CosTaL: an accurate and scalable graph-based clustering algorithm for high-dimensional single-cell data analysis.

With the aim of analyzing large-sized multidimensional single-cell datasets, we are describing a method for Cosine-based Tanimoto similarity-refined graph for community detection using Leiden's algorithm (CosTaL). As a graph-based clustering method, CosTaL transforms the cells with high-dimensional features into a weighted k-nearest-neighbor (kNN) graph. The cells are represented by the vertices of the graph, while an edge between two vertices in the graph represents the close relatedness between the two cells. Specifically, CosTaL builds an exact kNN graph using cosine similarity and uses the Tanimoto coefficient as the refining strategy to re-weight the edges in order to improve the effectiveness of clustering. We demonstrate that CosTaL generally achieves equivalent or higher effectiveness scores on seven benchmark cytometry datasets and six single-cell RNA-sequencing datasets using six different evaluation metrics, compared with other state-of-the-art graph-based clustering methods, including PhenoGraph, Scanpy and PARC. As indicated by the combined evaluation metrics, Costal has high efficiency with small datasets and acceptable scalability for large datasets, which is beneficial for large-scale analysis.

Multiparametric identification of putative senescent cells in skeletal muscle via mass cytometry.

Senescence is an irreversible arrest of the cell cycle that can be characterized by markers of senescence such as p16, p21, and KI-67. The characterization of different senescence-associated phenotypes requires selection of the most relevant senescence markers to define reliable cytometric methodologies. Mass cytometry (a.k.a. Cytometry by time of flight, CyTOF) can monitor up to 40 different cell markers at the single-cell level and has the potential to integrate multiple senescence and other phenotypic markers to identify senescent cells within a complex tissue such as skeletal muscle, with greater accuracy and scalability than traditional bulk measurements and flow cytometry-based measurements. This article introduces an analysis framework for detecting putative senescent cells based on clustering, outlier detection, and Boolean logic for outliers. Results show that the pipeline can identify putative senescent cells in skeletal muscle with well-established markers such as p21 and potential markers such as GAPDH. It was also found that heterogeneity of putative senescent cells in skeletal muscle can partly be explained by their cell type. Additionally, autophagy-related proteins ATG4A, LRRK2, and GLB1 were identified as important proteins in predicting the putative senescent population, providing insights into the association between autophagy and senescence. It was observed that sex did not affect the proportion of putative senescent cells among total cells. However, age did have an effect, with a higher proportion observed in fibro/adipogenic progenitors (FAPs), satellite cells, M1 and M2 macrophages from old mice. Moreover, putative senescent cells from muscle of old and young mice show different expression levels of senescence-related proteins, with putative senescent cells of old mice having higher levels of p21 and GAPDH, whereas putative senescent cells of young mice had higher levels of IL-6. Overall, the analysis framework prioritizes multiple senescence-associated proteins to characterize putative senescent cells sourced from tissue made of different cell types.

Numerical modeling reveals improved organelle separation for dielectrophoretic ratchet migration.

Organelle size varies with normal and abnormal cell function. Thus, size-based particle separation techniques are key to assessing the properties of organelle subpopulations differing in size. Recently, insulator-based dielectrophoresis (iDEP) has gained significant interest as a technique to manipulate sub-micrometer-sized particles enabling the assessment of organelle subpopulations. Based on iDEP, we recently reported a ratchet device that successfully demonstrated size-based particle fractionation in combination with continuous flow sample injection. Here, we used a numerical model to optimize the performance with flow rates a factor of three higher than previously and increased the channel volume to improve throughput. We evaluated the amplitude and duration of applied low-frequency DC-biased AC potentials improving separation efficiency. A separation efficiency of nearly 0.99 was achieved with the optimization of key parameters-improved from 0.80 in previous studies (Ortiz et al. Electrophoresis, 2022;43;1283-1296)-demonstrating that fine-tuning the periodical driving forces initiating the ratchet migration under continuous flow conditions can significantly improve the fractionation of organelles of different sizes.

Combining Isoprenoid Probes with Antibody Markers for Mass Cytometric Analysis of Prenylation in Single Cells.

Protein prenylation is an essential post-translational modification that plays a key role in facilitating protein localization. Aberrations in protein prenylation have been indicated in multiple disease pathologies including progeria, some forms of cancer, and Alzheimer's disease. While there are single-cell methods to study prenylation, these methods cannot simultaneously assess prenylation and other cellular changes in the complex cell environment. Here, we report a novel method to monitor, at the single-cell level, prenylation and expression of autophagy markers. An isoprenoid analogue containing a terminal alkyne, substrate of prenylation enzymes, was metabolically incorporated into cells in culture. Treatment with a terbium reporter containing an azide functional group, followed by copper-catalyzed azide-alkyne cycloaddition, covalently attached terbium ions to prenylated proteins within cells. In addition, simultaneous treatment with a holmium-containing analogue of the reporter, without an azide functional group, was used to correct for non-specific retention at the single-cell level. This procedure was compatible with other mass cytometric sample preparation steps that use metal-tagged antibodies. We demonstrate that this method reports changes in levels of prenylation in competitive and inhibitor assays, while tracking autophagy molecular markers with metal-tagged antibodies. The method reported here makes it possible to track prenylation along with other molecular pathways in single cells of complex systems, which is essential to elucidate the role of this post-translational modification in disease, cell response to pharmacological treatments, and aging.

Continuous organelle separation in an insulator-based dielectrophoretic device.

Heterogeneity in organelle size has been associated with devastating human maladies such as neurodegenerative diseases or cancer. Therefore, assessing the size-based subpopulation of organelles is imperative to understand the biomolecular foundations of these diseases. Here, we demonstrated a ratchet migration mechanism using insulator-based dielectrophoresis in conjunction with a continuous flow component that allows the size-based separation of submicrometer particles. The ratchet mechanism was realized in a microfluidic device exhibiting an array of insulating posts, tailoring electrokinetic and dielectrophoretic transport. A numerical model was developed to elucidate the particle migration and the size-based separation in various conditions. Experimentally, the size-based separation of a mixture of polystyrene beads (0.28 and 0.87 µ$\umu $ m) was accomplished demonstrating good agreement with the numerical model. Furthermore, the size-based separation of mitochondria was investigated using a mitochondria mixture isolated from HepG2 cells and HepG2 cells carrying the gene Mfn-1 knocked out, indicating distinct size-related migration behavior. With the presented continuous flow separation device, larger amounts of fractionated organelles can be collected in the future allowing access to the biomolecular signature of mitochondria subpopulations differing in size.

Nonspecific Binding Correction for Single-Cell Mass Cytometric Analysis of Autophagy and Myoblast Differentiation.

Satellite cells provide regenerative capacity to the skeletal muscle after injury. In this process, termed myogenesis, satellite cells get activated, proliferate, and differentiate. Myogenesis is recapitulated in the tissue culture of myoblasts that differentiate by fusion and then by the formation of myotubes. Autophagy plays an important role in myogenesis, but the asynchronous and unique trajectory of differentiation of each myoblast along the myogenic lineage complicates teasing apart at what stages of differentiation autophagy plays a critical role. In this report, we describe a mass cytometric, multidimensional, individual cell analysis of differentiating myoblasts that characterizes autophagy flux (i.e., autophagy rate) at separate myogenesis stages. Because mass cytometry uses a set of lanthanide-tagged antibodies, each being specific for a desired molecular target, quantification of each molecular target could be exaggerated by nonspecific binding of its respective antibody to other nontarget cellular regions. In this report, we used lanthanide-tagged isotypes, which allowed for correction for nonspecific binding at the single-cell level. Using this approach, myoblasts were phenotypically identified by their position in the myogenic lineage, simultaneously with the quantification of autophagic flux in each identified subset. We found that generally autophagy flux is upregulated specifically during myoblast fusion and declines in myotubes. We also observed that mitophagy (i.e., selective autophagic degradation of mitochondria) is also active after myotube formation. The ability to track different types of autophagy is another feature of this methodology, which could be key to expand the current understanding of autophagy regulation in regenerating the skeletal muscle.

Quantifying Heterogeneity of Individual Organelles in Mixed Populations via Mass Cytometry.

Macroautophagy is a complex degradative intracellular process by which long-lived proteins and damaged organelles are cleared. Common methods for the analysis of autophagy are bulk measurements which mask organelle heterogeneity and complicate the analysis of interorganelle association and trafficking. Thus, methods for individual organelle quantification are needed to address these deficiencies. Current techniques for quantifying individual autophagy organelles are either low through-put or are dimensionally limited. We make use of the multiparametric capability of mass cytometry to investigate phenotypic heterogeneity in autophagy-related organelle types that have been isolated from murine brain, liver, and skeletal muscle. Detection and phenotypic classification of individual organelles were accomplished through the use of a lanthanide-chelating membrane stain and organelle-specific antibodies. Posthoc sample matrix background correction and nonspecific antibody binding corrections provide measures of interorganelle associations and heterogeneity. This is the first demonstration of multiparametric individual organelle analysis via mass cytometry. The method described here illustrates the potential for further investigation of the inherently complex interorganelle associations, trafficking, and heterogeneity present in most eukaryotic biological systems.

Deterministic Ratchet for Sub-micrometer (Bio)particle Separation.

Resolving the heterogeneity of particle populations by size is important when the particle size is a signature of abnormal biological properties leading to disease. Accessing size heterogeneity in the sub-micrometer regime is particularly important to resolve populations of subcellular species or diagnostically relevant bioparticles. Here, we demonstrate a ratchet migration mechanism capable of separating sub-micrometer sized species by size and apply it to biological particles. The phenomenon is based on a deterministic ratchet effect, is realized in a microfluidic device, and exhibits fast migration allowing separation in tens of seconds. We characterize this phenomenon extensively with the aid of a numerical model allowing one to predict the speed and resolution of this method. We further demonstrate the deterministic ratchet migration with two sub-micrometer sized beads as model system experimentally as well as size-heterogeneous mouse liver mitochondria and liposomes as model system for other organelles. We demonstrate excellent agreement between experimentally observed migration and the numerical model.

Sizing lipid droplets from adult and geriatric mouse liver tissue via nanoparticle tracking analysis.

The significance of lipid droplets in lipid metabolism, cell signaling, and regulating longevity is increasingly recognized, yet the lipid droplet's unique properties and architecture make it difficult to size and study using conventional methods. To begin to address this issue, we demonstrate the capabilities of nanoparticle tracking analysis (NTA) for sizing of lipid droplets. NTA was found to be adequate to assess lipid droplet stability over time, indicating that lipid droplet preparations are stable for up to 24 h. NTA had the ability to compare the size distributions of lipid droplets from adult and geriatric mouse liver tissue, suggesting an age-related decrease in lipid droplet size. This is the first report on the use of NTA to size intracellular organelles. Graphical Abstract Light scattering reveals the temporal positions of individual lipid droplets, which are recorded with a camera. The two-dimensional diffusion constant of each lipid droplet is extracted from the data set, which is then used to calculate a hydrodynamic radius using the Stokes-Einstein equation.

Deterministic Absolute Negative Mobility for Micro- and Submicrometer Particles Induced in a Microfluidic Device.

Efficient separations of particles with micron and submicron dimensions are extremely useful in preparation and analysis of materials for nanotechnological and biological applications. Here, we demonstrate a nonintuitive, yet efficient, separation mechanism for µm and subµm colloidal particles and organelles, taking advantage of particle transport in a nonlinear post array in a microfluidic device under the periodic action of electrokinetic and dielectrophoretic forces. We reveal regimes in which deterministic particle migration opposite to the average applied force occurs for a larger particle, a typical signature of deterministic absolute negative mobility (dANM), whereas normal response is obtained for smaller particles. The coexistence of dANM and normal migration was characterized and optimized in numerical modeling and subsequently implemented in a microfluidic device demonstrating at least 2 orders of magnitude higher migration speeds as compared to previous ANM systems. We also induce dANM for mouse liver mitochondria and envision that the separation mechanisms described here provide size selectivity required in future separations of organelles, nanoparticles, and protein nanocrystals.

Identification and Characterization of Mitochondrial Subtypes in Caenorhabditis elegans via Analysis of Individual Mitochondria by Flow Cytometry.

Mitochondrial bioenergetics has been implicated in a number of vital cellular and physiological phenomena, including aging, metabolism, and stress resistance. Heterogeneity of the mitochondrial membrane potential (Δψ), which is central to organismal bioenergetics, has been successfully measured via flow cytometry in whole cells but rarely in isolated mitochondria from large animal models. Similar studies in small animal models, such as Caenorhabditis elegans (C. elegans), are critical to our understanding of human health and disease but lack analytical methodologies. Here we report on new methodological developments that make it possible to investigate the heterogeneity of Δψ in C. elegans during development and in tissue-specific studies. The flow cytometry methodology described here required an improved collagenase-3-based mitochondrial isolation procedure and labeling of mitochondria with the ratiometric fluorescent probe JC-9. To demonstrate feasibility of tissue-specific studies, we used C. elegans strains expressing blue-fluorescent muscle-specific proteins, which enabled identification of muscle mitochondria among mitochondria from other tissues. This methodology made it possible to observe, for the first time, critical changes in Δψ during C. elegans larval development and provided direct evidence of the elevated bioenergetic status of muscle mitochondria relative to their counterparts in the rest of the organism. Further application of these methodologies can help tease apart bioenergetics and other biological complexities in C. elegans and other small animal models used to investigate human disease and aging.

Capillary Electrophoresis with Laser-Induced Fluorescent Detection of Immunolabeled Individual Autophagy Organelles Isolated from Liver Tissue.

Macroautophagy is a cellular degradation process responsible for the clearance of excess intracellular cargo. Existing methods for bulk quantification of autophagy rely on organelle markers that bind to multiple autophagy organelle types, making it difficult to tease apart the subcellular mechanisms implicated in autophagy dysfunction in liver and other pathologies. To address this issue, methods based on individual organelle measurements are needed. Capillary electrophoresis with laser-induced fluorescent detection (CE-LIF) was previously used to count and determine properties of individual autophagy organelles isolated from an LC3-GFP expressing cell line, but has never been used on autophagy organelles originating from a tissue sample. Here, we used DyLight488-labeled anti-LC3 antibodies to label endogenous LC3 present on organelles isolated from murine liver tissue prior to CE-LIF analysis. We evaluated the ability of this method to detect changes in a known model system of altered autophagy, as well as confirmed the specificity and reproducibility of the antibody in the labeling of autophagy organelles from liver tissue. This is both the first demonstration of CE-LIF to analyze individual organelles labeled with fluorophore-conjugated antibodies, and the first application of individual organelle CE-LIF to measure the properties of autophagy organelles isolated from tissue. The observations described here demonstrate that CE-LIF of immunolabeled autophagy organelles is a powerful technique useful to investigate the complexity of autophagy in any tissue sample of interest.

Nanohole Array-Directed Trapping of Mammalian Mitochondria Enabling Single Organelle Analysis.

We present periodic nanohole arrays fabricated in free-standing metal-coated nitride films as a platform for trapping and analyzing single organelles. When a microliter-scale droplet containing mitochondria is dispensed above the nanohole array, the combination of evaporation and capillary flow directs individual mitochondria to the nanoholes. Mammalian mitochondria arrays were rapidly formed on chip using this technique without any surface modification steps, microfluidic interconnects, or external power sources. The trapped mitochondria were depolarized on chip using an ionophore with results showing that the organelle viability and behavior were preserved during the on-chip assembly process. Fluorescence signal related to mitochondrial membrane potential was obtained from single mitochondria trapped in individual nanoholes revealing statistical differences between the behavior of polarized vs depolarized mammalian mitochondria. This technique provides a fast and stable route for droplet-based directed localization of organelles-on-a-chip with minimal limitations and complexity, as well as promotes integration with other optical or electrochemical detection techniques.

Proinflammatory cytokines differentially regulate adipocyte mitochondrial metabolism, oxidative stress, and dynamics.

Proinflammatory cytokines differentially regulate adipocyte mitochondrial metabolism, oxidative stress, and dynamics. Macrophage infiltration of adipose tissue and the chronic low-grade production of inflammatory cytokines have been mechanistically linked to the development of insulin resistance, the forerunner of type 2 diabetes mellitus. In this study, we evaluated the chronic effects of TNFα, IL-6, and IL-1ß on adipocyte mitochondrial metabolism and morphology using the 3T3-L1 model cell system. TNFα treatment of cultured adipocytes led to significant changes in mitochondrial bioenergetics, including increased proton leak, decreased ΔΨm, increased basal respiration, and decreased ATP turnover. In contrast, although IL-6 and IL-1ß decreased maximal respiratory capacity, they had no effect on ΔΨm and varied effects on ATP turnover, proton leak, or basal respiration. Only TNFα treatment of 3T3-L1 cells led to an increase in oxidative stress (as measured by superoxide anion production and protein carbonylation) and C16 ceramide synthesis. Treatment of 3T3-L1 adipocytes with cytokines led to decreased mRNA expression of key transcription factors and control proteins implicated in mitochondrial biogenesis, including PGC-1α and eNOS as well as deceased expression of COX IV and Cyt C. Whereas each cytokine led to effects on expression of mitochondrial markers, TNFα exclusively led to mitochondrial fragmentation and decreased the total level of OPA1 while increasing OPA1 cleavage, without expression of levels of mitofusin 2, DRP-1, or mitofilin being affected. In summary, these results indicate that inflammatory cytokines have unique and specialized effects on adipocyte metabolism, but each leads to decreased mitochondrial function and a reprogramming of fat cell biology.

Simultaneous measurement of individual mitochondrial membrane potential and electrophoretic mobility by capillary electrophoresis.

Mitochondrial membrane potential varies, depending on energy demand, subcellular location, and morphology and is commonly used as an indicator of mitochondrial functional status. Electrophoretic mobility is a heterogeneous surface property reflective of mitochondrial surface composition and morphology, which could be used as a basis for separation of mitochondrial subpopulations. Since these properties are heterogeneous, methods for their characterization in individual mitochondria are needed to better design and understand electrophoretic separations of subpopulations of mitochondria. Here we report on the first method for simultaneous determination of individual mitochondrial membrane potential and electrophoretic mobility by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). Mitochondria were isolated from cultured cells, mouse muscle, or liver, and then polarized, labeled with JC-1 (a ratiometric fluorescent probe, which indicates changes in membrane potential), and separated with CE-LIF. Red/green fluorescence intensity ratios from individual mitochondria were used as an indicator of mitochondrial membrane potential. Reproducible distributions of individual mitochondrial membrane potential and electrophoretic mobility were observed. Analysis of polarized and depolarized regions of interest defined using red/green ratios and runs of depolarized controls allowed for the determination of membrane potential and comparison of electrophoretic mobility distributions in preparations containing depolarized mitochondria. Through comparison of these regions of interest, we observed dependence of electrophoretic mobility on membrane potential, with polarized regions of interest displaying decreased electrophoretic mobility. This method could be applied to investigate mitochondrial heterogeneity in aging or disease models where membrane potential is an important factor.

Monitoring subcellular biotransformation of N-L-leucyldoxorubicin by micellar electrokinetic capillary chromatography coupled to laser-induced fluorescence detection.

Development of prodrugs is a promising alternative to address cytotoxicity and nonspecificity of common anticancer agents. N-L-leucyldoxorubicin (LeuDox) is a prodrug that is biotransformed to the anticancer drug doxorubicin (Dox) in the extracellular space; however, its biotransformation may also occur intracellularly in endocytic organelles. Such organelle-specific biotransformation is yet to be determined. In this study, magnetically enriched endocytic organelle fractions from human uterine sarcoma cells were treated with LeuDox. Micellar electrokinetic chromatography with laser-induced fluorescence detection (MEKC-LIF) was used to determine that 10% of LeuDox was biotransformed to Dox, accounting for ~43% of the biotransformation occurring in the post-nuclear fraction. This finding suggests that endocytic organelles also participate in the intracellular biotransformation of LeuDox to Dox.

Analysis of individual mitochondria via fluorescent immunolabeling with Anti-TOM22 antibodies.

Mitochondria are responsible for maintaining a variety of cellular functions. One such function is the interaction and subsequent import of proteins into these organelles via the translocase of outer membrane (TOM) complex. Antibodies have been used to analyze the presence and function of proteins comprising this complex, but have not been used to investigate variations in the abundance of TOM complex in mitochondria. Here, we report on the feasibility of using capillary cytometry with laser-induced fluorescence to detect mitochondria labeled with antibodies targeting the TOM complex and to estimate the number of antibodies that bind to these organelles. Mitochondria were fluorescently labeled with DsRed2, while antibodies targeting the TOM22 protein, one of nine proteins comprising the TOM complex, were conjugated to the Atto-488 fluorophore. At typical labeling conditions, 94% of DsRed2 mitochondria were also immunofluorescently labeled with Atto-488 Anti-TOM22 antibodies. The calculated median number of Atto-488 Anti-TOM22 antibodies bound to the surface of mitochondria was ∼2,000 per mitochondrion. The combination of fluorescent immunolabeling and capillary cytometry could be further developed to include multicolor labeling experiments, which enable monitoring several molecular targets at the same time in the same or different organelle types.

Describing autophagy via analysis of individual organelles by capillary electrophoresis with laser induced fluorescence detection.

Autophagy is a cellular process responsible for the degradation of intracellular cargo. Its dynamic nature and the multiple types of autophagy organelles present at a given time make current measurements, such as those done by Western blotting, insufficient to understand autophagy and its roles in aging and disease. Capillary electrophoresis coupled to laser induced fluorescence detection (CE-LIF) has been used previously to count and determine properties of individual organelles, but has never been used on autophagy organelles or for determination of changes of such properties. Here we used autophagy organelles isolated from L6 cells expressing GFP-LC3, which is an autophagy marker, to develop a CE-LIF method for the determination of the number of autophagy organelles, their individual GFP-LC3 fluorescence intensities, and their individual electrophoretic mobilities. These properties were compared under basal and rapamycin-driven autophagy, which showed differences in the number of detected organelles and electrophoretic mobility distributions of autophagy organelles. Vinblastine treatment was also used to halt autophagy and further characterize changes and provide additional insight on the nature of autophagy organelles. This approach revealed dramatic and opposite directions in changes of organelle numbers, GFP-LC3 contents, and electrophoretic mobilities during the duration of the vinblastine treatment. These trends suggested the identity of organelle types being detected. These observations demonstrate that individual organelle analysis by CE-LIF is a powerful technology to investigate the complexity and nature of autophagy, a process that plays critical roles in response to drug treatments, aging, and disease.

Predicting isoelectric points of nonfunctional mitochondria from Monte Carlo simulations of surface compositions.

Mitochondria are heterogeneous organelles involved in energy production, metabolism, and cellular signaling that oftentimes are isolated from cells for chemical characterization (e.g., proteomic analysis). The chemical composition of the mitochondrial outer membrane is one of the factors defining the mitochondrial isoelectric point (pI), which is a property useful for the analysis and characterization of isolated mitochondria. We previously used capillary isoelectric focusing (cIEF) with laser-induced-fluorescence detection to determine the experimental pI of individual mitochondria after their isolation under depolarizing conditions. This technique revealed that, when kept nonfunctional, mitochondrial pI is heterogeneous as displayed by the observed distributions of pI. To model the effect of surface composition on pI heterogeneity of these mitochondria, we devised a method to predict mitochondrial pI values using simulated surface compositions. The method was initially validated by predicting the pI values of known mitochondrial outer membrane proteins and was then extended to isolated mitochondria, in which both ionizable amino acids and phospholipids contribute to mitochondrial pI. After using a Monte Carlo method to generate a library of over 2 million possible mitochondrial surface compositions, sufficient compositions to match the frequency of occurrence of experimental mitochondrial pI values were randomly selected. This comparison allows for association of a given individual mitochondrial pI with thousands of randomly chosen compositions. The method predicts significant changes in the percentages of some amino acids and phospholipids for observed pI differences between individual mitochondria, which is an important advancement toward explaining the observed heterogeneity of mitochondrial pI.

Capillary electrophoretic analysis of hydroxyl radicals produced by respiring mitochondria.

Here, we report the use of a capillary electrophoretic method with laser-induced fluorescence detection to evaluate hydroxyl radicals produced by respiring mitochondria. The probe, hydroxyphenylfluorescein (HPF), is separated from the product, fluorescein, in under 5 min with zeptomole and attomole limits of detection for fluorescein and HPF, respectively. Purification of the probe with a C-18 SPE column is necessary to reduce the fluorescein impurity in the probe stock solution from 0.4% to less than 0.001%. HPF was responsive to hydroxyl radicals produced by isolated mitochondria from L6 cells, and this signal was blunted when DMSO was added to scavenge hydroxyl radicals and when carbonyl cyanide m-chlorophenylhydrazone was added to depolarize the mitochondria. The method was used to compare hydroxyl radical levels in mitochondria isolated from brown adipose tissue of lean and obese mice. Mitochondria from obese mice produced significantly more hydroxyl radicals than those from lean mice.