Isotope Tracer Experiments Research Articles

Diazotrophs: Overlooked Key Players within the Coral Symbiosis and Tropical Reef Ecosystems?

Coral reefs are highly productive ecosystems thriving in nutrient-poor waters. Their productivity depends largely on the availability of nitrogen, the proximate limiting nutrient for primary production. In reefs, the major nitrogen pathways include regeneration, nitrification, ammonification and dinitrogen (N2) fixation. N2 fixation is performed by prokaryotes called ‘diazotrophs’ that abound in coral rubbles, sandy bottoms, microbial mats or seagrass meadows. Corals, which are the main reef builders, have developed a partnership with dinoflagellates which transform dissolved inorganic nitrogen into amino acids and protein, and with diazotrophs to gain diazotrophically-derived nitrogen (DDN). Pioneering studies found active diazotrophic cyanobacteria in the corals’ mucus and/or tissue, and later high throughput sequencing efforts have described diverse communities of non-cyanobacterial diazotrophs associated with scleractinian corals. Yet, the metabolic processes behind these associations and how they benefit corals is currently not well understood. While genomic studies describe the diversity of diazotrophs and isotopic tracer experiments quantify N2 fixation rates, combined advanced methods are needed to elucidate the mechanisms behind the transfer of DDN to the coral holobiont and whether these mechanisms change according to the identity of the diazotrophs or coral species. Here we review our current knowledge on N2 fixation in corals: the diversity and localization of diazotrophs in the coral holobiont, the environmental factors controlling N2 fixation, the fate of DDN within the coral symbiosis as well as its potential role in coral resilience. We finally summarize the unknowns: are the diversity, abundance and localization of diazotrophs within the holobiont species- and/or site-specific? Do they have an impact on DDN production? What are the metabolic mechanisms implicated? Do they change spatially, temporally or according to environmental factors? We encourage scientists to undertake research efforts to tackle these questions in order to shed light on nitrogen cycling in reef ecosystems and to understand if coral-associated N2 fixation can improve coral’s resilience in the face of climate change.

Reef sponges facilitate the transfer of coral-derived organic matter to their associated fauna via the sponge loop

The high biodiversity of coral reefs results in complex trophic webs where energy and nutrients are transferred between species through a multitude of pathways. Here, we hypothesize that reef sponges convert the dissolved organic matter released by benthic primary producers (e.g. corals) into particulate detritus that is transferred to sponge-associated detritivores via the sponge loop pathway. To test this hypothesis, we conducted stable isotope (C-13 and N-15) tracer experiments to investigate the uptake and transfer of coral-derived organic matter from the sponges Mycale fistulifera and Negombata magnifica to 2 types of detritivores commonly associated with sponges: ophiuroids (Ophiothrix savignyi and Ophiocoma scolopendrina) and polychaetes (Polydorella smurovi). Findings revealed that the organic matter naturally released by the corals was indeed readily assimilated by both sponges and rapidly released again as sponge detritus. This detritus was subsequently consumed by the detritivores, demonstrating transfer of coral-derived organic matter from sponges to their associated fauna and confirming all steps of the sponge loop. Thus, sponges provide a trophic link between corals and higher trophic levels, thereby acting as key players within reef food webs.

Characterization of Metabolic Response to AG-348, an Allosteric Activator of Red Cell Pyruvate Kinase, in Healthy Volunteers and Pyruvate Kinase Deficiency Patients

Pyruvate kinase (PK) deficiency is a glycolytic enzymopathy that causes lifelong chronic hemolytic anemia. AG-348 is an allosteric activator of the red cell isoform of pyruvate kinase (PK-R) that is in clinical development to treat PK deficiency. Phase 1 studies of AG-348 in healthy volunteers (NCT02108106, NCT02149966) have been completed, and a phase 2 study in patients with PK deficiency is in progress (DRIVE PK, NCT02476916). We have previously reported that in the healthy volunteer studies, AG-348 induced changes in levels of the metabolites adenosine triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) that are consistent with PK-R activation. A preliminary analysis of data showed that nine out of 18 DRIVE PK patients achieved a maximal increase in hemoglobin (Hb) levels of >1.0 g/dL.In this study, we characterize whole blood metabolism of healthy human subjects (from the multiple-ascending dose study NCT02149966) as well as PK-deficient patients (from DRIVE PK), before and after administration of AG-348, with a focus on the flux through the PK-R reaction. Whole blood taken from healthy subjects/DRIVE PK patients on each respective study was incubated with a stable isotope tracer, [U-13C6]-glucose. Glycolytic flux through PK-R was estimated by kinetic flux profiling based on the isotope labeling, as well as by the specific lactate production rate, with the two methods giving consistent results.Blood cells from healthy subjects exhibited classic red blood cell (RBC) metabolism, with the majority of glucose catabolized through glycolysis. AG-348 was shown to significantly increase the maximal PK-R protein activity in these subjects, and also to have metabolic effects consistent with PK-R activation, marked by decreased concentrations of glycolytic intermediates such as 2,3-DPG and phosphoenolpyruvate (PEP). Increases in ATP concentrations were also observed, with the magnitude and kinetics of the increase strongly suggesting enhanced adenosine salvage or synthesis. The overall glycolytic rates, however, did not change significantly after two weeks of AG-348 dosing, revealing the homeostatic regulation of RBC glycolysis in healthy blood.Five of the first 18 DRIVE PK patients underwent an extensive sampling protocol for metabolic analysis. This analysis revealed a number of distinct metabolic qualities in PK-deficient patients at baseline compared with healthy subjects. These included significantly reduced rates of lactate production and high concentrations of nucleotides, amino acids, and Krebs cycle intermediates. 13C labeling was observed in Krebs cycle intermediates, demonstrating significant respiratory metabolism in whole blood cells from PK-deficient patients, while incomplete labeling of glycolytic intermediates suggested the presence of a metabolically inactive cell subpopulation. These observations are most consistent with the hypothesis that PK-deficient whole blood is dominated metabolically by immature erythrocytes that retain residual mitochondrial activity.With AG-348 treatment, three of the five DRIVE PK patients had increases in Hb of >1.0 g/dL. In those three patients, we observed increased incorporation of 13C label into glycolytic intermediates such as 2,3-DPG, suggesting an increase in metabolically active erythrocytes. First order flux estimates based on either 2,3-DPG labeling kinetics or lactate production rates showed a >0.1 mmol/L/hr (>50%) increase in glycolytic flux. Neither of the two DRIVE PK patients that did not have an Hb increase of >1.0 g/dL showed significant metabolic changes.In conclusion, metabolic profiling and stable isotope tracing experiments in blood from healthy subjects treated with AG-348 revealed strong homeostatic regulation of glycolysis even in the presence of activated PK-R. Analysis of data from a small number (n=5) of PK-deficient patients treated with AG-348 for two weeks showed that the three patients with Hb increases >1.0 g/dL also had increased glycolytic flux. While the small number of patients makes these results preliminary, it is the first demonstration of a direct link between increased red cell glycolysis induced by the PK-R activator AG-348 and the resulting hematological response as assessed by increases in Hb levels. Updated analyses including additional patients will be presented as more data are collected in the ongoing study. DisclosuresChubukov:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Johnson:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Kosinski:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Clasquin:Agios Pharmaceuticals, Inc.: Other: former employee and stock holder; Pfizer, Inc.: Employment. Jha:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Kim:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Roddy:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Merica:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Barbier:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Dang:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Silverman:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership. Kung:Agios Pharmaceuticals, Inc.: Employment, Equity Ownership.

Interfacial SiO2 scavenging kinetics in HfO2 gate stack

In this paper, Si and oxygen diffusions as well as SiO2 reduction reaction in HfO2/SiO2/Si stacks are examined in detail for fully understanding the SiO2-IL scavenging process. A 29Si isotope tracing experiment confirms that Si in SiO2-IL is mostly emitted from the HfO2 surface. In addition, the reaction of oxygen from SiO2 with VO in HfO2 is suggested. Furthermore, it is reported that the Si substrate is not changed at all in the SiO2-IL scavenging by simultaneously comparing a Si surface between with and without scavenged parts. Based on these results, a kinetic model for the SiO2-IL scavenging in HfO2/SiO2/Si stacks is proposed.

Microbes make methane from coal

Microbial Physiology Methane associated with coal beds is an important global resource of natural gas. Much of the methane in coal comes from microbial methanogenesis. Mayumi et al. characterized a strain of Methermicoccus shengliensis that, unexpectedly, is capable of making methane from the dozens of methoxylated aromatic compounds found in a variety of coal types (see the Perspective by Welte). Isotope tracer experiments showed that this organism could also incorporate carbon dioxide into methane. Science , this issue p. [222][1]; see also p. [184][2] [1]: /lookup/doi/10.1126/science.aaf8821 [2]: /lookup/doi/10.1126/science.aai8101

Methane production from coal by a single methanogen.

Coal-bed methane is one of the largest unconventional natural gas resources. Although microbial activity may greatly contribute to coal-bed methane formation, it is unclear whether the complex aromatic organic compounds present in coal can be used for methanogenesis. We show that deep subsurface-derived Methermicoccus methanogens can produce methane from more than 30 types of methoxylated aromatic compounds (MACs) as well as from coals containing MACs. In contrast to known methanogenesis pathways involving one- and two-carbon compounds, this "methoxydotrophic" mode of methanogenesis couples O-demethylation, CO2 reduction, and possibly acetyl-coenzyme A metabolism. Because MACs derived from lignin may occur widely in subsurface sediments, methoxydotrophic methanogenesis would play an important role in the formation of natural gas not limited to coal-bed methane and in the global carbon cycle.

Accurate and efficient measurement of impurity (dilute) diffusion coefficients without isotope tracer experiments

Most impurity diffusion coefficients were measured in the 1960s and 1970s using tracer experiments with only 14 sets of tracer measurements performed worldwide in the past 15years. It is thus impractical to rely on tracer experiments to obtain the missing thousands of impurity diffusion coefficients. A recently developed forward-simulation analysis can change the scenario by reliably extracting impurity diffusion coefficients from regular diffusion couple profiles. A comparison of 51 values obtained from the forward-simulation analysis with tracer experiment values in the literature shows an excellent agreement, thus validating the reliability of the forward-simulation analysis in obtaining accurate impurity diffusion coefficients.

A Cu-Zn Intermetallic Catalyst Utilized for Electrochemical, Photoelectrochemical and Photocatalytic CO2 Reduction

Introduction To reduce the carbon dioxide concentration in the atmosphere and to solve the energy shortage issue, CO2 converting technologies, such as electrochemical, photoelectrochemical, and photocatalytic CO2 reduction, have been paid big attention in the last several decades. Researches in this field aim to convert CO2 into useful fuels such as formic acid, carbon monoxide, methane and methanol using electric or solar energy by means of CO2 reduction catalysis, in which the products can be directly used or easily stored. To achieve high conversion efficiency, the development of CO2reduction catalyst is essential. In the present research, a Cu-Zn intermetallic catalyst was developed on the basis of the understanding of the reaction mechanism of CO2 reduction on the metal surface in an aqueous media. It was reported that the adsorption or desorption strength of CO2 and CO anion radicals on the surface of the metal is the essential factor on the reaction pathway of CO2 reduction. 1,2 Among numerous candidates for catalyst materials, the Cu-Zn intermetallic catalyst is supposed to have a very appropriate adsorption-desorption strengths to achieve high efficiency and selectivity for converting CO2into formic acid. Besides, the Cu-Zn intermetallic catalyst is consisted of abundant and eco-friendly metals, also the synthesis method is very simple and low-cost. Experimental and Evaluation A Cu-Zn intermetallic electrode was prepared by sputtering and vacuum sealing methods. Its electrocatalytic properties were evaluated in an electrochemical cell and the composition was optimized according to the electrochemical performance. The optimized Cu-Zn electrode was then used to construct a photoelectrochemical (PEC) cell with a mesoporous SrTiO3photoanode prepared by screen printing method. According to the performance in the PEC cell, Cu-Zn intermetallic nanoparticles loaded SrTiO3powder photocatalyst was also synthesized by chemical reduction and vacuum sealing methods and its photocatalytic property was evaluated. Results and Discussion According to the electrochemical evaluation, the Cu-Zn intermetallic electrode behaved lower onset potential than pure copper and pure zinc. After the optimization of the Zn concentration, we measured the lowest onset potential (-0.65V vs. Ag/AgCl) when the Zn mass ratio was 53%. In the PEC cell, the optimized Cu-Zn electrode was used as the cathode in a CO2 purged KHCO3 electrolyte. When the SrTiO3 photoanode was irradiated by UV light, CO2 reduction was catalyzed on the surface of Cu-Zn electrode. Figure 1 shows the products detected at the rest potential, and HCOOH, CO, CH4 and H2 were produced. When we purged Ar gas in the electrolyte solution at cathodic side, the products amount was much lower than that of CO2 purged condition, indicating that most of the products were originated from catalytic CO2reduction. Further, products amounts from Cu-Zn intermetallic were two times higher than those from pure Cu. It is noteworthy that the faradic efficiency for HCOOH was as high as 79.72%. The turnover number (TON) of this electrode reached 1458.9, which proved that the Cu-Zn electrode performed high stability. In the photocatalytic evaluation, the Cu-Zn intermetallic nanoparticles loaded SrTiO3 powder was dispersed into CO2 purged KHCO3 solution and irradiated under UV light. Significant amount of HCOOH was also detected, while H2 production was almost negligible, indicating that the Cu-Zn also behaves high selectivity as co-catalyst in a CO2photocatalytic reduction system. The carbon source of the products was also confirmed by our isotope tracing experiment. Conclusion The Cu-Zn intermetallic electrode can catalyze CO2 reduction under low bias-potential in electrochemical and photoelectrochemical CO2 reduction systems with high conversion efficiency and selectivity for HCOOH. Our Cu-Zn electrode was very stable under bias application as well as photon irradiation conditions. The Cu-Zn intermetallic nanoparticles loaded STO powder can also convert CO2into HCOOH and other products under UV irradiation. Acknowledgement This work has been supported by a grant from Advanced Catalytic Transformation program for Carbon utilization (ACT-C), Japan Science and Technology Agency (JST). References Kuhl, Kendra P., et al. J. Am. Chem. Soc., 136,14107-14113 (2014)Hori, Y. Modern aspects of electrochemistry. Springer New York, 2008. 89-189. Figure 1

Activation of the Carbon Nitride Surface by Silica in a CO-Evolving Hybrid Photocatalyst.

Photocatalytic reduction of CO2 to CO proceeded by visible light (λ>400 nm) using mesoporous graphitic carbon nitride (C3 N4 ) coupled with a RuII -ReI binuclear complex (RuRe) containing a photosensitizer and catalytic units. The selectivity to CO exceeded 90 % during the initial stage. Photocatalytic reactions (including isotope tracer experiments) and electrochemical measurements revealed that the reaction proceeded according to a two-step photoexcitation of C3 N4 and the RuII photosensitizer unit, that is, it followed the Z-Scheme mechanism. Modification of C3 N4 with highly dispersed silica was found to improve the ability of C3 N4 to accommodate RuRe, which enhanced the photocatalytic activity for CO production.

Inaccurate quantitation of palmitate in metabolomics and isotope tracer studies due to plastics.

Palmitate, the typical end product released from fatty acid synthase, is of interest to many researchers performing metabolomics. Although palmitate can be readily detected by using mass spectrometry, many metabolomic platforms involve the use of plastic consumables that introduce a competing background signal of palmitate. The goal of this study was to quantify palmitate contamination in metabolomics and isotope tracer studies and to examine the reliability of approaches for reducing error. We measured the quantitative error introduced by palmitate contamination from 4 vendors of plastic consumables used in combination with several different extraction solvents. The background palmitate signal was as much as sixfold higher than the biological palmitate signal from 4 million 3T3-L1 cells. Importantly, the palmitate contamination signal was highly variable between plastic consumables (even within the same lot) and therefore could not be accurately removed by subtracting the background as measured from a blank. In addition to affecting relative and absolute quantitation, the palmitate background signal from disposable plastics also led to the underestimation of labeled palmitate in isotope tracer experiments. When measuring palmitate standard solutions, the best results were obtained when glass vials and glass pipettes were used. However, much of the palmitate background signal could be eliminated by pre-rinsing plastic vials and plastic pipette tips with methanol prior to sample introduction. For isotope tracer studies, error could also be minimized by estimating palmitate enrichment from palmitoylcarnitine, which does not have a competing contamination signal from plastic consumables.

Patterns of carbon processing at the seafloor: the role of faunal and microbial communities in moderating carbon flows

Abstract. Marine sediments, particularly those located in estuarine and coastal zones, are key locations for the burial of organic carbon (C). However, organic C delivered to the sediment is subjected to a range of biological C-cycling processes, the rates and relative importance of which vary markedly between sites, and which are thus difficult to predict. In this study, stable isotope tracer experiments were used to quantify the processing of C by microbial and faunal communities in two contrasting Scottish estuarine sites: a subtidal, organic C rich site in Loch Etive with cohesive fine-grained sediment, and an intertidal, organic C poor site on an Ythan estuary sand flat with coarse-grained permeable sediments. In both experiments, sediment cores were recovered and amended with 13C labelled phytodetritus to quantify whole community respiration of the added C and to trace the isotope label into faunal and bacterial biomass. Similar respiration rates were found in Loch Etive and on the Ythan sand flat (0.64 ± 0.04 and 0.63 ± 0.12 mg C m−2h−1, respectively), which we attribute to the experiments being conducted at the same temperature. Faunal uptake of added C over the whole experiment was markedly greater in Loch Etive (204 ± 72 mg C m−2) than on the Ythan sand flat (0.96 ± 0.3 mg C m−2), and this difference was driven by a difference in both faunal biomass and activity. Conversely, bacterial C uptake over the whole experiment in Loch Etive was much lower than that on the Ythan sand flat (1.80 ± 1.66 and 127 ± 89 mg C m−2, respectively). This was not driven by differences in biomass, indicating that the bacterial community in the permeable Ythan sediments was particularly active, being responsible for 48 ± 18 % of total biologically processed C. This type of biological C processing appears to be favoured in permeable sediments. The total amount of biologically processed C was greatest in Loch Etive, largely due to greater faunal C uptake, which was in turn a result of higher faunal biomass. When comparing results from this study with a wide range of previously published isotope tracing experiments, we found a strong correlation between total benthic biomass (fauna plus bacteria) and total biological C processing rates. Therefore, we suggest that the total C-cycling capacity of benthic environments is primarily determined by total biomass.

Density of Key-Species Determines Efficiency of Macroalgae Detritus Uptake by Intertidal Benthic Communities.

Accumulating evidence shows that increased biodiversity has a positive effect on ecosystem functioning, but the mechanisms that underpin this positive relationship are contentious. Complete extinctions of regional species pools are comparatively rare whereas compositional changes and reductions in abundance and biomass are common, although seldom the focus of biodiversity-ecosystem functioning studies. We use natural, small-scale patchiness in the density of two species of large bivalves with contrasting feeding modes (the suspension-feeding Austrovenus stutchburyi and deposit-feeding Macomona liliana) to examine their influence on the uptake of nitrogen from macroalgae detritus (i.e. measure of ecosystem function and food web efficiency) by other infauna in a 10-d laboratory isotope-tracer experiment. We predicted that densities of these key bivalve species and functional group diversity (calculated as Shannons H, a density-independent measure of community composition) of the intact infaunal community will be critical factors explaining variance in macroalgal per capita uptake rates by the community members and hence determine total uptake by the community. Results show that only two species, M. liliana and a large orbiniid polychaete (Scoloplos cylindrifer) dominated macroalgal nitrogen taken up by the whole community due to their large biomass. However, their densities were mostly not important or negatively influenced per capita uptake by other species. Instead, the density of a head-down deposit-feeder (the capitellid Heteromastus filiformis), scavengers (mainly nemertines and nereids) and species and functional group diversity, best explained per capita uptake rates in community members. Our results demonstrate the importance of species identity, density and large body size for ecosystem functioning and highlight the complex interactions underlying loss of ecological functions with declining biodiversity and compositional changes.

An Alkaline-Stable, Metal Hydroxide Mimicking Metal–Organic Framework for Efficient Electrocatalytic Oxygen Evolution

Postsynthetic ion exchange of [Co2(μ-Cl)2(btta)] (MAF-X27-Cl, H2bbta =1H,5H-benzo(1,2-d:4,5-d')bistriazole) possessing open metal sites on its pore surface yields a material [Co2(μ-OH)2(bbta)] (MAF-X27-OH) functionalized by both open metal sites and hydroxide ligands, giving drastically improved electrocatalytic activities for the oxygen evolution reaction (an overpotential of 292 mV at 10.0 mA cm(-2) in 1.0 M KOH solution). Isotope tracing experiments further confirm that the hydroxide ligands are involved in the OER process to provide a low-energy intraframework coupling pathway.

Effect of Precursor Selection on the Photocatalytic Performance of Indium Oxide Nanomaterials for Gas-Phase CO2 Reduction

Nonstoichiometric indium oxide nanoparticles, In2O3–x(OH)y, have been shown to function as active photocatalysts for gas-phase CO2 reduction under simulated solar irradiation. Herein we demonstrate that the choice of starting material has a strong effect on the photocatalytic activity of indium oxide nanoparticles. We examine three indium oxide materials prepared via the thermal decomposition of either indium(III) hydroxide or indium(III) nitrate and correlate their stability and photocatalytic activity to the number and type of defect present in the material. Further, we use 13CO2 isotope-tracing experiments to clearly identify the origins of the observed carbon-containing products. Significantly, we find that the oxidizing nature of the precursor anion has a substantial impact on the defect formation within the sample. This study demonstrates the importance of surface defects in designing an active heterogeneous photocatalyst and provides valuable insight into key parameters for the precursor design, se...

Community composition and activity of anaerobic ammonium oxidation bacteria in the rhizosphere of salt-marsh grass Spartina alterniflora.

Anaerobic ammonium oxidation (anammox) as an important nitrogen removal pathway has been investigated in intertidal marshes. However, the rhizosphere-driven anammox process in these ecosystems is largely overlooked so far. In this study, the community dynamics and activities of anammox bacteria in the rhizosphere and non-rhizosphere sediments of salt-marsh grass Spartina alterniflora (a widely distributed plant in estuaries and intertidal ecosystems) were investigated using clone library analysis, quantitative PCR assay, and isotope-tracing technique. Phylogenetic analysis showed that anammox bacterial diversity was higher in the non-rhizosphere sediments (Scalindua and Kuenenia) compared with the rhizosphere zone (only Scalindua genus). Higher abundance of anammox bacteria was detected in the rhizosphere (6.46×10(6)-1.56×10(7)copiesg(-1)), which was about 1.5-fold higher in comparison with that in the non-rhizosphere zone (4.22×10(6)-1.12×10(7)copiesg(-1)). Nitrogen isotope-tracing experiments indicated that the anammox process in the rhizosphere contributed to 12-14% N2 generation with rates of 0.43-1.58nmolNg(-1)h(-1), while anammox activity in the non-rhizosphere zone contributed to only 4-7% N2 production with significantly lower activities (0.28-0.83nmolNg(-1)h(-1)). Overall, we propose that the rhizosphere microenvironment in intertidal marshes might provide a favorable niche for anammox bacteria and thus plays an important role in nitrogen cycling.

Cholesterol homeostasis alters one carbon metabolism in vitro and in vivo

Cholesterol essential for the synthesis of steroid hormones, bile acids, and lipoproteins. Hyperhomocysteinemia and hypercholesterolemia are both risk factors for human cardiovascular diseases. Previous studies reported that cholesterol biosynthesis can be regulated by homocysteine‐induced endoplasmic reticulum stress in primary human umbilical vein endothelial cells (HUVEC), hepatocellular carcinoma cell line (HepG2), and in murine models. However, the physiological impact of excessive cholesterol exposure on one‐carbon metabolism is unknown. We hypothesize that cholesterol may alter methionine adenosyltransferase 1A (MAT1a) expression via sterol regulatory element‐binding proteins (SREBPs) that have been shown to target the promoter of MAT1a gene. Physiological doses of cholesterol or mevastatin, an inhibitor of cholesterol synthesis, were used to alter cellular cholesterol contents in liver derived cell lines L02. Mice fed high cholesterol diets or cholesterol‐lowering drugs were used as in vivo models. We discovered that ratios of nuclear/ total SREBPs tended were altered by cholesterol and mevestatin treatments in the cell models. Furthermore, reporter assay demonstrated that cholesterol treatment but not mevastatin significantly increased MAT1a promoter activity, supporting our hypothesis. Preliminary data from stable isotopic tracer experiments also showed consistent results that cholesterol alters MAT expression and S‐adenosyl‐ methionine synthesis in vitro. We demonstrated novel findings on how cholesterol homeostasis interacts with methyl group kinetics in mammalian cells. The in vivo kinetics studies on how cholesterol affect one carbon metabolism are underway.Support or Funding InformationMOST 104‐2320‐B005‐010‐MY3; MOST104‐2911‐I005‐301; Ministry of Education, Taiwan, R.O.C. under the ATU plan

Phospholipid transfer protein deficiency in mice impairs macrophage reverse cholesterol transport invivo.

Phospholipid transfer protein is expressed in various cell types and secreted into plasma, where it transfers phospholipids between lipoproteins and modulates the composition of high-density lipoprotein particles. Phospholipid transfer protein deficiency invivo can lower high-density lipoprotein cholesterol level significantly and impact the biological quality of high-density lipoprotein. Considering high-density lipoprotein was a critical determinant for reverse cholesterol transport, we investigated the role of systemic phospholipid transfer protein deficiency in macrophage reverse cholesterol transport invivo After the littermate phospholipid transfer protein KO and WT mice were fed high-fat diet for one month, they were injected intraperitoneally with (3)H-cholesterol-labeled and acLDL-loaded macrophages. Then the appearance of (3)H-tracer in plasma, liver, bile, intestinal wall, and feces over 48 h was determined. Plasma lipid analysis indicated phospholipid transfer protein deficiency lowered total cholesterol, high-density lipoprotein-C and apolipoprotein A1 levels significantly but increased triglyceride level in mice. The isotope tracing experiment showed (3)H-cholesterol of plasma was decreased by 68% for male and 62% for female, and (3)H-tracer of bile was decreased by 37% for male and 21% for female in phospholipid transfer protein KO mice compared with WT mice. However, there was no difference in liver, and (3)H-tracer of intestinal wall was increased by 43% for male and 27% for female. Finally, (3)H-tracer of fecal excretion in phospholipid transfer protein KO mice was reduced significantly by 36% for male and 43% for female during 0-24 h period, but there was no significant difference during 24-48 h period. Meanwhile, Western Blot analysis showed the expressions of reverse cholesterol transport -related protein liver X receptor α (LXRα), ATP binding cassette transporter A1, and cholesterol 7α-hydroxylase A1 were upregulated in liver of phospholipid transfer protein KO mice compared with WT mice. These data reveal that systemic phospholipid transfer protein deficiency in mice impairs macrophage-specific reverse cholesterol transport invivo.

Improved PCR primers to amplify 16S rRNA genes from NC10 bacteria.

Anaerobic oxidation of methane (AOM) coupled to nitrite reduction (AOM-NIR) is ecologically significant for mitigating the methane-induced greenhouse effect. The microbes responsible for this reaction, NC10 bacteria, have been widely detected in diverse ecosystems. However, some defects were discovered in the commonly used NC10-specific primers, 202F and qP1F. In the present work, the primers were redesigned and improved to overcome the defects found in the previous primers. A new nested PCR method was developed using the improved primers to amplify 16S ribosomal RNA (rRNA) genes from NC10 bacteria. In the new nested PCR method, the qP1mF/1492R and 1051F/qP2R primer sets were used in the first and second rounds, respectively. The PCR products were sequenced, and more operational taxonomic units (OTUs) of the NC10 phylum were obtained using the new primers compared to the previous primers. The sensitivity of the new nested PCR was tested by the serial dilution method, and the limit of detection was approximately 10(3)copiesg(-1) dry sed. for the environmental samples compared to approximately 10(5)copiesg(-1) dry sed. by the previous method. Finally, the improved primer, qP1mF, was used in quantitative PCR (qPCR) to determine the abundance of NC10 bacteria, and the results agreed well with the activity of AOM-NIR measured by isotope tracer experiments. The improved primers are able to amplify NC10 16S rRNA genes more efficiently than the previous primers and useful to explore the microbial community of the NC10 phylum in different systems.

Celastrus Orbiculatus Thunb. Reduces Lipid Accumulation by Promoting Reverse Cholesterol Transport in Hyperlipidemic Mice.

Previously, we found that Celastrus orbiculatus Thunb. (COT) decreases athero-susceptibility in lipoproteins and the aorta of guinea pigs fed a high-fat diet, and increases high-density lipoprotein (HDL). In the present study, we investigated the effect of COT in reducing lipid accumulation and promoting reverse cholesterol transport (RCT) in vivo and vitro. Healthy male mice were treated with high-fat diet alone, high-fat diet with COT (10.0g/kg/d), or general fodder for 6weeks. Serum levels of total cholesterol (TC), triglyceride (TG), HDL-C, non-HDL-C, and (3)H-cholesterol in plasma, liver, bile, and feces were determined. Pathological changes and the levels of TC and TG in liver were examined. The expression of hepatic genes and protein associated with RCT were analyzed. COT administration reduced lipid accumulation in the liver, ameliorated the pathological changes, and lessened liver injury, the levels of TG, TC, and non-HDL-C in plasma were decreased significantly, and COT led to a significant increase in plasma HDL-C and apolipoproteinA (apoA1). (3)H-cholesterol in plasma, liver, bile, and feces was also significantly increased in COT-treated mice compared to controls. Both mRNA and protein expression of SRB1, CYP7A1, LDLR, ATP-binding cassette transporters ABCA1, ABCG5, and LXRα were improved in COT-treated mice. An in vitro isotope tracing experiment showed that COT and its bioactive ingredients, such as celastrol, ursolic acid, oleanolic acid, and quercetin, significantly increased the efflux of (3)H-cholesterol. They also increased the expression of SRB1, ABCA1, and ABCG1 significantly in macrophages. Our findings provided a positive role of COT in reducing lipid accumulation by promoting RCT. These effects may be achieved by activating the SRB1 and ABC transporter pathway and promoting cholesterol metabolism via the CYP7A1 pathway in vivo. The effective ingredients in vitro are celastrol, ursolic acid, oleanolic acid, and quercetin.

Nitrogen removal by Providencia rettgeri strain YL with heterotrophic nitrification and aerobic denitrification

ABSTRACTProvidencia rettgeri strain YL shows the capability of nitrogen removal under sole aerobic conditions. By using isotope ratio mass spectrometry, 15N-labelled N2O and N2 were detected in aerobic batch cultures containing , or . Strain YL converted , and to produce more N2O than N2 in the presence of . An 15N isotope tracing experiment confirmed that the nitrogen removal pathway of strain YL was heterotrophic nitrification-aerobic denitrification. The optimal treatment conditions for nitrogen removal were pH of 8, C/N ratio of 12, temperature of 25°C and shaking speed of 105 rpm. A continuous aerobic bioreactor inoculated with strain YL was developed. With an influent concentration of 90–200 mg/L, the removal efficiency ranged from 80% to 97% and the total nitrogen removal efficiency ranged from 72% to 95%. The nitrogen balance in the continuous bioreactor revealed that approximately 35–52% of influent was denitrified aerobically to form gaseous nitrogen. These findings show that the P. rettgeri strain YL has potential application in wastewater treatment for nitrogen removal under sole aerobic conditions.