13C Labeling Research Articles

Measuring 15N and 13C Enrichment Levels in Sparsely Labeled Proteins Using High-Resolution and Tandem Mass Spectrometry.

Isotope labeling of both 15N and 13C in selected amino acids in a protein, known as sparse labeling, is an alternative to uniform labeling and is particularly useful for proteins that must be expressed using mammalian cells, including glycoproteins. High levels of enrichment in the selected amino acids enable multidimensional heteronuclear NMR measurements of glycoprotein three-dimensional structure. Mass spectrometry provides a means to quantify the degree of enrichment. Mass spectrometric measurements of tryptic peptides of a selectively labeled glycoprotein expressed in HEK293 cells revealed complicated isotope patterns which consisted of many overlapping isotope patterns from intermediately labeled peptides, which complicates the determination of the label incorporation. Two challenges are uncovered by these measurements. Metabolic scrambling of amino groups can reduce the 15N content of enriched amino acids or increase the 15N in nontarget amino acids. Also, undefined, unlabeled medium components may dilute the enrichment level of labeled amino acids. The impact of this unexpected metabolic scrambling was overcome by simulating isotope patterns for all isotope-labeled peptide states and generating linear combinations to fit to the data. This method has been used to determine the percent incorporation of 15N and 13C labels and has identified several metabolic scrambling effects that were previously undetected in NMR experiments. Ultrahigh mass resolution is also utilized to obtain isotopic fine structure, from which enrichment levels of 15N and 13C can be assigned unequivocally. Finally, tandem mass spectrometry can be used to confirm the location of heavy isotope labels in the peptides.

The origin of methyl group in methanogen-mediated mercury methylation: From the Wolfe cycle

Methylmercury (MeHg) is a bioaccumulating neurotoxin mainly produced by anaerobic microorganisms, with methanogen being one of the important methylators. A critical aspect for understanding the mechanism for microbial mercury (Hg) methylation is the origin of the methyl group. However, the origin of methyl group in methanogen-mediated Hg methylation remains unclear. This study aims to identify the source of methyl group for MeHg synthesis in methanogens. Our study revealed that Hg methylation in Methanospirillum hungatei JF-1 is closely related to methanogenesis process, according to the results of proteomic study and substrate limitation study. Next, we proved that nearly all methyl group in MeHg derives from the Wolfe cycle in this species, rather than the previously demonstrated acetyl-coenzyme A pathway, based on the results of 13C labeling study. We then proposed the Wolfe cycle-dependent Hg methylation mechanism in this species. Further genome analyses and 13C labeling experiments indicated that the involvement of the Wolfe cycle in Hg methylation is probably a universal feature among Hg-methylating methanogens. These findings reveal a unique Hg methylation mechanism in methanogens. Our study broadens the carbon substrates and controlling factors for MeHg synthesis in the environment, which can inform the prediction of MeHg production potential and remediation strategies for MeHg contamination.

Integrating Umpolung and CO2 Shuttling Strategies for the Synthesis of 12C- and 13C-α-Ketoacids from Aldehydes.

The direct carboxylation of aldehydes with CO2 is rare due to the polarity mismatch between these two electrophilic substrates. To address this challenge, we propose a sequential approach for synthesizing α-ketoacids from commercially available aldehydes by integrating umpolung and CO2 shuttling strategies. This transition metal-free shuttle carboxylation method enables the transfer of CO2 from triphenylacetic acid potassium salt to thioacetal, eliminating the need for handling pressurized CO2 gas or using specialized equipment, while also enhancing the reaction's functional group tolerance. Furthermore, the use of stoichiometric or slightly excess amounts of triphenylacetic acid potassium salt as a formal CO2 donor makes it suitable for complete 13C labeling of α-ketoacids.

Rubisco supplies pyruvate for the 2-C-methyl-D-erythritol-4-phosphate pathway.

RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE (Rubisco) produces pyruvate in the chloroplast through β-elimination of the aci-carbanion intermediate1. Here we show that this side reaction supplies pyruvate for isoprenoid, fatty acid and branched-chain amino acid biosynthesis in photosynthetically active tissue. 13C labelling studies of intact Arabidopsis plants demonstrate that the total carbon commitment to pyruvate is too large for phosphoenolpyruvate to serve as a precursor. Low oxygen stimulates Rubisco carboxylase activity and increases pyruvate production and flux through the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, which supplies the precursors for plastidic isoprenoid biosynthesis2,3. Metabolome analysis of mutants defective in phosphoenolpyruvate or pyruvate import and biochemical characterization of isolated chloroplasts further support Rubisco as the main source of pyruvate in chloroplasts. Seedlings incorporated exogenous,13C-labelled pyruvate into MEP pathway intermediates, while adult plants did not, underscoring the developmental transition in pyruvate sourcing. Rubisco β-elimination leading to pyruvate constituted 0.7% of the product profile in in vitro assays, which translates to 2% of the total carbon leaving the Calvin-Benson-Bassham cycle. These insights solve the "pyruvate paradox"4, improve the fit of metabolic models for central metabolism and connect the MEP pathway directly to carbon assimilation.

Bioupcycling Methane and CO2 for Succinate Production by an Engineered Type I Methanotrophic Bacterium.

Methane, a byproduct of agricultural activities, has shown potential as a nonedible substrate for biomanufacturing. The production of succinate by a methanotrophic bacterium utilizing methane presents an innovative route for the sustainable synthesis of chemicals. In this study, Methylotuvimicrobium buryatense 5GB1S was genetically modified through the reconstruction of an artificial serine cycle to enable the bioconversion of both methane and CO2 into succinate. The 13C labeling analysis confirmed the CO2 fixing in M. buryatense 5GB1S, leading to a 46% improvement in carbon conversion efficiency and a 107% increase in succinate production compared to the wild-type strain. The transcriptome data on carbon metabolisms was assessed to guide future optimizations for strengthening the overall carbon flux from methane to succinate. Finally, the maximum succinate titer of 299.36 mg/L was achieved under oxygen-limited conditions in 3 L bioreactors, which resulted in the volumetric productivity of 199.60 mg/L/day, representing a 23-fold enhancement compared to the wild-type strain. This study offers a new strategy for upcycling greenhouse gases into succinate in a sustainable manner through methanotrophic-based biomanufacturing.

1H, 13C and 15N backbone resonance assignment of the calcium-activated EndoU endoribonuclease.

The catalytic domain of the calcium-dependent endoribonuclease EndoU from Homo sapiens was expressed in E. coli with 13C and 15N labeling. A nearly complete assignment of backbone 1H, 15N, and 13C resonances was obtained, as well as a secondary structure prediction based on the assigned chemical shifts. The predicted secondary structures were almost identical to the published crystal structure of calcium-activated EndoU. This is the first NMR study of an eukaryotic member of the EndoU-like superfamily of ribonucleases.

Parasitic plants regulate C and N distribution among common mycorrhizal networks linking host and neighboring plants.

Common mycorrhizal networks (CMNs) can link multiple plants and distribute nutrients among them. However, how parasitic plants regulate the carbon and nutrient exchange between CMNs and the linked plants is unknown. Thus, we conducted a container experiment with two Trifolium pratense grown in two plastic cores and connected only by CMNs using a 25-μm nylon fabric in each container. Host T. pratense was parasitized or not parasitized by Cuscuta gronovii. CMNs were left intact or broken by rotating the cores with the host or neighboring T. pratense. The dual 15N and 13C labeling method was used to evaluate the N distributed by CMNs to the host and neighboring T. pratense and the recently fixed C from the host and neighboring T. pratense to CMNs. The results showed that CMNs distributed more 15N to unparasitized neighboring T. pratense than the parasitized host T. pratense. Moreover, the unparasitized neighboring T. pratense provides more recently fixed C to CMNs than the parasitized host T. pratense. These results revealed that the parasite regulated C and nutrient exchange between CMNs and the linked plants following the reciprocal rewards rule. Moreover, this study highlights the importance of parasitic plants in the regulation of mutualistic interactions in ecological webs.

Redox Transformations of the OX063 Radical in Biological Media: Oxidative Decay of Initial Trityl with Further Formation of Structurally-Modified TAM.

Being a low-toxic and hydrophilic representative of TAM, OX063 has shown its suitability for in-vivo and in-cell EPR experiments and design of spin labels. Using 13C labeling, we investigated the course of oxidative degradation of OX063 into quinone-methide (QM) under the influence of superoxide as well as further thiol-promoted reduction of QM into TAM radical, which formally corresponds to substitution of a carboxyl function by a hydroxyl group. We found these transformations being quantitative in model reactions mimicking specific features of biological media and confirmed the presence of these reactions in the blood and liver homogenate of mice in vitro. The emergence of the trityl with the hydroxyl group can be masked by an initial TAM in EPR spectra and may introduce distortions into EPR-derived oximetry data if they have been obtained for objects under hypoxia. 13C labeling allows one to detect its presence, considering its different hyperfine splitting constant on 13C1 (2.04 mT) as compared to OX063 (2.30 mT). The potential involvement of these reactions should be considered when using TAM in spin-labeling of biopolymers intended for subsequent EPR experiments, as well as in the successful application of TAM in experiments in vivo and in cell.

Synthesis of condensed tannin model compounds regioselectively labeled with a 13C-stable isotope

Condensed tannins exhibit diverse bioactivities that render them promising for applications in the food and medical fields. For the analysis and monitoring of condensed tannins, 13C-labeled derivatives would provide a useful tool. In this study, condensed tannin polymers regioselectively labeled with a stable isotope were synthesized using 13C-labeled acetic acid or 13C-labeled dimethylformamide as the starting material. The resulting synthetic polymers were labeled with 13C at the C-4 or C-2 positions. A GPC analysis revealed that all model polymers comprised mainly tetramer to hexamer structures. According to the 13C-NMR data of the synthetic tannin models and natural condensed tannin obtained from sugi bark, the chemical structure of both compounds was very similar. Furthermore, compared with the natural condensed tannin and unlabeled synthetic polymer, the labeled compound showed more intense C-4 and C-2 13C signals, indicating that the 13C labeling proceeded regioselectively. These compounds are useful for analyzing the chemical reactions of condensed tannins and monitoring structural transformation processes in vivo.

Getting to the root of the problem: Soil carbon and microbial responses to root inputs within a buried paleosol along an eroding hillslope in southwestern Nebraska, USA

Large quantities of soil carbon (C) can persist within paleosols for millennia due to burial and subsequent isolation from plant-derived inputs, atmospheric conditions, and microbial activity at the modern surface. Erosion exposes buried soils to modern root-derived C influx via root exudation and root turnover, thus stimulating microbial activity leading to SOC decomposition and accumulation through organo-mineral stabilization of modern C. With this study we aim to quantify how modern root-derived C inputs impact paleosol C decomposition and stabilization across varying degrees of isolation from modern surface conditions in southwestern Nebraska, USA, where hillslope erosion is bringing a buried Late-Pleistocene-early Holocene paleosol (the “Brady Soil”) closer to the modern surface. We collected Brady Soil samples from 0.2 m, 0.4 m, and 1.2 m below the modern surface and conducted two lab-based incubations. Soils were amended with either (1) a lab-synthesized mixture of low molecular weight compounds (12 atom% 13C), or (2) 13C enriched root residues (92 atom% 13C), in 30-day and 240-day incubation experiments, respectively. We determined microbial responses to synthetic root exudates and residues by partitioning the 13C label from Brady Soil C, including measurements of total, root, and primed C respiration, microbial biomass C (MBC), microbial C use efficiency (CUE). To assess the capacity of isolated paleosols to accrue modern plant C, we used Nano-scale Secondary Ion Mass Spectrometry imaging. We found that: (1) adding root-derived C inputs primed Brady Soil C across all depths, and was mediated by depth and composition of root additions; (2) root-derived C inputs stimulated microbial biomass C (MBC) growth similarly across depths, but the magnitude of CUE and MBC varied by chemistry of root-derived additions; (3) new particulate organic matter was incorporated into mineral-associated pools over time; (4) material from the added root residues was found in association with bacterial cells and fungal hyphae as well as with soil aggregate and mineral surfaces. Our study shows that paleosols defy expectations of C content and reactivity with depth, and changes in land cover and climate will expose buried paleosols to modern surface conditions, increasing respired C. This work highlights the importance of evaluating the role resurfacing buried soils through landscape change plays in C cycle feedbacks to the climate system.

Proteomic stable isotope probing with an upgraded Sipros algorithm for improved identification and quantification of isotopically labeled proteins

BackgroundProteomic stable isotope probing (SIP) is used in microbial ecology to trace a non-radioactive isotope from a labeled substrate into de novo synthesized proteins in specific populations that are actively assimilating and metabolizing the substrate in a complex microbial community. The Sipros algorithm is used in proteomic SIP to identify variably labeled proteins and quantify their isotopic enrichment levels (atom%) by performing enrichment-resolved database searching.ResultsIn this study, Sipros was upgraded to improve the labeled protein identification, isotopic enrichment quantification, and database searching speed. The new Sipros 4 was compared with the existing Sipros 3, Calisp, and MetaProSIP in terms of the number of identifications and the accuracy and precision of atom% quantification on both the peptide and protein levels using standard E. coli cultures with 1.07 atom%, 2 atom%, 5 atom%, 25 atom%, 50 atom%, and 99 atom% 13C enrichment. Sipros 4 outperformed Calisp and MetaProSIP across all samples, especially in samples with ≥ 5 atom% 13C labeling. The computational speed on Sipros 4 was > 20 times higher than Sipros 3 and was on par with the overall speed of Calisp- and MetaProSIP-based pipelines. Sipros 4 also demonstrated higher sensitivity for the detection of labeled proteins in two 13C-SIP experiments on a real-world soil community. The labeled proteins were used to trace 13C from 13C-methanol and 13C-labeled plant exudates to the consuming soil microorganisms and their newly synthesized proteins.ConclusionOverall, Sipros 4 improved the quality of the proteomic SIP results and reduced the computational cost of SIP database searching, which will make proteomic SIP more useful and accessible to the border community.-J6PT921fQgCkWKyfrRUdgVideo

Sucrose promotes cone enlargement via the TgNGA1-TgWRKY47-TgEXPA2 module in Torreya grandis.

Cone enlargement is a crucial process for seed production and reproduction in gymnosperms. Most of our knowledge of cone development is derived from observing anatomical structure during gametophyte development. Therefore, the exact molecular mechanism underlying cone enlargement after fertilization is poorly understood. Here, we demonstrate that sucrose promotes cone enlargement in Torreya grandis, a gymnosperm species with relatively low rates of cone enlargement, via the TgNGA1-TgWRKY47-TgEXPA2 pathway. Cell expansion plays a significant role in cone enlargement in T. grandis. 13C labeling and sucrose feeding experiments indicated that sucrose-induced changes in cell size and number contribute to cone enlargement in this species. RNA-sequencing analysis, transient overexpression in T. grandis cones, and stable overexpression in tomato (Solanum lycopersicum) suggested that the expansin gene TgEXPA2 positively regulates cell expansion in T. grandis cones. The WRKY transcription factor TgWRKY47 directly enhances TgEXPA2 expression by binding to its promoter. Additionally, the NGATHA transcription factor TgNGA1 directly interacts with TgWRKY47. This interaction suppresses the DNA-binding ability of TgWRKY47, thereby reducing its transcriptional activation on TgEXPA2 without affecting the transactivation ability of TgWRKY47. Our findings establish a link between sucrose and cone enlargement in T. grandis and elucidate the potential underlying molecular mechanism.

1H, 13C and 15N backbone resonance assignment of Cel45A from Phanerochaete chrysosporium

A glycoside hydrolase family 45 (GH45) enzyme from the white-rot basidiomycete fungus Phanerochaete chrysosporium (PcCel45A) was expressed in Pichia pastoris with 13C and 15N labelling. A nearly complete assignment of 1H, 13C and 15N backbone resonances was obtained, as well as the secondary structure prediction based on the assigned chemical shifts using the TALOS-N software. The predicted secondary structure was almost identical to previously published crystal structures of the same enzyme, except for differences in the termini of the sequence. This is the first NMR study using an isotopically labelled GH45 enzyme.

Seven-year long-term inoculation with Funneliformis mosseae increases maize yield and soil carbon storage evidenced by in situ 13C-labeling in a dryland

Arbuscular mycorrhizal fungi (AMF) establish symbiotic relationships with roots of most plants, contributing to plant water uptake and soil carbon (C) sequestration. However, the interactive contribution and of long-term field AMF inoculation and water conservation on maize yield and soil organic carbon (SOC) sequestration in drylands remain largely unknown. After 7-year long-term field inoculation with AMF Funneliformis mosseae, AMF suppression by fungicide benomyl, and no-AMF/no-benomyl control, and two water conservation practices of half-film and full-film mulching (∼50 % and ∼100 crop planted area covered with plastic film), this study thus applied in situ 13CO2-C labeling and high-throughput sequencing to quantify newly photosynthetically assimilated C into different soil C pools including soil aggregates and respiration, and their effects on maize growth and productivity. Results showed that 7-year long-term AMF inoculation significantly increased the relative abundance of F. mosseae in rhizosphere soil and root AMF colonization, indicating that F. mosseae successfully dominated in AMF communities. Compared to no-AMF/no-benomyl control, AMF colonization significantly increased shoot biomass and maize yield by 17.9 % and 20.3 % while mitigated the less water conservation effects of half-film mulching on maize performance. The SOC content under field AMF inoculation SOC was increased from 7.9 to 8.4 g kg−1 and also the mean weight diameter of aggregates (1.21 to 1.35), e.g. aggregate stability. After 1 and/or 40 days 13C labeling, the enhanced 13C translocations into macro-aggregates with decreased 13C emissions from microbial decomposition under field AMF inoculation had contributed to SOC conservation in bulk soil. These results suggest that AMF inoculation in dryland crops is promising to increase crop yield while promoting more atmospheric CO2 fixation in soil aggregates. A long-term field AMF inoculation will enhance our understanding of applying beneficial mycorrhizal fungi to enhance soil C sequestration and also crop yield via plant-fixed atmospheric CO2 in semi-arid and arid farmlands.

Carbon-14 Labeling Synthesis of RORγt Inhibitor JNJ-61803534.

Carbon-14 labeling synthesis of RORγt inhibitor JNJ-61803534 (1) was accomplished in four steps with the C14 label located at the thiazole-2-carboxamide carbon. The synthesis featured a highly efficient conversion of nitrile [14C]-12 to ester [14C]-17 under mild conditions via an imidate intermediate, overcoming the unsuccessful direct hydrolysis of nitrile 12 under either acidic or basic conditions. Since carbon-14 labeling via [14C]-nitrile installation and subsequent conversion to [14C]-carboxylic acid derivatives is a common labeling strategy, an efficient conversion of a nitrile to an ester under mild conditions could be of use for the future C14 labeling syntheses.

In vivo X-ray microtomography locally affects stem radial growth with no immediate physiological impact.

Microcomputed tomography (µCT) is a nondestructive X-ray imaging method used in plant physiology to visualize in situ plant tissues that enables assessments of embolized xylem vessels. Whereas evidence for X-ray-induced cellular damage has been reported, the impact on plant physiological processes such as carbon (C) uptake, transport, and use is unknown. Yet, these damages could be particularly relevant for studies that track embolism and C fluxes over time. We examined the physiological consequences of µCT scanning for xylem embolism over 3 mo by monitoring net photosynthesis (Anet), diameter growth, chlorophyll (Chl) concentration, and foliar nonstructural carbohydrate (NSC) content in 4 deciduous tree species: hedge maple (Acer campestre), ash (Fraxinus excelsior), European hornbeam (Carpinus betulus), and sessile oak (Quercus petraea). C transport from the canopy to the roots was also assessed through 13C labeling. Our results show that monthly X-ray application did not impact foliar Anet, Chl, NSC content, and C transport. Although X-ray effects did not vary between species, the most pronounced impact was observed in sessile oak, marked by stopped growth and stem deformations around the irradiated area. The absence of adverse impacts on plant physiology for all the tested treatments indicates that laboratory-based µCT systems can be used with different beam energy levels and doses without threatening the integrity of plant physiology within the range of tested parameters. However, the impacts of repetitive µCT on the stem radial growth at the irradiated zone leading to deformations in sessile oak might have lasting implications for studies tracking plant embolism in the longer-term.

Indium(III)-Catalyzed Haloalkynylation Reaction of Alkynes.

Green chemistry strives for sustainability at the molecular level and is gaining increasing relevance in the development of chemical reactions. The haloalkynylation reaction is a highly atom-economical C-C coupling reaction that was previously only achieved using transition metal catalysts. It enables the introduction of an alkyne unit and a halogen atom into the target molecule. Herein, we present a haloalkynylation reaction catalyzed by indium(III) halides. The use of indium(III) bromide as a catalyst leads exclusively to the cis addition products with yields up to 86 %. In addition, iodoacetylenes can be applied for the first time for the haloalkynylation reaction of internal alkynes which is an important step forward in the development of industrially relevant and sustainable catalysts. In contrast to gold catalysis, which proceeds via a similar mechanism, the use of alkyl-substituted haloacetylenes as reagents is also possible. Based on 13C labeling experiments and quantum chemical calculations, we postulate two possible mechanisms for the indium(III)-catalyzed haloalkynylation reactions.

Post-synthetic Metalation on the Ionic TiO2 Surface to Enhance Metal-CO2 Interaction During Photochemical CO2 Reduction.

During the photochemical CO2 reduction reaction, CO2 adsorption on the catalyst's surface is a crucial step where the binding mode of the [metal-CO2] adduct directs the product selectivity and efficiency. Herein, an ionic TiO2 nanostructure stabilized by polyoxometalates (POM), ([POM]x@TiO2), is prepared and the sodium counter ions present on the surface to balance the POMs' charge are replaced with copper(II) ions, (Cux[POM]@TiO2). The microscopic and spectroscopic studies affirm the copper exchange without altering the TiO2 core and weak coordination of copper (II) ions to the POMs' surface. Band structure analysis suggests the photo-harvesting efficiency of the TiO2 core with the conduction band edge higher than the reduction potential of CuII/I and multi-electron CO2 reduction potentials. Photochemical CO2 reduction with Cux[POM]@TiO2 results in 30 μmol gcat. -1 CO (79 %) and 8 μmol gcat -1 of CH4 (21 %). Quasi-in-situ Raman study provides evidence in support of CO2 adsorption on the Cux[POM]@TiO2 surface. 13C and D2O labeling studies affirm the {Cu-[CO2]-} adduct formation. Despite the photo-harvesting ability of Nax[POM]@TiO2 itself, the poor CO2 adsorption ability of sodium ions highlights the crucial role of copper ion CO2 photo-reduction. Characterization of the {M-[η2-CO2]-} species via surface tuning validates the CO2 activation and photochemical reduction pathway proposed earlier.

Species-specific responses of C and N allocation to N addition: evidence from dual 13C and 15N labeling in three tree species

Soil nitrogen (N) availability affects plant carbon (C) utilization. However, it is unclear how various tree functional types respond to N addition in terms of C assimilation, allocation, and storage. Here, a microcosm experiment with dual 13C and 15N labeling was conducted to study the effects of N addition (i.e., control, 0 g N kg−1; moderate N addition, 1.68 g N kg−1; and high N addition, 3.36 g N kg−1 soil) on morphological traits, on changes in nonstructural carbohydrates (NSC) in different organs, as well as on C and N uptake and allocation in three European temperate forest tree species (i.e., Acer pseudoplatanus, Picea abies and Abies alba). Our results demonstrated that root N uptake rates of the three tree species increased by N addition. In A. pseudoplatanus, N uptake by roots, N allocation to aboveground organs, and aboveground biomass allocation significantly improved by moderate and high N addition. In A. alba, only the high N addition treatment considerably raised aboveground N and C allocation. In contrast, biomass as well as C and N allocation between above and belowground tissues were not altered by N addition in P. abies. Meanwhile, NSC content as well as C and N coupling (represented by the ratio of relative 13C and 15N allocation rates in organs) were affected by N addition in A. pseudoplantanus and P. abies but not in A. alba. Overall, A. pseudoplatanus displayed the highest sensitivity to N addition and the highest N requirement among the three species, while P. abies had a lower N demand than A. alba. Our findings highlight that the responses of C and N allocation to soil N availability are species-specific and vary with the amount of N addition.

Abstract 6612: Therapy-resistant endocycling cancer cells alter mitochondrial structure and metabolism to survive therapeutic stress

Abstract Therapy resistance is responsible for 90% of cancer-related deaths. We identified a novel mechanism of therapy resistance in which cancer cells enter an endocycling cell state, undergoing repeated S and G phases without dividing. As a result, resistant endocycling cells are 40 times larger than mitotic cancer cell controls. Interestingly, we observed that these non-proliferative endocycling cells can eventually give rise to proliferative progeny weeks after chemotherapy is removed, modeling cancer recurrence. We hypothesize that this endocycling cancer cell state is a critical driver of therapy resistance and cancer lethality. We evaluated oxidative stress in endocycling cells that survived the days and weeks following chemotherapy treatment using DCF-DA staining. We found increased levels of reactive oxygen species (ROS) in endocycling prostate cancer cells compared to untreated mitotic cells when normalized to cell size. Given that mitochondria are major producers and regulators of ROS, we hypothesize that cells surviving in this resistant state alter mitochondrial structure and function in response to increased oxidative stress. We performed immunofluorescence and confocal microscopy on mitochondria in untreated cells and cells following chemotherapy treatment to observe their morphology. Surviving endocycling cells had increased mitochondrial fragmentation when compared to the untreated group, a result that was further supported by reduced expression of phospho-DRP1 Ser637 via western blot. To assess changes in glucose metabolism, we then utilized isotope tracing-based metabolomics with [U-13C]glucose on untreated and surviving endocycling cells following chemotherapy. Endocycling cells had decreased labeling of 13C in TCA cycle metabolites, suggesting a decrease in mitochondrial function when compared to untreated cells. Ongoing work involves further studying metabolic reprogramming as cells enter this resistant state with parallel labeling experiments using [1,2-13C]glucose and [U-13C]glutamine to perform mathematical modeling-based 13C-metabolic flux analysis. In addition, we engineered a cell line to track mitochondria in real-time to quantify mitochondrial dynamics underlying the morphology observed in untreated and surviving cells. Our goal is to characterize how this therapy-resistant cell state alters its mitochondrial structure and function to survive therapeutic stress. Identifying vulnerabilities of this phenotype will enable new approaches for eliminating drug resistance in cancer. Citation Format: Melvin Li, Sarah R. Amend, Kenneth J. Pienta. Therapy-resistant endocycling cancer cells alter mitochondrial structure and metabolism to survive therapeutic stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6612.