Stable Isotope Tracer Research Articles

Investigating β‐Lapachone Mediated Metabolic Disruption Using Stable Isotope Tracers

β‐lapachone has been found to be a highly potent anti‐cancer agent in phase 1 clinical trials, but has dose limiting toxicity in red blood cells [Gerber et. al Br J Cancer. 2018 Oct;119(8):928‐936]. Identifying a safe and quantitative method of monitoring treatment efficacy will be highly beneficial to establishing an adaptive dosing platform. Adaptative dosing can allow personalized treatments that significantly reduce off target effects while maintaining an effective therapeutic regimen. In this study we present the use of a safe and noninvasive tracer strategy utilizing both [2H7]glucose and [U‐13C]glucose tracers to assess the metabolic effects caused by β‐lapachone treatment in vitro. Treating cancers with β‐lapachone bioactivates NAD(P)H: quinone oxidoreductase 1 (NQO1) to produce a highly unstable hydroquinone intermediate that reestablishes itself through an aggressive futile cycle, generating reactive oxygen species (ROS) in the form of hydrogen peroxide. Accelerated accumulation of ROS causes large amounts of DNA damage, triggers poly‐ADP‐ribose polymerase‐I hyperactivation, induces global depletion of NAD+ and ATP, and hinders overall glycolytic flux [Moore et. al Cell Death Dis. 2015 Jan; 6(1): e159]. Cancer cells that overexpress NQO1 subsequently die through NAD+‐keresis. Measuring changes in glycolysis and downstream metabolism caused by the bioactivation of NQO1 would establish a platform for assessing the efficacy of treatment, potentially allowing the reduction of chemotherapeutic dosage thereby diminishing off‐target toxicities. Changes in central carbon metabolism caused by NQO1 bioactivation were detected and assessed in various cancer cells using both tracer strategies. Deuterated lactate and HDO (deuterated water) were easily quantified, and the production of these metabolic products were linearly correlated with [2H7]glucose consumption. Mass isotopologue distribution analysis by gas chromatography‐mass spectrometry demonstrated downregulated energy metabolism with a reduction in 2H and 13C labeling in NQO1+ cancer cells treated with β‐lapachone. These methods are highly beneficial for measuring rates of glycolytic metabolism, identifying potential therapeutic synergies, the efficacy of chemotherapeutic agents that hamper central carbon metabolism, and can be potentially translated to in vivo models.

Metabolic outliers in human disease

Many human diseases are caused by mutations that perturb metabolism at the cellular level and result in tissue dysfunction. Some metabolic perturbations result in disease by interrupting the canonical functions of the metabolic network: producing energy, generating precursors for macromolecular synthesis, maintaining redox balance, disposing of waste, etc. Others interfere with processes beyond the conventional metabolic network, interfering with signaling and gene expression networks. Understanding these pathological states of metabolic perturbation may help us develop rational approaches to normalize metabolism and restore health. We study two types of diseases characterized by metabolic dysfunction: inborn errors of metabolism and cancer. I will discuss ongoing work in these diseases that seeks to characterize abnormal metabolic states directly in human subjects, then uses experimental models to explore disease mechanisms and propose potential therapies. I will emphasize methods in metabolomics and stable isotope tracing that allow us observe metabolic phenotypes in intact systems relevant to physiology and disease, highlighting recent work on tumor metabolism in patients and genetically‐defined metabolic anomalies that interfere with mammalian developmental programs.

Glycine decarboxylase maintains mitochondrial protein lipoylation to support tumor growth

The folic acid cycle mediates the transfer of one-carbon (1C) units to support nucleotide biosynthesis. While the importance of serine as a mitochondrial and cytosolic donor of folate-mediated 1C units in cancer cells has been thoroughly investigated, a potential role of glycine oxidation remains unclear. We developed an approach for quantifying mitochondrial glycine cleavage system (GCS) flux by combining stable and radioactive isotope tracing with computational flux decomposition. We find high GCS flux in hepatocellular carcinoma (HCC), supporting nucleotide biosynthesis. Surprisingly, other than supplying 1C units, we found that GCS is important for maintaining protein lipoylation and mitochondrial activity. Genetic silencing of glycine decarboxylase inhibits the lipoylation and activity of pyruvate dehydrogenase and impairs tumor growth, suggesting a novel drug target for HCC. Considering the physiological role of liver glycine cleavage, our results support the notion that tissue of origin plays an important role in tumor-specific metabolic rewiring.

Hydrogeochemistry and Isotope Hydrology of Surface Water and Groundwater in the Mountain Watersheds of Daqing River, North China

Surface water and groundwater interaction variations in time and space are crucial for effective water management, especially in low-precipitation regions. To comprehensively determine the hydrochemical characteristics and interaction processes of surface water and groundwater and to investigate the decreasing causes of water resources in semi-arid mountainous watersheds under changing environments, intensive field surveys were conducted in the Daqing River watershed, a tributary of the Haihe River basin in northern China, during two different times of the year: after the rainy season (September 2018) and before the rainy season (July 2019). Sixty surface water and groundwater samples were collected along the mountainous watershed. Using a combination method of hydrogen and oxygen stable isotope tracing and hydrochemical analysis, the hydrogen and oxygen isotopes and hydrochemical characteristics of surface water and groundwater in the mountainous watershed of the Daqing River were analyzed. Furthermore, the effect of elevation (altitude) on isotopes was discussed, and the correlation between hydrogen and oxygen isotope composition and hydrochemical characteristics was obtained. The results were processed using endmember mixing analysis to determine the amount of contribution of the surface water and groundwater interaction processes. The results show that the hydrochemical characteristics are relatively stable in the mountainous watersheds of the Daqing River, and the surface water and groundwater are mainly of the HCO3-Ca type. The slope of the local meteoric water line is smaller than the slope of the global meteoric water line, and the δD and δ18O in surface water and groundwater show a good linear relationship both before and after the rainy season. There is a decreasing trend of the value of δ18O in surface water samples with decreasing altitude, but a decreasing trend of the value of δ18O in groundwater samples is not obvious. The evaporation intensity of surface water is stronger after the rainy season than before the rainy season, and the connection between the surface water and the groundwater is stronger before the rainy season. Influenced by topographic conditions and other factors, the exchange of surface water and groundwater is frequent, and there is a large difference in the exchange ratio before and after the rainy season. The exchange ratio can be more than 50% after the rainy season. Thus, the reasons for decreasing water resources in the mountains can be implied to be due to the increasing hydraulic gradient between the mountains and the piedmont plains, and the water resources are discharged more in the form of groundwater to the downstream. The conclusions help to enhance the understanding of the water cycle in the mountainous watershed and can provide some theoretical basis for the sustainable development and utilization of water resources in the Haihe River basin and the regional water ecology of the Xiong’an New Area.

The combination of NRF1 and Nrf2 activators in myoblasts stimulate mechanisms of proteostasis without changes in mitochondrial respiration

Persistent oxidative stress contributes to hallmarks of aging, including impaired proteostasis and mitochondrial dysfunction, while acute oxidative challenges resolved swiftly contribute to beneficial adaptations. Adaptive homeostasis is where acute exposures to sub‐toxic stimuli kindle transient expansion of responses necessary to reestablish homeostasis. Elucidating mechanisms underlying adaptive homeostasis will provide novel targets for healthspan extension. Nrf2 is a key regulator of cytoprotective gene transcription for redox homeostasis; NRF1 is a transcription factor that regulates expression of genes necessary for mitochondrial function. Regulation of both is compromised with advancing age. We hypothesized that NRF1 (NRF1a) and Nrf2 (Nrf2a) activators might improve adaptive homeostasis in response to an oxidative challenge by promoting mitochondrial proteome maintenance and function in C2C12 myoblasts. Using deuterium stable isotope tracing, we assessed protein synthesis over a 16‐hr treatment with a CON (DMSO), NRF1a, Nrf2a, or both, with and without a hydrogen peroxide (H2O2) stress. We assessed mitochondrial function using high‐resolution respirometry. Co‐treatment of NRF1a and Nrf2a under H2O2 stress favored proteostatic maintenance (p<0.05) through maintained protein synthesis (p>0.05), but a decrease in cell proliferation (p<0.05). H2O2 stress significantly decreased mitochondrial maximal respiration, but this decrease was not rescued by NRF1a/Nrf2a co‐treatment indicating the same ATP availability. Interestingly, there were no differences in submaximal ADP stimulated respiration or ADP sensitivity between CON, H2O2, or the NRF1a/Nrf2a co‐treatment (p>0.05). Further, there were no differences in endogenous redox capacity or mitochondrial protein content between treatment groups. These results suggest that simultaneously targeting NRF1 and Nrf2 may be a viable approach for reestablishing mitochondrial protein homeostasis following a stress, but that this adaptation may not improve respiratory capacity.

Quantitative Stable Isotope Tracing of Water Uptake in Cynanchum sibiricum during Its Growth Period in Arid Areas of Northwest China

Vegetation root water uptake plays a significant role in water transport processes and has multiple effects along the soil–vegetation–atmosphere interface, particularly in semi-arid and arid ecosystems. The objective of this study was to quantitatively examine the water uptake in Cynanchum sibiricum, a common desert rattan plant in arid regions, during its growth period, as well as to assess the relevance of the contributions of various water sources via a multivariate linear mixed model based on water-stable isotopes and eliminating the significant short-term impact of precipitation pulses on root water uptake. The results indicated that when the influence of a precipitation event was minimized, 0–10 cm soil water was the dominant water source for Cynanchum sibiricum in its growth phase, and that the species’ relative uptake of rainfall was positively proportional to that of 10–70 cm soil water while negatively proportional to that of 0–10 cm soil water. We concluded that Cynanchum sibiricum relies on its active roots extending transversely to extract water from shallow soil to survive under extreme drought conditions and would be inseparable from the symbiosis formed with other deep-rooted vegetation. Additional research into disentangling the water transport processes and studying the ecohydrological feedback along the soil–vegetation interface in arid ecosystems would be beneficial and could contribute to the evidence-based management of water resources for ecological protection.

Studying Metabolism by NMR-Based Metabolomics.

During the past few decades, the direct analysis of metabolic intermediates in biological samples has greatly improved the understanding of metabolic processes. The most used technologies for these advances have been mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. NMR is traditionally used to elucidate molecular structures and has now been extended to the analysis of complex mixtures, as biological samples: NMR-based metabolomics. There are however other areas of small molecule biochemistry for which NMR is equally powerful. These include the quantification of metabolites (qNMR); the use of stable isotope tracers to determine the metabolic fate of drugs or nutrients, unravelling of new metabolic pathways, and flux through pathways; and metabolite-protein interactions for understanding metabolic regulation and pharmacological effects. Computational tools and resources for automating analysis of spectra and extracting meaningful biochemical information has developed in tandem and contributes to a more detailed understanding of systems biochemistry. In this review, we highlight the contribution of NMR in small molecule biochemistry, specifically in metabolic studies by reviewing the state-of-the-art methodologies of NMR spectroscopy and future directions.

Mass defect filter technique combined with stable isotope tracing for drug metabolite identification using high-resolution mass spectrometry

Metabolism studies are one of the important steps in pharmaceutical research. LC-MS combined with metabolomics data-processing approaches have been developed for rapid screening of drug metabolites. Mass defect filter (MDF) is one of the LC/MS-based metabolomics data processing approaches and has been applied to screen drug metabolites. Although MDF can remove most interference ions from an incubation sample, the true positive rate of the retaining ions is relatively low (approximately 10%). To improve the efficacy of MDF, we developed a two-stage data-processing approach by combining MDF and stable isotope tracing (SIT) for metabolite identification. Pioglitazone (PIO), which is an antidiabetic drug used to treat type 2 diabetes mellitus, was taken as an example drug. Our results demonstrated that this new approach could substantially increase the validated rate from about 10% to 74%. Most of these validated metabolite signals (13/14) could be verified as PIO structure-related metabolites. In addition, we applied this approach to identify uncommon metabolite signals (a mass change beyond the window of 50 Da around its parent drug, MDF1). SIT could remove most interference ions (approximately 98%) identified by MDF1, and four out of five validated metabolite signals could be verified as PIO structure-related metabolites. Interestingly, a lot of the verified metabolites (10/17) were novel PIO metabolites. Among these novel metabolites, nine were thiazolidinedione ring-opening signals that might be related to the toxicity of PIO. Our developed approach could significantly improve the efficacy in drug metabolite identification compared with that of MDF.

Consequences of an ecosystem state shift for nitrogen cycling in a desert stream

AbstractCessation of cattle grazing has resulted in the reestablishment of wetlands in some streams of the U.S. Southwest. Decades of cattle grazing prevented vascular plant growth in Sycamore Creek (Arizona, U.S.A.), resulting in stream reaches dominated by diatoms and filamentous green algae. Establishment of vascular plants can profoundly modify ecosystem processes; yet, the effects on nitrogen (N) cycling remain unexplored. We examined the consequences of this ecosystem state shift on N cycling in this N‐limited desert stream. We compared results from whole‐reach ammonium‐N stable isotope (15NH4+) tracer additions conducted before (pre‐wetland state) and 13 yr after (wetland state) free‐range cattle removal from the watershed. Water column estimations showed that in‐stream N uptake and storage were higher in the pre‐wetland than in the wetland state. N turnover was also higher in the pre‐wetland state, indicating that assimilated N was retained for shorter time in stream biomass. In addition, N uptake was mostly driven by assimilatory uptake regardless of the ecosystem state considered. Water column trends were mechanistically explained by the fact that the dominant primary uptake compartments in the pre‐wetland state (i.e., algae and diatoms) had higher assimilatory uptake and turnover rates than those in the wetland state (i.e., vascular plants). Overall, results show that the shift in the composition and dominance of primary producers induced by the cessation of cattle grazing within the stream‐riparian corridor changes in‐stream N processing from a dominance of intense and fast N recycling to a prevalence of N retention in biomass of primary producers.

Uncovering geochemical fractionation of the newly deposited Hg in paddy soil using a stable isotope tracer

The newly deposited mercury (Hg) is more readily methylated to methylmercury (MeHg) than native Hg in paddy soil. However, the biogeochemical processes of the newly deposited Hg in soil are still unknown. Here, a field experimental plot together with a stable Hg isotope tracing technique was used to demonstrate the geochemical fractionation (partitioning and redistribution) of the newly deposited Hg in paddy soils during the rice-growing period. We showed that the majority of Hg tracer (200Hg, 115.09 ± 0.36 μg kg−1) was partitioned as organic matter bound 200Hg (84.6–89.4%), followed by residual 200Hg (7.6–8.1%), Fe/Mn oxides bound 200Hg (2.8–7.2%), soluble and exchangeable 200Hg (0.05–0.2%), and carbonates bound 200Hg (0.04–0.07%) in paddy soils. Correlation analysis and partial least squares path modeling revealed that the coupling of autochthonous dissolved organic matter and poorly crystalline Fe (oxyhydr)oxides played a predominant role in controlling the redistribution of the newly deposited Hg among geochemical fractions (i.e., fraction changes). The expected aging processes of the newly deposited Hg were absent, potentially explaining the high bioavailability of these Hg in paddy soil. This study implies that other Hg pools (e.g., organic matter bound Hg) should be considered instead of merely soluble Hg pools when evaluating the environmental risks of Hg from atmospheric depositions.

Nitrogen biogeochemical reactions during bank filtration constrained by hydrogeochemical and isotopic evidence: A case study in a riverbank filtration site along the Second Songhua River, NE China

River eutrophication and nitrogen pollution poses a potential threat to the groundwater quality in riverbank filtration systems. The river filtration process is often accompanied by complex redox reactions. Therefore, identification of nitrogen biogeochemical processes is essential to scientifically characterize the cause of NO3− attenuation and NH4+ accumulation in groundwater, optimize the design of groundwater pumping wells, and design protection strategies in sudden environmental pollution accidents. Hydrogeochemical and stable isotope tracing techniques were employed in this study at a typical riverbank filtration site located in Songyuan, NE China, to explore the main geochemical reactions controlling the migration and transformation of nitrogen along the groundwater flow path during riverbank filtration. The results indicate that mixing, adsorption, organic nitrogen mineralization, denitrification, and dissimilatory nitrate reduction to ammonium (DNRA) represent the major geochemical reactions. Denitrification primarily occurs within 10–20 m from the riverbed surface along the filtration path and is the primary reaction accounting for NO3− attenuation, whereas DNRA typically occurs between 1.5 and 6 m from the riverbed surface along the filtration path and is more active in the wet season, characterized by high temperatures (>15–17 °C), low dissolved oxygen (DO, < 2–4 mg/L), and high carbon load i.e., organic carbon (OC):NO3− > 10–15. This work confirms that DNRA is an important nitrogen geochemical reaction during riverbank filtration, and emphasizes its role in NH4+ enrichment.

Metabolism of triglyceride-rich lipoproteins in health and dyslipidaemia.

Accumulating evidence points to the causal role of triglyceride-rich lipoproteins and their cholesterol-enriched remnants in atherogenesis. Genetic studies in particular have not only revealed a relationship between plasma triglyceride levels and the risk of atherosclerotic cardiovascular disease, but have also identified key proteins responsible for the regulation of triglyceride transport. Kinetic studies in humans using stable isotope tracers have been especially useful in delineating the function of these proteins and revealing the hitherto unappreciated complexity of triglyceride-rich lipoprotein metabolism. Given that triglyceride is an essential energy source for mammals, triglyceride transport is regulated by numerous mechanisms that balance availability with the energy demands of the body. Ongoing investigations are focused on determining the consequences of dysregulation as a result of either dietary imprudence or genetic variation that increases the risk of atherosclerosis and pancreatitis. The identification of molecular control mechanisms involved in triglyceride metabolism has laid the groundwork for a 'precision-medicine' approach to therapy. Novel pharmacological agents under development have specific molecular targets within a regulatory framework, and their deployment heralds a new era in lipid-lowering-mediated prevention of disease. In this Review, we outline what is known about the dysregulation of triglyceride transport in human hypertriglyceridaemia.

An enhanced rural anoxic/oxic biological contact oxidation process with air-lift reflux technique to strengthen total nitrogen removal and reduce sludge generation

Biological contact oxidation process is attractive and widely used in rural sewage treatment, due to its simplicity and relatively superior pollutants elimination efficiency. However, the effluent total nitrogen (TN) and excess sludge yield during the biological contact oxidation process still need to be intensified to stably satisfy the discharge standard. In the current study, an enhanced rural anoxic/oxic (A/O) biological contact oxidation process with air-lift reflux technique was properly established, which achieved better pollutant removal efficiency and lower excess sludge yield. Under the pathway of air-lift reflux technique, the TN and ammonia nitrogen (AN) removal capacity of A/O biological contact oxidation process were obviously increased from 66.5% and 73.8% to 95.3% and 80.6%, respectively, which should be attributed to the increasement of nitrifying bacteria (Nitrospira and Nitrosomonas) and denitrifying bacteria (Denitratisoma, Thauera, and Dechloromonas). The excess sludge yield was also decreased from 0.19 kg MLSS/kg COD (no reflux model) to 0.11 kg MLSS/kg COD (air-lift reflux model) since the degradation of returned oxic sludge in the anoxic tank, which was verified by stable isotope tracer analysis. The rural A/O biological contact oxidation process with air-lift reflux technique had great potential to strengthen TN removal and reduce sludge generation, which could provide a useful and easy-implement technology for rural sewage treatment.

Postprandial Plasma Lipidomics Reveal Specific Alteration of Hepatic-derived Diacylglycerols in Nonalcoholic Fatty Liver Disease

Hepatic energy metabolism is a dynamic process modulated by multiple stimuli. In nonalcoholic fatty liver disease (NAFLD), human studies typically focus on the static fasting state. We hypothesized that unique postprandial alterations in hepatic lipid metabolism are present in NAFLD. In a prospective clinical study, 37 patients with NAFLD and 10 healthy control subjects ingested a standardized liquid meal with pre- and postprandial blood sampling. Postprandial plasma lipid kinetics were characterized at the molecular lipid species level by untargeted lipidomics, cluster analysis, and lipid particle isolation, then confirmed in a mouse model. There was a specific increase of multiple plasma diacylglycerol (DAG) species at 4 hours postprandially in patients with NAFLD but not in controls. This was replicated in a nonalcoholic steatohepatitis mouse model, where postprandial DAGs increased in plasma and concomitantly decreased in the liver. The increase in plasma DAGs appears early in the disease course, is dissociated from NAFLD severity and obesity, and correlates with postprandial insulin levels. Immunocapture isolation of very low density lipoprotein in human samples and stable isotope tracer studies in mice revealed that elevated postprandial plasma DAGs reflect hepatic secretion of endogenous, rather than meal-derived lipids. We identified a selective insulin-related increase in hepatic secretion of endogenously derived DAGs after a mixed meal as a unique feature of NAFLD. DAGs are known to be lipotoxic and associated with atherosclerosis. Although it is still unknown whether the increased exposure to hepatic DAGs contributes to extrahepatic manifestations and cardiovascular risk in NAFLD, our study highlights the importance of extending NAFLD research beyond the fasting state.

Evaluation of Hepatic Glucose Production in a Polycystic Ovary Syndrome Mouse Model.

Polycystic ovary syndrome (PCOS) is a common disease that results in disorders of glucose metabolism, such as insulin resistance and glucose intolerance. Dysregulated glucose metabolism is an important manifestation of the disease and is the key to its pathogenesis. Therefore, studies involving evaluation of glucose metabolism in PCOS are of utmost importance. Very few studies have quantified hepatic glucose production directly in PCOS models using non-radioactive glucose tracers. In this study, we discuss step-by-step instructions for the quantification of the rate of hepatic glucose production in a PCOS mouse model by measuring M+2 enrichment of [6,6-2H2]glucose, a stable isotopic glucose tracer, via gas chromatography - mass spectrometry (GCMS). This procedure involves creation of stable isotopic glucose tracer solution, use of tail vein catheter placement and infusion of the glucose tracer in both fasting and glucose-rich states in the same mouse in tandem. The enrichment of [6,6-2H2]glucose is measured using pentaacetate derivative in GCMS. This technique can be applied to a wide variety of studies involving direct measurement of the rate of hepatic glucose production.

Threshold recognition for shallow groundwater recharge by precipitation using dual isotopes in a small subtropical hilly catchment

Understanding the origins of groundwater and its movement from hillslopes to valleys is critical for conserving water supplies, determining water-use policies and controlling pollution. These factors are also the keys for understanding the dominant processes in hydrological models. In this study, the sources of groundwater from hillslopes to valleys were explored by using a multiple-hillside observatory in an agricultural catchment in Southwest China. Stable isotope tracers (e.g., 18O and 2H) combined with the concept of line-conditioned excess (lc-excess), which describes the offset of the groundwater evaporation line from the local meteoric water line, were used to understand the development of kinetic fractionation at specific sampling sites and to further determine the threshold for precipitation recharge to groundwater. The results showed that the isotope kinetic fractionations of soil water and shallow groundwater in the rainy season were stronger than those in the dry season. The precipitation sources and temperatures were the key factors affecting groundwater isotope fractionation. The recharging processes and the increasing lc-excess values from upslope areas to the valleys indicated a greater contribution of lateral flow from the upslope. In the rainy season, precipitation amounts greater than 30 mm d−1 could effectively recharge the shallow groundwater in the study area. Our results confirmed that it was possible to elucidate the recharging mechanisms of shallow groundwater by using the dual-isotope method based on the lc-excess concept. It is anticipated that a broader application of these findings could be helpful for evaluating water resource sustainability in the subtropics, particularly in hilly regions.

A low postabsorptive whole body protein balance is associated with markers of poor daily physical functioning in Chronic Obstructive Pulmonary Disease

Postabsorptive whole body protein kinetics are related to age, gender, body mass index (BMI), and habitual protein intake level. It is unclear how protein synthesis, breakdown, and postabsorptive protein balance rates are affected in Chronic Obstructive Pulmonary Disease (COPD)) and whether these relate to disease severity, lifestyle characteristics and poor daily functioning. We studied 91 COPD (GOLD 1-4) and 56 age matched control subjects without COPD or other chronic or acute health disease/condition in the postabsorptive state and measured body composition by Dual-energy X-ray Absorptiometry, and disease severity and comorbidities by medical screening, blood analysis and questionnaires. We assessed whole body production rates of phenylalanine and tyrosine by pulse stable isotope tracer infusion to calculate whole body protein breakdown (PB) and hydroxylation of phenylalanine to tyrosine, representative of postabsorptive protein balance. We measured muscle and cognitive function, and physical performance by isokinetic dynamometry, cognitive assessments, and 6-min walk test. We assessed physical activity level, mood and dietary protein intake by questionnaires. We measured plasma enrichments by LC-MS/MS and statistics by Fisher's exact test or analysis of covariance. Data are mean [95% CI]. The COPD patients had moderate to severe airflow obstruction, multiple comorbidities, and elevated values for plasma high sensitivity c-reactive protein (hs-CRP) and glucose. Although PB (3630 [3361, 3900] vs 3504 [3297, 3711] umol/h, p=0.1649) was not different, postabsorptive protein balance was lower in COPD patients (274.2 [242.4, 306.1] vs 212.9 [194.7, 231.0] umol/h, p<0.0001), both compared to control subjects. A lower postabsorptive protein balance was associated with age (p<0.0001) and higher levels for systolic blood pressure (p=0.0051) and hs-CRP (p=0.0046) but not with lung function. Furthermore, a lower postabsorptive protein balance level was associated with a lower intake of total calories and protein (p<0.0001) and lower muscle strength (p=0.0248), while only in COPD with a lower physical performance (p=0.0343). We found no association with cognitive function or mood. For all subjects, a cumulative model that included group, gender, age, BMI, systolic blood pressure, hs-CRP, caloric intake, protein intake, and leg strength was able to explain 55% of the variation in postabsorptive protein balance. These data suggest that systemic inflammation, high blood pressure and low protein intake are risk factors of a lower postabsorptive protein balance in COPD patients. A lower postabsorptive protein balance is associated with markers of poor daily physical functioning.

Combining Isotope and Hydrogeochemistry Methods to Study the Seawater Intrusion: A Case Study in Longkou City, Shandong Province, China

In order to study hydrogeochemical effect in the process of seawater intrusion (SI), and provide scientific basis for comprehensive management of water resources and water ecological restoration, the Longkou city of Shandong province in China was taken as an example in this study. Based on the observed data, traditional hydrogeochemistry methods of hydrochemistry analysis, correlation analysis, principal component analysis, and reverse geochemical simulation was firstly comprehensively combined with stable isotope tracing in Longkou city, and this is the first study to use the isotope method to study SI in the study area. The results showed Cl− had high correlation with Na+, Mg2+, and K+. The hydrochemical types of groundwater in Longkou city were mainly HCO3.Cl-Na.Ca, and HCO3.Cl-Ca, showing the evolution of HCO3-Ca to HCO3.Cl-Na to Cl-Na from the inland to the coastline. Stable isotopes analysis with δ2H, δ18O and 87Sr/86Sr indicated the main source of groundwater was atmospheric precipitation. The SI degree was the strongest at the junction of the west and north coast zones, with high values of δ2H and δ18O. The high Sr2+ concentration of groundwater was mainly from SI and groundwater–rock interactions. In the SI process, the mixing of seawater and fresh water took place first, and then different degrees of cation exchange and mineral dissolution and sedimentation occurred. Results of reverse hydrogeochemical simulation showed dolomite and quartz precipitated, with negative migrated masses of 1.38 × 10−3 and 1.08 × 10−5 mol/L on simulation Path 1, respectively, where calcite, halite, and gypsum dissolved with positive migrated masses of 2.89 × 10−3, 3.52 × 10−3, and 4.66 × 10−4 mol/L, respectively, while dolomite and gypsum precipitated and calcite, halite, and quartz dissolved on simulation Path 2. On simulation Path 3, the dolomite, gypsum, halite, and quartz were dissolved, and calcite was precipitated, with a negative migrated mass of 1.77 × 10−4 mol/L.

Abstract P5-02-01: A FACS-free purification method to study estrogen signaling, organoid formation, and metabolic reprogramming in mammary epithelial cells

Abstract Mammary epithelial cells (MECs) are known to have their metabolism reprogrammed following pregnancy to allow the increased energy requirements for lactation. The estrogen receptor α (ERα) is mainly associated with the regulation of biological pathways linked to mammary gland development but its influence on MECs metabolism is still unknown. Our hypothesis is that ERα reprograms cell metabolism in normal MECs, a phenomenon that would be reprogrammed during breast carcinogenesis. Few in vitro models are used to study MECs, and most of them do not express ERα. Primary MECs can be used to overcome this issue, but methods to purify these cells generally require flow cytometry and fluorescence-activated cell sorting (FACS), which require specialized instruments and expertise. Herein, we present in detail a FACS-free protocol for purification and primary culture of mouse MECs to study ERα metabolic functions using mass spectrometry (MS). Purified MECs from nulliparous mice remain differentiated for up to six days with &amp;gt;85% luminal epithelial cells in two-dimensional culture. When seeded in Matrigel, they form organoids that recapitulate the mammary gland morphology in vivo by developing lumens, contractile cells, and lobular structures. MECs express a functional ERα signaling pathway in both two- and three-dimensional cell culture, as shown at the mRNA and protein levels and by the phenotypic characterization. Extracellular metabolic flux analysis showed that estrogens induce a metabolic switch favouring aerobic glycolysis over mitochondrial respiration in MECs grown in two-dimensions, a phenomenon known as the Warburg effect. We also performed (MS)-based metabolomics in organoids. Estrogens altered the levels of metabolites from various pathways, including aerobic glycolysis, citric acid cycle, urea cycle, and amino acid metabolism, demonstrating that ERα reprograms cell metabolism in mammary organoids. To further understand this reprogramming, stable isotope tracer analysis in primary culture organoids are currently performed. In addition, pregnancy and breast-feeding are known to be protective against breast carcinogenesis. Consequently, we also performed MEC purification and organoid culture using mammary glands from multiparous mice. Intriguingly, organoid phenotypic characterization indicated a difference in organoid structures between MECs from nulliparous and multiparous mice. Furthermore, we observe significant differences in estrogenic response between both conditions, suggesting that pregnancy and/or lactation promotes the establishment of specific epigenetic marks that are preserved even ex vivo. Chromatin immunoprecipitation of specific histone marks and MS-based metabolic studies are ongoing to better understand the different responses of mammary organoids to estrogens between nulliparous and multiparous mice. Overall, we have optimized mouse MEC isolation and purification for two- and three-dimensional cultures and for MS-based metabolomics. We demonstrated that these organoids retain a functional ERα pathway over time and that ERα significantly reprograms multiple metabolic pathways. This model represents a valuable tool to study how estrogens modulate mammary gland biology, and particularly how these hormones reprogram metabolism during lactation and breast carcinogenesis. Citation Format: Aurélie Lacouture, Cynthia Jobin, Alisson Clemenceau, Cindy Weidmann, Line Berthiaume, Dominic Bastien, Isabelle Laverdière, Martin Pelletier, Caroline Diorio, Francine Durocher, Étienne Audet-Walsh. A FACS-free purification method to study estrogen signaling, organoid formation, and metabolic reprogramming in mammary epithelial cells [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-02-01.

Themis is indispensable for IL-2 and IL-15 signaling in T cells.

To perform their antiviral and antitumor functions, T cells must integrate signals both from the T cell receptor (TCR), which instruct the cell to remain quiescent or become activated, and from cytokines that guide cellular proliferation and differentiation. In mature CD8+ T cells, Themis has been implicated in integrating TCR and cytokine signals. We investigated whether Themis plays a direct role in cytokine signaling in mature T cells. Themis was required for IL-2- and IL-15-driven CD8+ T cell proliferation both in mice and in vitro. Mechanistically, we found that Themis promoted the activation of the transcription factor Stat and mechanistic target of rapamycin signaling downstream of cytokine receptors. Metabolomics and stable isotope tracing analyses revealed that Themis deficiency reduced glycolysis and serine and nucleotide biosynthesis, demonstrating a receptor-proximal requirement for Themis in triggering the metabolic changes that enable T cell proliferation. The cellular, metabolic, and biochemical defects caused by Themis deficiency were corrected in mice lacking both Themis and the phosphatase Shp1, suggesting that Themis mediates IL-2 and IL-15 receptor-proximal signaling by restraining the activity of Shp1. Together, these results not only shed light on the mechanisms of cytokine signaling but also provide new clues on manipulating T cells for clinical applications.