Stable Isotope Tracer Research Articles

MFSD7c functions as a transporter of choline at the blood–brain barrier

Mutations in the orphan transporter MFSD7c (also known as Flvcr2), are linked to Fowler syndrome. Here, we used Mfsd7c knockout (Mfsd7c–/–) mice and cell-based assays to reveal that MFSD7c is a choline transporter at the blood–brain barrier (BBB). We performed comprehensive metabolomics analysis and detected differential changes of metabolites in the brains and livers of Mfsd7c–/–embryos. Particularly, we found that choline-related metabolites were altered in the brains but not in the livers of Mfsd7c–/– embryos. Thus, we hypothesized that MFSD7c regulates the level of choline in the brain. Indeed, expression of human MFSD7c in cells significantly increased choline uptake. Interestingly, we showed that choline uptake by MFSD7c is greatly increased by choline-metabolizing enzymes, leading us to demonstrate that MFSD7c is a facilitative transporter of choline. Furthermore, single-cell patch clamp analysis showed that the import of choline by MFSD7c is electrogenic. Choline transport function of MFSD7c was shown to be conserved in vertebrates, but not in yeasts. We demonstrated that human MFSD7c is a functional ortholog of HNM1, the yeast choline importer. We also showed that several missense mutations identified in patients exhibiting Fowler syndrome had abolished or reduced choline transport activity. Mice lacking Mfsd7c in endothelial cells of the central nervous system suppressed the import of exogenous choline from blood but unexpectedly had increased choline levels in the brain. Stable-isotope tracing study revealed that MFSD7c was required for exporting choline derived from lysophosphatidylcholine in the brain. Collectively, our work identifies MFSD7c as a choline exporter at the BBB and provides a foundation for future work to reveal the disease mechanisms of Fowler syndrome.

Mid‐Holocene rainfall seasonality and ENSO dynamics over the south‐western Pacific

AbstractEl Niño–Southern Oscillation dynamics affect global weather patterns, with regionally diverse hydrological responses posing critical societal challenges. The lack of seasonally resolved hydrological proxy reconstructions beyond the observational era limits our understanding of boundary conditions that drive and/or adjust El Niño–Southern Oscillation variability. Detailed reconstructions of past El Niño–Southern Oscillation dynamics can help modelling efforts, highlight impacts on disparate ecosystems and link to extreme events that affect populations from the tropics to high latitudes. Here, mid‐Holocene El Niño–Southern Oscillation and hydrological changes are reconstructed in the south‐west Pacific using a stalagmite from Niue Island, which represents the period 6.4–5.4 ka BP. Stable oxygen and carbon isotope ratios, trace elements and greyscale data from a U/Th‐dated and layer counted stalagmite profile are combined to infer changes in local hydrology at sub‐annual to multi‐decadal timescales. Principal component analysis reveals seasonal‐scale hydrological changes expressed as variations in stalagmite growth patterns and geochemical characteristics. Higher levels of host rock‐derived elements (Sr/Ca and U/Ca) and higher δ18O and δ13C values are observed in dark, dense calcite laminae deposited during the dry season, whereas during the wet season, higher concentrations of soil‐derived elements (Zn/Ca and Mn/Ca) and lower δ18O and δ13C values are recorded in pale, porous calcite laminae. The multi‐proxy record from Niue shows seasonal cycles associated with hydrological changes controlled by the positioning and strength of the South Pacific Convergence Zone. Wavelet analysis of the greyscale record reveals that El Niño–Southern Oscillation was continuously active during the mid‐Holocene, with two weaker intervals at 6–5.9 and 5.6–5.5 ka BP. El Niño–Southern Oscillation especially affects dry season rainfall dynamics, with increased cyclone activity that reduces hydrological seasonality during El Niño years.

Linking Transpiration to Reef Nitrogen Supply on a Tropical Coral Island

AbstractCoral reef islands are biodiversity hotspots with high conservation value, but we have a poor understanding of how island vegetation, through transpiration, influences groundwater nutrient supply to adjacent reef systems. Here we combine stable isotope tracing, geophysical surveys, and satellite analysis to unravel the links between transpiration and the discharge of groundwater nitrate to the waters surrounding a coral reef island (Lady Elliot Island in the Great Barrier Reef). Over a 2‐year study period (2020–2021) there was a net loss of freshwater from the island, that is, evapotranspiration (ET) exceeded rainfall, largely caused by high rates of transpiration (73% of ET). Transpiration was higher in forested areas, and groundwater salinity appeared to be higher there. By tracing the nitrogen stable isotope signature of groundwater nitrate into reef organism tissue we were able to map groundwater nitrogen discharge spatially and temporally. Groundwater nitrogen discharge was focussed on one side of the island and did not vary seasonally as expected, despite reduced rainfall and seabird guano inputs over the austral winter/spring. Based on our results, we propose that transpiration by island vegetation slows groundwater flow and concentrates nitrate in the groundwater being released to the surrounding reef system. High concentrations of nitrate in groundwater (up to 27 mmol L−) were observed on Lady Elliot Island and these seem to have increased since 2014, but further work is required to understand if this is a normal scenario for tropical coral islands or the result of the islands' revegetation program.

Ammonia-oxidizing bacteria and archaea exhibit differential nitrogen source preferences.

Ammonia-oxidizing microorganisms (AOM) contribute to one of the largest nitrogen fluxes in the global nitrogen budget. Four distinct lineages of AOM: ammonia-oxidizing archaea (AOA), beta- and gamma-proteobacterial ammonia-oxidizing bacteria (β-AOB and γ-AOB) and complete ammonia oxidizers (comammox), are thought to compete for ammonia as their primary nitrogen substrate. In addition, many AOM species can utilize urea as an alternative energy and nitrogen source through hydrolysis to ammonia. How the coordination of ammonia and urea metabolism in AOM influences their ecology remains poorly understood. Here we use stable isotope tracing, kinetics and transcriptomics experiments to show that representatives of the AOM lineages employ distinct regulatory strategies for ammonia or urea utilization, thereby minimizing direct substrate competition. The tested AOA and comammox species preferentially used ammonia over urea, while β-AOB favoured urea utilization, repressed ammonia transport in the presence of urea and showed higher affinity for urea than for ammonia. Characterized γ-AOB co-utilized both substrates. These results reveal contrasting niche adaptation and coexistence patterns among the major AOM lineages.

Discovering the strength of immunometabolism in cancer therapy: Employing metabolic pathways to enhance immune responses.

Immunometabolism, which studies cellular metabolism and immune cell function, is a possible cancer treatment. Metabolic pathways regulate immune cell activation, differentiation, and effector functions, crucial to tumor identification and elimination. Immune evasion and tumor growth can result from tumor microenvironment metabolic dysregulation. These metabolic pathways can boost antitumor immunity. This overview discusses immune cell metabolism, including glycolysis, oxidative phosphorylation, amino acid, and lipid metabolism. Amino acid and lipid metabolic manipulations may improve immune cell activity and antitumor immunity. Combination therapy using immunometabolism-based strategies may enhance therapeutic efficacy. The complexity of the metabolic network, biomarker development, challenges, and future approaches are all covered, along with a summary of case studies demonstrating the effectiveness of immunometabolism-based therapy. Metabolomics, stable isotope tracing, single-cell analysis, and computational modeling are also reviewed for immunometabolism research. Personalized and combination treatments are considered. This review adds to immunometabolism expertise and sheds light on metabolic treatments' ability to boost cancer treatment immunological response. Also, in this review, we discussed the immune response in cancer treatment and altering metabolic pathways to increase the immune response against malignancies.

Development and optimization of a triple quadrupole ICP-MS-based system for the quantification of pre-concentrated gaseous elementary mercury.

Understanding the redox reactions and transformation rates of mercury (Hg) species in the environment is important for predicting future gaseous elemental Hg (Hg0) levels and assessing the impacts of anthropogenic Hg0 emissions on human health. Stable Hg isotope tracers are a promising tool for estimating Hg0 production rates; however, traditional analytical approaches for quantifying Hg0, such as atomic fluorescence spectroscopy or atomic absorption spectrometry, cannot differentiate between Hg isotopes, and alternative approaches, such as inductively coupled plasma mass spectrometry (ICP-MS) with a typical aqueous sample introductory system, have relatively higher detection limit of Hg. Here, we developed and evaluated a custom-made thermal desorption unit coupled directly to a triple quadrupole ICP-MS (ICP-QQQ) for the quantification of Hg0 pre-concentrated on Au traps. The performance of the system was validated with measurements of a Hg standard gas and of Hg0 generated from aqueous Hg standards. Using our system, we were able to detect ultra-trace amounts of Hg0 and obtain precise Hg isotope measurements with an analytical error of ≤ 3.5%. Calibration curves with superb linearity (r2 > 0.999) were obtained for the Hg concentration range of 0-300pg. The method detection limit was approximately 0.01-0.03pg of Hg. Using the latest ICP-QQQ instrument (Agilent 8900; Agilent Technologies Ltd.) was far superior to using a previous model (Agilent 8800), with the Agilent 8900 showing approximately five times higher sensitivity than the Agilent 8800 as well as the ability to precisely and simultaneously analyze up to five Hg isotopes by time-resolved analysis.

Abstract PR10: Uridine-to-ribose axis supports glucose-restricted pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) is a lethal disease with a 5-year survival rate of 12% in the USA. PDA highly rely on glucose but is surrounded by a nutrient-limited microenvironment. How PDA survive such condition, and the metabolites enabling that process, is unclear. To determine how PDA adapt to starvation, we profiled the utility of >175 metabolites by 21 pancreatic cell lines cultured in glucose limited condition. This nutrient profiling assay revealed that PDA use uridine when glucose is depleted. Uridine utilization strongly correlated with uridine phosphorylase 1 (UPP1) – the enzyme that breaks down uridine to uracil and ribose-1-phosphate. Metabolomics, including stable isotope tracing, revealed that when glucose is limited, PDA cells use the ribose from uridine to sustain central carbon metabolism and that this process is blocked upon the knockout of UPP1. We found that UPP1 is regulated by KRAS, is high in PDA, and correlates with poor survival in patients. Moreover, deletion of UPP1 blunted tumor growth in mice. Our data identify the uridine-ribose axis as a crucial compensatory pathway in nutrient-deprived PDA, suggesting a potential therapeutic target. Citation Format: Zeribe C. Nwosu, Matthew H. Ward, Peter Sajjakulnukit, Pawan Poudel, Chanthirika Ragulan, Steven Kasperek, Megan Radyk, Damien Sutton, Rosa E. Menjivar, Anthony Andren, Juan J. Apiz-Saab, Zachary Tolstyka, Kristee Brown, Ho-Joon Lee, Lindsey Dzierozynski, Xi He, Hari Ps, Julia Ugras, Gift Nyamundanda, Li Zhang, Christopher J. Halbrook, Eileen Carpenter, Jiaqi Shi, Leah P. Shriver, Gary J. Patti, Alexander Muir, Marina Pasca di Magliano, Anguraj Sadanandam, Costas A. Lyssiotis. Uridine-to-ribose axis supports glucose-restricted pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr PR10.

Methylotrophic methanogenesis in the Archaeoglobi revealed by cultivation of Ca. Methanoglobus hypatiae from a Yellowstone hot spring.

Over the past decade, environmental metagenomics and polymerase chain reaction-based marker gene surveys have revealed that several lineages beyond just a few well-established groups within the Euryarchaeota superphylum harbor the genetic potential for methanogenesis. One of these groups are the Archaeoglobi, a class of thermophilic Euryarchaeota that have long been considered to live non-methanogenic lifestyles. Here, we enriched Candidatus Methanoglobus hypatiae, a methanogen affiliated with the family Archaeoglobaceae, from a hot spring in Yellowstone National Park. The enrichment is sediment-free, grows at 64-70°C and a pH of 7.8, and produces methane from mono-, di-, and tri-methylamine. Ca. M. hypatiae is represented by a 1.62Mb metagenome-assembled genome with an estimated completeness of 100% and accounts for up to 67% of cells in the culture according to fluorescence in situ hybridization. Via genome-resolved metatranscriptomics and stable isotope tracing, we demonstrate that Ca. M. hypatiae expresses methylotrophic methanogenesis and energy-conserving pathways for reducing monomethylamine to methane. The detection of Archaeoglobi populations related to Ca. M. hypatiae in 36 geochemically diverse geothermal sites within Yellowstone National Park, as revealed through the examination of previously published gene amplicon datasets, implies a previously underestimated contribution to anaerobic carbon cycling in extreme ecosystems.

Expression and function of the urea cycle in widely-used hepatic cellular models.

The group of rare metabolic defects termed urea cycle disorders (UCDs) occur within the ammonia elimination pathway and lead to significant neurocognitive sequelae for patients surviving decompensation episodes. Besides orthotopic liver transplantation, curative options are lacking for UCDs, with dietary management being the gold clinical standard. Novel therapeutic approaches are essential for UCDs; however, such effort presupposes preclinical testing in cellular models that effectively capture disease manifestation. Several cellular and animal models exist and aim to recapitulate the broad phenotypic spectrum of UCDs; however, the majority of those lack extensive molecular and biochemical characterization. The development of cellular models is emerging since animal models are extremely time and cost consuming, and subject to ethical considerations, including the 3R principle that endorses animal welfare over unchecked preclinical testing. The aim of this study was to compare the extent of expression and functionality of the urea cycle in two commercial hepatoma-derived cell lines, induced pluripotent stem cell hepatocytes (iPSC-Heps), primary human hepatocytes (PHHs) and human liver cell preparations. Using immunoblotting, immunocytochemistry, and stable isotope tracing of the urea cycle metabolites, we identified that the hepatoma-derived, 2-week differentiated HepaRG cells are urea cycle proficient and behave as cellular alternatives to PHHs. Furthermore, HepaRG cells were superior to iPSC-Heps, which are known to exhibit batch-to-batch variabilities in terms of hepatic maturity and enzyme expression. Finally, HepG2 cells lack the urea cycle enzymes ornithine transcarbamylase and arginase 1, the transporter ORNT1, which limits their suitability as model for the study of UCDs.

A multi‐method approach for the identification and quantification of water balance components of two lentic small water bodies

AbstractThe protection of the globally widespread lentic small water bodies (LSWB) must be based on detailed knowledge about their hydrological connectivity and water balance. The study aimed to identify and quantify water balance components as well as surface‐groundwater interaction of two LSWB in a characteristic lowland region with a combination of different methods. This includes the collection of hydrological data and the use of bromide and water stable isotopes (δ2H and δ18O) as tracers. With their help, mixing models were established, and daily water balances were assessed. The results show a strong bidirectional interaction of both LSWB systems with shallow groundwater. Bromide and stable isotope tracers allowed for the identification of the most relevant in‐ and outflow sources and pathways. Thereby, isotope data revealed isotopic enrichment typical for open‐water bodies and only minor precipitation inputs mainly relevant at the end of the dry season. Water balance calculations suggested accentuated seasonal dynamics that were strongly influenced by shallow groundwater, which represented large inputs into both LSWB. By that, different phases could be identified, with high inflow rates in winter and spring and decreasing fluxes in summer. In one LSWB, a drainage system was found to have a major impact next to the shallow groundwater interaction. The findings of this research provide detailed insights into the influence and importance of shallow groundwater for LSWB in lowland regions. This impacts the diffuse input of agricultural pollutants into these ecologically important landscape features.

River influence on mercury bioaccumulation in the coastal food web of Eeyou Istchee, James Bay, Canada

Spatial patterns of bioaccumulated mercury were evaluated in coastal marine food webs of east Hudson Bay and east James Bay in the boreal subarctic of Canada. Two marine species, blue mussels ( Mytilus edulis) and common eider ducks ( Somateria mollissima) that consume mussels, were collected by a regional community-based monitoring network established in five communities. Stable isotope tracers (carbon, nitrogen, sulfur, and mercury) were measured to evaluate environmental drivers of mercury spatial patterns. Mercury concentrations of blue mussels and common eiders were twofold and fivefold higher, respectively, on the James Bay coast near the community of Chisasibi compared to sites in east Hudson Bay. Liver and muscle mercury concentrations of eiders from James Bay are among the highest values reported for the circumpolar subarctic and Arctic. Multiple lines of evidence (mercury spatial patterns, crustal elements in blue mussels, and mercury isotope values of common eiders) suggest elevated mercury in the coastal food web of east James Bay may be due to mercury loading from the La Grande River, which drains one of the largest hydroelectric developments in the world. These findings highlight the importance of further research on environmental processes linking large rivers to mercury bioaccumulation in northern coastal food webs.

Fatty Acid Oxidation Promotes Apoptotic Resistance and Proinflammatory Phenotype of CD4+ Tissue-resident Memory T cells in Crohn’s Disease

Background and aimsAs the most abundant memory T cells and major source of tumor necrosis factor α (TNF-α) in the intestinal mucosa of Crohn’s disease (CD) patients, CD4+ tissue-resident memory T (TRM) cells play a critical role in CD pathogenesis. We investigated the role of metabolic reprogramming in the regulation of proinflammatory and apoptosis-resistant phenotype for CD4+ TRM cells. MethodsCD4+ TRM cells were collected from intestinal resection tissues from control and CD patients. Transcriptomic and metabolomic analysis were performed to identify metabolic characteristics of CD4+ TRM cells. ELISA and qPCR experiments were utilized to assess cytokines level in CD4+ TRM cells, activation-induced cell apoptosis (AICD) rate was evaluated by flow cytometry. Transwell assay and wound healing assay were performed to detect the effect of CD4+ TRM cells on the migration of normal intestinal epithelial cells. ResultsTranscriptomic data combined with unbiased metabolomic analysis revealed an increased fatty acid oxidation (FAO) phenotype existed in CD4+ TRM cells from CD patients. The lipidomic data and stable isotope tracer experiments demonstrated that CD4+ TRM cells upregulated their lipid lipolysis and fatty acid uptake to fuel FAO in CD patients. Mechanistically, the activated NF-κB signaling increased transcription of genes involved in lipid lipolysis, fatty acid uptake and oxidation in CD4+ TRM cells from CD patients. Targeting FAO of CD4+ TRM cells reversed their apoptosis-resistant and proinflammatory phenotype in CD Patients. ConclusionCD4+ TRM cells process an accelerated FAO mediated by activated NF-κB signaling in CD patients, targeting FAO could reverse their apoptosis-resistant and proinflammatory phenotype. These findings shed a new light on the pathogenic mechanism investigation and novel therapy development in CD patients.

Plant–plant interactions affect seasonal nitrogen uptake of subalpine conifer seedlings by altering root traits and soil nitrogen availabilities

AbstractAlthough it is known that plant–plant interaction is an important factor influencing plant nitrogen (N) uptake and biomass productivity, its effects on seasonal inorganic N uptake, preference, and allocation remain unclear. In this study, two conifer species (Picea asperata and Abies faxoniana) were planted in three different planting modes (i.e., single, intraspecific, and interspecific interaction). Using 15N stable isotope tracer, we quantified plant biomass, ammonium (NH4+), and nitrate (NO3−) uptake rate (mass) and allocation in the middle (July) and the end (September) of the growing season, respectively, followed by analyses of root traits and soil properties so as to explore the underlying mechanism. Across the two seasons, intraspecific interaction decreased plant biomass and inorganic N‐uptake rate, which triggered intense competition for both species. Intraspecific competition of P. asperata was stronger than that of A. faxoniana. In contrast, interspecific interaction revealed significant facilitative effects on A. faxoniana, particularly in September. From the middle to the late growing season, the inorganic N‐uptake rate of P. asperata reduced, whereas that of A. faxoniana increased under interspecific interaction. The seasonal variation in plant N uptake was regulated by changes in root traits (such as root nitrogen concentration, specific root length, and branching intensity) and soil N availabilities. Both species indicated a preference for NO3− across seasons. Furthermore, we observed that 15N allocation to shoots of A. faxoniana under interspecific interaction was higher than that of P. asperata and declined from July to September. These findings on how plant–plant interactions affect plant N uptake seasonally can facilitate our understanding of species co‐existence and community assembly in forest ecosystems.

Climatic factors and fertilization rates co-regulate anaerobic methane oxidation driven by multiple electron acceptors in Chinese paddy fields

Paddy fields constitute a substantial anthropogenic reservoir of methane, with their inundation management fostering an ideal habitat for anaerobic methane oxidation. Within this context, a novel clade of anaerobic methanotrophic (ANME) archaea, known as ANME-2d, has been identified as capable of catalyzing anaerobic methane oxidation in conjunction with nitrate and metal oxide reduction processes within paddy fields. Nevertheless, our comprehension of the mechanisms governing anaerobic methane oxidation and its pivotal role in regulating methane emissions within rice paddies remains limited. This study quantified the rates of nitrate- and iron (III)-driven anaerobic methane oxidation through 13C-labeled stable isotope tracing experiments in Chinese paddy fields spanning diverse climate zones. Additionally, it investigated the ANME-2d archaeal community using quantitative polymerase chain reaction and high-throughput sequencing techniques. The nitrate-driven anaerobic methane oxidation contributed 10.9% to methane emission reduction. This contribution is equal to the previously identified contribution of nitrite-driven anaerobic methane oxidation (11.2%) mediated via NC10 bacteria, but played more important roles than iron-driven one (4.1%). The rates of nitrate- and nitrite-driven anaerobic methane oxidation differed significantly among climate zones and showed positive correlation with the mean annual temperature. Furthermore, their rates were more sensitive to temperature increases at higher and lower latitudes, respectively, under both representative concentration pathways 2.6 and 8.5. The rate of anaerobic methane oxidation driven by nitrate exhibited a positive correlation with nitrogen fertilization rate but displayed a negative correlation with phosphorus fertilization rate. Conversely, the rate of anaerobic methane oxidation driven by iron showed no significant correlation with either nitrogen or phosphorus fertilization rates. This study underscores the great potential of anaerobic methane oxidation in mitigating global warming, particularly under the conditions of future climate change and elevated nitrogen loading. These findings underline the necessity of incorporating anaerobic methane oxidation as a crucial parameter in methane emission prediction models.

Serine synthesis via reversed SHMT2 activity drives glycine depletion and acetaminophen hepatotoxicity in MASLD

Metabolic dysfunction-associated steatotic liver disease (MASLD) affects one-third of the global population. Understanding the metabolic pathways involved can provide insights into disease progression and treatment. Untargeted metabolomics of livers from mice with early-stage steatosis uncovered decreased methylated metabolites, suggesting altered one-carbon metabolism. The levels of glycine, a central component of one-carbon metabolism, were lower in mice with hepatic steatosis, consistent with clinical evidence. Stable-isotope tracing demonstrated that increased serine synthesis from glycine via reverse serine hydroxymethyltransferase (SHMT) is the underlying cause for decreased glycine in steatotic livers. Consequently, limited glycine availability in steatotic livers impaired glutathione synthesis under acetaminophen-induced oxidative stress, enhancing acute hepatotoxicity. Glycine supplementation or hepatocyte-specific ablation of the mitochondrial SHMT2 isoform in mice with hepatic steatosis mitigated acetaminophen-induced hepatotoxicity by supporting de novo glutathione synthesis. Thus, early metabolic changes in MASLD that limit glycine availability sensitize mice to xenobiotics even at the reversible stage of this disease.

Glycine homeostasis requires reverse SHMT flux

The folate-dependent enzyme serine hydroxymethyltransferase (SHMT) reversibly converts serine into glycine and a tetrahydrofolate-bound one-carbon unit. Such one-carbon unit production plays a critical role in development, the immune system, and cancer. Using rodent models, here we show that the whole-body SHMT flux acts to net consume rather than produce glycine. Pharmacological inhibition of whole-body SHMT1/2 and genetic knockout of liver SHMT2 elevated circulating glycine levels up to eight-fold. Stable-isotope tracing revealed that the liver converts glycine to serine, which is then converted by serine dehydratase into pyruvate and burned in the tricarboxylic acid cycle. In response to diets deficient in serine and glycine, de novo biosynthetic flux was unaltered, but SHMT2- and serine-dehydratase-mediated catabolic flux was lower. Thus, glucose-derived serine synthesis is largely insensitive to systemic demand. Instead, circulating serine and glycine homeostasis is maintained through variable consumption, with liver SHMT2 a major glycine-consuming enzyme.

Setting new standards: Multiphasic analysis of microplastic mineralization by fungi

Plastic materials provide numerous benefits. However, properties such as durability and resistance to degradation that make plastic attractive for variable applications likewise foster accumulation in the environment. Fragmentation of plastics leads to the formation of potentially hazardous microplastic, of which a considerable amount derives from polystyrene. Here, we investigated the biodegradation of polystyrene by the tropical sooty mold fungus Capnodium coffeae in different experimental setups. Growth of C. coffeae was stimulated significantly when cultured in presence of plastic polymers rather than in its absence. Stable isotope tracing using 13C-enriched polystyrene particles combined with cavity ring-down spectroscopy showed that the fungus mineralized polystyrene traces. However, phospholipid fatty acid stable isotope probing indicated only marginal assimilation of polystyrene-13C by C. coffeae in liquid cultures. NMR spectroscopic analysis of residual styrene contents prior to and after incubation revealed negligible changes in concentration. Thus, this study suggests a plastiphilic life style of C. coffeae despite minor usage of plastic as a carbon source and the general capability of sooty mold fungi to stimulate polystyrene mineralization, and proposes new standards to identify and unambiguously demonstrate plastic degrading capabilities of microbes.

Evaluation of urinary limonene metabolites as biomarkers of exposure to greenness

Exposure to plants is known to improve physical and mental health and living in areas of high vegetation is associated with better health. The addition of quantitative measures of greenness exposure at individual-level to other objective and subjective study measures will help establish cause-and-effect relationships between greenspaces and human health. Because limonene is one of the most abundant biogenic volatile organic compounds emitted by plants, we hypothesized that urinary metabolites of inhaled limonene can serve as biomarkers of exposure to greenness. To test our hypothesis, we analyzed urine samples collected from eight human volunteers after limonene inhalation or after greenness exposure using liquid chromatography-high resolution mass spectrometry-based profiling. Eighteen isomers of nine metabolites were detected in urine after limonene inhalation, and their kinetic parameters were estimated using nonlinear mixed effect models. Urinary levels of most abundant limonene metabolites were elevated after brief exposure to a forested area, and the ratio of urinary limonene metabolites provided evidence of recent exposure. The identities and structures of these metabolites were validated using stable isotope tracing and tandem mass spectral comparison. Together, these data suggest that urinary metabolites of limonene, especially uroterpenol glucuronide and dihydroperillic acid glucuronide, could be used as individualized biomarkers of greenness exposure.

Tracers and Imaging of Fatty Acid and Energy Metabolism of Human Adipose Tissues.

White adipose tissue and brown adipose tissue (WAT and BAT) regulate fatty acid metabolism and control lipid fluxes to other organs. Dysfunction of these key metabolic processes contributes to organ insulin resistance and inflammation leading to chronic diseases such as type 2 diabetes, metabolic dysfunction-associated steatohepatitis, and cardiovascular diseases. Metabolic tracers combined with molecular imaging methods are powerful tools for the investigation of these pathogenic mechanisms. Herein, I review some of the positron emission tomography and magnetic resonance imaging methods combined with stable isotopic metabolic tracers to investigate fatty acid and energy metabolism, focusing on human WAT and BAT metabolism. I will discuss the complementary strengths offered by these methods for human investigations and current gaps in the field.

Metabolic Patterns of High-Invasive and Low-Invasive Oral Squamous Cell Carcinoma Cells Using Quantitative Metabolomics and 13C-Glucose Tracing.

Most current metabolomics studies of oral squamous cell carcinoma (OSCC) are mainly focused on identifying potential biomarkers for early screening and diagnosis, while few studies have investigated the metabolic profiles promoting metastasis. In this study, we aimed to explore the altered metabolic pathways associated with metastasis of OSCC. Here, we identified four OSCC cell models (CAL27, HN6, HSC-3, SAS) that possess different invasive heterogeneity via the transwell invasion assay and divided them into high-invasive (HN6, SAS) and low-invasive (CAL27, HSC-3) cells. Quantitative analysis and stable isotope tracing using [U-13C6] glucose were performed to detect the altered metabolites in high-invasive OSCC cells, low-invasive OSCC cells and normal human oral keratinocytes (HOK). The metabolic changes in the high-invasive and low-invasive cells included elevated glycolysis, increased fatty acid metabolism and an impaired TCA cycle compared with HOK. Moreover, pathway analysis demonstrated significant differences in fatty acid biosynthesis; arachidonic acid (AA) metabolism; and glycine, serine and threonine metabolism between the high-invasive and low-invasive cells. Furthermore, the high-invasive cells displayed a significant increase in the percentages of 13C-glycine, 13C-palmitate, 13C-stearic acid, 13C-oleic acid, 13C-AA and estimated FADS1/2 activities compared with the low-invasive cells. Overall, this exploratory study suggested that the metabolic differences related to the metastatic phenotypes of OSCC cells were concentrated in glycine metabolism, de novo fatty acid synthesis and polyunsaturated fatty acid (PUFA) metabolism, providing a comprehensive understanding of the metabolic alterations and a basis for studying related molecular mechanisms in metastatic OSCC cells.