PubMed

Methods Mol Biol. 2023;2625:141-148. doi: 10.1007/978-1-0716-2966-6_13.ABSTRACTAn optimized Bligh and Dyer protocol and subsequent derivatization is described in this chapter for the extraction of free cholesterol and cholesterol esters from tissue samples. Quantification analysis of lipid species is then described utilizing gas chromatography-mass spectrometry, the ideal method for analysis of volatile organic compounds and extraction of sterols.PMID:36653640 | DOI:10.1007/978-1-0716-2966-6_13

Methods Mol Biol. 2023;2625:103-106. doi: 10.1007/978-1-0716-2966-6_9.ABSTRACTLipids serve an essential role in multiple cellular functions including signaling, metabolism, energy storage, and membrane constitution. Lipidomics, the study of lipids using analytical chemistry, allows for the study of disease states and cellular metabolism. Shotgun lipidomics is a technique that involves direct-infusion electrospray ionization (ESI) and analysis with a triple quadrupole mass spectrometer. Triple quadrupole mass spectrometry is ideally suited for lipidomics analysis because it allows for class-specific identification of lipids. Individual lipid class can be identified by the adjustment of three parameters-collision energy, ion mode, and scan type. This chapter describes the use of a triple quadrupole mass spectrometer, the TSQ Quantum Access MAX, to perform lipidomics analysis with high sensitivity, accuracy, and precision.PMID:36653636 | DOI:10.1007/978-1-0716-2966-6_9

Methods Mol Biol. 2023;2625:79-88. doi: 10.1007/978-1-0716-2966-6_7.ABSTRACTPhospholipids are essential components of membrane lipid bilayers and serve as precursors of multiple signaling molecules, so alterations in their homeostasis are associated with the pathogenesis of numerous diseases. In this context, the application of mass spectrometry-based metabolomics has demonstrated great potential to comprehensively characterize the human phospholipidome. In this chapter, we describe an untargeted method for the determination of phospholipids and other related metabolites in a variety of biological matrices, including plasma/serum, erythrocytes, and tissues, based on the combination of high-throughput direct mass spectrometry fingerprinting and subsequent profiling by ultra-high-performance reversed-phase liquid chromatography coupled to mass spectrometry. Furthermore, we also review the characteristic fragmentation patterns of phospholipids with the aim of providing simple guidelines for their straightforward annotation.PMID:36653634 | DOI:10.1007/978-1-0716-2966-6_7

Methods Mol Biol. 2023;2625:65-69. doi: 10.1007/978-1-0716-2966-6_5.ABSTRACTThis chapter focuses on identifying gangliosides in the optic nerve of the mouse using mass spectrometry techniques. The described protocol will also permit the characterization of the sample's lipidome. Two deuterium-labeled ganglioside standards and a general lipid class standard will be utilized for extraction efficiency and quantification. Using reversed-phase high-performance liquid chromatography (HPLC) coupled to a Q Exactive mass spectrometer, the samples will be analyzed. The method will consist of both an untargeted approach and a targeted approach with a ganglioside-specific inclusion list.PMID:36653632 | DOI:10.1007/978-1-0716-2966-6_5

Methods Mol Biol. 2023;2625:57-63. doi: 10.1007/978-1-0716-2966-6_4.ABSTRACTLiquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is a powerful tool for identification and classification of lipids. Ultra-high performance liquid chromatography (UHPLC) allows for robust separations of complex mixtures, while high-resolution mass spectrometry (HRMS) identifies compounds with efficiency and accuracy (Zullig T and Kofeler HC, Mass Spectrom Rev 40:162-176, 2021). The high specificity and sensitivity of mass spectrometry makes it the method of choice when analyzing lipids (Kofeler HC, J Lipid Res 62:100138, 2021). Untargeted mass spectrometry identifies all lipids within a sample and is useful for identification and further discovery. This chapter describes the use of a Q Exactive mass spectrometer to perform an untargeted LC-MS/MS lipidomics analysis.PMID:36653631 | DOI:10.1007/978-1-0716-2966-6_4

Methods Mol Biol. 2023;2625:1-6. doi: 10.1007/978-1-0716-2966-6_1.ABSTRACTMitochondria participate in many important metabolic processes in the body. The lipid profile of mitochondria is especially important in membrane regulation and pathway signaling. The isolation and study of these lipids can provide unparalleled information about the mechanisms behind these cellular processes. In this chapter, we describe a protocol to isolate mitochondrial lipids from homogenized murine optic nerves. The lipid extraction was performed using butanol-methanol (BUME) and subsequently analyzed using liquid chromatography-mass spectrometry. Further analysis of the raw data was conducted using LipidSearch™ and MetaboAnalyst 4.0.PMID:36653628 | DOI:10.1007/978-1-0716-2966-6_1

Commun Biol. 2023 Jan 18;6(1):65. doi: 10.1038/s42003-023-04444-7.ABSTRACTHuman cancers often re-express germline factors, yet their mechanistic role in oncogenesis and cancer progression remains unknown. Here we demonstrate that DEAD-box helicase 4 (DDX4), a germline factor and RNA helicase conserved in all multicellular organisms, contributes to increased cell motility and cisplatin-mediated drug resistance in small cell lung cancer (SCLC) cells. Proteomic analysis suggests that DDX4 expression upregulates proteins related to DNA repair and immune/inflammatory response. Consistent with these trends in cell lines, DDX4 depletion compromised in vivo tumor development while its overexpression enhanced tumor growth even after cisplatin treatment in nude mice. Further, the relatively higher DDX4 expression in SCLC patients correlates with decreased survival and shows increased expression of immune/inflammatory response markers. Taken together, we propose that DDX4 increases SCLC cell survival, by increasing the DNA damage and immune response pathways, especially under challenging conditions such as cisplatin treatment.PMID:36653474 | DOI:10.1038/s42003-023-04444-7

Nat Commun. 2023 Jan 18;14(1):288. doi: 10.1038/s41467-023-35952-z.ABSTRACTDietary restriction (DR) delays aging and the onset of age-associated diseases. However, it is yet to be determined whether and how restriction of specific nutrients promote longevity. Previous genome-wide screens isolated several Escherichia coli mutants that extended lifespan of Caenorhabditis elegans. Here, using 1H-NMR metabolite analyses and inter-species genetics, we demonstrate that E. coli mutants depleted of intracellular glucose extend C. elegans lifespans, serving as bona fide glucose-restricted (GR) diets. Unlike general DR, GR diets don't reduce the fecundity of animals, while still improving stress resistance and ameliorating neuro-degenerative pathologies of Aβ42. Interestingly, AAK-2a, a new AMPK isoform, is necessary and sufficient for GR-induced longevity. AAK-2a functions exclusively in neurons to modulate GR-mediated longevity via neuropeptide signaling. Last, we find that GR/AAK-2a prolongs longevity through PAQR-2/NHR-49/Δ9 desaturases by promoting membrane fluidity in peripheral tissues. Together, our studies identify the molecular mechanisms underlying prolonged longevity by glucose specific restriction in the context of whole animals.PMID:36653384 | DOI:10.1038/s41467-023-35952-z

Cell Death Dis. 2023 Jan 18;14(1):35. doi: 10.1038/s41419-022-05542-w.ABSTRACTThe SIRT6 deacetylase has been implicated in DNA repair, telomere maintenance, glucose and lipid metabolism and, importantly, it has critical roles in the brain ranging from its development to neurodegeneration. Here, we combined transcriptomics and metabolomics approaches to characterize the functions of SIRT6 in mouse brains. Our analysis reveals that SIRT6 is a central regulator of mitochondrial activity in the brain. SIRT6 deficiency in the brain leads to mitochondrial deficiency with a global downregulation of mitochondria-related genes and pronounced changes in metabolite content. We suggest that SIRT6 affects mitochondrial functions through its interaction with the transcription factor YY1 that, together, regulate mitochondrial gene expression. Moreover, SIRT6 target genes include SIRT3 and SIRT4, which are significantly downregulated in SIRT6-deficient brains. Our results demonstrate that the lack of SIRT6 leads to decreased mitochondrial gene expression and metabolomic changes of TCA cycle byproducts, including increased ROS production, reduced mitochondrial number, and impaired membrane potential that can be partially rescued by restoring SIRT3 and SIRT4 levels. Importantly, the changes we observed in SIRT6-deficient brains are also occurring in aging human brains and particularly in patients with Alzheimer's, Parkinson's, Huntington's, and Amyotrophic lateral sclerosis disease. Overall, our results suggest that the reduced levels of SIRT6 in the aging brain and neurodegeneration initiate mitochondrial dysfunction by altering gene expression, ROS production, and mitochondrial decay.PMID:36653345 | DOI:10.1038/s41419-022-05542-w

Clin Transl Med. 2023 Jan;13(1):e1150. doi: 10.1002/ctm2.1150.ABSTRACTBACKGROUND: Low-density neutrophils (LDN) are a distinct subset of neutrophils rarely detected in healthy people but appear in the blood of patients with autoimmune diseases, including systemic lupus erythematosus (SLE), and are mobilised in response to granulocyte colony-stimulating factor (G-CSF). The aim of this study was to identify novel mechanisms responsible for the pathogenic capacity of LDN in SLE.METHODS: Neutrophils were isolated from donors treated with G-CSF, and whole-cell proteomic analysis was performed on LDN and normal-density neutrophils.RESULTS: CD98 is significantly upregulated in LDN from G-CSF donors and defines a subset of LDN within the blood of SLE patients. CD98 is a transmembrane protein that dimerises with L-type amino acid transporters. We show that CD98 is responsible for the increased bioenergetic capacity of LDN. CD98 on LDN mediates the uptake of essential amino acids that are used by mitochondria to produce adenosine triphosphate, especially in the absence of glucose. Inhibition of CD98 reduces the metabolic flexibility of this population, which may limit their pathogenic capacity. CD98+ LDN produce more proinflammatory cytokines and chemokines than their normal density counterparts and are resistant to apoptosis, which may also contribute to tissue inflammation and end organ damage in SLE.CONCLUSIONS: CD98 provides a phenotypic marker for LDN that facilitates identification of this population without density-gradient separation and represents a novel therapeutic target to limit its pathogenic capacity.PMID:36653319 | DOI:10.1002/ctm2.1150

Methods Cell Biol. 2023;173:91-107. doi: 10.1016/bs.mcb.2022.07.008. Epub 2022 Sep 29.ABSTRACTNatural Killer (NK) cells play a pivotal role in the elimination of tumor cells. The interactions that NK cells can establish with cancer cells in the tumor microenvironment (TME) are crucial for the outcome of the anti-tumor response, possibly resulting in mechanisms able to modulate NK cell effector functions on the one side, and to modify tumor cell phenotype and properties on the other side. This chapter will describe two different experimental approaches for the evaluation of NK-tumor cell interactions. First, a detailed protocol for the setting up of NK-tumor cell co-cultures will be illustrated, followed by information on cell imaging techniques, useful for assessing cell morphology and cytoskeletal changes. The second part will be focused on the description of a proteomic approach aimed at investigating the effect of this crosstalk from another point of view, i.e., characterizing the cellular and molecular pathways modulated in tumor cells following interaction with NK cells. The chapter centers on the interaction between NK and melanoma cells and refers to experimental approaches we set up to study the effects of this cross-talk on the process of the Epithelial-to-Mesenchymal Transition (EMT). Nevertheless, the described protocols can be quite easily adapted to study the interaction of NK cells with adherent tumor cell lines of different origin and histotype, as in our original study, we also analyzed possible NK-induced morphologic changes in the cervix adenocarcinoma HeLa cells and the colon cancer HT29 cells.PMID:36653088 | DOI:10.1016/bs.mcb.2022.07.008

Cell Rep Med. 2023 Jan 17;4(1):100897. doi: 10.1016/j.xcrm.2022.100897.ABSTRACTFeeding behavior must be continuously adjusted to match energy needs. Recent discoveries in murine models identified uridine as a regulator of energy balance. Here, we explore its contribution to the complex control of food intake in humans by administering a single dose of uridine monophosphate (UMP; 0.5 or 1 g) to healthy participants in two placebo-controlled studies designed to assess food behavior (registration: DRKS00014874). We establish that endogenous circulating uridine correlates with hunger and ensuing food consumption. It also dynamically decreases upon caloric ingestion, prompting its potential role in a negative feedback loop regulating energy intake. We further demonstrate that oral UMP administration temporarily increases circulating uridine and-when within the physiological range-enhances hunger and caloric intake proportionally to participants' basal energy needs. Overall, uridine appears as a potential target to tackle dysfunctions of feeding behavior in humans.PMID:36652907 | DOI:10.1016/j.xcrm.2022.100897

Plant Physiol Biochem. 2023 Jan 13;195:288-299. doi: 10.1016/j.plaphy.2023.01.020. Online ahead of print.ABSTRACTWild ginseng is thought to be superior in its medicinal quality to cultivated ginseng, potentially owing to the differences in active components. This study was designed accordingly to assess the differences in secondary metabolite components and their synthesis in wild and cultivated ginseng by using quantitative proteomics combined with secondary metabolomics approaches. A total of 72 secondary metabolites were found to be differentially abundant, of which dominant abundant in wild ginseng primarily included triterpenoid saponins (ginsenosides) and phytosterols. Ginsenoside diversity was increased in wild ginseng, particularly with respect to rare ginsenosides. Ginsenoside Rk1, F1, Rg5, Rh1, PPT, Rh2, and CK enriched in wild ginseng were validated by HPLC. In addition to ginsenosides, stigmasterol and β-sitosterol were accumulated in wild ginseng. 102 differentially expressed proteins between wild and cultivated ginseng were identified using iTRAQ labeling technique. Among them, 25 were related to secondary metabolism, mainly involved in sesquiterpene and triterpene biosynthesis, which was consistent with metabolomics results. Consistently, the activity levels of HMGR, FDPS, SS, SE, DS, CYP450, GT and CAS, which are key enzymes related to ginsenoside and phytosterol biosynthesis, were confirmed to be elevated in wild ginseng.The biosynthesis of ginsenosides and phytosterols in wild ginseng is higher than that in cultivated ginseng, which may be related to natural growth without artificial domestication. To some extent, this study explained the accumulation of pharmacodynamic components and overall quality of ginseng, which could provide reference for the germplasm improvement and planting of ginseng.PMID:36652850 | DOI:10.1016/j.plaphy.2023.01.020

Plant Physiol Biochem. 2023 Jan 13;195:275-287. doi: 10.1016/j.plaphy.2023.01.025. Online ahead of print.ABSTRACTThe pericarp of fruit can be differentiated into endocarp, mesocarp, and exocarp. To explore the differences in gene expression and metabolites in different tissues of the pericarp, the fruits of sumac (Toxicodendron vernicifluum) were separated into endocarp and mesocarp-exocarp. The metabolites and transcriptome of exocarp-mesocarp and endocarp of Toxicodendron vernicifluum were analyzed by HPLC-QTOF-MS/MS and RNA sequencing, respectively. A total of 52 phenolic compounds were identified, including 3 phenylpropane derivatives, 10 urushiol compounds and 39 flavonoids. The exocarp-mesocarp contained more urushiol compounds and flavonoid glycosides while the endocarp contained more biflavonoids, such as rhusflavone and dihydromorelloflavone. The characteristic component of endocarp was rhusflavone and the characteristic component of exocarp-mesocarp was urushiol (triene). Most of the genes involved in flavonoid synthesis pathway were upregulated in endocarp compared with exocarp-mesocarp and positively correlated with the content of flavonoids. The candidate genes related to the synthesis of components of flavonoid glycosides and biflavonoids were screened. Metabolomic and transcriptomic analyses provide new insights into the synthesis and distribution of flavonoid glycosides and biflavonoids in the fruits of Toxicodendron vernicifluum.PMID:36652849 | DOI:10.1016/j.plaphy.2023.01.025

J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Jan 11;1216:123603. doi: 10.1016/j.jchromb.2023.123603. Online ahead of print.ABSTRACTMyocardial ischemia/reperfusion (MI/R) injury is a life-threatening syndrome with high morbidity and mortality. Zhishi-Xiebai-Guizhi Decoction (ZSXBGZD) is a classic traditional Chinese medicine formula, used to treat cardiovascular diseases for centuries. However, its underlying medicinal mechanism has not been clearly elucidated, which hinders its widespread application. Here, the curative effects and therapeutic mechanism of ZSXBGZD against MI/R were addressed based on an integration of pharmaceutical evaluation and cellular metabolomics. First, a hypoxia/reoxygenation (H/R) model in H9c2 cells was employed to resemble MI/R and multiple pharmacological indicators were performed to assess the efficacy of ZSXBGZD. The results showed that ZSXBGZD possessed exceptional ability in attenuating cardiomyocyte injury, concerning oxidative stress, mitochondrial dysfunction, energy acquisition and cell apoptosis. Furthermore, a cell metabolomics approach based on HILIC and UPLC-Q-TOF-MS coupled with multivariate analysis was conducted to explore the metabolic regulation of ZSXBGZD. 38 differential polar metabolites related to H/R were uncovered, and 34 of them were reversed to normal state after the treatment of ZSXBGZD, revealing the perturbations of energy metabolism and amino acid metabolism. Moreover, formula decomposition justified the combination of single herbs to form ZSXBZGD and confirmed the pivotal status of Allii Macrostemonis Bulbus and Trichosanthis Fructus.PMID:36652817 | DOI:10.1016/j.jchromb.2023.123603

J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Jan 10;1216:123602. doi: 10.1016/j.jchromb.2023.123602. Online ahead of print.ABSTRACTKidney yang deficiency syndrome (KYDS) is a classic syndrome of traditional Chinese medicine (TCM). The salt-processed product of Semen Cuscuta (YP) is the monarch drug in Bushen Antai Mixture (BAM), can improve the reproductive dysfunction caused by KYDS, and the effect is better than that of raw products of Semen Cuscuta (SP). However, its mechanism is not completely clear yet. In this study, an integrated strategy combining untargeted metabolomics with microbiology was used to explore the mechanism of YP in the BAM improving KYDS. 16S rDNA gene sequencing showed that BAM containing YP (Y-BAM) had a significantly better regulatory effect on Desulfobacterota and Desulfovibrionaceae_unclassified than BAM containing SP (S-BAM). Untargeted metabolomics studies showed that Y-BAM significantly regulated 4 metabolites and 4 metabolic pathways. In addition, multi-index analysis showed that the effect of Y-BAM on arachidonic acid metabolism, tyrosine metabolism, purine metabolism, fructose and mannose metabolism and total metabolism was closer to that of the control group compared to S-BAM. The analysis of serum biochemical indexes showed that Y-BAM had more significant regulating effect on the levels of luteinizing hormone (LH), follicle stimulating hormone (FSH), testosterone (T) and superoxide dismutase (SOD) in serum of KYDS rats compared to S-BAM. Spearman correlation analysis showed that there was a significant correlation between intestinal microorganisms and metabolites and serum biochemical indexes. For example, Desulfovibrionaceae_unclassified was positively correlated with arachidonic acid, and negatively correlated with SOD and LH. This study suggests that YP may enhance the regulation of intestinal flora and endogenous metabolism of KYDS, so that BAM shows a better therapeutic effect on KYDS, which also reasonably explains why BAM uses Semen Cuscuta stir-baked with salt solution.PMID:36652816 | DOI:10.1016/j.jchromb.2023.123602

Food Chem. 2023 Jan 9;410:135441. doi: 10.1016/j.foodchem.2023.135441. Online ahead of print.ABSTRACTThe effects of superfine yak bone powder (YBP) on human gut microbiota (HGM) were investigated by in vitro digestion and fermentation combined with microbiomics and metabolomics. Results showed that size reduction and protein structural degradation during digestion allowed superfine YBP to release more Ca2+ than CaCO3 powders with similar particle size. Moreover, the indigestible YBP further influenced HGM and was associated with increased occurrence of beneficial bacteria such as Megasphaera spp., Megamonas spp., Acidaminococcus spp., and Prevotella spp. The altered HGM was associated with greater production of short-chain fatty acids with 4-6 carbon atoms. Furthermore, the indigestible YBP was associated with up-regulation of many lipid-related metabolites, including taurine, secondary bile acids, saturated long-chain fatty acids, and ω-3/ω-6 polyunsaturated fatty acids, which modulated favorably lipid metabolic pathways. These findings implied the potential activity of superfine YBP as a food fortifier in favorably altering HGM community structure and regulating lipid metabolism.PMID:36652799 | DOI:10.1016/j.foodchem.2023.135441

Curr Opin Plant Biol. 2023 Jan 16;73:102332. doi: 10.1016/j.pbi.2022.102332. Online ahead of print.ABSTRACTFlowering plants have evolved extraordinarily diverse metabolites that underpin the floral visual and olfactory signals enabling plant-pollinator interactions. In some cases, these metabolites also provide unusual rewards that specific pollinators depend on. While some metabolites are shared by most flowering plants, many have evolved in restricted lineages in response to the specific selection pressures encountered within different niches. The latter are designated as specialized metabolites. Recent investigations continue to uncover a growing repertoire of unusual specialized metabolites. Increased accessibility to cutting-edge multi-omics technologies (e.g. genome, transcriptome, proteome, metabolome) is now opening new doors to simultaneously uncover the molecular basis of their synthesis and their evolution across diverse plant lineages. Drawing upon the recent literature, this perspective discusses these aspects and, where known, their ecological and evolutionary relevance. A primer on omics-guided approaches to discover the genetic and biochemical basis of functional specialized metabolites is also provided.PMID:36652780 | DOI:10.1016/j.pbi.2022.102332

Clin Cancer Res. 2023 Jan 18:CCR-22-2263. doi: 10.1158/1078-0432.CCR-22-2263. Online ahead of print.ABSTRACTPURPOSE: Inhibition of monocarboxylate transporter (MCT) 1-mediated lactate transport may have cytostatic/cytotoxic effects on tumour cells. We report results from the dose-escalation part of a first‑in‑human trial of AZD3965, a first-in-class MCT1 inhibitor, in advanced cancer.EXPERIMENTAL DESIGN: This multicentre, Phase 1, dose-escalation and dose-expansion trial enrolled patients with advanced solid tumours or lymphoma and no standard therapy options. Exclusion criteria included history of retinal/cardiac disease, due to MCT1 expression in the eye and heart. Patients received daily oral AZD3965 according to a 3+3 then rolling 6 design. Primary objectives were to assess safety and determine the maximum tolerated dose and/or recommended Phase 2 dose (RP2D). Secondary objectives for dose-escalation included measurement of pharmacokinetics and pharmacodynamic activity. Exploratory biomarkers included tumour expression of MCT1 and MCT4, functional imaging of biological impact and metabolomics.RESULTS: During dose-escalation, 40 patients received AZD3965 at 5-30 mg once daily or 10 or 15 mg twice daily (BD). Treatment‑emergent adverse events were primarily Grade 1/2, most commonly electroretinogram changes (retinopathy), fatigue, anorexia and constipation. Seven patients receiving ≥20 mg daily experienced dose-limiting toxicities (DLTs): Grade 3 cardiac troponin rise (n=1), asymptomatic ocular DLTs (n=5) and Grade 3 acidosis (n=1). Plasma pharmacokinetics demonstrated attainment of target concentrations; pharmacodynamic measurements indicated on‑target activity.CONCLUSIONS: AZD3965 is tolerated at doses that produce target engagement. DLTs were on-target and primarily dose-dependent, asymptomatic, reversible ocular changes. An RP2D of 10 mg BD was established for use in dose-expansion in cancers that generally express high MCT1/low MCT4 (not yet published).PMID:36652553 | DOI:10.1158/1078-0432.CCR-22-2263

PLoS One. 2023 Jan 18;18(1):e0280491. doi: 10.1371/journal.pone.0280491. eCollection 2023.ABSTRACTDiabetes mellitus is a multiorgan systemic disease impacting numerous ocular structures that results in significant ocular morbidity and often results in more frequent corneal and glaucoma surgeries for affected individuals. We hypothesize that the systemic metabolic and proteomic derangement observed in the progression of diabetes influences the composition of the aqueous humor (AH), which ultimately impacts the anterior segment health of the eye. To identify changes associated with diabetes progression, we mapped the metabolite profile and proteome of AH samples from patients with varying severities of type II diabetes (T2DM). Patients were classified as nondiabetic (ND or control), non-insulin-dependent diabetic without advanced features of disease (NAD-ni), insulin-dependent diabetic without advanced features (NAD-i), or diabetic with advanced features (AD). AH samples collected from the anterior chamber during elective ophthalmic surgery were evaluated for metabolite and protein expression changes associated with diabetic severity via gas chromatography/mass spectrometry and ultra-high performance liquid chromatography tandem mass spectrometry, respectively. Metabolic and proteomic pathway analyses were conducted utilizing MetaboAnalyst 4.0 and Ingenuity Pathway Analysis. A total of 14 control, 12 NAD-ni, 4 NAD-I, and 14 AD samples were included for analysis. Elevated levels of several branched amino acids (e.g., valine, leucine, isoleucine), and lipid metabolites (e.g., palmitate) were found only with increasing diabetic severity (i.e., the AD group). Similar proteomic trends were noted in amino acid and fatty acid metabolism and the unfolded protein/stress response. These results represent the first report of both metabolomic and proteomic evaluation of aqueous humor. Diabetes results in metabolic and proteomic perturbations detectable in the AH, and unique changes become manifest as T2DM severity worsens. Changes in AH composition may serve as an indicator of disease severity, risk assessment of anterior segment cells and structures, and potential future therapies.PMID:36652491 | DOI:10.1371/journal.pone.0280491