Labelling Of Fatty Acids Research Articles

Lipid availability influences ferroptosis sensitivity in cancer cells by regulating polyunsaturated fatty acid trafficking.

Ferroptosis is a form of cell death caused by lipid peroxidation that is emerging as a target for cancer therapy, highlighting the need to identify factors that govern ferroptosis susceptibility. Lipid peroxidation occurs primarily on phospholipids containing polyunsaturated fatty acids (PUFAs). Here, we show that even though extracellular lipid limitation reduces cellular PUFA levels, lipid-starved cancer cells are paradoxically more sensitive to ferroptosis. Using mass spectrometry-based lipidomics with stable isotope fatty acid labeling, we show that lipid limitation induces a fatty acid trafficking pathway in which PUFAs are liberated from triglycerides to synthesize highly unsaturated PUFAs such as arachidonic and adrenic acid. These PUFAs then accumulate in phospholipids, including ether phospholipids, to promote ferroptosis sensitivity. Therefore, PUFA levels within cancer cells do not necessarily correlate with ferroptosis susceptibility. Rather, how cancer cells respond to extracellular lipid levels by trafficking PUFAs into proper phospholipid pools contributes to their sensitivity to ferroptosis.

Drought resistance and resilience of rhizosphere communities in forest soils from the cellular to ecosystem scale - insights from 13C pulse labeling.

The link between above- and belowground communities is a key uncertainty in drought and rewetting effects on forest carbon (C) cycle. In young beech model ecosystems and mature naturally dry pine forest exposed to 15-yr-long irrigation, we performed 13C pulse labeling experiments, one during drought and one 2 wk after rewetting, tracing tree assimilates into rhizosphere communities. The 13C pulses applied in tree crowns reached soil microbial communities of the young and mature forests one and 4 d later, respectively. Drought decreased the transfer of labeled assimilates relative to the irrigation treatment. The 13C label in phospholipid fatty acids (PLFAs) indicated greater drought reduction of assimilate incorporation by fungi (-85%) than by gram-positive (-43%) and gram-negative bacteria (-58%). 13C label incorporation was more strongly reduced for PLFAs (cell membrane) than for microbial cytoplasm extracted by chloroform. This suggests that fresh rhizodeposits are predominantly used for osmoregulation or storage under drought, at the expense of new cell formation. Two weeks after rewetting, 13C enrichment in PLFAs was greater in previously dry than in continuously moist soils. Drought and rewetting effects were greater in beech systems than in pine forest. Belowground C allocation and rhizosphere communities are highly resilient to drought.

Carnitine octanoyltransferase is important for the assimilation of exogenous acetyl-L-carnitine into acetyl-CoA in mammalian cells

In eukaryotes, carnitine is best known for its ability to shuttle esterified fatty acids across mitochondrial membranes for β-oxidation. It also returns to the cytoplasm, in the form of acetyl-L-carnitine (LAC), some of the resulting acetyl groups for posttranslational protein modification and lipid biosynthesis. While dietary LAC supplementation has been clinically investigated, its effects on cellular metabolism are not well understood. To explain how exogenous LAC influences mammalian cell metabolism, we synthesized isotope-labeled forms of LAC and its analogs. In cultures of glucose-limited U87MG glioma cells, exogenous LAC contributed more robustly to intracellular acetyl-CoA pools than did β-hydroxybutyrate, the predominant circulating ketone body in mammals. The fact that most LAC-derived acetyl-CoA is cytosolic is evident from strong labeling of fatty acids in U87MGcells by exogenous 13C2-acetyl-L-carnitine. We found that the addition of d3-acetyl-L-carnitine increases the supply of acetyl-CoA for cytosolic posttranslational modifications due to its strong kinetic isotope effect on acetyl-CoA carboxylase, the first committed step in fatty acid biosynthesis. Surprisingly, whereas cytosolic carnitine acetyltransferase is believed to catalyze acetyl group transfer from LAC to coenzyme A, CRAT-/- U87MGcells were unimpaired in their ability to assimilate exogenous LAC into acetyl-CoA. We identified carnitine octanoyltransferase as the key enzyme in this process, implicating a role for peroxisomes in efficient LAC utilization. Our work has opened the door to further biochemical investigations of a new pathway for supplying acetyl-CoA to certain glucose-starved cells.

Microscopy-Based Assessment of Fatty Acid Uptake and Lipid Accumulation in Cultured Cells.

The heart relies predominantly on the use of fatty acids to derive energy. Metabolic disorders such as obesity, insulin resistance, and diabetes pose a major risk factor for the development of heart failure. Dysregulation of lipid metabolism observed in these diseases manifests as cardiac lipotoxicity, and is associated with cardiac dysfunction. The alarming rise in the incidence of these metabolic disorders warrants the need for tools to investigate the underlying molecular mechanisms. In this article, we describe a confocal microscopy-based approach to monitor fatty acid uptake and lipid accumulation in vitro, in neonatal murine cardiomyocytes and H9c2 cells. The protocol for assessment of fatty acid uptake relies on the use of BODIPY FL C 12™ to study the kinetics of fatty acid uptake via real-time imaging of fatty acid uptake in live cells. Importantly, it circumvents the need for radioactive labeling of fatty acids to evaluate their uptake. Similarly, the protocol for assessment of lipid accumulation relies on the use of BODIPY™ 493/503 to stain the cytosolic neutral lipid population in fixed cells. We couple these confocal microscopy-based approaches with fluorescence intensity analysis using FIJI to quantify fatty acid uptake and lipid accumulation in vitro. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Assessment of fatty acid uptake Basic Protocol 2: Assessment of lipid accumulation.

Inhibition of Stearoyl-CoA Desaturase Has Anti-Leukemic Properties in Acute Myeloid Leukemia

Metabolic rewiring, a hallmark of acute myeloid leukemia (AML), contributes to the development of resistance to intensive chemotherapy and has been shown to play an essential role in leukemic cell maintenance. Several metabolic alterations have been described in AML, such as dependence on amino acids to fuel oxidative phosphorylation or ability of leukemic cells to switch energy dependence to fatty acid oxidation in response to treatment. However, little is known about the functional significance of the fatty acid synthesis (FAS) pathway in AML. Here we have identified stearoyl-CoA desaturase (SCD), a key enzyme responsible for the conversion of saturated fatty acids (SFA) into monounsaturated fatty acids (MUFA), as a novel therapeutic target in AML. High SCD expression predicts worse survival across several patient datasets, even after correction for gender, age and AML European LeukemiaNet risk category. Moreover, genetic and chemical inhibition of SCD reduces growth and induces cell death in AML cells. Since targeting SCD with currently available inhibitors has been challenging because of their toxicity profile in vivo, we have accessed a novel pharmacological SCD inhibitor with a more favorable toxicity profile, SSI-4 (Modulation Therapeutics Inc., Morgantown, USA). SSI-4 reduces viability in several human and mouse AML models in vitro and in about half of the 36 primary AML samples tested ex vivo. Moreover, SSI-4 displays anti-leukemic effects in AML murine models and patient-derived xenografts (PDX). Since we could not correlate susceptibility to SCD inhibition with any genetic characteristic of AML cells, we aimed to determine the molecular basis of cell sensitivity using phosphoproteomic, proteomic and transcriptomic approaches coupled with metabolic analyses of sensitive and resistant cell lines and primary AML cells. Sensitivity to SCD inhibition directly correlates with rate of de novo FAS, as measured by fatty acid labeling with 13C-glucose. Moreover, modulation of FAS by the AMPK signaling activity dictates sensitivity to SCD inhibition and AMPK activation rescues the anti-leukemic effects of SSI-4. The anti-leukemic effects of SCD inhibition are secondary to the induction of SFA mediated lipotoxicity, as demonstrated by the increased sensitivity of previously resistant cells to SSI-4, when grown in the presence of the SFA palmitate. Conversely, growing cells in the presence of the MUFA oleate, as well as inhibition of FAS by targeting enzymes upstream of SCD, results in resistance to SSI-4, thus confirming that the increased SFA/MUFA ratio drives leukemic cell death. SFA mediated lipotoxicity leads to both increased lipid peroxidation and activation of the unfolded protein response (UPR), namely its PERK branch, which in turn leads to apoptotic cell death. Indeed, inhibition of PERK, but not of other branches of UPR, rescues SCD-mediated cytotoxicity. Despite causing disease debulking in vivo, SSI-4 does not prolong survival in murine AML models as a single agent and the toxic effects of SSI-4 are decreased when AML cells are grown in co-culture with stroma, suggesting a protective role of tumor microenvironment. However, we consistently detect increased lipid peroxidation and DNA damage both in vitro and in vivo following SCD inhibition. We therefore combined SSI-4 with doxorubicin, a known inducer of lipid peroxidation and DNA damage, which is routinely used in AML induction therapy. Indeed, both genetic depletion and pharmacological inhibition of SCD renders AML cells more sensitive to doxorubicin, and addition of SSI-4 to a standard AML chemotherapy protocol consisting of doxorubicin and cytarabine significantly extends survival in AML mouse models. In summary, we identify and validate SCD as a therapeutic target in AML. The novel SCD inhibitor SSI-4 displays anti-leukemic properties both in vitro and in vivo across different AML models and particularly when combined with standard chemotherapy while showing minimal toxicity in vivo. We show that sensitivity to SCD inhibition is driven by metabolic rather than genetic AML features, thus making this therapeutic approach potentially applicable across several AML subtypes, including high risk ones. Finally, the mechanism of toxicity following SCD inhibition suggests that this can be further enhanced by modulating the levels of SFA/MUFA through dietary interventions or by combining it with DNA damaging agents. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Use of Fatty Acids From Aquatic Prey Varies With Foraging Strategy

Across ecosystems, resources vary in their nutritional composition and thus their dietary value to consumers. Animals can either access organic compounds, such as fatty acids, directly from diet or through internal biosynthesis, and the extent to which they use these two alternatives likely varies based on the availability of such compounds across the nutritional landscape. Cross-ecosystem subsidies of important dietary nutrients, like omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA), may provide consumers with the opportunity to relax the demands of synthesis and rely upon dietary flexibility rather than internal metabolic processes. Here, we examined how dietary flexibility and distance from a lake influenced the degree to which generalist insectivores relied upon dietary n-3 LC-PUFA from emergent aquatic insects versus n-3 LC-PUFA synthesized from precursor compounds found in terrestrial insects. We used bulk and compound-specific stable isotope analyses to understand spider and insectivorous bird (Blue Tit;Cyanistes caeruleus) reliance on aquatic and terrestrial resources, including dietary PUFA sources, along a riparian to upland gradient from a lake. We simultaneously investigated n-3 LC-PUFA synthesis ability in nestlings using13C fatty acid labeling. We found that riparian spiders took advantage of emergent aquatic insect subsidies, deriving their overall diet and their n-3 PUFA from aquatic resources whereas nestling birds at all distances and upland spiders relied upon terrestrial resources, including PUFA. Our13C labeling experiment demonstrated that nestling tits were able to synthesize the n-3 LC-PUFA docosahexaenoic acid from the dietary precursor α-linolenic acid, suggesting that they are not limited by aquatic resources to satisfy their LC-PUFA requirements. Overall, this study suggests that habitat generalist insectivores vary in the degree to which they can shift diet to take advantage of high-quality aquatic resources depending upon both their foraging flexibility and internal synthesis capacity.

Cost-efficient and user-friendly 17O/18O labeling procedures of fatty acids using mechanochemistry.

Two mechanochemical procedures for 17O/18O-isotope labeling of fatty acids are reported: a carboxylic acid activation/hydrolysis approach and a saponification approach. The latter route allowed first-time enrichment of important polyunsaturated fatty acids (PUFAs) including docosahexaenoic acid (DHA). Overall, a total of 9 pure labeled products were isolated in high yields (≥80%) and with high enrichment levels (≥37% average labeling of C=O and C-OH carboxylic oxygen atoms), under mild conditions, and in short time (<half day).

Abstract LB-295: Discovery of potent selective oral ACSS2 inhibitors for the treatment of acetate avid tumors

Abstract Under the low oxygen and lipid-depleted conditions, often encountered in the tumor microenvironment, acetate is used as nutrient by cancer cells for the biosynthesis of fatty acids. ACSS2, the enzyme responsible for acetate capture within the cell and its conversion into Acetyl-CoA, is upregulated under metabolically stressed conditions and its silencing is shown to inhibit tumor growth in xenografts. Two independent HTS campaigns identified the same small molecule ACSS2 inhibitor series that exhibited uncompetitive binding behavior in respect to ATP. A co-crystal structure of human ACSS2 in complex with a congener of the series revealed that it binds in the acetate pocket and forms favorable interactions with ATP, which is bound adjacently. These compounds were over 1000-fold times selective for ACSS2 over the mitochondrial ACSS1 and other Acyl-CoA synthetases. Medicinal chemistry optimization of this series resulted in advanced compounds with sub-nanomolar biochemical potency and single digit nanomolar cellular EC50s in inhibiting de novo fatty acid synthesis from labeled acetate and excellent pharmacokinetic properties. In vivo, in mice, these compounds inhibited labeling of fatty acids in various tissues after administration of labelled acetate or ethanol. They exhibited in vivo anti-tumor efficacy after oral administration in xenograft models that are acetate avid, whereas had no effect in tumors that show low acetate uptake. MTB-9655, one of the compounds from this series with optimally balanced properties, is currently entering IND-enabling studies to allow its testing in cancer patients. Citation Format: Andreas Goutopoulos, Omri Erez, Philippe Nakache, Iris Alchanati, Avital Hay-Koren, Dikla Paz, Dimitri Kovalerchik, Hannah Oppenheimer, Eyal Gottlieb, Simone Botti, Inbal Mor, Nina Pastukh. Discovery of potent selective oral ACSS2 inhibitors for the treatment of acetate avid tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-295.

Carbon processing by the benthic ecosystem and benthic C fixation in methane-rich sediments on the South Georgia margin

AbstractAs bottom water warms, destabilisation of gas hydrates may increase the extent of methane-rich sediments. The authors present an assessment of organic carbon processing by the benthic community in methane-rich sediments, including one of the first investigations of inorganic C fixation in a non-hydrothermal vent setting. This topic was previously poorly studied, and there is much need to fill the gaps in knowledge of such ecosystems. The authors hypothesized that benthic C fixation would occur, and that a high biomass macrofaunal community would play a substantial role in organic C cycling. Experiments were conducted at a 257 m deep site off South Georgia. Sediment cores were amended with13C and15N labelled algal detritus, or13C labelled bicarbonate solution. In the bicarbonate experiment, labelling of bacteria-specific phospholipid fatty acids provided direct evidence of benthic C fixation, with transfer of fixed C to macrofauna and dissolved organic carbon (DOC). In the algae experiment, macrofauna played an active role in organic carbon cycling. Compared to similar experiments, low temperature supressed the rates of community respiration and macrofaunal C uptake. While benthic C fixation occurred, the biological processing of organic carbon was dominantly controlled by low temperature and high photic zone productivity.

Mild and Direct Multiple Deuterium‐Labeling of Saturated Fatty Acids

AbstractWe have established a mild and direct platinum on carbon (Pt/C)‐catalyzed multi‐deuterium labeling of various saturated fatty acids including bioactive compounds with high deuterium efficiencies in a mixed solvent of isopropyl alcohol and deuterium oxide (i‐PrOH)/D2O under neutral conditions at 120 °C without the external addition of deuterium or hydrogen gas.magnified image

Activation of Exogenous Fatty Acids to Acyl-Acyl Carrier Protein Cannot Bypass FabI Inhibition in Neisseria

Neisseria is a Gram-negative pathogen with phospholipids composed of straight chain saturated and monounsaturated fatty acids, the ability to incorporate exogenous fatty acids, and lipopolysaccharides that are not essential. The FabI inhibitor, AFN-1252, was deployed as a chemical biology tool to determine whether Neisseria can bypass the inhibition of fatty acid synthesis by incorporating exogenous fatty acids. Neisseria encodes a functional FabI that was potently inhibited by AFN-1252. AFN-1252 caused a dose-dependent inhibition of fatty acid synthesis in growing Neisseria, a delayed inhibition of growth phenotype, and minimal inhibition of DNA, RNA, and protein synthesis, showing that its mode of action is through inhibiting fatty acid synthesis. Isotopic fatty acid labeling experiments showed that Neisseria encodes the ability to incorporate exogenous fatty acids into its phospholipids by an acyl-acyl carrier protein-dependent pathway. However, AFN-1252 remained an effective antibacterial when Neisseria were supplemented with exogenous fatty acids. These results demonstrate that extracellular fatty acids are activated by an acyl-acyl carrier protein synthetase (AasN) and validate type II fatty acid synthesis (FabI) as a therapeutic target against Neisseria.

Potential of trans fats policies to reduce socioeconomic inequalities in mortality from coronary heart disease in England: cost effectiveness modelling study

Objectives To determine health and equity benefits and cost effectiveness of policies to reduce or eliminate trans fatty acids from processed foods, compared with consumption remaining at most recent levels...

Microbial ecology associated to soil-plant interactions: exemplifying recent progress in East Asia

s for potential papers were invited from the symposium presentations and posters, from which 19 full papers were invited. The papers subsequently accepted for publication include a review and three research articles. The review paper focused on the application of stable isotope labelling of phospholipid fatty acids (SIP-PLFA) to trace C fluxes in soil-plant ecosystems (Yao et al. 2015). It concluded that the SIP-PLFA method provides an excellent tool to study the incorporation of rhizodeposited C into soil microbial biomass and understand the plant-microbe-soil interactions in the rhizosphere. The three research articles used labeling techniques to assess the flow of C photosynthetates in paddy soils and focused on molecular biological techniques to determine the microbial community Plant Soil (2015) 392:1–2 DOI 10.1007/s11104-015-2521-6 Responsible Editor: Paul Bodelier. H. Yao (*) :Y. Zhu Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China e-mail: hyyao@iue.ac.cn Y. Zhu e-mail: ygzhu@iue.ac.cn H. Yao :Y. Zhu Ningbo Urban Environment Observation and Research Station, Chinese Academy of Sciences, Ningbo 315830, China changes in response to biological invasion, elevated CO2 and temperature. Ge et al. (2015) demonstrated that nitrogen fertilization can promote the incorporation of photosynthesis derived C into soil organic C pools. Overall, the amount of CO2 that was converted into SOC was proportional to the soil N concentration, indicating that N fertilization promoted rhizodeposition. However, the proportion of C in the microbial biomass C was highest at the lower fertilization rate. Higher microbial activity was found in areas invaded by Phragmites australis compared to areas dominated by native species, and plant diversity was found to be the most important regulator of microbial community structure and diversity in these areas (Song et al. 2015). Ren et al. (2015) found distinct response patterns of soil and leaf-associated bacterial communities to elevated CO2 and temperature. In this study it was observed that the dominance of Enterobacteriaceae in leaf tissue may be of great importance in rice growth. These papers highlight the current focus of understanding the interactions between crop plants and microbial communities with respect to nutrient cycling. Future research is likely to increasingly focus on problems affecting food security. Over fertilization, pesticide and industrial pollution are major concerns in many parts of East Asia, in addition to understanding the long-term consequences of climate change. We would like to thank all the authors who attended the symposium, and contributed abstracts and full manuscripts for their active responses, encouragement and cooperation. Thanks are also due to all the reviewers and to Professor Hans Lambers, the Editor-in-chief and his editorial team at Plant and Soil for handling these papers.

Complexity of fatty acid distribution inside human macrophages on single cell level using Raman micro-spectroscopy.

Macrophages are phagocytic cells which are involved in the non-specific immune defense. Lipid uptake and storage behavior of macrophages also play a key role in the development of atherosclerotic lesions within walls of blood vessels. The allocation of exogenous lipids such as fatty acids in the blood stream dictates the accumulation and quantity of lipids within macrophages. In case of an overexposure, macrophages transform into foam cells because of the large amount of lipid droplets in the cytoplasm. Raman micro-spectroscopy is a powerful tool for studying single cells due to the combination of microscopic imaging with spectral information. With a spatial resolution restricted by the diffraction limit, it is possible to visualize lipid droplets within macrophages. With stable isotopic labeling of fatty acids with deuterium, the uptake and storage of exogenously provided fatty acids can be investigated. In this study, we present the results of time-dependent Raman spectroscopic imaging of single THP-1 macrophages incubated with deuterated arachidonic acid. The polyunsaturated fatty acid plays an important role in the cellular signaling pathway as being the precursor of icosanoids. We show that arachidonic acid is stored in lipid droplets but foam cell formation is less pronounced as with other fatty acids. The storage efficiency in lipid droplets is lower than in cells incubated with deuterated palmitic acid. We validate our results with gas chromatography and gain information on the relative content of arachidonic acid and its metabolites in treated macrophages. These analyses also provide evidence that significant amounts of the intracellular arachidonic acid is elongated to adrenic acid but is not metabolized any further. The co-supplementation of deuterated arachidonic acid and deuterated palmitic acid leads to a non-homogenous storage pattern in lipid droplets within single cells.

Soil warming alters microbial substrate use in alpine soils

Will warming lead to an increased use of older soil organic carbon (SOC) by microbial communities, thereby inducing C losses from C-rich alpine soils? We studied soil microbial community composition, activity, and substrate use after 3 and 4 years of soil warming (+4 °C, 2007-2010) at the alpine treeline in Switzerland. The warming experiment was nested in a free air CO2 enrichment experiment using depleted (13)CO2 (δ(13)C = -30‰, 2001-2009). We traced this depleted (13)C label in phospholipid fatty acids (PLFA) of the organic layer (0-5 cm soil depth) and in C mineralized from root-free soils to distinguish substrate ages used by soil microorganisms: fixed before 2001 ('old'), from 2001 to 2009 ('new') or in 2010 ('recent'). Warming induced a sustained stimulation of soil respiration (+38%) without decline in mineralizable SOC. PLFA concentrations did not reveal changes in microbial community composition due to soil warming, but soil microbial metabolic activity was stimulated (+66%). Warming decreased the amount of new and recent C in the fungal biomarker 18:2ω6,9 and the amount of new C mineralized from root-free soils, implying a shift in microbial substrate use toward a greater use of old SOC. This shift in substrate use could indicate an imbalance between C inputs and outputs, which could eventually decrease SOC storage in this alpine ecosystem.

Fatty Acid Labeling from Glutamine in Hypoxia Can Be Explained by Isotope Exchange without Net Reductive Isocitrate Dehydrogenase (IDH) Flux

Acetyl-CoA is an important anabolic precursor for lipid biosynthesis. In the conventional view of mammalian metabolism, acetyl-CoA is primarily derived by the oxidation of glucose-derived pyruvate in mitochondria. Recent studies have employed isotope tracers to show that in cancer cells grown in hypoxia or with defective mitochondria, a major fraction of acetyl-CoA is produced via another route, reductive carboxylation of glutamine-derived α-ketoglutarate (catalyzed by reverse flux through isocitrate dehydrogenase, IDH). Here, we employ a quantitative flux model to show that in hypoxia and in cells with defective mitochondria, oxidative IDH flux persists and may exceed the reductive flux. Therefore, IDH flux may not be a net contributor to acetyl-CoA production, although we cannot rule out net reductive IDH flux in some compartments. Instead of producing large amounts of net acetyl-CoA reductively, the cells adapt by reducing their demand for acetyl-CoA by importing rather than synthesizing fatty acids. Thus, fatty acid labeling from glutamine in hypoxia can be explained by spreading of label without net reductive IDH flux.

Cancer cells incorporate and remodel exogenous palmitate into structural and oncogenic signaling lipids

De novo lipogenesis is considered the primary source of fatty acids for lipid synthesis in cancer cells, even in the presence of exogenous fatty acids. Here, we have used an isotopic fatty acid labeling strategy coupled with metabolomic profiling platforms to comprehensively map palmitic acid incorporation into complex lipids in cancer cells. We show that cancer cells and tumors robustly incorporate and remodel exogenous palmitate into structural and oncogenic glycerophospholipids, sphingolipids, and ether lipids. We also find that fatty acid incorporation into oxidative pathways is reduced in aggressive human cancer cells, and instead shunted into pathways for generating structural and signaling lipids. Our results demonstrate that cancer cells do not solely rely on de novo lipogenesis, but also utilize exogenous fatty acids for generating lipids required for proliferation and protumorigenic lipid signaling. This article is part of a special issue entitled Lipid Metabolism in Cancer.

Relevance and Diversity of Nitrospira Populations in Biofilters of Brackish RAS

Lithoautotrophic nitrite-oxidizing bacterial populations from moving-bed biofilters of brackish recirculation aquaculture systems (RAS; shrimp and barramundi) were tested for their metabolic activity and phylogenetic diversity. Samples from the biofilters were labeled with 13C-bicarbonate and supplemented with nitrite at concentrations of 0.3, 3 and 10 mM, and incubated at 17 and 28°C, respectively. The biofilm material was analyzed by fatty acid methyl ester - stable isotope probing (FAME-SIP). High portions of up to 45% of Nitrospira-related labeled lipid markers were found confirming that Nitrospira is the major autotrophic nitrite oxidizer in these brackish systems with high nitrogen loads. Other nitrite-oxidizing bacteria such as Nitrobacter or Nitrotoga were functionally not relevant in the investigated biofilters. Nitrospira-related 16S rRNA gene sequences were obtained from the samples with 10 mM nitrite and analyzed by a cloning approach. Sequence studies revealed four different phylogenetic clusters within the marine sublineage IV of Nitrospira, though most sequences clustered with the type strain of Nitrospira marina and with a strain isolated from a marine RAS. Three lipids dominated the whole fatty acid profiles of nitrite-oxidizing marine and brackish enrichments of Nitrospira sublineage IV organisms. The membranes included two marker lipids (16∶1 cis7 and 16∶1 cis11) combined with the non-specific acid 16∶0 as major compounds and confirmed these marker lipids as characteristic for sublineage IV species. The predominant labeling of these characteristic fatty acids and the phylogenetic sequence analyses of the marine Nitrospira sublineage IV identified organisms of this sublineage as main autotrophic nitrite-oxidizers in the investigated brackish biofilter systems.

FadD Is Required for Utilization of Endogenous Fatty Acids Released from Membrane Lipids

FadD is an acyl coenzyme A (CoA) synthetase responsible for the activation of exogenous long-chain fatty acids (LCFA) into acyl-CoAs. Mutation of fadD in the symbiotic nitrogen-fixing bacterium Sinorhizobium meliloti promotes swarming motility and leads to defects in nodulation of alfalfa plants. In this study, we found that S. meliloti fadD mutants accumulated a mixture of free fatty acids during the stationary phase of growth. The composition of the free fatty acid pool and the results obtained after specific labeling of esterified fatty acids with a Δ5-desaturase (Δ5-Des) were in agreement with membrane phospholipids being the origin of the released fatty acids. Escherichia coli fadD mutants also accumulated free fatty acids released from membrane lipids in the stationary phase. This phenomenon did not occur in a mutant of E. coli with a deficient FadL fatty acid transporter, suggesting that the accumulation of fatty acids in fadD mutants occurs inside the cell. Our results indicate that, besides the activation of exogenous LCFA, in bacteria FadD plays a major role in the activation of endogenous fatty acids released from membrane lipids. Furthermore, expression analysis performed with S. meliloti revealed that a functional FadD is required for the upregulation of genes involved in fatty acid degradation and suggested that in the wild-type strain, the fatty acids released from membrane lipids are degraded by β-oxidation in the stationary phase of growth.

N-Alkylpyridinium isotope quaternization for matrix-assisted laser desorption/ionization Fourier transform mass spectrometric analysis of cholesterol and fatty alcohols in human hair

Isotope-coded reagents have been developed for labeling of amino acids, phenols and fatty acids, but not for alcohols. In this work, a simple reaction based on direct N-alkylpyridinium isotope quaternization (NAPIQ) was developed for mild derivatization of cholesterol and fatty alcohols. Different from the conventional quaternary reagents with cations on themselves, two simple and charge-neutral reagents: pyridine and d 5-pyridine directly attached N-cationic tag onto the target compounds in the presence of trifluoromethanesulfonic anhydride (Tf 2O) without tedious sample preparation. The derivatization completed in 5 min and achieved charge labeling of the target compounds, which significantly improved the detection limits of analytes by 10 3-folds in further analysis by matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI–FTMS). The use of commercially available d 0/ d 5-pyridine pairs facilitated isotope-coded chemical derivatization and avoided the use of isotope-labeled internal standards; the excess pyridine did not affect the signals of analytes. Utility of the NAPIQ method was examined in the identification of cholesterol and fatty alcohols in small amount of human hair sample (<0.5 mg). The fluctuation of total cholesterol in human body was profiled during time by quantitatively comparing the different segments of a single strand of hair. This study combines the direct pyridinium quaternization with MALDI–FTMS, which offers a perspective and an alternative tool for the identification and quantification of substances in biological matrix by comparing d 0/ d 5 pairs, especially when isotope-labeled internal standards are unavailable.