Ether phospholipids are required for mitochondrial reactive oxygen species homeostasis.

Mitochondria are hubs where bioenergetics, redox homeostasis, and anabolic metabolism pathways integrate through a tightly coordinated flux of metabolites. The contributions of mitochondrial metabolism to tumor growth and therapy resistance are evident, but drugs targeting mitochondrial metabolism have repeatedly failed in the clinic. Our study in pancreatic ductal adenocarcinoma (PDAC) finds that cellular and mitochondrial lipid composition influence cancer cell sensitivity to pharmacological inhibition of electron transport chain complex I. Profiling of patient-derived PDAC models revealed that monounsaturated fatty acids (MUFAs) and MUFA-linked ether phospholipids play a critical role in maintaining ROS homeostasis. We show that ether phospholipids support mitochondrial supercomplex assembly and ROS production; accordingly, blocking de novo ether phospholipid biosynthesis sensitized PDAC cells to complex I inhibition by inducing mitochondrial ROS and lipid peroxidation. These data identify ether phospholipids as a regulator of mitochondrial redox control that contributes to the sensitivity of PDAC cells to complex I inhibition.

Mucus-degrading Bacteroides link carbapenems to aggravated graft-versus-host disease.

The intestinal microbiota is an important modulator of graft-versus-host disease (GVHD), which often complicates allogeneic hematopoietic stem cell transplantation (allo-HSCT). Broad-spectrum antibiotics such as carbapenems increase the risk for intestinal GVHD, but mechanisms are not well understood. In this study, we found that treatment with meropenem, a commonly used carbapenem, aggravates colonic GVHD in mice via the expansion of Bacteroides thetaiotaomicron (BT). BT has a broad ability to degrade dietary polysaccharides and host mucin glycans. BT in meropenem-treated allogeneic mice demonstrated upregulated expression of enzymes involved in the degradation of mucin glycans. These mice also had thinning of the colonic mucus layer and decreased levels of xylose in colonic luminal contents. Interestingly, oral xylose supplementation significantly prevented thinning of the colonic mucus layer in meropenem-treated mice. Specific nutritional supplementation strategies, including xylose supplementation, may combat antibiotic-mediated microbiome injury to reduce the risk for intestinal GVHD in allo-HSCT patients.

The bacterial microbiota regulates normal hematopoiesis via metabolite-induced type 1 interferon signaling.

Antibiotic therapy, especially when administered long term, is associated with adverse hematologic effects such as cytopenia. Signals from the intestinal microbiota are critical to maintain normal hematopoiesis, and antibiotics can cause bone marrow suppression through depletion of the microbiota. We reported previously that STAT1 signaling is necessary for microbiota-dependent hematopoiesis, but the precise mechanisms by which the gut microbiota signals to the host bone marrow to regulate hematopoiesis remain undefined. We sought to identify the cell type(s) through which STAT1 promotes microbiota-mediated hematopoiesis and to elucidate which upstream signaling pathways trigger STAT1 signaling. Using conditional knockout and chimeric mice, we found that the microbiota induced STAT1 signaling in non-myeloid hematopoietic cells to support hematopoiesis and that STAT1 signaling was specifically dependent on type I interferons (IFNs). Indeed, basal type I IFN signaling was reduced in hematopoietic progenitor cells with antibiotic treatment. In addition, we discovered that oral administration of a commensal-derived product, NOD1 ligand, rescues the hematopoietic defects induced by antibiotics in mice. Using metabolomics, we identified additional microbially produced candidates that can stimulate type I IFN signaling to potentially rescue the hematopoietic defects induced by antibiotics, including phosphatidylcholine and γ-glutamylalanine. Overall, our studies define a signaling pathway through which microbiota promotes normal hematopoiesis and identify microbial metabolites that may serve as therapeutic agents to ameliorate antibiotic-induced bone marrow suppression and cytopenia.

Lipidomic Profiles of Plasma Exosomes Identify Candidate Biomarkers for Early Detection of Hepatocellular Carcinoma in Patients with Cirrhosis.

Novel biomarkers for HCC surveillance in cirrhotic patients are urgently needed. Exosomes and their lipid content in particular represent potentially valuable noninvasive diagnostic biomarkers. We isolated exosomes from plasma of 72 cirrhotic patients, including 31 with HCC. Exosomes and unfractionated plasma were processed for untargeted lipidomics using ultra-high-resolution mass spectrometry. A total of 2,864 lipid species, belonging to 52 classes, were identified. Both exosome fractionation and HCC diagnosis had significant impact on the lipid profiles. Ten lipid classes were enriched in HCC exosomes compared with non-HCC exosomes. Dilysocardiolipins were detected in 35% of the HCC exosomes but in none of the non-HCC exosomes (P < 0.001). Cardiolipins and sphingosines had the highest differential effects (fold change of 133.08, q = 0.001 and 38.57, q < 0.001, respectively). In logistic regression analysis, high abundances of exosomal sphingosines, dilysocardiolipins, lysophosphatidylserines, and (O-acyl)-1-hydroxy fatty acids were strongly associated with HCC [OR (95% confidence interval (CI)), 271.1 (14.0-5,251.9), P < 0.001; 46.5 (2.3-939.9), P = 0.012; 14.9 (4.3-51.2), P < 0.001; 10.3 (3.2-33.1), P < 0.001]. Four lipid classes were depleted in HCC exosomes compared with non-HCC exosomes. In logistic regression analysis, lack of detection of sulfatides and acylGlcSitosterol esters was strongly associated with HCC [OR (95% CI): 215.5 (11.5-4,035.9), P < 0.001; 26.7 (1.4-528.4), P = 0.031]. These HCC-associated changes in lipid composition of exosomes reflected alterations in glycerophospholipid metabolism, retrograde endocannabinoid signaling, and ferroptosis. In conclusion, this study identified candidate biomarkers for early detection of HCC as well as altered pathways in exosomes that may contribute to tumor development and progression. PREVENTION RELEVANCE: This study identifies lipids in circulating exosomes, that could serve as biomarkers for the early detection of hepatocellular carcinoma as well as altered pathways in exosomes that may contribute to tumor development and progression.

Circulating Fatty Acids Associated with Advanced Liver Fibrosis and Hepatocellular Carcinoma in South Texas Hispanics.

BACKGROUND: Hispanics in South Texas have high rates of hepatocellular carcinoma (HCC) and nonalcoholic fatty liver disease (NAFLD). Liver fibrosis severity is the strongest predictive factor of NAFLD progression to HCC. We examined the association between free fatty acids (FA) and advanced liver fibrosis or HCC in this population. METHODS: We quantified 45 FAs in plasma of 116 subjects of the Cameron County Hispanic Cohort, 15 Hispanics with HCC, and 56 first/second-degree relatives of Hispanics with HCC. Liver fibrosis was assessed by FibroScan. RESULTS: Advanced liver fibrosis was significantly associated with low expression of very long chain (VLC) saturated FAs (SFA), odd chain SFAs, and VLC n-3 polyunsaturated FAs [PUFA; AOR; 95% confidence interval (CI), 10.4 (3.7-29.6); P < 0.001; 5.7 (2.2-15.2); P < 0.001; and 3.7 (1.5-9.3); P = 0.005]. VLC n3-PUFAs significantly improved the performance of the noninvasive markers for advanced fibrosis - APRI, FIB-4, and NFS. Plasma concentrations of VLC SFAs and VLC n-3 PUFAs were further reduced in patients with HCC. Low concentrations of these FAs were also observed in relatives of patients with HCC and in subjects with the PNPLA3 rs738409 homozygous genotype. CONCLUSIONS: Low plasma concentrations of VLC n-3 PUFAs and VLC SFAs were strongly associated with advanced liver fibrosis and HCC in this population. Genetic factors were associated with low concentrations of these FAs as well. IMPACT: These results have implications in identifying those at risk for liver fibrosis progression to HCC and in screening this population for advanced fibrosis. They also prompt the evaluation of VLC n-3 PUFA or VLC SFA supplementation to prevent cirrhosis and HCC.

Fecal Microbiome, Metabolites, and Stem Cell Transplant Outcomes: A Single-Center Pilot Study.

BACKGROUND: Accumulating evidence suggests that the intestinal microbiome may dramatically affect the outcomes of hematopoietic stem cell transplant (HSCT) recipients. Providing 16S ribosomal RNA based microbiome characterization in a clinically actionable time frame is currently problematic. Thus, determination of microbial metabolites as surrogates for microbiome composition could offer practical biomarkers. METHODS: Longitudinal fecal specimens (n = 451) were collected from 44 patients before HSCT through 100 days after transplantation, as well as 1-time samples from healthy volunteers (n = 18) as controls. Microbiota composition was determined using 16S ribosomal RNA V4 sequencing. Fecal indole and butyrate levels were determined using liquid chromatography tandem mass spectrometry. RESULTS: Among HSCT recipients, both fecal indole and butyrate levels correlated with the Shannon diversity index at baseline (P = .02 and P = .002, respectively) and directly after transplantation (P = .006 and P < .001, respectively). Samples with high butyrate levels were enriched for Clostridiales, whereas samples containing high indole were also enriched for Bacteroidales. A lower Shannon diversity index at the time of engraftment was associated with increased incidence of acute intestinal graft-vs-host disease (iGVHD) (P = .02) and transplant-related deaths (P = .03). Although fecal metabolites were not associated with acute iGVHD or overall survival, patients contracting bloodstream infections within 30 days after transplantation had significantly lower levels of fecal butyrate (P = .03). CONCLUSIONS: Longitudinal analysis of fecal microbiome and metabolites after HSCT identified butyrate and indole as potential surrogate markers for microbial diversity and specific taxa. Further studies are needed to ascertain whether fecal metabolites can be used as biomarkers of acute iGVHD or bacteremia after HSCT.

Glycosylation Changes in Brain Cancer.

Protein glycosylation is a posttranslational modification that affects more than half of all known proteins. Glycans covalently bound to biomolecules modulate their functions by both direct interactions, such as the recognition of glycan structures by binding partners, and indirect mechanisms that contribute to the control of protein conformation, stability, and turnover. The focus of this Review is the discussion of aberrant glycosylation related to brain cancer. Altered sialylation and fucosylation of N- and O-glycans play a role in the development and progression of brain cancer. Additionally, aberrant O-glycan expression has been implicated in brain cancer. This Review also addresses the clinical potential and applications of aberrant glycosylation for the detection and treatment of brain cancer. The viable roles glycans may play in the development of brain cancer therapeutics are addressed as well as cancer-glycoproteomics and personalized medicine. Glycoprotein alterations are considered as a hallmark of cancer while high expression in body fluids represents an opportunity for cancer assessment.

Isomeric Separation of Permethylated Glycans by Porous Graphitic Carbon (PGC)-LC-MS/MS at High Temperatures.

Permethylation is a common derivatization method for MS-based glycomic analyses. Permethylation enhances glycan ionization efficiency in positive MS analysis and improves glycan structural stability. Recent biological glycomic studies have added to the growing body of knowledge and suggest the need for complete structural analysis of glycans. However, reverse phase LC analysis of permethylated glycans usually results in poor isomeric separation. To achieve isomeric separation of permethylated glycans, a porous graphitic carbon (PGC) column was used. PGC columns are well-known for their isomeric separation capability for hydrophilic analyses. In this study, we have optimized temperature conditions to overcome the issues encountered while separating permethylated glycans on a PGC column and found that the highest temperature examined, 75 °C, was optimal. Additionally, we utilized tandem MS to elucidate detailed structural information for the isomers separated. Glycan standards were also utilized to facilitate structural identifications through MS/MS spectra and retention time comparison. The result is an efficient and sensitive method capable of the isomeric separation of permethylated glycans. This method was successfully applied for the isomeric characterization of N-glycans released from the breast cancer cell lines MDA-MB-231 and MDA-MB-231BR (brain seeking). A total of 127 unique glycan structures were identified with 39 isobaric structures, represented as 106 isomers, with 21 nonisomeric glycans. Thirty seven structures exhibited significant differences in isomeric distribution (P < 0.05). Additionally, alterations in the distribution of isomeric sialylated glycans, structures known to be involved in cell attachment to the blood-brain barrier during brain metastasis, were observed.

Recent advances in mass spectrometric analysis of glycoproteins.

Glycosylation is one of the most common posttranslational modifications of proteins that plays essential roles in various biological processes, including protein folding, host-pathogen interaction, immune response, and inflammation and aberrant protein glycosylation is a well-known event in various disease states including cancer. As a result, it is critical to develop rapid and sensitive methods for the analysis of abnormal glycoproteins associated with diseases. Mass spectrometry (MS) in conjunction with different separation methods, such as capillary electrophoresis (CE), ion mobility (IM), and high performance liquid chromatography (HPLC) has become a popular tool for glycoprotein analysis, providing highly informative fragments for structural identification of glycoproteins. This review provides an overview of the developments and accomplishments in the field of glycomics and glycoproteomics reported between 2014 and 2016.

Quantitative LC-MS/MS Glycomic Analysis of Biological Samples Using AminoxyTMT.

Protein glycosylation plays an important role in various biological processes, such as modification of protein function, regulation of protein-protein interactions, and control of turnover rates of proteins. Moreover, glycans have been considered as potential biomarkers for many mammalian diseases and development of aberrant glycosylation profiles is an important indicator of the pathology of a disease or cancer. Hence, quantitation is an important aspect of a comprehensive glycomics study. Although numerous MS-based quantitation strategies have been developed in the past several decades, some issues affecting sensitivity and accuracy of quantitation still exist, and the development of more effective quantitation strategies is still required. Aminoxy tandem mass tag (aminoxyTMT) reagents are recently commercialized isobaric tags which enable relative quantitation of up to six different glycan samples simultaneously. In this study, liquid chromatography and mass spectrometry conditions have been optimized to achieve reliable LC-MS/MS quantitative glycomic analysis using aminoxyTMT reagents. Samples were resuspended in 0.2 M sodium chloride solution to promote the formation of sodium adduct precursor ions, which leads to higher MS/MS reporter ion yields. This method was first evaluated with glycans from model glycoproteins and pooled human blood serum samples. The observed variation of reporter ion ratios was generally less than 10% relative to the theoretical ratio. Even for the highly complex minor N-glycans, the variation was still below 15%. This strategy was further applied to the glycomic profiling of N-glycans released from blood serum samples of patients with different esophageal diseases. Our results demonstrate the benefits of utilizing aminoxyTMT reagents for reliable quantitation of biological glycomic samples.

Remodeling of marrow hematopoietic stem and progenitor cells by non-self ST6Gal-1 sialyltransferase.

Glycans occupy the critical cell surface interface between hematopoietic cells and their marrow niches. Typically, glycosyltransferases reside within the intracellular secretory apparatus, and each cell autonomously generates its own cell surface glycans. In this study, we report an alternate pathway to generate cell surface glycans where remotely produced glycosyltransferases remodel surfaces of target cells and for which endogenous expression of the cognate enzymes is not required. Our data show that extracellular ST6Gal-1 sialyltransferase, originating mostly from the liver and released into circulation, targets marrow hematopoietic stem and progenitor cells (HSPCs) and mediates the formation of cell surface α2,6-linked sialic acids on HSPCs as assessed by binding to the specific lectins Sambucus nigra agglutinin and Polysporus squamosus lectin and confirmed by mass spectrometry. Marrow HSPCs, operationally defined as the Lin-c-Kit+ and Lin-Sca-1+c-Kit+ populations, express negligible endogenous ST6Gal-1. Animals with reduced circulatory ST6Gal-1 have marrow Lin-Sca-1+c-Kit+ cells with reduced S. nigra agglutinin reactivity. Bone marrow chimeras demonstrated that α2,6-sialylation of HSPCs is profoundly dependent on circulatory ST6Gal-1 status of the recipients and independent of the ability of HSPCs to express endogenous ST6Gal-1. Biologically, HSPC abundance in the marrow is inversely related to circulatory ST6Gal-1 status, and this relationship is recapitulated in the bone marrow chimeras. We propose that remotely produced, rather than the endogenously expressed, ST6Gal-1 is the principal modifier of HSPC glycans for α2,6-sialic acids. In so doing, liver-produced ST6Gal-1 may be a potent systemic regulator of hematopoiesis.