13C Enrichment Research Articles

Stable Isotope Tracing Reveals an Altered Fate of Glucose in N-Acetyltransferase 1 Knockout Breast Cancer Cells.

Breast cancer is one of the leading causes of cancer death. Recent studies found that arylamine N-acetyltransferase 1 (NAT1) is frequently upregulated in breast cancer, further suggesting NAT1 could be a potential therapeutic target for breast cancer. Previous publications have established that NAT1 knockout (KO) in breast cancer cell lines leads to growth reduction both in vitro and in vivo and metabolic changes. These reports suggest that NAT1 contributes to the energy metabolism of breast cancer cells. Proteomic analysis and non-targeted metabolomics suggested that NAT1 KO may change the fate of glucose as it relates to the TCA/KREB cycle of the mitochondria of breast cancer cells. In this current study, we used [U-13C]-glucose stable isotope resolved metabolomics to determine the effect of NAT1 KO on the metabolic profile of MDA-MB-231 breast cancer cells. We incubated breast cancer cells (MDA-MB-231 cells) and NAT1 Crispr KO cells (KO#2 and KO#5) with [U-13C]-glucose for 24 h. Tracer incubation polar metabolites from the cells were extracted and analyzed by 2DLC-MS, and metabolite differences were compared between the parental and NAT1 KO cells. Differences consistent between the two KO cells were considered changes due to the loss of NAT1. The data revealed decreases in the 13C enrichment of TCA/Krebs cycle intermediates in NAT1 KO cells compared to the MDA-MB-231 cells. Specifically, 13C-labeled citrate, isocitrate, a-ketoglutarate, fumarate, and malate were all decreased in NAT1 KO cells. We also detected increased 13C-labeled L-lactate levels in the NAT1 KO cells and decreased 13C enrichment in some nucleotides. Pathway analysis showed that arginine biosynthesis, alanine, aspartate and glutamate metabolism, and the TCA cycle were most affected. These data provide additional evidence supporting the impacts of NAT1 knockout on cellular energy metabolism. The data suggest that NAT1 expression is important for the proper functioning of mitochondria and the flux of glucose through the TCA/Krebs cycle in breast cancer cells. The metabolism changes in the fate of glucose in NAT1 KO breast cancer cells offer more insight into the role of NAT1 in energy metabolism and the growth of breast cancer cells. These data provide additional evidence that NAT1 may be a useful therapeutic target for breast cancer.

Long-term different fertilization regimes impact on the fate of root-derived C and microbial community structure in paddy soil

It is known that fertilization pattern could alter soil nutrient and organic matter status. However, it is still unclear how photosynthesized C is distributed in rice–rhizosphere soil-microbial system and its influencing factors due to fertilization pattern and subsequent changes in soil fertility. This study examined the allocation of rice photosynthetic C in rice–rhizosphere soil–microbial systems and the changes in microbial community under three long-term fertilizer regimes. Rice was grown in soils with 8-year history of no fertilizer, inorganic fertilizer (NPK) or NPK fertilizer plus cattle manure (NPK + CM), and was pulse–labelled with 13CO2 at rice tillering stage. The results showed that 13C incorporation in rice-rhizosphere soil system was the highest in the NPK + CM treatment, increased by 489% and 28.9% compared to unfertilized control and the NPK alone. In the >2 mm aggregate fraction, the 13C enrichment was higher in NPK + CM compared to no-fertilized control and NPK treatments. Total PLFA and PLFA groups contents in all aggregate size fractions were the greatest in the NPK + CM treatment. Partial least squares path modelling analysis revealed that microbial community in large macroaggregate (>2 mm) was significantly (P < 0.05) affected by root properties, while soil fertility had a significant (P < 0.01) impact on microbial community in microaggregate (<0.25 mm). The redundancy analysis showed that soil parameters except soil alkali-hydro nitrogen (AN) significantly impacted the soil microbial communities. Through canonical variation partitioning analysis, soil available K was the most important factor leading to variance (11.4%) in microbial community composition. On the other hand, soil pH significantly (P < 0.05) affected the distribution of 13C in microbe community. More than 20% of 13C was incorporated into G– and G+ bacteria across all treatments. Overall, it was observed the NPK fertilizer plus cattle manure treatment was most conducive to retain C from root exudates in the rice-rhizosphere soil-microbial system.

Novel 13C enrichment technique reveals early turnover of DHA in peripheral tissues

The brain is rich in DHA, which plays important roles in regulating neuronal function. Recently, using compound-specific isotope analysis that takes advantage of natural differences in carbon-13 content (13C/12C ratio or δ13C) of the food supply, we determined the brain DHA half-life. However, because of methodological limitations, we were unable to capture DHA turnover rates in peripheral tissues. In the current study, we applied compound-specific isotope analysis via high-precision GC combustion isotope ratio mass spectrometry to determine half-lives of brain, liver, and plasma DHA in mice following a dietary switch experiment. To model DHA tissue turnover rates in peripheral tissues, we added earlier time points within the diet switch study and took advantage of natural variations in the δ13C-DHA of algal and fish DHA sources to maintain DHA pool sizes and used an enriched (uniformly labeled 13C) DHA treatment. Mice were fed a fish-DHA diet (control) for 3 months, then switched to an algal-DHA treatment diet, the 13C enriched-DHA treatment diet, or they stayed on the control diet for the remainder of the study time course. In mice fed the algal and 13C enriched-DHA diets, the brain DHA half-life was 47 and 46 days, the liver half-life was 5.6 and 7.2 days, and the plasma half-life was 4.7 and 6.4 days, respectively. By using improved methodologies, we calculated DHA turnover rates in the liver and plasma, and our study for the first time, by using an enriched DHA source (very high δ13C), validated its utility in diet switch studies.

Role of root hair elongation in rhizosheath aggregation and in the carbon flow into the soil

One of the most prominent changes in the rhizospheric soil structure is associated with the formation of a strongly bound soil layer in the surroundings of the root, which is named rhizosheath. In this study, we investigated how root hair elongation, a ubiquitous root morphological trait, affect the stability of rhizosheath aggregates. Using 13CO2 pulse labeling, we tracked the fate of root-derived 13C inputted into the rhizosheath of two Zea mays L. genotypes with contrasting root hair elongation: a mutant with root hair defective elongation (rth3) and a corresponding wild type (WT). In addition, we also investigated the differences between two 13CO2 labeling approaches (single vs. multiple pulse labeling) in the distribution of 13C in the rhizosheath aggregates. We were able to demonstrate that the rhizosheath aggregate stability and the resulting aggregate size distribution follows the same mechanisms irrespective of the root hair elongation. This result reinforces the assumption that other soil properties are more decisive for the soil structure formation in the rhizosheath in comparison to root hair elongation. The majority of recently deposited root-derived C (57%) was found in the macroaggregates. Increasing the number of pulses (multiple pulse labeling approach) resulted in a higher 13C enrichment of the rhizosheath aggregates fractions in comparison to the application of a single pulse. While both labeling approaches resulted in a similar distribution of 13C in the rhizosheath aggregates, the higher enrichment given by multiple pulse labeling allowed the separation of significant differences between the genotypes in plant C allocation in the rhizosheath.

13C surface characterization of midplane and crown collector probes on DIII-D

A dual collector probe system has been implemented on DIII-D for scrape-off-layer (SOL) impurity transport studies. These experiments injected isotopically enriched methane (13CD4) and sampled the impurities from this extrinsic, primary source with graphite collector probes at the outboard midplane and crown of upper single null L-mode plasmas. Using a stable isotopic mixing model, results suggest that 13C from methane injections prior to these experiments has built up on the walls of DIII-D to act as a secondary, intrinsic source of enriched 13C to the collector probes. This secondary source accounts for nearly 60% of the deposits on the midplane collector probes and nearly 90% of the deposition on the collector probes in the crown. These results lay the foundation for future impurity transport models and suggest that further simulation of impurity transport during the methane injection experiments will require two sources of enriched impurities in order to accurately model the SOL impurity profiles of 13C.

Production of Large Amounts of [13C]Leucine-Enriched Milk Proteins by Lactating Cows

During protein metabolism kinetic studies, oral tracers are administered as labeled free amino acids and do not necessarily represent the metabolic fate of amino acids ingested as proteins. However, sufficient quantities of 13C-labeled proteins are not currently available. We here present a new methodology for producing large amounts of milk proteins intrinsically labeled with [13C]leucine. After surgical preparation, two lactating cows were infused with 80–90 g of L-[1-13C]leucine for 24–32 h, and milk was collected during and after the infusion. Casein and whey protein fractions were purified by membrane separation techniques. Arteriovenous balance across the udder indicated a very efficient extraction of leucine by the mammary gland. Five batches of pure casein and whey proteins, totaling 3854 g of protein of excellent bacteriological quality, were obtained. Two thirds of these proteins had [13C]leucine enrichments ranging from 10.5 to 19.4% ([13C]leucine atom percent excess). The overall tracer recoveries were 22 and 27% (cows 1 and 2, respectively). Thus, pure milk proteins were produced in large amounts with sufficient 13C enrichment to be used in human protein metabolism studies.

Recently fixed carbon fuels microbial activity several meters below the soil surface

Abstract. The deep soil, &gt;1 m, harbors a substantial share of the global microbial biomass. Currently, it is not known whether microbial activity several meters below the surface is fueled by recently fixed carbon or by old carbon that persisted in soil for several hundred years. Understanding the carbon source of microbial activity in deep soil is important to identify the drivers of biotic processes in the critical zone. Therefore, we explored carbon cycling in soils in three climate zones (arid, mediterranean, and humid) of the Coastal Cordillera of Chile down to a depth of 6 m, using carbon isotopes. Specifically, we determined the 13C : 12C ratio (δ13C) of soil and roots and the 14C : 12C ratio (Δ14C) of soil organic carbon and CO2–C respired by microorganisms. We found that the Δ14C of the respired CO2–C was significantly higher than that of the soil organic carbon in all soils. Further, we found that the δ13C of the soil organic carbon changed only in the upper decimeters (by less than 6 ‰). Our results show that microbial activity several meters below the soil surface is mostly fueled by recently fixed carbon that is on average much younger than the total soil organic carbon present in the respective soil depth increments, in all three climate zones. Further, our results indicate that most decomposition that leads to enrichment of 13C occurs in the upper decimeters of the soils, which is possibly due to stabilization of organic carbon in the deep soil. In conclusion, our study demonstrates that microbial processes in the deep soil several meters below the surface are closely tied to input of recently fixed carbon.

Carbon stabilization pathways in soil aggregates during long-term forest succession: Implications from δ13C signatures

Forest restoration increases organic carbon (OC) sequestration mainly via the additional litter input and improvements in soil structure that result in biochemical and physical C stabilization over the short term. However, the pathways of long-term C stabilization in soil aggregates during forest succession are unclear. To characterize the long-term C fluxes, the aggregate-associated OC content and C flow pathways were examined over 160 years of secondary successional chronosequence from Lespedeza bicolor to Quercus liaotungensis. The contribution of plant-derived C (litters and fine roots) to aggregate-associated OC was assessed using 13C natural abundance. The proportion of macroaggregates increased but microaggregates decreased along forest succession, and the macroaggregate-associated OC content increased from 4.6 to 28 g kg−1 during succession. The 13C enrichment trend was common in all aggregate size classes: macroaggregates to silt and clay size classes. The maximum δ13C was −23‰ in the silt and clay size classes in the pioneer weed stage at 20−30 cm soil depth. The C pathways followed the trend from macroaggregates to silt and clay size classes. The intensity of the C flows decreased in the topsoil (<10 cm), but plant-derived C stocks within the aggregates increased during forest succession over the 160 years. Notably, fine roots made a greater contribution to the OC accumulation within aggregates than aboveground litter did. The microbial biomass was an important factor affecting fine root-derived C stocks in the aggregates. Concluding, this analysis of natural δ13C signature provides detailed insights into long-term C stabilization pathways associated with soil aggregates during forest succession.

Effect of light conditions on trophic level and gene expression of partially mycoheterotrophic orchid, Cymbidium goeringii

ABSTRACT Partial mycoheterotrophic i.e., mixotrophic, plants are the species which partially depend on mycorrhizal fungi for its nutrients. Although some of these plants are known to show plasticity in the degree of fungal dependence induced by the changes in light condition, the genetic background of this plasticity is largely unsolved. Here, we investigated the relationships between environmental conditions and nutrient sources based on 13C and 15N enrichment in mixotrophic orchid Cymbidium goeringii. We also shaded them for 2 months and evaluated the effect of light condition on the nutrient sources based on the abundance of 13C and 15N and the gene expressions by RNA-seq based de novo assembly. The shading had no effect on isotope enrichment, possibly because of the translocation of carbon and nitrogen from the storage organs. Gene expression analysis showed the upregulation of genes involved in jasmonic acid response in leaves of the shaded plants, which suggests that the jasmonic acid played an important role in regulation of degree of dependence against the mycorrhizal fungi. Our results suggest that mixotrophic plants might be controlling their dependency against the mycorrhizal fungi by a common mechanism with the autotrophic plants.

Turnover of the extracellular polymeric matrix of granules performing biological phosphate removal

Polyphosphate accumulating organisms (PAOs) are responsible for enhanced biological phosphate removal (EBPR) from wastewater, where they grow embedded in a matrix of extracellular polymeric substances (EPS). EPSs comprise a mixture of biopolymers like polysaccharides or (glyco)proteins. Despite previous studies, little is known about the dynamics of EPS in mixed cultures, and their production by PAOs and potential consumption by flanking microbes. EPSs are biodegradable and have been suggested to be a substrate for other organisms in the community. Studying EPS turnover can help elucidate their biosynthesis and biodegradation cycles. We analyzed the turnover of proteins and polysaccharides in the EPS of an enrichment culture of PAOs relative to the turnover of internal proteins. An anaerobic-aerobic sequencing batch reactor (SBR) simulating EBPR conditions was operated to enrich for PAOs. After achieving a stable culture, carbon source was switched to uniformly 13C-labeled acetate. Samples were collected at the end of each aerobic phase. EPSs were extracted by alkaline treatment. 13C enrichment in proteins and sugars (after hydrolysis of polysaccharides) in the extracted EPS were measured by mass spectrometry. The average turnover rate of sugars and proteins (0.167 and 0.192 d−1 respectively) was higher than the expected value based on the solid removal rate (0.132 d−1), and no significant difference was observed between intracellular and extracellular proteins. This indicates that EPS from the PAO enriched community is not selectively degraded by flanking populations under stable EBPR process conditions. Instead, we observed general decay of biomass, which corresponds to a value of 0.048 d−1.Key Points• Proteins showed a higher turnover rate than carbohydrates.• Turnover of EPS was similar to the turnover of intracellular proteins.• EPS is not preferentially consumed by flanking populations.

Response of zooplankton to inputs of terrestrial dissolved organic matter: Food quality constraints induced by microbes

AbstractTerrestrial inputs of dissolved organic matter (DOM) stimulate microbial growth by supplying organic carbon and DOM‐associated nutrients, and thus increase food availability for zooplankton. Terrestrial inputs of DOM might decrease the content of eicosapentaenoic acid (EPA) in particular organic matter (POM) due to an increase in biomass of EPA‐poor microbes, for example, heterotrophic bacteria and autotrophic cyanobacteria, thereby reducing food quality and filter‐feeding zooplankton's biomass. We performed a 26‐d mesocosm experiment where terrestrial DOM was manipulated by adding (treatments) and kept without adding (controls) 13C‐labeled maize leachate to evaluate the effect of food quality changes induced by terrestrial DOM inputs on zooplankton. DOM addition treatments had significantly higher concentrations of POM and Chlorophyll a relative to controls, but significant lower EPA content of POM, responding to increased biomass of bacteria and chlorophytes. Biomass and EPA content of filter‐feeding cladoceran Chydorus sphaericus was significantly reduced by terrestrial DOM addition. Stable isotope analyses showed 13C enrichments in bacteria and cryptophytes in the treatment of 13C‐labeled maize leachate, indicating a growth stimulation of heterotrophic microbes by organic carbon addition. Although the 13C enrichment in Chydorus evidenced the extensive use of terrestrial DOM by this cladoceran, positive relationships between Chydorus biomass and EPA content of POM suggest that terrestrial DOM inputs increase food quality constrains on filter‐feeding zooplankton. Our study suggests that the response of filter‐feeding zooplankton to inputs of terrestrial DOM depends on how the terrestrial inputs change the microbial communities and thus the food quality in the receiving aquatic ecosystems.

Using Kinetic Modelling to Infer Adaptations in Saccharomyces cerevisiae Carbohydrate Storage Metabolism to Dynamic Substrate Conditions.

Microbial metabolism is strongly dependent on the environmental conditions. While these can be well controlled under laboratory conditions, large-scale bioreactors are characterized by inhomogeneities and consequently dynamic conditions for the organisms. How Saccharomyces cerevisiae response to frequent perturbations in industrial bioreactors is still not understood mechanistically. To study the adjustments to prolonged dynamic conditions, we used published repeated substrate perturbation regime experimental data, extended it with proteomic measurements and used both for modelling approaches. Multiple types of data were combined; including quantitative metabolome, 13C enrichment and flux quantification data. Kinetic metabolic modelling was applied to study the relevant intracellular metabolic response dynamics. An existing model of yeast central carbon metabolism was extended, and different subsets of enzymatic kinetic constants were estimated. A novel parameter estimation pipeline based on combinatorial enzyme selection supplemented by regularization was developed to identify and predict the minimum enzyme and parameter adjustments from steady-state to dynamic substrate conditions. This approach predicted proteomic changes in hexose transport and phosphorylation reactions, which were additionally confirmed by proteome measurements. Nevertheless, the modelling also hints at a yet unknown kinetic or regulation phenomenon. Some intracellular fluxes could not be reproduced by mechanistic rate laws, including hexose transport and intracellular trehalase activity during substrate perturbation cycles.

Neogene burial of organic carbon in the global ocean.

Organic carbon buried in marine sediment serves as a net sink for atmospheric carbon dioxide and a source of oxygen1,2. The rate of organic carbon burial through geologic history is conventionally established by using the mass balance between inorganic and organic carbon, each with distinct carbon isotopic values (δ13C)3,4. This method is complicated by large uncertainties, however, and has not been tested with organic carbon accumulation data5,6. Here we report a 'bottom-up' approach for calculating the rate of organic carbon burial that is independent from mass balance calculations. We use data from 81 globally distributed sites to establish the history of organic carbon burial during the Neogene (roughly 23-3 Ma). Our results show larger spatiotemporal variability of organic carbon burial than previously estimated7-9. Globally, the burial rate is high towards the early Miocene and Pliocene and lowest during the mid-Miocene, with the latter period characterized by the lowest ratio of organic-to-carbonate burial rates. This is in contrast to earlier work that interpreted enriched carbonate 13C values of the mid-Miocene as massive organic carbon burial (that is, the Monterey Hypothesis)10,11. Suppressed organic carbon burial during the warm mid-Miocene is probably related to temperature-dependent bacterial degradation of organic matter12,13, suggesting that the organic carbon cycle acted as positive feedback of past global warming.

Interpreting δ13C Values Obtained on SOM from Ancient Maya Reservoirs and Depressions

Elemental analyzer (EA) Isotope Ratio Mass Spectrometry was used to measure ∂13C values on soil organic matter from reservoirs and depressions at the ancient Maya urban centers of Tikal, Guatemala and Yaxnohcah, Mexico. Variation in δ13C values on soil organic matter were &gt; −2.0‰, which suggests enrichment from C4 plants including maize, other tropical grasses (Poaceae), and tropical sedges (Cyperaceae), CAM plants (Clusia sp.), and cyanobacteria (blue-green algae). Cyanobacteria were likely a major contributor to the 13C enrichment of soil organic matter in Maya reservoirs and depressions, which has obfuscated our understanding of ancient Maya maize production. It is possible that the Maya used cyanobacteria as a fertilizer, which enriched agricultural field soil organic matter.

Shade and drought increase fungal contribution to partially mycoheterotrophic terrestrial orchids Goodyera pubescens and Tipularia discolor

Many photosynthetic plants supplement photosynthetic carbon with fungal carbon, but the mechanisms that govern dependence on mycoheterotrophic carbon are poorly understood. We used exclusion shelters to manipulate water and light availability to plants of the terrestrial orchids Goodyera pubescens and Tipularia discolor. We tracked changes in δ13C from photosynthesis and δ15N acquired from soil-derived inorganic nitrogen versus mycoheterotrophy, along with direct measures of photosynthesis in T. discolor. We hypothesized that shade would increase dependence on mycoheterotrophy compared to reference plants, while drought would decrease both photosynthesis and the abundance of potential mycorrhizal fungi. Drought and shade enriched 13C and 15N in both G. pubescens and T. discolor, compared to control plants, indicating increased fungal contribution to orchid tissues. Physiological measurements of T. discolor leaves showed that dark respiration, water use efficiency, and relative electron transport rate did not vary significantly, but shaded plants had greater quantum efficiency, suggesting they were light-limited. Light saturated photosynthesis of T. discolor leaves was lower in both shaded and drought-treated plants, indicating lower photosynthetic capacity, and likely greater dependence on mycoheterotrophy and corresponding enrichment in 13C and 15N. This study documented changes in orchid dependence on fungal carbon in response to manipulated environmental conditions. Both shade and drought increased the dependence of both orchids on mycoheterotrophically derived carbon and nitrogen.

Simultaneous Inhibition of SIRT3 and Cholesterol Homeostasis Targets AML Stem Cells By Perturbing Fatty Acid β-Oxidation and Inducing Lipotoxicity

Outcomes for patients with acute myeloid leukemia (AML) remain poor due to the inability of current therapies to fully eradicate disease initiating leukemia stem cells (LSCs) (Shlush et al., 2017). Oxidative phosphorylation (OXPHOS) is a highly targetable and essential process for LSC energy production and survival (Lagadinou et al., 2013). Sirtuin 3 (SIRT3), a mitochondrial deacetylase with a multi-faceted role in metabolic regulation (Finley et al., 2016), has been specifically shown to regulate OXPHOS in cancer models (Li et al., 2019, Xu et al., 2019). Thus, we sought to identify if SIRT3 is essential for LSC function. To determine if SIRT3 is essential for LSCs, we measured cell viability, colony forming potential, and engraftment potential of primary AML specimens upon perturbation of SIRT3 function using both RNAi and a small molecule inhibitor (YC8-02) (Li et al., 2019). SIRT3 reduction significantly decreased viability, colony forming potential and engraftment of LSCs (Fig. 1A), suggesting that SIRT3 inhibition decreases LSC function. Importantly, neither SIRT3 knockdown nor inhibition impacted the viability, colony forming potential, or engraftment potential of normal hematopoietic stem and progenitor cells (HSPCs). Altogether, these data suggest that SIRT3 is essential for LSCs and not HSPCs. To determine the molecular mechanisms by which SIRT3 is essential for LSC function, we performed proximity-dependent biotin labeling (BioID) in 293 T-Rex cells to map the interactome of SIRT3. Strikingly, our BioID analysis identified several fatty acid oxidation (FAO) enzymes as proximity interactors including CPT2, ACADVL, ACADM, ECHI, HADH, and ACAA2. To corroborate these findings in LSCs, we performed RNA-sequencing upon SIRT3 inhibition in primary LSCs derived from three primary AML specimens. Gene set enrichment analysis revealed a significant decrease in OXPHOS and FAO gene signatures upon SIRT3 inhibition. Importantly, FAO is known to support OXPHOS which suggests that SIRT3 may regulate OXPHOS through mediation of FAO. To determine if protein interactions and gene expression changes result in functional changes in mitochondrial biology, we measured OXPHOS and mitochondrial ATP production in LSCs upon SIRT3 perturbation in LSCs using a seahorse assay. This analysis revealed that SIRT3 perturbation resulted in decreased OXPHOS and ATP production. To determine if SIRT3 regulates OXPHOS via FAO mediation, we performed mass spectrometry-based lipidomic and metabolic interrogation on LSCs from three AML samples treated with YC8-02, which showed an accumulation of fatty acid upon SIRT3 inhibition. To interrogate fatty acid metabolism further, we performed stable isotope tracing analysis using 13C16-palmitate on LSC enriched from three patient specimens upon SIRT3 inhibition. Tracing studies revealed a reduction in 13C enrichment in tricarboxylic acid (TCA) cycle metabolites in the YC8-02-treated LSCs (Fig. 1B), suggesting an impairment to fatty acid utilization upon SIRT3 inhibition. Decreasing availability of TCA products impairs OXPHOS and ultimately causes cell death. Importantly, viability of LSCs was partially rescued through addition of the TCA intermediate α-ketoglutarate, demonstrating reduced FAO to be an essential part of the mechanism by which SIRT3 targets LSCs. In addition to decreasing OXPHOS, the accumulation of fatty acids should induce cell death by causing lipotoxicity. However, phenocopy experiments revealed that LSCs are protected from lipotoxicity unlike AML blasts or AML cell lines. Lipidomic analysis suggested that fatty acids may be converted to cholesterol esters, which can then be stored or excreted from cells, specifically in LSCs and not in AML cell lines. Therefore, we postulated that targeting cholesterol homeostasis using dipyridamole would enhance the effect of SIRT3 inhibition (Esquejo et al., 2021). The combination treatment significantly reduced the viability of LSCs compared to individual agents, indicating that co-inhibition of SIRT3 and cholesterol homeostasis enhances the toxic effects of FAO inhibition on LSCs. In all, we identify SIRT3 as a key regulator of LSC survival, through the mediation of lipid metabolism. We further demonstrate that LSCs are tolerant to FAO inhibition which can be overcome by concurrent inhibition of FAO and cholesterol metabolism, which has the potential to lead to improved treatment response. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Helicobacter pylori infection and UBT-13C values are associated with changes in body mass index in children and adults.

The urea breath test (UBT-13C) is a non-invasive technique that allows the diagnosis and confirmation of eradication of Helicobacter pylori infection. To evaluate H. pylori positivity and values of UBT-13C among infected Chilean children and adults, and to analyze its variation in relation to sex, nutritional status, and age of the patients. Retrospective study of 1141 patients aged 6 to 94 years, with an indication for a UBT-13C either for diagnosis or for confirmation of eradication of H. pylori infection. 13C enrichment was measured using an infrared spectrometer calculating the delta 13C values before and after the ingestion of 13C marked urea. The clinical data of the patients were obtained at the time of the examination. We included 241 children and 900 adults. Infected children obtained lower UBT-13C delta values than infected adults (16.1 ± 8.7 and 37 ± 52.9, respectively). The rates of infection were higher in males who were recruited for diagnosis. Significant differences were obtained between positivity for H. pylori in overweight and obese children but not adults. UBT-13C titers were significantly associated with the body mass index (BMI) only in adults. H. pylori infection rates are similar between sexes and are higher in children probably because of selection bias. In children, H. pylori positivity is associated with higher BMI and excess malnutrition although with similar UBT-13C values. In adults, H. pylori infection is not related with BMI, but a higher BMI impacts UBT-13C titers.

Tracing Carbon Metabolism with Stable Isotope Metabolomics Reveals the Legacy of Diverse Carbon Sources in Soil.

Tracking the metabolic activity of whole soil communities can improve our understanding of the transformation and fate of carbon in soils. We used stable isotope metabolomics to trace 13C from nine labeled carbon sources into the water-soluble metabolite pool of an agricultural soil over time. Soil was amended with a mixture of all nine sources, with one source isotopically labeled in each treatment. We compared changes in the 13C enrichment of metabolites with respect to carbon source and time over a 48-day incubation and contrasted differences between soluble sources (glucose, xylose, amino acids, etc.) and insoluble sources (cellulose and palmitic acid). Whole soil metabolite profiles varied singularly by time, while the composition of 13C-labeled metabolites differed primarily by carbon source (R2 = 0.68) rather than time (R2 = 0.07), with source-specific differences persisting throughout incubations. The 13C labeling of metabolites from insoluble carbon sources occurred slower than that from soluble sources but yielded a higher average atom percent (atom%) 13C in metabolite markers of biomass (amino acids and nucleic acids). The 13C enrichment of metabolite markers of biomass stabilized between 5 and 15 atom% 13C by the end of incubations. Temporal patterns in the 13C enrichment of tricarboxylic acid cycle intermediates, nucleobases (uracil and thymine), and by-products of DNA salvage (allantoin) closely tracked microbial activity. Our results demonstrate that metabolite production in soils is driven by the carbon source supplied to the community and that the fate of carbon in metabolites do not generally converge over time as a result of ongoing microbial processing and recycling. IMPORTANCE Carbon metabolism in soil remains poorly described due to the inherent difficulty of obtaining information on the microbial metabolites produced by complex soil communities. Our study demonstrates the use of stable isotope probing (SIP) to study carbon metabolism in soil by tracking 13C from supplied carbon sources into metabolite pools and biomass. We show that differences in the metabolism of sources influence the fate of carbon in soils. Heterogeneity in 13C-labeled metabolite profiles corresponded with compositional differences in the metabolically active populations, providing a basis for how microbial community composition correlates with the quality of soil carbon. Our study demonstrates the application of SIP-metabolomics in studying soils and identifies several metabolite markers of growth, activity, and other aspects of microbial function.

Chemical character and structure of uraniferous bitumens (Vrchlabí, Czech Republic)

Black shales from the Permian rocks at Vrchlabí in the Krkonoše Piedmont Basin (Czech Republic) contain bitumens with uraninite grain inclusions and bulk uranium concentrations up to 4.8 wt%, determined by instrumental neutron activation analysis. The carbonaceous matter in the bitumens has been characterised by gas chromatography/mass spectrometry, attenuated total reflection Fourier transform infrared spectroscopy, scanning electron microscopy, and stable carbon isotopic composition. Progressive radiolytic alteration was documented by changes in the chemical composition and structure of both bulk samples and on the microscale. Condensation of aromatic structures, increase in content of oxygenated compounds, and enrichment in 13C were documented in the uranium-rich bulk samples. Micro-ATR-FTIR area mapping throughout the bright halos around uraninite inclusions revealed detailed structural changes, particularly a decrease in aliphatic bond intensities and an increase in the aromaticity of the structures close to the inclusions. A radiolytic alteration pathway has been suggested assuming a complex of reactions between organic matter and uranyl containing hydrothermal solutions, and oxidation by OH radicals produced in radiolysis of water. The organic matter transformations observed in the uraniferous bitumen correspond to the coalification processes, changing the bitumen composition from type I to type III and IV kerogens. Detailed characterisation of the radiation-induced chemical and structural changes in the naturally occurring uraniferous bitumens will contribute to the general understanding of the nature and behaviour of organic matter in other uraniferous environments or in the radioactive waste repositories.

Exploring methane cycling in an arctic lake in Kangerlussuaq Greenland using stable isotopes and 16S rRNA gene sequencing

Lakes are currently responsible for a significant amount of total natural methane emission. Microbial oxidation of methane plays a central role in Arctic carbon cycling, potentially reducing methane emissions from lakes, though little is known about methane cycling in the water column of Arctic lakes. We previously detected surprisingly large enrichments of heavy carbon and hydrogen isotopes of methane in three small lakes in Greenland suggesting unusually efficient methanotrophic communities in these Arctic lakes. Using stable isotope and 16S rRNA gene sequencing we determined carbon and hydrogen isotopes and microbial community composition down the water column of Teardrop lake, under open-water conditions. We found that isotopic values of methane in Teardrop lake were again highly enriched 13C and 2H at 4 m depth with −13.2‰ and −27.1‰ values for carbon and hydrogen isotopes, respectively. Methane concentrations slightly increased at the depth interval with isotope enrichment, not typical of classic methanotrophy. Consistent with isotopic enrichment of the heavy isotopes we detected the highest relative abundance of putative methanotrophs, in particular Methylovulum at 4 m. The highest relative abundance of putative methanogens was detected at 3 m as well as at 5 m. At the same depth interval, temperature and oxidation reduction potential also increase, supporting increased microbial activity within the water column. Based on geochemical and microbial observations, we suggest that the methane cycling in Teardrop lake is decoupled from a traditional depth dependent model where the dominant source of methane is in the anoxic sediments. Instead, methane in the water column is likely from a combination of anoxic sediment, littoral transport and oxic methanogenesis in the mid-water column, and recycling of carbon within the water column is leading to extreme isotope enrichments. Thus, understanding linkages between depth-dependent microbial dynamics and methane biogeochemistry are necessary to constrain the sensitivity of the methane cycle to future climate change.