Isotope Tracer Experiments Research Articles

Spatiotemporally Resolved Approach for Profiling Ferroptosis-Associated Metabolic Vulnerabilities in Tumors Using Mass Spectrometry Imaging and Stable Isotope Tracing.

Ferroptosis, as an iron-dependent cell death mediated by lipid peroxidation, has sparked great interest in the tumor research community. Targeting ferroptosis has been proven to be a new therapeutic opportunity for inhibiting tumor growth. However, it is challenging to precisely characterize the metabolic pattern of ferroptosis in heterogeneous tumors and further identify ferroptosis-associated metabolic vulnerabilities for tumor treatment. In this work, we developed a spatiotemporally resolved method to image ferroptosis-associated metabolic alterations in 3D tumor spheroids by combining mass spectrometry imaging and stable isotope tracing techniques. The construction of a 3D tumor spheroid model allows for a more accurate simulation of ferroptosis, and the introduction of MALDI-MSI enables in situ screening of abnormal molecules in tumor tissues. Using this method, we showed that the expression proportion of nC═C = 4 polyunsaturated fatty acids, including arachidonic acid (FA-20:4) and adrenic acid (FA-22:4), were upregulated in RSL3-induced 3D tumor ferroptosis models. And the isotope tracing experiment revealed that the absorption of fatty acids and the biosynthesis of polyunsaturated fatty acids were significantly increased during ferroptosis. In addition, we discovered that arachidonic acid and adrenic acid supplementation render tumor cell sensitive to ferroptosis, thereby limiting the growth of tumor cells and the formation of 3D tumor spheroids. Such findings provide significant clues for understanding the metabolic signatures of tumor ferroptosis and raise the possibility to screen potential metabolic vulnerabilities for better tumor treatment in combination with ferroptosis.

Parameter Estimation and Identifiability in Kinetic Flux Profiling Models of Metabolism

Metabolic fluxes are the rates of life-sustaining chemical reactions within a cell and metabolites are the components. Determining the changes in these fluxes is crucial to understanding diseases with metabolic causes and consequences. Kinetic flux profiling (KFP) is a method for estimating flux that utilizes data from isotope tracing experiments. In these experiments, the isotope-labeled nutrient is metabolized through a pathway and integrated into the downstream metabolite pools. Measurements of proportion labeled for each metabolite in the pathway are taken at multiple time points and used to fit an ordinary differential equations model with fluxes as parameters. We begin by generalizing the process of converting diagrams of metabolic pathways into mathematical models composed of differential equations and algebraic constraints. The scaled differential equations for proportions of unlabeled metabolite contain parameters related to the metabolic fluxes in the pathway. We investigate flux parameter identifiability given data collected only at the steady state of the differential equation. Next, we give criteria for valid parameter estimations in the case of a large separation of timescales with fast–slow analysis. Bayesian parameter estimation on simulated data from KFP experiments containing both irreversible and reversible reactions illustrates the accuracy and reliability of flux estimations. These analyses provide constraints that serve as guidelines for the design of KFP experiments to estimate metabolic fluxes.

Quantifying protein kinetics in vivo: Influence of precursor dynamics on product labeling.

Protein kinetics can be quantified by coupling stable isotope tracer methods with mass spectrometry readouts; however, inter-connected decision points in the experimental design affect the complexity of the workflow and impact data interpretations. For example, choosing between a single bolus (pulse-chase) or a continuous exposure protocol influences subsequent decisions regarding when to measure and how to model the temporal labeling of a target protein. Herein, we examine the merits of in vivo tracer protocols, we direct attention towards stable isotope tracer experiments that rely on administering a single bolus since these are generally more practical to use as compared to continuous administration protocols. We demonstrate how the interplay between precursor and product kinetics impacts downstream analytics and calculations by contrasting fast vs slow turnover precursors (e.g. 13C-leucine vs 2H-water, respectively). Although the data collected here underscore certain advantages of using longer lived precursors (e.g. 2H- or 18O-water) the results also highlight the influence of tracer recycling on measures of protein turnover. We discuss the impact of tracer recycling and consider how the sampling interval is critical for interpreting studies. Finally, we demonstrate that tracer recycling does not limit the ability to perform back-to-back studies of protein kinetics. It is possible to run experiments in which subjects are used as their own controls even though the precursor and product remain labeled following an initial tracer dosing.

The glutamate/aspartate transporter EAAT1 is crucial for T-cell acute lymphoblastic leukemia proliferation and survival.

T-cell acute lymphoblastic leukemia (T-ALL) is a cancer of the immune system. Approximately 20% of pediatric and 50% of adult T-ALL patients have refractory disease or relapse and die from the disease. To improve patient outcome new therapeutics are needed. With the aim to identify new therapeutic targets, we combined the analysis of T-ALL gene expression and metabolism to identify the metabolic adaptations that T-ALL cells exhibit. We found that glutamine uptake is essential for T-ALL proliferation. Isotope tracing experiments showed that glutamine fuels aspartate synthesis through the tricarboxylic acid cycle and that glutamine and glutamine-derived aspartate together supply three nitrogen atoms in purines and all but one atom in pyrimidine rings. We show that the glutamate-aspartate transporter EAAT1 (SLC1A3), which is normally expressed in the central nervous system, is crucial for glutamine conversion to aspartate and nucleotides and that T-ALL cell proliferation depends on EAAT1 function. Through this work, we identify EAAT1 as a novel therapeutic target for T-ALL treatment.

Hollow TiO2/Bi2MoO6 Janus-nanofibers for photo-reforming of antibiotics into carbon monoxide

Photo-reforming is an environmentally friendly technology for converting organic wastewater into carbon monoxide (CO), a potential energy source and an important industrial material. Achieving efficient photo-reforming of CO production remains a challenge due to the limitation on fast photogenerated carrier recombination. Here, the TiO2/Bi2MoO6 hetero-nanofibers are well-designed to possess unique Janus interface and ingenious hollow structure. The photo-deposition experiments demonstrated that the Janus binary interface can realize the directional migration of interfacial charges and the spatial separation of redox sites. Moreover, the hollow framework allows full exposure of the bicomponent to reactants and provides excellent optical absorption through multiple light scattering inside the tube, as evidenced by theoretical simulations via COMSOL Multiphysics. The photo-reforming yield of CO from 6000 ppm tetracycline hydrochloride (TCH) solution reaches 2.69 μmol∙g−1∙h−1 (corresponding degradation rate of 44.55 % after 5 h), about 1.44-fold superior to that of the TiO2/Bi2MoO6 core-shell hetero-nanofibers. 13C isotope tracing experiments showed that the efficiency of CO production in the photocatalytic TCH oxidation process is superior to that in carbon dioxide reduction process. Furthermore, when oxygen was introduced into the photocatalytic system, the photo-reforming CO yield from the TCH solution was significantly increased to 60.45 μmol∙g−1∙h−1, which was 22.47 and 5.12 times higher than in carbon dioxide and air atmosphere, respectively. This work provides a promising interfacial engineering to design efficient photo-reforming catalysts for energy production and environmental remediation.

Enhancement of nitrogen removal in constructed wetlands through boron mud and elemental sulfur addition: Regulation of sulfur and oxygen cycling

Constructed wetlands (CWs) utilizing elemental sulfur (S0) as an electron donor are effective for nitrate (NO3–-N) removal from low C/N wastewater. However, the limited bioavailability of chemical S0 (chem-S0) due to low water solubility and inadequate mass transfer impedes complete denitrification. In this study, boron mud waste and S0 (S0&BM) were processed and combined to convert chem-S0 to biological S0 (bio-S0) in CWs. The employment of S0&BM improved NO3–-N and total nitrogen (TN) removal rates by 15.36 % and 10.55 % compared to S0 alone. The 15N isotope tracing experiment unveiled the pathways of N2O production and demonstrated that S0&BM facilitated full denitrification, resulting in a higher percentage of nitrogen being degraded in a non-polluting form (54.70 %). Releasing trace amounts of boron and magnesium in S0&BM mitigated the excessive acidification caused by the sulfur autotrophic process and promoted biofilm formation, thereby pronouncing vertical oxygen fluctuations. Meanwhile, oxygen vacancies generated in the S0&BM promoted the closed-loop sulfur cycle, starting from the oxidation of chem-S0 to sulfate (SO42-), the important process of SO42--reducing bacteria using partial organic compounds as electron donors to reduce SO42- to sulfide (S2-) was realized, and finally the S2- was oxidized to bio-S0 with better properties. S0&BM enriched functional microbial communities in the biofilm related to nitrogen and sulfur transformation. Functional gene analysis showed an increase in the narG, nirS, norB, and nosZ genes supporting denitrification. The dsrA and sqr genes responsible for the sulfur cycle exhibited the highest abundance. This realization allowed for the utilization of waste materials for treatment, enhancing the energy efficiency of carbon reduction and pollution reduction in CWs.

Tissue Inhibitor of Metalloproteinase 3 (TIMP3) mutations increase glycolytic activity and dysregulate glutamine metabolism in RPE cells

ObjectivesMutations in Tissue Inhibitor of Metalloproteinases 3 (TIMP3) cause Sorsby's Fundus Dystrophy (SFD), a dominantly inherited, rare form of macular degeneration that results in vision loss. TIMP3 is synthesized primarily by retinal pigment epithelial (RPE) cells, which constitute the outer blood-retinal barrier. One major function of RPE is the synthesis and transport of vital nutrients, such as glucose, to the retina. Recently, metabolic dysfunction in RPE cells has emerged as an important contributing factor in retinal degenerations. We set out to determine if RPE metabolic dysfunction was contributing to SFD pathogenesis. MethodsQuantitative proteomics was conducted on RPE of mice expressing the S179C variant of TIMP3, known to be causative of SFD in humans. Proteins found to be differentially expressed (P < 0.05) were analyzed using statistical overrepresentation analysis to determine enriched pathways, processes, and protein classes using g:profiler and PANTHER Gene Ontology. We examined the effects of mutant TIMP3 on RPE metabolism using human ARPE-19 cells expressing mutant S179C TIMP3 and patient-derived induced pluripotent stem cell-derived RPE (iRPE) carrying the S204C TIMP3 mutation. RPE metabolism was directly probed using isotopic tracing coupled with GC/MS analysis. Steady state [U–13C6] glucose isotopic tracing was preliminarily conducted on S179C ARPE-19 followed by [U–13C6] glucose and [U–13C5] glutamine isotopic tracing in SFD iRPE cells. ResultsQuantitative proteomics and enrichment analysis conducted on RPE of mice expressing mutant S179C TIMP3 identified differentially expressed proteins that were enriched for metabolism-related pathways and processes. Notably these results highlighted dysregulated glycolysis and glucose metabolism. Stable isotope tracing experiments with [U–13C6] glucose demonstrated enhanced glucose utilization and glycolytic activity in S179C TIMP3 APRE-19 cells. Similarly, [U–13C6] glucose tracing in SFD iRPE revealed increased glucose contribution to glycolysis and the TCA cycle. Additionally, [U–13C5] glutamine tracing found evidence of altered malic enzyme activity. ConclusionsThis study provides important information on the dysregulation of RPE glucose metabolism in SFD and implicates a potential commonality with other retinal degenerative diseases, emphasizing RPE cellular metabolism as a therapeutic target.

Deciphering the Lithium-Ion Conduction Mechanism of LiH in Solid-Electrolyte Interphase.

Lithium hydride (LiH) has been widely recognized as the critical component of the solid-electrolyte interphase (SEI) in Li batteries. Although the formation mechanism and structural model of LiH in SEI have been extensively reported, the role in electro-performance of LiH in SEI is stillambiguous and has proven challenging to explored due to the complicated structure SEI and the lack of advanced in situ experimental technology. In this study, the isotopic exchange experiments combined with isotopic tracer experiments is applied to solidly illustrate the superior conductivity and Li+ conduction behavior of the LiH in natural SEI. Importantly, in situ transmission electron microscopy analysis is utilized to visualize the self-electrochemical decomposition of LiH, which is significantly distinctive from LiF and Li2O. The critical experimental evidence discovered by the work demonstrates ion transport behaviors of key components in the SEI, which is imperative for designing novel SEI and augurs a new area in optimizing the performance of lithium batteries.

Enhanced Degradation of Micropollutants in a Peracetic Acid/Mn(II) System with EDDS: An Investigation of the Role of Mn Species.

Extensive research has been conducted on the utilization of a metal-based catalyst to activate peracetic acid (PAA) for the degradation of micropollutants (MPs) in water. Mn(II) is a commonly employed catalyst for homogeneous advanced oxidation processes (AOPs), but its catalytic performance with PAA is poor. This study showed that the environmentally friendly chelator ethylenediamine-N,N'-disuccinic acid (EDDS) could greatly facilitate the activation of Mn(II) in PAA for complete atrazine (ATZ) degradation. In this process, the EDDS enhanced the catalytic activity of manganese (Mn) and prevented disproportionation of transient Mn species, thus facilitating the decay of PAA and mineralization of ATZ. By employing electron spin resonance detection, quenching and probe tests, and 18O isotope-tracing experiments, the significance of high-valent Mn-oxo species (Mn(V)) in the Mn(II)-EDDS/PAA system was revealed. In particular, the involvement of the Mn(III) species was essential for the formation of Mn(V). Mn(III) species, along with singlet oxygen (1O2) and acetyl(per)oxyl radicals (CH3C(O)O•/CH3C(O)OO•), also contributed partially to ATZ degradation. Mass spectrometry and density functional theory methods were used to study the transformation pathway and mechanism of ATZ. The toxicity assessment of the oxidative products indicated that the toxicity of ATZ decreased after the degradation reaction. Moreover, the system exhibited excellent interference resistance toward various anions and humid acid (HA), and it could selectively degrade multiple MPs.

ATP-dependent citrate lyase Drives Left Ventricular Dysfunction by Metabolic Remodeling of the Heart.

Metabolic remodeling is a hallmark of the failing heart. Oncometabolic stress during cancer increases the activity and abundance of the ATP-dependent citrate lyase (ACL, Acly ), which promotes histone acetylation and cardiac adaptation. ACL is critical for the de novo synthesis of lipids, but how these metabolic alterations contribute to cardiac structural and functional changes remains unclear. We utilized human heart tissue samples from healthy donor hearts and patients with hypertrophic cardiomyopathy. Further, we used CRISPR/Cas9 gene editing to inactivate Acly in cardiomyocytes of MyH6-Cas9 mice. In vivo, positron emission tomography and ex vivo stable isotope tracer labeling were used to quantify metabolic flux changes in response to the loss of ACL. We conducted a multi-omics analysis using RNA-sequencing and mass spectrometry-based metabolomics and proteomics. Experimental data were integrated into computational modeling using the metabolic network CardioNet to identify significantly dysregulated metabolic processes at a systems level. Here, we show that in mice, ACL drives metabolic adaptation in the heart to sustain contractile function, histone acetylation, and lipid modulation. Notably, we show that loss of ACL increases glucose oxidation while maintaining fatty acid oxidation. Ex vivo isotope tracing experiments revealed a reduced efflux of glucose-derived citrate from the mitochondria into the cytosol, confirming that citrate is required for reductive metabolism in the heart. We demonstrate that YAP inactivation facilitates ACL deficiency. Computational flux analysis and integrative multi-omics analysis indicate that loss of ACL induces alternative isocitrate dehydrogenase 1 flux to compensate. This study mechanistically delineates how cardiac metabolism compensates for suppressed citrate metabolism in response to ACL loss and uncovers metabolic vulnerabilities in the heart.

Spatiotemporal patterns and co-occurrence patterns of dissimilatory nitrate reduction to ammonium community in sediments of the Lancang River cascade reservoirs.

Dissimilatory nitrate reduction to ammonium (DNRA) is an important nitrate reduction pathway in freshwater sediments. Many studies have focused on the DNRA process in various natural habitats. However, the joint operation of cascade reservoirs will affect the physical and chemical properties of sediments, which may change the DNRA process and bacterial community pattern in the surface sediments of cascade reservoirs. Our study was the first to investigate the spatiotemporal distribution patterns of potential DNRA rate, nrfA gene abundances, and DNRA bacterial community diversity in surface sediments of the Lancang River cascade reservoirs. The results of slurry incubation experiments combined with the 15N isotope tracer experiment ascertained that the potential rates of DNRA were 0.01-0.15 nmol-N cm-3 h-1, and qPCR results indicated that the abundance range of nrfA was 1.08 × 105-2.51 × 106 copies g-1 dry weight. High throughput sequencing of the nrfA gene revealed that the relative abundance of Anaeromyxobacter (4.52% on average), Polyangium (4.09%), Archangium (1.86%), Geobacter (1.34%), and Lacunisphaera (1.32%) were high. Pearson and RDA correlation analysis exhibited that nrfA gene abundance was positively correlated with altitude, pH, OC, and sand concentration. Anaeromyxobacter was positively correlated with reservoir age and DNRA potential rate. The deterministic environmental selection process plays a crucial role in the formation of the DNRA bacterial community. Network analysis displayed that the dominant DNRA genus was the key population of the DNRA microbial community in the sediments of Lancang River cascade reservoirs. This study reveals that the variation of DNRA bacterial activity and community structure is largely driven by the construction of cascade reservoirs, and provides a new idea for further understanding the characteristics of the DNRA community in the cascade reservoir ecosystem.

UnitedMet harnesses RNA-metabolite covariation to impute metabolite levels in clinical samples.

Comprehensively studying metabolism requires the measurement of metabolite levels. However, in contrast to the broad availability of gene expression data, metabolites are rarely measured in large molecularly-defined cohorts of tissue samples. To address this basic barrier to metabolic discovery, we propose a Bayesian framework ("UnitedMet") which leverages the empirical strength of RNA-metabolite covariation to impute otherwise unmeasured metabolite levels from widely available transcriptomic data. We demonstrate that UnitedMet is equally capable of imputing whole pool sizes as well as the outcomes of isotope tracing experiments. We apply UnitedMet to investigate the metabolic impact of driver mutations in kidney cancer, identifying a novel association between BAP1 and a highly oxidative tumor phenotype. We similarly apply UnitedMet to determine that advanced kidney cancers upregulate oxidative phosphorylation relative to early-stage disease, that oxidative metabolism in kidney cancer is associated with inferior outcomes to combination therapy, and that kidney cancer metastases themselves demonstrate elevated oxidative phosphorylation relative to primary tumors. UnitedMet therefore enables the assessment of metabolic phenotypes in contexts where metabolite measurements were not taken or are otherwise infeasible, opening new avenues for the generation and evaluation of metabolite-centered hypotheses. UnitedMet is open source and publicly available (https://github.com/reznik-lab/UnitedMet).

Recovering palladium and gold by peroxydisulfate-based advanced oxidation process.

Palladium (Pd) and gold (Au) are the most often used precious metals (PMs) in industrial catalysis and electronics. Green recycling of Pd and Au is crucial and difficult. Here, we report a peroxydisulfate (PDS)-based advanced oxidation process (AOPs) for selectively recovering Pd and Au from spent catalysts. The PDS/NaCl photochemical system achieves complete dissolution of Pd and Au. By introducing Fe(II), the PDS/FeCl2·4H2O solution functioned as Fenton-like system, enhancing the leaching efficiency without xenon (Xe) lamp irradiation. Electron paramagnetic resonance (EPR), 18O isotope tracing experiments, and density functional theory calculations revealed that the reactive oxidation species of SO4·-, ·OH, and Fe(IV)═O were responsible for the oxidative dissolution process. Lixiviant leaching and one-step electrodeposition recovered high-purity Pd and Au. Strong acids, poisonous cyanide, and volatile organic solvents were not used during the whole recovery, which enables an efficient and sustainable precious metal recovery approach and encourage AOP technology for secondary resource recycling.

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.

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.

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.

Inferring mitochondrial and cytosolic metabolism by coupling isotope tracing and deconvolution

The inability to inspect metabolic activities within distinct subcellular compartments has been a major barrier to our understanding of eukaryotic cell metabolism. Previous work addressed this challenge by analyzing metabolism in isolated organelles, which grossly bias metabolic activity. Here, we describe a method for inferring physiological metabolic fluxes and metabolite concentrations in mitochondria and cytosol based on isotope tracing experiments performed with intact cells. This is made possible by computational deconvolution of metabolite isotopic labeling patterns and concentrations into cytosolic and mitochondrial counterparts, coupled with metabolic and thermodynamic modelling. Our approach lowers the uncertainty regarding compartmentalized fluxes and concentrations by one and three orders of magnitude compared to existing modelling approaches, respectively. We derive a quantitative view of mitochondrial and cytosolic metabolic activities in central carbon metabolism across cultured cell lines without performing cell fractionation, finding major variability in compartmentalized malate-aspartate shuttle fluxes. We expect our approach for inferring metabolism at a subcellular resolution to be instrumental for a variety of studies of metabolic dysfunction in human disease and for bioengineering.

Capturing the Dynamic Processes of Porosity Clogging

AbstractUnderstanding mineral precipitation induced porosity clogging and being able to quantify its non‐linear feedback on transport properties is fundamental for predicting the long‐term evolution of energy‐related subsurface systems. Commonly applied porosity‐diffusivity relations used in numerical simulations on the continuum‐scale predict the case of clogging as a final state. However, recent experiments and pore‐scale modeling investigations suggest dissolution‐recrystallization processes causing a non‐negligible inherent diffusivity of newly formed precipitates. To verify these processes, we present a novel microfluidic reactor design that combines time‐lapse optical microscopy and confocal Raman spectroscopy, providing real‐time insights of mineral precipitation induced porosity clogging under purely diffusive transport conditions. Based on 2D optical images, the effective diffusivity was determined as a function of the evolving porous media, using pore‐scale modeling. At the clogged state, Raman isotopic tracer experiments were conducted to visualize the transport of deuterium through the evolving microporosity of the precipitates, demonstrating the non‐final state of clogging. The evolution of the porosity‐diffusivity relationship in response to precipitation reactions shows a behavior deviating from Archie's law. The application of an extended power law improved the description of the evolving porosity‐diffusivity, but still neglected post‐clogging features. Our innovative combination of microfluidic experiments and pore‐scale modeling opens new possibilities to validate and identify relevant pore‐scale processes, providing data for upscaling approaches to derive key relationships for continuum‐scale reactive transport simulations.

Effects of 13C isotope-labeled allelochemicals on the growth of the invasive plant Alternantheraphiloxeroides

The secondary metabolites of indigenous plants have significant allelopathic inhibitory effects on the growth and development of invasive alien plants. Methyl palmitate (MP) and methyl linolenate (ML) were used as exogenous allelopathic substances. The research investigated the differences of inhibitory effects of MP and ML on the growth of seedlings of Alternantheraphiloxeroides, and calculated their morphological characteristics, biomass, physiological indicators and the response index (RI). The synthetical allelopathic index (SE) of 1 mmol/L MP was the smallest (− 0.26) and the allelopathic inhibition was the strongest; therefore, it was selected as a 13C-labeled allelochemical. The distribution of 1 mmol/L MP in different parts of A.philoxeroides and the correlation between the biomass ratios of roots, stems and leaves and the 13C content were studied by 13C stable isotope tracing experiments. Atom percent excess (APE) between roots, stems and leaves of A.philoxeroides treated with 1 mmol/L MP were significantly different in terms of magnitude, with leaves (0.17%) > roots (0.12%) > stems (0.07%). The root, stem and leaf biomass ratios of invasive weeds had great significant positive correlation with 13C content (p < 0.01, R2 between 0.96 and 0.99). This current research provides a new idea and method for the control of A.philoxeroides, but large-scale popularization remains to be studied.

Human acute leukemia uses branched-chain amino acid catabolism to maintain stemness through regulating PRC2 function.

Cancer-specific metabolic activities play a crucial role in the pathogenesis of human malignancies. To investigate human acute leukemia-specific metabolic properties, we comprehensively measured the cellular metabolites within the CD34+ fraction of normal hematopoietic stem progenitor cells (HSPCs), primary human acute myelogenous leukemia (AML), and acute lymphoblastic leukemia (ALL) cells. Here, we show that human leukemia cells are addicted to the branched-chain amino acid (BCAA) metabolism to maintain their stemness, irrespective of myeloid or lymphoid types. Human primary acute leukemias had BCAA transporters for BCAA uptake, cellular BCAA, α-ketoglutarate (α-KG), and cytoplasmic BCAA transaminase-1 (BCAT1) at significantly higher levels than control HSPCs. Isotope-tracing experiments showed that in primary leukemia cells, BCAT1 actively catabolizes BCAA using α-KG into branched-chain α-ketoacids, whose metabolic processes provide leukemia cells with critical substrates for the trichloroacetic acid cycle and the synthesis of nonessential amino acids, both of which reproduce α-KG to maintain its cellular level. In xenogeneic transplantation experiments, deprivation of BCAA from daily diet strongly inhibited expansion, engraftment and self-renewal of human acute leukemia cells. Inhibition of BCAA catabolism in primary AML or ALL cells specifically inactivates the function of the polycomb repressive complex 2, an epigenetic regulator for stem cell signatures, by inhibiting the transcription of PRC components, such as zeste homolog 2 and embryonic ectoderm development. Accordingly, BCAA catabolism plays an important role in the maintenance of stemness in primary human AML and ALL, and molecules related to the BCAA metabolism pathway should be critical targets for acute leukemia treatment.