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Ganglioside GM3 synthase is a key enzyme involved in the biosynthesis of gangliosides. GM3 synthase deficiency (GM3SD) causes an absence of GM3 and all downstream biosynthetic derivatives, including all the a-, b-, c-series gangliosides, commonly found in neural tissues. The affected individuals manifest with severe irritability, intractable seizures, hearing loss, blindness, and profound intellectual disability. It has been reported that oral ganglioside supplementation has achieved some significant improvements in clinical symptoms, growth parameters, and developmental and cognitive scores in GM3SD patients. To gain insight into the molecular mechanisms of this supplementation, we performed supplementation of oral bovine milk gangliosides to GM3 synthase-deficient mice from early weaning periods. The oral milk ganglioside preparations were dominated by GM3 and GD3 gangliosides. Oral milk ganglioside supplementation improved the decreased cognitive function observed in GM3 synthase-deficient mice. The improvement in cognitive function was accompanied by increased ganglioside levels and neurogenesis in the hippocampus in the supplemented animals., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The authors declare that this study received funding from Fonterra co-operative Group Ltd. The funder had the following involvement in the study: study design, analysis and interpretation of data, the writing of this article and the decision to submit it for publication., (Copyright © 2024 Inokuchi, Go, Suzuki, Nakagawasai, Odaira-Satoh, Veillon, Nitta, McJarrow, Kanoh, Inamori, Tan-No and Collett.)

Ion suppression is a major problem in mass spectrometry (MS)-based metabolomics; it can dramatically decrease measurement accuracy, precision, and signal-to-noise sensitivity. Here we report a new method, the IROA TruQuant Workflow, that uses a stable isotope-labeled internal standard (IROA-IS) plus novel companion algorithms to 1) measure and correct for ion suppression, and 2) perform Dual MSTUS normalization of MS metabolomic data. We have evaluated the method across ion chromatography (IC), hydrophilic interaction liquid chromatography (HILIC), and reverse phase liquid chromatography (RPLC)-MS systems in both positive and negative ionization modes, with clean and unclean ion sources, and across different biological matrices. Across the broad range of conditions tested, all detected metabolites exhibited ion suppression ranging from 1% to 90+% and coefficient of variations ranging from 1% to 20%, but the Workflow and companion algorithms were highly effective at nulling out that suppression and error. Overall, the Workflow corrects ion suppression across diverse analytical conditions and produces robust normalization of non-targeted metabolomic data.

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., Competing Interests: Declaration of interests R.R.J. has served as a consultant or advisory board member for Merck, Microbiome DX, Karius, MaaT Pharma, LisCure, Seres, Kaleido, and Prolacta and has received patent license fee or stock options from Seres and Kaleido. E.H., M.A.J., J.L.K., and R.R.J. are inventors on a patent application by the University of Texas MD Anderson Cancer Center, supported by the results of the current study entitled “Methods and Compositions for Treating Cancer therapy-induced Neutropenic Fever and/or GVHD.”, (Copyright © 2022 Elsevier Inc. All rights reserved.)

Carboxylesterase 1d (Ces1d) is a crucial enzyme with a wide range of activities in multiple tissues. It has been reported to localize predominantly in ER. Here, we found that Ces1d levels are significantly increased in obese patients with type 2 diabetes. Intriguingly, a high level of Ces1d translocates onto lipid droplets where it digests the lipids to produce a unique set of fatty acids. We further revealed that adipose tissue-specific Ces1d knock-out (FKO) mice gained more body weight with increased fat mass during a high fat-diet challenge. The FKO mice exhibited impaired glucose and lipid metabolism and developed exacerbated liver steatosis. Mechanistically, deficiency of Ces1d induced abnormally large lipid droplet deposition in the adipocytes, causing ectopic accumulation of triglycerides in other peripheral tissues. Furthermore, loss of Ces1d diminished the circulating free fatty acids serving as signaling molecules to trigger the epigenetic regulations of energy metabolism via lipid-sensing transcriptional factors, such as HNF4α. The metabolic disorders induced an unhealthy microenvironment in the metabolically active tissues, ultimately leading to systemic insulin resistance., (© 2022 Li et al.)

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., (© 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

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., (©2021 American Association for Cancer Research.)

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., (©2021 American Association for Cancer Research.)

Innate immune signaling via TLR4 plays critical roles in pathogenesis of metabolic disorders, but the contribution of different lipid species to metabolic disorders and inflammatory diseases is less clear. GM3 ganglioside in human serum is composed of a variety of fatty acids, including long-chain (LCFA) and very-long-chain (VLCFA). Analysis of circulating levels of human serum GM3 species from patients at different stages of insulin resistance and chronic inflammation reveals that levels of VLCFA-GM3 increase significantly in metabolic disorders, while LCFA-GM3 serum levels decrease. Specific GM3 species also correlates with disease symptoms. VLCFA-GM3 levels increase in the adipose tissue of obese mice, and this is blocked in TLR4-mutant mice. In cultured monocytes, GM3 by itself has no effect on TLR4 activation; however, VLCFA-GM3 synergistically and selectively enhances TLR4 activation by LPS/HMGB1, while LCFA-GM3 and unsaturated VLCFA-GM3 suppresses TLR4 activation. GM3 interacts with the extracellular region of TLR4/MD2 complex to modulate dimerization/oligomerization. Ligand-molecular docking analysis supports that VLCFA-GM3 and LCFA-GM3 act as agonist and antagonist of TLR4 activity, respectively, by differentially binding to the hydrophobic pocket of MD2. Our findings suggest that VLCFA-GM3 is a risk factor for TLR4-mediated disease progression., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Introduction: IgA nephropathy (IgAN) can be associated with spondyloarthritis (SpA). The course of SpA-associated IgAN remains largely unknown due to the absence of large cohorts., Methods: This retrospective study included patients with biopsy-proven IgAN and definite SpA. Kidney biopsies were centrally examined and scored according to the IgAN Oxford Classification. Thirty-two patients fulfilled the inclusion criteria, with a male:female ratio of 9:1 and median age of 27 and 37 years at SpA and IgAN diagnosis, respectively. HLA-B27 was positive in 90% of cases, and most patients (60%) presented with ankylosing spondylitis. The mean baseline estimated glomerular filtration rate (eGFR) was 84 ± 26 ml/min per 1.73 m 2 , and the urine protein-to-creatinine ratio was 0.19 g/mmol., Results: Renal biopsy revealed frequent presence of crescents (33%) and interstitial inflammation (18%). Despite almost constant use of renin-angiotensin system inhibitors, combined with steroids in 13 of 32 patients, renal outcome was particularly poor. After a median follow-up of 5.9 years, 4 patients (12.5%) reached end-stage renal disease and 41% of patients experienced a >50% decrease of eGFR. The mean annual eGFR decline rate was -4.3 ± 6.7 ml/min per 1.73 m 2 . The risk of reaching class IV or V chronic kidney disease (CKD) stage during follow-up was associated with the presence of hypertension, level of proteinuria, and baseline S- and T-scores of the Oxford., Conclusion: SpA-associated IgAN is associated with a poor renal outcome, despite frequent use of steroids. Tumor necrosis factor (TNF)-α blockade did not appear to influence the rate of eGFR decline in this setting., (© 2020 International Society of Nephrology. Published by Elsevier Inc.)

Glycosylation, an essential post-translational protein modification, is known to be altered in a variety of diseases, including neurodegenerative diseases such as Alzheimer's disease (AD), which is one of the most common neurodegenerative disorders that results in cognitive and memory impairments. To investigate the progression of such a condition, cerebrospinal fluid (CSF), a unique biofluid that may possess significant biochemical and neurochemical changes due to the disease, is utilized. However, due to the low concentration of proteins in CSF, a large volume of the biofluid is often required to comprehensively characterize the glycome in CSF. In this work, a glycomic study of CSF was performed using as little as 10 μL of CSF. This approach was executed with permethylation of released N-glycans with minimal sample cleanup, in conjunction with an online purification system attached to liquid chromatography and a high-resolution mass spectrometer. This technique was then applied to clinical samples. Preliminary data suggest that fucosylated and bisecting GlcNAc structures were higher in abundances in females with AD, while both females and males exhibited lower abundances of high-mannose structures. Although there seems to be statistically significant differences between disease state and disease-free CSF, due to the lack of number of samples, further validation study should be conducted.

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.

Intestinal cholesterol absorption is a key regulator of systemic cholesterol homeostasis. Excessive dietary cholesterol and its intestinal uptake lead to hypercholesterolemia, a major risk factor for cardiovascular disease. Intestinal cholesterol uptake is mediated by Niemann-Pick C1-like 1 (NPC1L1), a transmembrane protein localized in membrane microdomains (lipid rafts) enriched in gangliosides and cholesterol. The roles of gangliosides, such as monosialodihexosylganglioside (GM3) and its synthesizing enzyme GM3 synthase (GM3S), in NPC1L1-dependent cholesterol uptake have not been examined previously. Here, we examined NPC1L1-dependent cholesterol uptake in a cell model as well as in wild-type and apoE-deficient mice fed normal or high-cholesterol diets. We showed that NPC1L1-dependent cholesterol uptake was impaired in GM3S-deficient cells and that GM3S deficiency promoted resistance to hypercholesterolemia in both wild-type and apoE-deficient mice fed the high-cholesterol but not the normal diet. Our findings suggest that GM3 and related gangliosides are essential for NPC1L1-mediated intestinal cholesterol absorption and are potential targets for hypercholesterolemia therapy., (Copyright © 2018 Nihei et al.)

Glycomics continues to be a highly dynamic and interesting research area due to the need to comprehensively understand the biological attributes of glycosylation in many important biological functions such as the immune response, cell development, cell differentiation/adhesion, and host-pathogen interactions. Although matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) has proven to be suitable for glycomic profiling studies, there is a need for improved sensitivity in the detection of native glycans, which ionize inefficiently. In this study, we investigated the efficiencies of graphene nanosheets (GNs) and carbon nanoparticles (CNPs) as MALDI matrices and co-matrices in glycan profiling. Our results indicated an enhancement of signal intensity by several orders of magnitude upon using GNs and CNPs in MALDI analysis of N-glycans derived from a variety of biological samples. Interestingly, increasing the amounts of CNPs and GNs improved not only the signal intensities but also prompted in-source decay (ISD) fragmentations, which produced extensive glycosidic and cross-ring cleavages. Our results indicated that the extent of ISD fragmentation could be modulated by CNP and GN concentrations, to obtain MS 2 and pseudo-MS 3 spectra. The results for glycan profiling in high salt solutions confirmed high salt-tolerance capacities for both CNPs and GNs. Finally, the results showed that by using CNPs and GNs as co-matrices, DHB crystal formation was more homogeneous which improved shot-to-shot reproducibility and sensitivity. Graphical Abstract ᅟ.

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.

Protein glycosylation is a common post-translational modification that has significant impacts on protein folding, lifespan, conformation, distribution and function. N-Glycans, which are attached to asparagine residues of proteins, are studied most often due to their compatibility with enzymatic release. Despite the ease of N-glycan release, compositional and structural complexity coupled with poor ionization efficiency during liquid chromatography mass spectrometry (LC-MS) make quantitative glycomic studies a significant challenge. To overcome these challenges, glycans are almost always derivatized prior to LC-MS analyses to impart favorable characteristics, such as improved ionization efficiency, increased LC separation efficiency and the production of more informative fragments during tandem MS. There are a number of derivatization methods available for LC-MS analysis of glycans, each of which imparts different properties that affect both glycan retention on LC columns and MS analyses. To provide guidance for the proper selection of derivatizing reagents and LC columns, herein, we describe a comprehensive assessment of 2-aminobenzamide, procainamide, aminoxyTMT, RapiFluor-MS (RFMS) labeling, reduction and reduction with permethylation for N-glycan analysis. Of the derivatization strategies examined, RFMS provided the highest MS signal enhancement for neutral glycans, while permethylation significantly enhanced the MS intensity and structural stability of sialylated glycans.

Background: In experiments with Formosan subterranean termites (Coptotermes formosanus Shirakii), myo-inositol-2-monophosphate as the dicyclohexylammonium salt was tested among other sugar derivatives, and was found to be toxic to C. formosanus when added to a moistened filter paper food source in plastic Petri dishes., Results: Curiously, over a nine-day period, the moniliform (beaded) antenna of C. formosanus deteriorated in a stepwise fashion with the most distal pseudosegment (bead) turning brown and falling off, followed by the penultimate pseudosegment, sequentially, until 7-9 days when only a stub of the antenna remained. Termites became increasingly moribund with the loss of antennae, and quit normal behavior including consuming cellulose food, and died. sn-Glycerol-3-phosphate as the dicyclohexylammonium salt also gave the same results. Dicyclohexylammonium hydrogen phosphate and monocyclohexylammonium dihydrogen phosphate were synthesized, to find a low-cost form for application to baits, both of which also showed similar toxicity. In a trial with Fibonacci series dilutions of neat cyclohexylamine, the antenna-affecting activity became apparent in the LD 30 (14 days) to LD 70 range of concentrations. At the higher concentrations, darkening of the most distal parts of leg extremities was noticed., Conclusion: Cyclohexylamine appears to be a novel termiticide with a previously unreported mechanism of toxicity. Its hydrogen phosphate salts retain the toxic effect and are inexpensive and easily synthesized. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

The characterization of glycosylation is critical for obtaining a comprehensive view of the regulation and functions of glycoproteins of interest. Due to the complex nature of oligosaccharides, stemming from variable compositions and linkages, and ion suppression effects, the chromatographic separation of glycans, including isomeric structures, is necessary for exhaustive characterization by MS. This review introduces the fundamental principles underlying the techniques in LC utilized by modern day glycomics researchers. Recent advances in porous graphitized carbon, reverse phase, ion exchange, and hydrophilic interaction LC utilized in conjunction with MS, for the characterization of protein glycosylation, are described with an emphasis on methods capable of resolving isomeric glycan structures., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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.

Gangliosides (sialic acid-containing glycosphingolipids) help regulate many important biological processes, including cell proliferation, signal transduction, and differentiation, via formation of functional microdomains in plasma membranes. The structural diversity of gangliosides arises from both the ceramide moiety and glycan portion. Recently, differing molecular species of a given ganglioside are suggested to have distinct biological properties and regulate specific and distinct biological events. Elucidation of the function of each molecular species is important and will provide new insights into ganglioside biology. Gangliosides are also suggested to be involved in skeletal muscle differentiation; however, the differential roles of ganglioside molecular species remain unclear. Here we describe striking changes in quantity and quality of gangliosides (particularly GM3) during differentiation of mouse C2C12 myoblast cells and key roles played by distinct GM3 molecular species at each step of the process., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The biosynthesis of glycans is a template-free process; hence compositionally identical glycans may contain highly heterogeneous structures. Meanwhile, the functions of glycans in biological processes are significantly influenced by the glycan structure. Structural elucidation of glycans is an essential component of glycobiology. Although NMR is considered the most powerful approach for structural glycan studies, it suffers from low sensitivity and requires highly purified glycans. Although mass spectrometry (MS)-based methods have been applied in numerous glycan structure studies, there are challenges in preserving glycan structure during ionization. Permethylation is an efficient derivatization method that improves glycan structural stability. In this report, permethylated glycans are isomerically separated; thus facilitating structural analysis of a mixture of glycans by LC-MS/MS. Separation by porous graphitic carbon liquid chromatography at high temperatures in conjunction with tandem mass spectrometry (PGC-LC-MS/MS) was utilized for unequivocal characterization of glycan isomers. Glycan fucosylation sites were confidently determined by eliminating fucose rearrangement and assignment of diagnostic ions, achieved by permethylation and PGC-LC at high temperatures, respectively. Assigning monosaccharide residues to specific glycan antennae was also achieved. Galactose linkages were also distinguished from each other by CID/HCD tandem MS. This was attainable because of the different bond energies associated with monosaccharide linkages. Graphical Abstract LC-MS and tandem MS of terminal galactose isomers.

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.

Serum GM3 molecular species were quantified in 125 Japanese residents using tandem mass spectrometry multiple reaction monitoring. Individuals were categorized by the presence or absence of metabolic disease risk factors including visceral fat accumulation, hyperglycemia and dyslipidemia. A total of 23 GM3 molecular species were measured, of these, eight were found to be significantly elevated in individuals with visceral fat accumulation and metabolic disease, defined as the presence of hyperglycemia and dyslipidemia. All of the GM3 molecular species were composed of the sphingoid base sphingosine (d18:1 (Δ4)) and, interestingly, six of the eight elevated GM3 molecular species contained a hydroxylated ceramide moiety. The hydroxylated GM3 species were, in order of decreasing abundance, d18:1-h24:0 ≈ d18:1-h24:1 > d18:1-h22:0 » d18:1-h20:0 > d18:1-h21:0 > d18:1-h18:1. Univariate and multiple linear regression analyses were conducted using a number of clinical health variables associated with obesity, type 2 diabetes, metabolic disease, atherosclerosis and hypertension. GM3(d18:1-h24:1) was identified as the best candidate for metabolic screening, proving to be significantly correlated with intima-media thickness, used for the detection of atherosclerotic disease in humans, and a number of metabolic disease risk factors including autotaxin, LDL-c and homeostatic model assessment insulin resistance (HOMA-IR).

Several rare and common monosaccharides were screened for toxic effects on the Formosan subterranean termite, Coptotermes formosanus Shiraki, with the aim of identifying environmentally friendly termiticides. myo-Inositol and phytic acid, which are nontoxic to mammals, were identified as potential termite control compounds. Feeding bioassays with termite workers, where both compounds were supplied on filter paper in concentrations from 160.2 to 1,281.7 μg/mm(3), showed concentration-dependent toxicity within 2 wk. Interestingly myo-inositol was nontoxic when administered to termites in agar (40 mg/ml) in the absence of a cellulosic food source, an unexplained phenomenon. In addition, decreased populations of termite hindgut protozoa were observed upon feeding on myo-inositol but not phytate-spiked filter paper. Radiotracer feeding studies using myo-inositol-[2-(3)H] with worker termites showed no metabolism after ingestion over a 2-d feeding period, ruling out metabolites responsible for the selective toxicity., (© 2014 Entomological Society of America.)

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.

Glycan structures on glycoproteins and glycolipids play critical roles in biological recognition, targeting, and modulation of functions in animal systems. Many classes of glycan structures are capped with terminal sialic acid residues, which contribute to biological functions by either forming or masking glycan recognition sites on the cell surface or secreted glycoconjugates. Sialylated glycans are synthesized in mammals by a single conserved family of sialyltransferases that have diverse linkage and acceptor specificities. We examined the enzymatic basis for glycan sialylation in animal systems by determining the crystal structures of rat ST6GAL1, an enzyme that creates terminal α2,6-sialic acid linkages on complex-type N-glycans, at 2.4 Å resolution. Crystals were obtained from enzyme preparations generated in mammalian cells. The resulting structure revealed an overall protein fold broadly resembling the previously determined structure of pig ST3GAL1, including a CMP-sialic acid-binding site assembled from conserved sialylmotif sequence elements. Significant differences in structure and disulfide bonding patterns were found outside the sialylmotif sequences, including differences in residues predicted to interact with the glycan acceptor. Computational substrate docking and molecular dynamics simulations were performed to predict and evaluate the CMP-sialic acid donor and glycan acceptor interactions, and the results were compared with kinetic analysis of active site mutants. Comparisons of the structure with pig ST3GAL1 and a bacterial sialyltransferase revealed a similar positioning of donor, acceptor, and catalytic residues that provide a common structural framework for catalysis by the mammalian and bacterial sialyltransferases.

In the interest of developing interventions to infestations by Formosan subterranean termites, Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae), several rare sugars were tested for effects on the termites and symbionts. Among these, the D-galactose analog, 2-deoxy-D-galactose (2deoxyGal) showed promise as a potential control chemical. At a test concentration of 2deoxyGal (320.4 microg/mm3) in water applied to 5-cm filter paper, in bioassays with 20 termite workers, we found that worker termite mortality was significantly affected over a 2-wk period. Subsequent dose-mortality feeding studies confirmed these findings. In addition, consumption of the sugar-treated filter paper by termites caused a significant decrease in hindgut protozoan populations. 2deoxyGal caused dose-dependent termite mortality, taking on average 1 wk to begin killing workers, indicating that it may have promise as a delayed action toxin, which, if added to baits, could allow time after bait discovery for an entire colony to be affected.

A case of noninfectious polyarthritis of over one year's duration with calcaneal enthesopathy in a patient with visceral tuberculosis is reported. This pattern, termed Poncet's disease, shares pathophysiologic mechanisms with Freund's complete adjuvant-induced arthritis. Future studies should include polymerase chain reaction studies to look for the tubercle bacillus in joint fluid or synovial biopsy specimens.