylase (ACC) needs to be considered while explaining the measured non-stochastic 13C pattern within fatty acids. These data combined with steady-state calculations give a new description of metabolic steps responsible for the typical 13C intramolecular distribution of acetogenic lipids. In addition, the non-stochastic pattern measured in these plant fatty acids is similar to those previously detected within long-chain n-alkanes suggesting a preservation through geological time and demonstrating the interest of position-specific isotope analysis for studying the evolution of metabolic pathways.

Zhao (1) considers our examination (2) of the relationship between deuterium offset (hereafter “Δ”) and stem gravimetric water content (hereafter “ W ”) across all the study species to be misleading, and instead suggest that data should be analyzed by individual habitat type based on a vague argument that “different habitats/species will behave differently.” Such a suggestion is unjustified as it ignores the fact that the results of our experiment have already ruled out root uptake-related fractionation (which, if present, would justify the use of a habitat type-based approach) as a mechanism underpinning Δ. As a matter of fact, given our demonstration that extraction-associated deuterium depletion … [↵][1]1To whom correspondence may be addressed. Email: songxbnu{at}gmail.com. [1]: #xref-corresp-1-1

In their reexamination of an earlier study (1) in which water isotope data from globally distributed sites were compiled, Evaristo et al. (2) show that, after accounting for extraction artifacts, precipitation offsets of xylem water ( d xylem_c) are still statistically distinct from those of stream ( d stream)/groundwater ( d gw) in a majority of the sampling sites. In agreement, our own analysis on the dataset of ref. 1 also revealed a significant d xylem_c versus d gw/ d stream difference at the global scale (Table 1). However, we would like to point out that these analyses miss a crucial point: By removing the ∼8‰ stem water extraction-associated error, there is as much “ecohydrological separation” from xylem water … [↵][1]1To whom correspondence may be addressed. Email: songxbnu{at}gmail.com. [1]: #xref-corresp-1-1

The

Dinoflagellates are important primary producers and major causative agents of harmful algal blooms in the global ocean. Despite the great ecological significance, the photosynthetic carbon acquisition by dinoflagellates is still poorly understood. The pathways of photosynthetic carbon assimilation in a marine dinoflagellate Prorocentrum donghaiense under both in situ and laboratory-simulated bloom conditions were investigated using a combination of metaproteomics, qPCR, stable carbon isotope and targeted metabolomics approaches. A rapid consumption of dissolved CO

Dinoflagellate blooming periods are paradoxically characterized by high biomass growth rate and low ambient dissolved CO

RATIONALE Quantitatively relating 13 C/12 C, 2 H/1 H and 18 O/16 O ratios of plant α-cellulose and 2 H/1 H of n-alkanes to environmental conditions and metabolic status should ideally be based on the leaf, the plant organ most sensitive to environmental change. The fact that leaf organic matter is composed of isotopically different heterotrophic and autotrophic components means that it is imperative that one be able to disentangle the relative heterotrophic and autotrophic contributions to leaf organic matter. METHODS We tackled this issue by two-dimensional sampling of leaf water and α-cellulose, and specific n-alkanes from greenhouse-grown immature and mature and field-grown mature banana leaves, taking advantage of their large areas and thick waxy layers. Leaf water, α-cellulose and n-alkane isotope ratios were then characterized using elemental analysis isotope ratio mass spectrometry (IRMS) or gas chromatography IRMS. A three-member (heterotrophy, autotrophy and photoheterotrophy) conceptual linear mixing model was then proposed for disentangling the relative contributions of the three trophic modes. RESULTS We discovered distinct spatial leaf water, α-cellulose and n-alkane isotope ratio patterns that varied with leaf developmental stages. We inferred from the conceptual model that, averaged over the leaf blade, only 20% of α-cellulose in banana leaf is autotrophically laid down in both greenhouse-grown and field-grown banana leaves, while approximately 60% and 100% of n-alkanes are produced autotrophically in greenhouse-grown and field-grown banana leaves, respectively. There exist distinct lateral (edge to midrib) gradients in autotrophic contributions of α-cellulose and n-alkanes. CONCLUSIONS Efforts to establish quantitative isotope-environment relationships should take into account the fact that the evaporative leaf water 18 O and 2 H enrichment signal recorded in autotrophically laid down α-cellulose is significantly diluted by the heterotrophically formed α-cellulose. The δ2 H value of field-grown mature banana leaf n-alkanes is much more sensitive than α-cellulose as a recorder of the growth environment. Quantitative isotope-environment relationship based on greenhouse-grown n-alkane δ2 H values may not be reliable.

Information about plant photosynthetic carbon assimilation, physiology, and biochemistry is locked in the

RATIONALE Although the 2 H/1 H ratio of the carbon-bound hydrogens (C-Hs) in α-cellulose extracted from higher plants has long been used successfully for climate, environmental and metabolic studies, the assumption that bleaching with acidified NaClO2 to remove lignin before pure α-cellulose can be obtained does not alter the 2 H/1 H ratio of α-cellulose C-Hs has nonetheless not been tested. METHODS For reliable application of the 2 H/1 H ratio of α-cellulose C-H, we processed plant materials representing different phytochemistries and photosynthetic carbon assimilation modes in isotopically contrasting bleaching media (with an isotopic difference of 273 mUr). All the isotope ratios were measured by elemental analyzer/isotope ratio mass spectrometry (EA/IRMS). RESULTS Our results show that H from the bleaching medium does appear in the final pure α-cellulose product, although the isotopic alteration to the C-H in α-cellulose due to the incorporation of processing H from the medium is small if isotopically "natural" water is used to prepare the processing medium. However, under prolonged bleaching such an isotope effect can be significant, implying that standardizing the bleaching process is necessary for reliable 2 H/1 H measurement. CONCLUSIONS The currently adopted method for removing lignin for α-cellulose extraction from higher plant materials with acidified NaClO2 bleaching is considered acceptable in terms of preserving the isotopic fidelity if isotopically "natural" water is used to prepare the bleaching solution.

Two new C31 branched alkanes (botryococcanes) presumably produced by the B race of Botryococcus braunii were isolated and purified from the Maoming Basin shales using column chromatography and preparative gas chromatography and structurally characterized with HR-EI-MS and 1D and 2D NMR. Interpretation of their EI mass spectral and 1D and 2D NMR (HMBC and HSQC) data led to the firm assignments of the two alkanes as diastereoisomeric 2,3,6,7,10,12,15,16,19,20-decamethylhenicosanes (DMHs). The structural assignments were further confirmed by the close match of the measured 13C NMR chemical shifts with those predicted by Lindeman-Adams 13C chemical shift modeling. The skeletons of these two DMHs are virtually identical to that of the recently identified C33 botryococcane/botryococcanone in the same sample. It is proposed that these two DMHs share a precursor C33 botryococcene biochemically formed by condensing two farnesyl diphosphates involving an unusual cyclobutanation, a retro-Prins reaction and a tetramethylation. A photo-mediated geochemical oxidation of the double bond in the ethenyl group connected to the sole quaternary carbon C-10 is also proposed to be responsible for the formation of the co-occurring DMHs and C33 botryococcane/botryococcanone.

A novel C33 botryococcane in a sedimentary sample from the Maoming Basin in China was found to have a methyl group positioned β to the quaternary carbon C-10 in its structure based on IR, MS and NMR analyses and Lindeman-Adams modelling. This structure is inconsistent with the prediction from the currently accepted botryococcene biosynthetic pathway and different from the skeleton for the C33 botryococcanes suggested in the literature. A C33 botryococcan-24-one with the same carbon skeleton as the C33 botryococcane was also found to co-occur in the sediment sample. The location of the carbonyl group at C-24 is different from that of a previously reported botryococcenone from an Australian collection of race B of Botryococcus braunii where it is positioned at C-15. We propose a biogeochemical pathway to explain the occurrence of this unique skeleton in the Maoming Basin sediment. The biochemical component of the pathway involves electrophilic or nucleophilic attack between two farnesyl diphosphates (FPPs) leading to direct or indirect formation of a C30 botryococcene via a cyclobutane ring intermediate (1′-2-3-2′ cyclisation). A Retro-Prins reaction and subsequent methylation gives rise to C29 and C33 botryococcenes in sequence. The geochemical component involves first in-water-column (photo)-oxidation of the alkene to a C33 botryococcanone and subsequent reduction (hydrogenation) to stabilize the ketone as an alkane.

The 18O/16O ratio at both molecular and positional levels in the carbohydrates of higher plants is a reliable proxy for the plant growth environment, and a potential indicator of the plant photosynthetic carbon assimilation mode, and its physiological, biochemical and metabolic status. The lack of exploitable nuclear resonance in 18O and 16O and the extremely low 17O abundance make the NMR-based PSIA (position-specific isotopic analysis) a significant challenge. In this Article, an alternative three-step wet chemistry based method for accessing the 18O/16O of glucose O-3 is presented. The O atoms (OH groups) at positions 1, 2, 5, and 6 were first protected by acetonation (converting glucose to 1,2;5,6-di-O-isopropylidene-glucofuranose). The protected glucose was then esterified at O-3 by thionoformylation. Subsequent Barton–McCombie deoxygenation quantitatively removed the O-3 from the protected sugar. Mass balance was then applied to calculate the 18O/16O of O-3 using the isotopic values of the protected...

Compartmentation of C4 photosynthetic biochemistry into bundle sheath (BS) and mesophyll (M) cells, and photorespiration in C3 plants is predicted to have hydrogen isotopic consequences for metabolites at both molecular and site-specific levels. Molecular-level evidence was recently reported (Zhou et al., 2016), but evidence at the site-specific level is still lacking. We propose that such evidence exists in the contrasting 2H distribution profiles of glucose samples from naturally grown C3, C4 and CAM plants: photorespiration contributes to the relative 2H enrichment in H5 and relative 2H depletion in H1 & H6 (the average of the two pro-chiral Hs and in particular H6, pro-R) in C3 glucose, while 2H-enriched C3 mesophyll cellular (chloroplastic) water most likely contributes to the enrichment at H4; export of (transferable hydrogen atoms of) NADPH from C4 mesophyll cells to bundle sheath cells (via the malate shuttle) and incorporation of 2H-relatively unenriched BS cellular water contribute to the relative depletion of H4 & H5 respectively; shuttling of triose-phosphates (PGA: phosphoglycerate dand DHAP: dihydroacetone phosphate) between C4 bundle sheath and mesophyll cells contributes to the relative enrichment in H1 & H6 (in particular H6, pro-R) in C4 glucose.

An unknown compound dominant in the aliphatic hydrocarbon fraction of the solvent extract of a Dominican amber sample was isolated and purified using column chromatography and preparative gas chromatography. 1D and 2D NMR analyses allowed its structure to be assigned as 15- nor- cleroda-3,12-diene. The bridgehead methyl group (CH 3 at C-19) has a 13 C chemical shift of 19.6 ppm, typical for a trans -decalin framework (Nogueira, 2001), indicating the diterpenoid has a trans A/B ring structure. ROESY correlations indicate that the methyl groups at C-19, C-20 were axial and the C-17 methyl group was equatorial, while the H-10 and H-8 were axial and on the other side of Ring B. Taken together, a 5 R ,8 R ,9 S ,10 R -configuration for the new nor-clerodadiene can be established. The absolute configuration of 5 R ,8 R ,9 S ,10 R was further confirmed by comparing the experimental and calculated electronic circular dichroism (ECD) spectra of this diterpenoid. The identified diene appears to originate from a Hymenaea species ( Fabaceae ). Non-oxidative decarboxylation of cleroda-3,13-diene-15-oic acid (kovalenic acid) present in Hymenaea species or oxidative decarboxylation of 3-cleroden-15-oic acid in Dominican amber are considered to be the two viable pathways leading to the formation of the identified 15- nor -cleroda-3,12-diene, although the former pathway is more likely.

The chemical linkage between α-cellulose and lignin in plant cell walls has long been a controversial topic and crucial to devising effective strategies for sustainable biomass and bioenergy utilization. In this new contribution, we surveyed 80 Poaceae (Gramineae) species grown in tropical Hainan Island (China) to test the hypothesis that the presence of oxygen-containing chemical linkage in Poaceae species is widespread. Our innovative natural abundance oxygen isotopic analysis allowed us to infer that more than 1/3 of the species investigated has chemical (ether) linkages between α-cellulose and lignin in their leaf cell walls, with a species-specific 2–89 oxygen-containing bonds for every 1000 glucose units. However, the presence of such linkage appears to be phylogeny-dependent. On average, species of C3 photosynthetic mode are found to have more extensive oxygen-containing linkages than those of C4 photosynthetic mode. Our finding challenges the conventional view that no chemical bonds between α-cell...

Although theFor reliable application of theOur results show that H from the bleaching medium does appear in the final pure α-cellulose product, although the isotopic alteration to the C-H in α-cellulose due to the incorporation of processing H from the medium is small if isotopically "natural" water is used to prepare the processing medium. However, under prolonged bleaching such an isotope effect can be significant, implying that standardizing the bleaching process is necessary for reliableThe currently adopted method for removing lignin for α-cellulose extraction from higher plant materials with acidified NaClO

Liver X receptors (LXR) α and β serve important roles in cholesterol homeostasis, anti-inflammatory processes and the activation of hepatic stellate cells (HSCs). However, the development of therapies for liver fibrosis based on LXR agonists have been hampered due to side-effects such as liver steatosis. In this study, we demonstrated that HSCs expressed high levels of LXRβ, but not LXRα, and that overexpression of LXRβ suppressed fibrosis and HSC activation in a carbon tetrachloride (CCl4)-induced fibrosis mouse model, without resulting in liver steatosis. Furthermore, Hedgehog (Hh)-regulated proteins, markedly increased in the CCl4-affected liver and mainly expressed in activated HSCs, were repressed under conditions of LXRβ overexpression. In addition, LXRβ knockout led to activation of Hh signaling and triggering of HSC activation, while overexpression of LXRβ led to the inhibition of the Hh pathway and suppression of HSC activation. These results suggest that LXRβ suppresses the activation mechanism of HSCs by inhibiting Hh signaling. In conclusion, LXRβ, by restoring the differentiation of HSCs, may be a promising therapeutic target for liver fibrosis without the adverse side-effects of LXRα activation.

The 2 H/1 H ratio of carbon-bound H in biolipids holds potential for probing plant lipid biosynthesis and metabolism. The biochemical mechanism underlying the isotopic differences between lipids from C3 and C4 plants is still poorly understood. GC-pyrolysis-IRMS (gas chromatography-pyrolysis-isotope ratio mass spectrometry) measurement of the 2 H/1 H ratio of leaf lipids from controlled and field grown plants indicates that the biochemical isotopic fractionation (e2 Hlipid_biochem ) differed between C3 and C4 plants in a pathway-dependent manner: e2 HC4 > e2 HC3 for the acetogenic pathway, e2 HC4

Realizing the potential of terpenoid biomarkers as an unequivocal land ecosystem and floral diversity indicator calls for work to establish a direct link between the terpenoids extracted from the plants and those found in the underlying soils. To this end, we have characterized the triterpenoid profile of soils and vegetation along two altitudinal transects representing very different ecosystems. The first transect, comprising 31 sample sites, was on the eastern slopes of Mt Gongga (eastern Tibetan Plateau) rising from 1230 m to 4500 m above sea level, an area with a temperate monsoonal climate. The second transect, sampling 29 sites, was on the northern slopes of Mt West Kunlun, a region characterized by montane desert, meadow and alpine steppe (located between 1300 m and 5050 m above sea level). The dominant terpenoid biomarkers (polycyclic triterpenoids of the taraxerane, oleanane, friedelane, ursane, lupane, glutinane and lanostane series) identified in the soils were found to be consistent with the overlying vegetation. However, the discovery of some unusual triterpenoids, including the pentacyclic triterpene 3-methyl ethers (PTMEs), D:A-friedo-2,3-secoolean-2,3-dioic acid, methyl 3β-acetoxylup-20(29)-en-28-oate, 24-methylenecycloartan-3-one, 9,19-cyclolanostan-3-yl 24-methylene acetate, 3α-methoxy-9β-lanosta-7,24-dien-26,23-olide and 3α-methoxy-9β-lanost-7-en-26,23-olide in the Gongga soils, does not match the current vegetation and may be indicative of a long vanished forest-type vegetation. Thus, soil biomarkers may be a useful tool in the reconstruction of palaeo-vegetation.

Further to our recent identification of a C33 botryococcane, a C33 botryococcan-24-one and two C31 botryococcanes (2,3,6,7,10,12,15,16,19,20-decamethylhenicosanes: C31 DMHs) with a methyl group β-positioned to the sole quaternary carbon in the skeleton in a sediment sample from the Maoming Basin, we report the NMR-based structural identification of a new C32 botryococcane (2,3,6,7,10,10,12,15,16,19,20-undecamethylhenicosane: C32 UMH), and MS-based structural identification of a C32 botryococcanal (C32 UMH-al) and two C31 epimeric botryococcanols (C31 DMH-ols) in the same sediment sample after trimethylsilylation. These three oxygenated botryococcanoids are speculated to possess the same β-positioned methyl group (to the sole quaternary carbon). We propose that the C32 UMH-al and two epimeric C31 DMH-ols are degradation products of the C C double bond in the ethenyl group connected to the sole quaternary carbon in their biological precursor C33 botryococcene with a unique β-positioned methyl group (to the quaternary carbon), and are geochemical precursors for the C32 UMH and previously identified C31 DMHs, respectively. Since it is known that abiotic degradation (photo-mediated or autoxidation) preferentially acts on the internal double bond while sedimentary bacteria preferentially degrade the terminal double bond, our finding of a degraded terminal C24-25 double bond and an intact internal double bond (at C11-12) is indicative of bacterially mediated degradation. A detailed bacterially mediated degradation pathway is proposed to explain the formation of C31–C33 botryococcanes and their oxygenated derivatives.

We report the spectroscopy-based (NMR, HRMS, IR) reassignment of the structures of two previously mis-identified saturated methyl ketones (formerly C15 ketone: 5,9-dimethyl-6-isopropyl-2-decanone and C18 ketone: 4,9,11-trimethyl-6-isopropyl-2-dodecanone) in the Maoming oil shale to: 5,6,9,10-tetramethylundecan-2-one and 4,7,8,11,12-pentamethyltridecan-2-one, respectively. A diagenetic pathway is proposed to explain the isotopic and structural closeness between these two methyl ketones and recently identified saturated C31 alcohols, C32 aldehydes and C33 ketones (all with a unique methyl group β to the sole quaternary carbon in their skeletons) obtained from the same Maoming Basin sediments. The pathway involves, firstly, epoxidation, hydration, oxidation and double decarboxylation of the C33 botryococcene to C31 botryococcene. Subsequent photo- or autoxidation of the internal double bond around the quaternary carbon in the C31 botryococcene in an oxic environment leads to the formation of a C31 hydroperoxy-alkene. A heterolytic (Hock-type) or homolytic cleavage of the hydroperoxy-alkene around the quaternary carbon then gives rise to the C15 and C18 unsaturated methyl ketones, respectively. The so-formed unsaturated ketones are then geochemically stabilized by hydrogenation in an anoxic environment.

The adulteration of rice using synthetic aromatic flavorings to fraudulently imitate commercially valuable fragrant rice varieties has attracted extensive attention from regulatory authorities around the world. In order to get convincing evidence of adulteration, appropriate scientific analytical methods need to be developed. In this study, a simple and efficient headspace solid-phase microextraction (HS SPME) technique coupled with gas chromatography mass spectrometry using selected ion monitoring (GC–MS–SIM) for the determination of four food flavoring compounds which are possibly used as adulterants is proposed. The HS SPME operating under optimized conditions increased the selectivity and sensitivity of the analysis by eliminating matrix interferences. The method presented adequate precision and linearity with limits of detection ranging from 0.5 to 10 ng/mL. This HS SPME/GC–MS-SIM method is directly applicable to the analysis of volatiles in rice and has the advantages of minimal pretreatment. It was applied successfully to the analysis of six rice flavoring essences, ten fragrant rice and four artificially scented rice samples.

The

Non-alcoholic fatty liver disease (NAFLD) is a manifestation of metabolic syndrome in the liver and is closely associated with diabetes; however, its pathogenesis remains to be elucidated. Carbohydrate responsive element binding protein (ChREBP), the hub of glucolipid metabolism, regulates the induction of fatty acid synthase (FASN), the key enzyme of de novo lipogenesis, by directly binding to carbohydrate response element (ChoRE) in its promoter. Investigations of histone modifications on NAFLD remain in their infancy. In the present study, by using ChIP, the association between histone modifications and FASN transcription was investigated and histone modifications in FASN modulated by ChREBP were measured. It was demonstrated that ChREBP induced FASN ChREBP-ChoRE binding to accelerate the expression of FASN, leading to hepatocellular steatosis by facilitating H3 and H4 acetylation, H3K4 trimethylation and the phosphorylation of H3S10, but inhibiting the trimethylation of H3K9 and H4K20 in FASN promoter regions of HepG2 and L02 cells. It was also found that ChREBP-ChoRE binding of FASN relied on histone acetylation and that the transcriptional activity of ChREBP on FASN is required, based on the premise that histone acetylation causes conformational changes in FASN chromatin. This indicated histone acetylation as a crucial mechanism involved in the transcription of FASN modulated by ChREBP. Consequently, the present study provides further insight into the pathophysiology and a novel therapeutic potential of NAFLD based on epigenetic mechanisms.

Background The aim of this study was to investigate how the signaling pathway downstream of mTOR/S6K1 contributes to the regulation of SREBP-1c expression during lipogenesis in HepG2 cells. Methods The model of steatosis was established using human hepatocytes HepG2 and inducting them with sodium palmitate. mTOR, S6K1 and LXRα were inhibited by rapamycin, PF-4708671 and siRNA-LXRα, respectively. After a variety of different treatment, the levels of intracellular triglycerides, the accumulation of lipid droplets and the expression levels of related genes were detected. Results Rapamycin, PF-4708671 and siRNA-LXRα treatment could decrease the accumulation of triglycerides and lipid droplets induced by sodium palmitate in HepG2 cells, and the inhibitory effect could be enhanced by the combination of them. Sodium palmitate stimulated the expression of genes encoding mTOR, S6K1, LXRα, SREBP-1c and SREBP-1c target enzymes (FAS and ACC1) in HepG2 cells. Moreover, these genes were sensitive to rapamycin. PF-4708671 also decreased the expression of these genes, except for the mTOR gene, and the extent of reduction could be enhanced by combination with rapamycin. Knockdown of LXRα decreased the expression of SREBP-1c, FAS and ACC1, but it had no effect on the expression of mTOR or S6K1. Furthermore, rapamycin and PF-4708671 enhanced the inhibitory effect of siRNA-LXRα. Conclusions mTOR/S6K1 regulates the SREBP-1c signaling pathway through LXRα in sodium palmitate-induced HepG2 cells, suggesting LXRα might be a potential therapeutic target for NAFLD.

Although deemed important to δ 18 O measurement by on-line high-temperature conversion techniques, how the GC conditions affect δ 18 Omeasurement israrelyexaminedadequately.WethereforedirectlyinjecteddifferentvolumesofCOorCO–N2mixonto the GC column by a six-port valve and examined the CO yield, CO peak shape, CO–N2 separation, and δ 18 O value under different GC temperatures and carrier gas flow rates. The results show the CO peak area decreases when the carrier gas flow rate increases. The GC temperature has no effect on peak area. The peak width increases with the increase of CO injection volume but decreases with the increase of GC temperature and carrier gas flow rate. The peak intensity increases with the increase of GC temperature and CO injection volume but decreases with the increase of carrier gas flow rate. The peak separation time between N2 and CO decreases with an increase of GC temperature and carrier gas flow rate. δ 18 O value decreases with the increase of CO injection volume (when half m/z 28 intensity is

Lipidomic approaches are now widely used to investigate the relationship between lipid metabolism, health and disease. Large-scale lipidomics studies typically aim to quantify hundreds to thousands of lipid molecular species in a large number of samples. Consequently, high throughput methodology that can efficiently extract a wide range of lipids from biological samples is required. Current methods often rely on extraction in chloroform:methanol with or without two phase partitioning or other solvents, which are often incompatible with liquid chromatography electrospray ionization-tandem mass spectrometry (LC ESI-MS/MS). Here, we present a fast, simple extraction method that is suitable for high throughput LC ESI-MS/MS. Plasma (10 μL) was mixed with 100 μL 1-butanol:methanol (1:1 v/v) containing internal standards resulting in efficient extraction of all major lipid classes (including sterols, glycerolipids, glycerophospholipids and sphingolipids). Lipids were quantified using positive-ion mode LC ESI-MS/MS. The method showed high recovery (&gt, 90%) and reproducibility (%CV &lt, 20%). It showed a strong correlation of all lipid measures with an established chloroform:methanol extraction method (R2 = 0.976). This method uses non-halogenated solvents, requires no drying or reconstitution steps and is suitable for large-scale LC ESI-MS/MS-based lipidomic analyses in research and clinical laboratories.

It has long been theorized that carbon allocation, in addition to the carbon source and to kinetic isotopic effects associated with a particular lipid biosynthetic pathway, plays an important role in shaping the carbon isotopic composition ((13)C/(12)C) of lipids (Park and Epstein, 1961). If the latter two factors are properly constrained, valuable information about carbon allocation during lipid biosynthesis can be obtained from carbon isotope measurements. Published work of Chikaraishi et al. (2004) showed that leaf lipids isotopic shifts from bulk leaf tissue Δδ(13)C(bk-lp) (defined as δ(13)C(bulkleaftissue)-δ(13)C(lipid)) are pathway dependent: the acetogenic (ACT) pathway synthesizing fatty lipids has the largest isotopic shift, the mevalonic acid (MVA) pathway synthesizing sterols the lowest and the phytol synthesizing 1-deoxy-D-xylulose 5-phosphate (DXP) pathway gives intermediate values. The differences in Δδ(13)C(bk-lp) between C3 and C4 plants Δδ(13)C(bk-lp,C4-C3) are also pathway-dependent: Δδ(13)C(ACT)(bk-lp,C4-C3) > Δδ(13)C(DXP(bk-lp,C4-C3) > Δδ(13)C(MVA)(bk-lp,C4-C3). These pathway-dependent differences have been interpreted as resulting from kinetic isotopic effect differences of key but unspecified biochemical reactions involved in lipids biosynthesis between C3 and C4 plants. After quantitatively considering isotopic shifts caused by (dark) respiration, export-of-carbon (to sink tissues) and photorespiration, we propose that the pathway-specific differences Δδ(13)C(bk-lp,C4-C3) can be successfully explained by C4-C3 carbon allocation (flux) differences with greatest flux into the ACT pathway and lowest into the MVA pathways (when flux is higher, isotopic shift relative to source is smaller). Highest carbon allocation to the ACT pathway appears to be tied to the most stringent role of water-loss-minimization by leaf waxes (composed mainly of fatty lipids) while the lowest carbon allocation to the MVA pathway can be largely explained by the fact that sterols act as regulatory hormones and membrane fluidity modulators in rather low concentrations.

Compartmentation of C