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The oolemma of the second metaphase mouse oocyte has a relatively large domain in the peri-polar body region that is non-permissive for sperm attachment. In this study, certain biochemical components of this non-permissive domain were examined and compared with permissive regions to investigate the molecular basis of an oolemma that becomes functionally polarized during fertilization; an attempt was also made to determine whether similarities exist in the formation of abnormally occurring non-permissive domains in human oocytes. In the present study, microdomains composed of lipid rafts enriched in the ganglioside GM1 provided a platform for sperm docking at the initial stage of fertilization and their disruption led to sperm attachment defects resembling those associated with fertilization failure in human IVF. The regulation of GM1 microdomain density is suggested to involve the oolemmal protein annexin 2 and the magnitude of the inner mitochondrial membrane potential (ΔΨm) in the adjacent subplasmalemmal cytoplasm. These studies also showed that a rapid focal reduction in GM1, f-actin, annexin 2 and cholesterol occurs at the site of sperm docking and attachment whereas the distribution of fertilization-associated proteins CD9 and FOLR4 remains unchanged, suggesting that they may not participate in the earliest phases of fertilization. [ABSTRACT FROM AUTHOR]

Fragile X mental retardation protein (FMRP), fragile X related 1 protein (FXR1P) and FXR2P are the members of the FMR protein family. These proteins contain two KH domains and a RGG box, which are characteristic of RNA binding proteins. The absence of FMRP, causes fragile X syndrome (FXS), the leading cause of hereditary mental retardation. FXR1P is expressed throughout the body and important for normal muscle development, and its absence causes cardiac abnormality. To investigate the functions of FXR1P, a screen was performed to identify FXR1P-interacting proteins and determine the biological effect of the interaction. The current study identified CMP-N-acetylneuraminic acid synthetase (CMAS) as an interacting protein using the yeast two-hybrid system, and the interaction between FXR1P and CMAS was validated in yeast using a β-galactosidase assay and growth studies with selective media. Furthermore, co-immunoprecipitation was used to analyze the FXR1P/CMAS association and immunofluorescence microscopy was performed to detect expression and intracellular localization of the proteins. The results of the current study indicated that FXR1P and CMAS interact, and colocalize in the cytoplasm and the nucleus of HEK293T and HeLa cells. Accordingly, a fragile X related 1 (FXR1) gene overexpression vector was constructed to investigate the effect of FXR1 overexpression on the level of monosialotetrahexosylganglioside 1 (GM1). The results of the current study suggested that FXR1P is a tissue-specific regulator of GM1 levels in SH-SY5Y cells, but not in HEK293T cells. Taken together, the results initially indicate that FXR1P interacts with CMAS, and that FXR1P may enhance the activation of sialic acid via interaction with CMAS, and increase GM1 levels to affect the development of the nervous system, thus providing evidence for further research into the pathogenesis of FXS. [ABSTRACT FROM AUTHOR]

Monosialoganglioside (GM1) is a glycosphingolipid that protects against some neurological conditions, such as seizures and ischemia. Glutaric acidemia type I (GA-I) is an inherited disease characterized by striatal degeneration, seizures, and accumulation of glutaric acid (GA). In this study, we show that GA inhibits Na+,K+-ATPase activity and increases oxidative damage markers (total protein carbonylation and thiobarbituric acid-reactive substances—TBARS) production in striatal homogenates from rats in vitro and ex vivo. It is also shown that GM1 (50 mg/kg, i.p., twice) protects against GA-induced (4 μmol/striatum) seizures, protein carbonylation, TBARS increase, and inhibition of Na+,K+-ATPase activity ex vivo. Convulsive episodes induced by GA strongly correlated with Na+,K+-ATPase activity inhibition in the injected striatum but not with oxidative stress marker measures. Muscimol (46 pmol/striatum), but not MK-801 (3 nmol/striatum) and DNQX (8 nmol/striatum) prevented GA-induced convulsions, increase of TBARS and protein carbonylation and inhibition of Na+,K+-ATPase activity. The protection of GM1 and muscimol against GA-induced seizures strongly correlated with Na+,K+-ATPase activity maintenance ex vivo. In addition, GM1 (50–200 μM) protected against Na+,K+-ATPase inhibition induced by GA (6 mM) but not against oxidative damage in vitro. GM1 also decreased pentylenetetrazole (PTZ)-induced (1.8 μmol/striatum) seizures, Na+,K+-ATPase inhibition, and increase of TBARS and protein carbonyl in the striatum. These data suggest that Na+,K+-ATPase and GABAA receptor-mediated mechanisms may play important roles in GA-induced seizures and in their prevention by GM1.

Gangliosides (sialic acid-containing glycosphingolipids) are abundant in neurons of all animal species and play important roles in many cell physiological processes, including differentiation, memory control, cell signaling, neuronal protection, neuronal recovery, and apoptosis. Gangliosides also function as anchors or entry points for various toxins, bacteria, viruses, and autoantibodies. GM1, a ganglioside component of mammalian brains, is present mainly in neurons. GM1 is one of the best studied gangliosides, and our understanding of its properties is extensive. Simple and rapid procedures are available for preparation of GM1 as a natural compound on a large scale, or as a derivative containing an isotopic radionuclide or a specific probe. Great research interest in the properties of GM1 arose from the discovery in the early 1970s of its role as receptor for the bacterial toxin responsible for cholera pathogenesis. [ABSTRACT FROM AUTHOR]

Objective: To observe the effects of GM1 on apomorphine (APO)-induced rotational behavior and the expression of inflammatory factors in 6-hydroxydopamine-induced Parkinson's disease (PD) rat models.Methods: Mature and healthy Wistar rats of either sex with body weight of 150–200 g were randomly divided into control group, PD+APO group and PD+APO+GM1 group (10 mg/kg of GM1, intraperitoneally, once a day, for 14 days; each group with 15 rats). PD rat models were prepared by injecting 6-hydroxydopamine into rat's right striatum, and then rotational behavior was induced by intraperitoneal injection of APO 7 days after operation. Rat rotational behavior was observed, and mRNA and protein levels of interleukin-1β (IL-1β) and interleukin-1Ra (IL-1Ra) were determined, respectively, by RT-PCR and Western blot.Results: Compared with PD+APO group, the rotational behavior was significantly relieved in PD+APO+GM1 group (p< 0.05). Compared with control group, mRNA and protein expressions of IL-1βin the striatum significantly increased in PD+APO group (p< 0.05). However, mRNA and protein expressions of IL-1βsignificantly decreased in PD+APO+GM1 group compared with PD+APO group (p< 0.05), but mRNA and protein expressions of IL-1βwere also higher in PD+APO+GM1 group than in control group (p< 0.05). mRNA and protein expressions of IL-1Ra in the striatum were significantly higher in PD+APO+GM1 group than in PD+APO group (p< 0.05).Conclusion: GM1 can inhibit inflammatory reaction through decreasing mRNA and protein expressions of IL-1β and increasing mRNA and protein expressions of IL-1Ra with the therapeutic effects on PD. [ABSTRACT FROM PUBLISHER]

FITC-labeled cholera toxin subunit B (CTB) stained the surfaces of cells of mucous acini in the submandibular gland. CTB, also called choleragenoid, binds to the GM1 glycolipid in the cell membrane. The binding in most acini was inhibited by periodic acid oxidation of the sections, while some acini remained unaffected even after increased oxidation. Staining with the subunit was also reduced significantly by adding galactose to the incubation medium. Binding of CTB to cell surfaces apparently requires intact sialic groups on most, but not all, cell surfaces. Oxidation of the sialic acid residues may influence the structure of the sialylated GM1 molecules on the cell surface in different ways. It is possible that both the sialic acid residue and the terminal galactose are oxidized. Alternatively, the sialic acid may be resistant to acid hydrolysis in gangliosides in which the sialic acid is attached to the internal galactose residue linked to GalNAc, as in the GM1 glycolipid. Inhibition of the GM1 receptor binding to cholera toxin has potential for protection of humans against cholera. Galactose and agents that modify sialic acid inhibit the accessibility of the toxin to the GM1 carbohydrate receptor. Human milk contains high levels of sialic acid glycoconjugates that may provide defense mechanisms. [ABSTRACT FROM AUTHOR]

Studies on the structure and dynamics of interfacial water, emphasizing on the properties of water near the surface of biomolecules, are well reported, but there is a lack of evidence on the behavior of water near a comparatively rough surface containing molecules with a bulky head group like GM1. In this report we comparatively analyze the structure and dynamics of water as a function of distance from the lipid head group in GM1 containing lipid bilayers, with the lipid bilayers where GM1 is not present. This approach effectively demonstrates the behavioral difference and hence delayed convergence from bound water to bulk water in the presence of GM1 compared to a relatively smooth surface. [ABSTRACT FROM AUTHOR]

GM1 ganglioside plays a role in essential neuronal processes, including differentiation, survival, and signaling. Yet, little is known about GM1 species with different sphingosine bases, such as the most abundant species containing 18 carbon atoms in the sphingosine chain (GM1d18:1), and the less abundant containing 20 carbon atoms (GM1d20:1). While absent in the early fetal brain, GM1d20:1 continues to increase throughout pre- and postnatal development and into old age, raising questions about the functional relevance of the GM1d18:1 to GM1d20:1 ratio. Matrix-assisted laser desorption/ionization imaging mass spectrometry is a novel technology that allows differentiation between these two GM1 species and quantification of their expression within an anatomical context. Using this technology, we find GM1d18:1/d20:1 expression ratios are highly specific to defined anatomical brain regions in adult rats. Thus, the ratio was significantly different among different thalamic nuclei and between the corpus callosum and internal capsule. Differential GM1d18:1/GM1d20:1 ratios measured in hippocampal subregions in rat brain complement previous studies conducted in mice. Across layers of the sensory cortex, opposing expression gradients were found for GM1d18:1 and GM1d20:1. Superficial layers demonstrated lower GM1d18:1 and higher GM1d20:1 signal than other layers, while in deep layers GM1d18:1 expression was relatively high and GM1d20:1 expression low. By far the highest GM1d18:1/d20:1 ratio was found in the amygdala. Differential expression of GM1 with d18:1- or d20:1-sphingosine bases in the adult rat brain suggests tight regulation of expression and points toward a distinct functional relevance for each of these GM1 species in neuronal processes. [ABSTRACT FROM AUTHOR]

Alzheimer’s disease (AD) is a progressive dementia associated with loss of memory and cognitive dysfunction. In a previous study, we demonstrated a decrease in b-series gangliosides along with a change in ganglioside molecular species in the hippocampal grey matter of patients with AD. The present study demonstrates the use of imaging mass spectrometry for analyzing the spatial arrangement of ganglioside GM1 (GM1) molecular species in the hippocampus. In AD patients, we found a decrease in the ratio of GM1(d20:1/C18:0) to GM1 d18:1/C18:0) in the outer molecular layer (ML) of the dentate gyrus. Because the outer ML is the region of main input into the hippocampus, our findings may have a direct relationship to the mechanism of dysfunction in AD. [ABSTRACT FROM AUTHOR]

Cholera toxin (CTX) is a virulent factor of Vibrio cholerae that causes life-threatening diarrheal disease. Its non-toxic subunit CTB has been extensively studied for vaccine delivery. In immune cells, CTB induces a number of signaling molecules related to cellular activation and cytokine production. The mechanisms by which CTB exerts its immunological effects are not understood. We report here the immunological targets of CTB. The unexpected finding that GM1 ganglioside inhibited NF-κB activation in human monocytes stimulated with CTX and agonists of Toll-like receptors (TLR) suggests the possibility of CTX–TLR interaction. Indeed, CTX-induced IL-6 production was substantially reduced in MyD88 −/− or TLR4 −/− macrophages. Ectopic expression of TLR4 was required for CTX-induced NF-κB activation in HEK 293 cells. Furthermore, the inflammatory capacity of CTB was lost in the absence of TLR4, adaptor protein FcRγ, or its downstream signaling molecule CARD9. Attempts have been made to identify CTB-binding targets from various C-type lectin and immunoglobulin-like receptors. CTB targeted not only GM1 and TLR4 but also TREM2 and LMIR5/CD300b. CTB–TREM2 interaction initiated signal transduction through adaptor protein DAP12. The binding of CTB inhibited LMIR5 activation induced by its endogenous ligand 3- O -sulfo-β- d -galactosylceramide C24:1. In summary, CTB targets TLR4, FcRγ–CARD9, TREM2, and LMIR5. These findings provide new insights into the immunobiology of cholera toxin. [ABSTRACT FROM AUTHOR]

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Various procedures have been proposed to reduce parasitemia during Chagas disease. Here we analyze in detail models developed to predict the possible outcomes of two of these treatments. The model solutions reproduce available experimental population data and can be used as tools to help researchers to find a cure or, at least, an optimal treatment to reduce cardiac cell damage. In particular, we study how the phase diagram describing the infection outcome is modified by the application of the ganglioside GM1, finding the optimum concentration of the drug in terms of the parameters characterizing its influence on the parasite-host interaction. We also investigate the use of the non-pathogenic parasite T. rangeli to reduce T. cruzi load in a mixed infection, finding that this method is not effective against all T. cruzi strains. Furthermore, we compare phase portraits of the evolution of the disease for a single and a mixed infection, and evaluate the cell damage as a function of the time elapsed between both infections, remarking on the temporary protective effect of the reaction to T. rangeli. [ABSTRACT FROM AUTHOR]

The B-subunits of heat-labile enterotoxins LT-I (LT-IB) and LT-IIa (LT-IIaB) are strong adjuvants that bind to cell-surface receptors, including gangliosides G M1 and GD1b, respectively. LT-IIaB also binds TLR-2. We demonstrate for the first time that co-incubation with the B-subunits induces significant clustering of B cells after only 4 h, and B and T cells in 24 h. Clustering was dependent on intact B-subunits, but not on the TLR-2 binding activity of LT-IIaB, indicating it was ganglioside-mediated. Treatment of B cells with LT-IB, a mixture of LT-IB + LT-IIaB, but not LT-IIaB alone, caused a delay in T cell division following ovalbumin endocytosis. B cell receptor-mediated uptake in presence of each treatment caused an arrest, but with increased production of IL-2. Further, treatments differentially increased the proportion of macrophages expressing MHC class-II. These results highlight the outcomes of interplay between signals involving different receptors and implicate a novel mechanism of adjuvanticity. [ABSTRACT FROM AUTHOR]

Gangliosides are compounds that are abundant throughout the CNS, participating actively in neuroplasticity. We previously described that exogenous GM1 ganglioside pretreatment enhances the rewarding properties of cocaine, evidenced by a lower number of sessions and/or dosage necessary to induce conditioned place preference (CPP). Since GM1 pretreatment did not modify cocaine's pharmacokinetic parameters, we suspected that the increased rewarding effect found might be mediated by BDNF, a neurotrophic factor closely related to cocaine addiction. This study was performed to investigate the possibility that GM1 may induce changes in BDNF levels in the nucleus accumbens (NAc), a core structure in the brain's reward circuitry, of rats submitted to three conditioning sessions with cocaine (10 mg/kg, i.p.). The results demonstrate that GM1 administration, which showed no rewarding effect by itself in the CPP, induced a significant increase of BDNF protein levels in the NAc, which may account for the increased rewarding effect of cocaine shown in the CPP paradigm. [ABSTRACT FROM AUTHOR]

Age-related alterations of membrane lipids in brain cell membranes together with high blood cholesterol are considered as major risk factors for Alzheimer’s disease. Yet the molecular mechanisms by which these factors increase Alzheimer’s risk are mostly unknown. In lipid raft domains of the plasma membrane, neurotoxic Alzheimer’s beta-amyloid (Abeta) peptides interact with both cholesterol and ganglioside GM1. Recent data also suggested that cholesterol could stimulate the binding of Abeta to GM1 through conformational modulation of the ganglioside headgroup. Here we used a combination of physicochemical and molecular modelling approaches to decipher the mechanisms of cholesterol-assisted binding of Abeta to GM1. With the aim of decoupling the effect of cholesterol on GM1 from direct Abeta-cholesterol interactions, we designed a minimal peptide (Abeta5-16) containing the GM1-binding domain but lacking the amino acid residues involved in cholesterol recognition. Using the Langmuir technique, we showed that cholesterol (but not phosphatidylcholine or sphingomyelin) significantly accelerates the interaction of Abeta5-16 with GM1. Molecular dynamics simulations suggested that Abeta5-16 interacts with a cholesterol-stabilized dimer of GM1. The main structural effect of cholesterol is to establish a hydrogen-bond between its own OH group and the glycosidic-bond linking ceramide to the glycone part of GM1, thereby inducing a tilt in the glycolipid headgroup. This fine conformational tuning stabilizes the active conformation of the GM1 dimer whose headgroups, oriented in two opposite directions, form a chalice-shaped receptacle for Abeta. These data give new mechanistic insights into the stimulatory effect of cholesterol on Abeta/GM1 interactions. They also support the emerging concept that cholesterol is a universal modulator of protein-glycolipid interactions in the broader context of membrane recognition processes.

Gangliosides and the urokinase plasminogen activator receptor (u PAR) tipically partition in specialized membrane microdomains called lipid-rafts. u PAR becomes functionally important in fostering angiogenesis in endothelial progenitor cells ( EPCs) upon recruitment in caveolar-lipid rafts. Moreover, cell membrane enrichment with exogenous GM1 ganglioside is pro-angiogenic and opposite to the activity of GM3 ganglioside. On these basis, we first checked the interaction of u PAR with membrane models enriched with GM1 or GM3, relying on the adoption of solid-supported mobile bilayer lipid membranes with raft-like composition formed onto solid hydrophilic surfaces, and evaluated by surface plasmon resonance ( SPR) the extent of u PAR recruitment. We estimated the apparent dissociation constants of u PAR- GM1/ GM3 complexes. These preliminary observations, indicating that u PAR binds preferentially to GM1-enriched biomimetic membranes, were validated by identifying a pro-angiogenic activity of GM1-enriched EPCs, based on GM1-dependent u PAR recruitment in caveolar rafts. We have observed that addition of GM1 to EPCs culture medium promotes matrigel invasion and capillary morphogenesis, as opposed to the anti-angiogenesis activity of GM3. Moreover, GM1 also stimulates MAPKinases signalling pathways, typically associated with an angiogenesis program. Caveolar-raft isolation and Western blotting of u PAR showed that GM1 promotes caveolar-raft partitioning of u PAR, as opposed to control and GM3-challenged EPCs. By confocal microscopy, we have shown that in EPCs u PAR is present on the surface in at least three compartments, respectively, associated to GM1, GM3 and caveolar rafts. Following GM1 exogenous addition, the GM3 compartment is depleted of u PAR which is recruited within caveolar rafts thereby triggering angiogenesis. [ABSTRACT FROM AUTHOR]

It has been proposed that GM1 ganglioside promotes neuronal growth, phenotypic expression, and survival by modulating tyrosine kinase receptors for neurotrophic factors. Our studies tested the hypothesis that GM1 exerts its neurotrophic action on dopaminergic neurons, in part, by interacting with the GDNF (glia cell-derived neurotrophic factor) receptor complex, Ret tyrosine kinase and GFRα1 co-receptor. GM1 addition to striatal slices in situ increased Ret activity in a concentration- and time-dependent manner. GM1-induced Ret activation required the whole GM1 molecule and was inhibited by the kinase inhibitors PP2 and PP1. Ret activation was followed by Tyr1062 phosphorylation and PI3 kinase/Akt recruitment. The Src kinase was associated with Ret and GM1 enhanced its phosphorylation. GM1 responses required the presence of GFRα1, and there was a GM1 concentration-dependent increase in the binding of endogenous GDNF which paralleled that of Ret activation. Neutralization of the released GDNF did not influence the Ret response to GM1, and GM1 had no effect on GDNF release. Our in situ studies suggest that GM1 via GFRα1 modulates Ret activation and phosphorylation in the striatum and provide a putative mechanism for its effects on dopaminergic neurons. Indeed, chronic GM1 treatment enhanced Ret activity and phosphorylation in the striatum of the MPTP-mouse and kinase activation was associated with recovery of dopamine and DOPAC deficits. [ABSTRACT FROM AUTHOR]

Abstract: Lipid rafts are assumed to undergo biologically important size-modulations from nanorafts to microrafts. Due to the complexity of cellular membranes, model systems become important tools, especially for the investigation of the factors affecting “raft-like” Lo domain size and the search for Lo nanodomains as precursors in Lo microdomain formation. Because lipid compositional change is the primary mechanism by which a cell can alter membrane phase behavior, we studied the effect of the ganglioside GM1 concentration on the Lo/Ld lateral phase separation in PC/SM/Chol/GM1 bilayers. GM1 above 1mol % abolishes the formation of the micrometer-scale Lo domains observed in GUVs. However, the apparently homogeneous phase observed in optical microscopy corresponds in fact, within a certain temperature range, to a Lo/Ld lateral phase separation taking place below the optical resolution. This nanoscale phase separation is revealed by fluorescence spectroscopy, including C12NBD-PC self-quenching and Laurdan GP measurements, and is supported by Gaussian spectral decomposition analysis. The temperature of formation of nanoscale Lo phase domains over an Ld phase is determined, and is shifted to higher values when the GM1 content increases. A “morphological” phase diagram could be made, and it displays three regions corresponding respectively to Lo/Ld micrometric phase separation, Lo/Ld nanometric phase separation, and a homogeneous Ld phase. We therefore show that a lipid only-based mechanism is able to control the existence and the sizes of phase-separated membrane domains. GM1 could act on the line tension, “arresting” domain growth and thereby stabilizing Lo nanodomains. [Copyright &y& Elsevier]

Abstract: Alzheimer's disease (AD) is a neurodegenerative disorder and the aggregation of amyloid β peptides (Aβ) is the hallmark of this disease. Recent experimental evidences suggested that a unique complex, mono-sialo ganglioside (GM1) bound Aβ, possesses a high potential to facilitate this protein assembly, which is considered to be the seed for such aggregation process. There are no specific atomic molecular level studies done to characterize this complex computationally to give more insight into the conformational changes and interactions between them. In this work, we have done a simulation study on amyloid β peptide (Aβ42) with GM1 ganglioside in DPPC lipid membrane. An all atom molecular dynamics simulation is performed between the above peptide and GM1 in the membrane. Our simulation analysis explores the structural changes which occur in the protein by the influence of GM1 and lipids in the membrane. Secondary structure of the peptide in Aβ42 complex shows significant changes in C-terminal region especially at the helical segment. The complex is stabilized by a number of intra- and inter-hydrogen bonds within the desired cutoff distance. Thus, studying the conformations and interactions of beta amyloid protein and GM1 gives a starting point to investigate the so called ‘GAβ42’ in the membrane. [Copyright &y& Elsevier]

Abstract: The incretin hormone Glucagon-like peptide 1 (GLP-1) requires delivery by injection for the treatment of Type 2 diabetes mellitus. Here, we test if the properties of glycosphingolipid trafficking in epithelial cells can be applied to convert GLP-1 into a molecule suitable for mucosal absorption. GLP-1 was coupled to the extracellular oligosaccharide domain of GM1 species containing ceramides with different fatty acids and with minimal loss of incretin bioactivity. When applied to apical surfaces of polarized epithelial cells in monolayer culture, only GLP-1 coupled to GM1-ceramides with short- or cis-unsaturated fatty acids trafficked efficiently across the cell to the basolateral membrane by transcytosis. In vivo studies showed mucosal absorption after nasal administration. The results substantiate our recently reported dependence on ceramide structure for trafficking the GM1 across polarized epithelial cells and support the idea that specific glycosphingolipids can be harnessed as molecular vehicles for mucosal delivery of therapeutic peptides. [Copyright &y& Elsevier]

Highlights: [•] Cell-bound galectin-1 sequesters Lck from tumor cell/T-cell contact. [•] CD45 is required for Lck sequestration and the subsequent apoptotic response. [•] The GM1 ganglioside composition determines relocalization of Lck. [•] Membrane level of GM1 also affects the Gal-1 induced apoptosis of activated T-cells. [ABSTRACT FROM AUTHOR]

Direct electrospray ionization mass spectrometry (ESI-MS) assay was used to investigate the stepwise binding of the GM1 pentasaccharide β- D-Gal p-(1→3)-β-D-Gal pNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β- D-Gal p-(1→4)-β-D-Glc p (GM1os) to the cholera toxin B subunit homopentamer (CTB) and to establish conclusively whether GM1os binding is cooperative. Apparent association constants were measured for the stepwise addition of one to five GM1os to CTB at pH 6.9 and 22 °C. The intrinsic association constant, which was established from the apparent association constant for the addition of a single GM1os to CTB, was found to be (3.2 ± 0.2) × 10 M. This is in reasonable agreement with the reported value of (6.4 ± 0.3) × 10 M, which was measured at pH 7.4 and 25 °C using isothermal titration calorimetry (ITC). Analysis of the apparent association constants provides direct and unambiguous evidence that GM1os binding exhibits small positive cooperativity. Binding was found to be sensitive to the number of ligand-bound nearest neighbor subunits, with the affinities enhanced by a factor of 1.7 and 2.9 when binding occurs next to one or two ligand-bound subunits, respectively. These findings, which provide quantitative support for the binding model proposed by Homans and coworkers [], highlight the unique strengths of the direct ESI-MS assay for measuring cooperative ligand binding. [Figure not available: see fulltext.] [ABSTRACT FROM AUTHOR]

The roles of oolemmal lipid raft microdomains enriched in the ganglioside GM1 and the tetraspanin protein CD9 were investigated as causative agents in fertilization failure in human IVF where spermatozoa progress to the oolemma but fail to attach or, if attached, to penetrate. The findings show that specific configurations of GM1 lipid raft microdomains are consistent with attachment and penetration, while microdomains composed of CD9 lipid rafts, a protein known to be critical for penetration, do not appear to have a central role in the initial stages of attachment. The relative magnitude of the potential difference across the inner membrane (ΔΨm) in mitochondria localized to a stable subplasmalemmal domain appears to influence the organization of GM1 but not CD9 lipid raft microdomains in the corresponding oolemma. The findings present a novel view of how fertilization competence may be established in the human oocyte and a means by which certain fertilization failures that occur after conventional clinical IVF can be identified and explained in the unfortunate instance of fertilization arrest at the oolemma. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is a neurodegenerative disorder whose pathogenesis involves production and aggregation of amyloid-β peptide (Aβ). Aβ-induced toxicity is believed to involve alterations on as Na,K-ATPase and acetylcholinesterase (AChE) activities, prior to neuronal death. Drugs able to prevent or to reverse these biochemical changes promote neuroprotection. GM1 is a ganglioside proposed to have neuroprotective roles in AD models, through mechanisms not yet fully understood. Therefore, this study aimed to investigate the effect of Aβ1-42 infusion and GM1 treatment on recognition memory and on Na,K-ATPase and AChE activities, as well as, on antioxidant defense in the brain cortex and the hippocampus. For these purposes, Wistar rats received i.c.v. infusion of fibrilar Aβ1-42 (2 nmol) and/or GM1 (0.30 mg/kg). Behavioral and biochemical analyses were conducted 1 month after the infusion procedures. Our results showed that GM1 treatment prevented Aβ-induced cognitive deficit, corroborating its neuroprotective function. Aβ impaired Na,K-ATPase and increase AChE activities in hippocampus and cortex, respectively. GM1, in turn, has partially prevented Aβ-induced alteration on Na,K-ATPase, though with no impact on AChE activity. Aβ caused a decrease in antioxidant defense, specifically in hippocampus, an effect that was prevented by GM1 treatment. GM1, both in cortex and hippocampus, was able to increase antioxidant scavenge capacity. Our results suggest that Aβ-triggered cognitive deficit involves region-specific alterations on Na,K-ATPase and AChE activities, and that GM1 neuroprotection involves modulation of Na,K-ATPase, maybe by its antioxidant properties. Although extrapolation from animal findings is difficult, it is conceivable that GM1 could play an important role in AD treatment. [ABSTRACT FROM AUTHOR]

Abstract: Melittin is a bee venom toxin that can act as antimicrobial peptide. Gangliosides are glycosphingolipids that help maintain membrane structure and organization as well as act as anchors for lectins, toxins, pathogens and antimicrobial peptides. Here we investigate interaction of melittin with fast tumbling isotropic control DMPC/CHAPS bicelles and ganglioside doped DMPC/CHAPS/GM1 bicelles. DOSY result shows that larger percentage of peptide binds to GM1 containing bicelles than that of the control PC bicelles. Bound peptide induces leakage of the bicelles entrapped carboxyfluorescein. Percentage of leakage is higher from control PC bicelles than that of the GM1 containing bicelles. In the presence of control PC bicelles melittin acquired fully α-helical structure. But in the presence of GM1 containing bicelles the peptide is not fully α-helical i.e., some random coil structure is present in this folded form. The present study shows that GM1 has an effect on membrane active antimicrobial peptide melittin. [Copyright &y& Elsevier]

Age-related alterations of membrane lipids in brain cell membranes together with high blood cholesterol are considered as major risk factors for Alzheimer's disease. Yet the molecular mechanisms by which these factors increase Alzheimer's risk are mostly unknown. In lipid raft domains of the plasma membrane, neurotoxic Alzheimer's beta-amyloid (Abeta) peptides interact with both cholesterol and ganglioside GM1. Recent data also suggested that cholesterol could stimulate the binding of Abeta to GM1 through conformational modulation of the ganglioside headgroup. Here we used a combination of physicochemical and molecular modeling approaches to decipher the mechanisms of cholesterol-assisted binding of Abeta to GM1. With the aim of decoupling the effect of cholesterol on GM1 from direct Abeta-cholesterol interactions, we designed a minimal peptide (Abeta5-16) containing the GM1-binding domain but lacking the amino acid residues involved in cholesterol recognition. Using the Langmuir technique, we showed that cholesterol (but not phosphatidylcholine or sphingomyelin) significantly accelerates the interaction of Abeta5-16 with GM1. Molecular dynamics simulations suggested that Abeta5-16 interacts with a cholesterol-stabilized dimer of GM1. The main structural effect of cholesterol is to establish a hydrogen-bond between its own OH group and the glycosidic-bond linking ceramide to the glycone part of GM1, thereby inducing a tilt in the glycolipid headgroup. This fine conformational tuning stabilizes the active conformation of the GM1 dimer whose headgroups, oriented in two opposite directions, form a chalice-shaped receptacle for Abeta. These data give new mechanistic insights into the stimulatory effect of cholesterol on Abeta/GM1 interactions. They also support the emerging concept that cholesterol is a universal modulator of protein-glycolipid interactions in the broader context of membrane recognition processes. [ABSTRACT FROM AUTHOR]

Highlights: [•] Co-localization of GM1 with Aβ1–42 and amylin aggregates supported by in situ PLA. [•] Tracking of single GM1 reveals interaction with Aβ oligomers and amylin aggregates. [•] Amyloid aggregates interact generically with GM1 and decrease its mobility. [Copyright &y& Elsevier]

GM1 gangliosidosis is a rare, inherited neurodegenerative disorder caused by mutations in the GLB1 gene, which encodes the lysosomal hydrolase acid β-galactosidase (β-gal). β-gal deficiency leads to toxic accumulation of GM1 ganglioside, predominantly in the central nervous system (CNS), resulting in progressive neurodegeneration. LYS-GM101 is an AAVrh.10-based gene therapy vector carrying the human GLB1 cDNA. The efficacy of intra-cerebrospinal fluid injection of LYS-GM101 analogs was demonstrated in GM1 mouse and cat models with widespread diffusion of β-gal and correction of GM1 ganglioside accumulation in the CNS without observable adverse effects. Clinical dose selection was performed, based on a good-laboratory-practice study, in nonhuman primates (NHPs) using the clinical LYS-GM101 vector. A broadly distributed increase of β-gal activity was observed in NHP brain 3 months after intra-cisterna magna injection of LYS-GM101 at 1.0 × 10 12 vg/mL CSF and 4.0 × 10 12 vg/mL CSF, with 20% and 60% increases compared with vehicle-treated animals, respectively. Histopathologic examination revealed asymptomatic adverse changes in the sensory pathways of the spinal cord and dorsal root ganglia in both sexes and at both doses. Taken as a whole, these pre-clinical data support the initiation of a clinical study with LYS-GM101 for the treatment of GM1 gangliosidosis., Competing Interests: These studies were funded by grants from Lysogene, who were also involved in study design. M.H., X.M., L.G., and R.L. are full-time employees and hold equity of Lysogene. M.S.-E. and D.R.M. are shareholders in Lysogene. M.S.-E., H.L.G.-E., A.N.R., A.L.G., and D.R.M. are beneficiaries of a licensing agreement with Sio Gene Therapies for a separate technology., (© 2022 The Authors.)

Abstract: Antibodies to a ganglioside complex consisting of GM1 and GalNAc-GD1a (GM1/GalNAc-GD1a) are found in sera from patients with Guillain–Barré syndrome (GBS). To elucidate the clinical significance of anti-GM1/GalNAc-GD1a antibodies in GBS, clinical features of 58 GBS patients with IgG anti-GM1/GalNAc-GD1a antibodies confirmed by enzyme-linked immunosorbent assay and thin layer chromatography immunostaining were analyzed. Compared to GBS patients without anti-GM1/GalNAc-GD1a antibodies, anti-GM1/GalNAc-GD1a-positive patients more frequently had a preceding respiratory infection (n=38, 66%, p<0.01) and were characterized by infrequency of cranial nerve deficits (n=9, 16%, p<0.01) and sensory disturbances (n=26, 45%, p<0.01). Of the 28 anti-GM1/GalNAc-GD1a-positive patients for whom electrophysiological data were available, 14 had conduction blocks (CBs) at intermediate segments of motor nerves, which were not followed by evident remyelination. Eight of 10 bedridden cases were able to walk independently within one month after the nadir. These results show that the presence of anti-GM1/GalNAc-GD1a antibodies correlated with pure motor GBS characterized by antecedent respiratory infection, fewer cranial nerve deficits, and CBs at intermediate sites of motor nerves. The CB may be generated through alteration of the regulatory function of sodium channels in the nodal axolemma. [Copyright &y& Elsevier]

Abstract: The present single center, double-blind, delayed start study was conducted to examine possible symptomatic and disease-modifying effects of GM1 ganglioside in Parkinson''s disease (PD). Seventy-seven subjects with PD were randomly assigned to receive GM1 for 120weeks (early-start group) or placebo for 24weeks followed by GM1 for 96weeks (delayed-start group). Washout evaluations occurred at 1 and 2years after the end of treatment. Seventeen additional subjects who received standard-of-care were followed for comparative information about disease progression. Primary outcome was change from baseline Unified Parkinson''s Disease Rating Scale (UPDRS) motor scores. At week 24, the early-start group had significant improvement in UPDRS motor scores vs. a significant worsening of scores in the delayed-start group. The early-start group also showed a sustained benefit vs. the delayed-start group at week 72 and at week 120. Both groups had significant symptom worsening during washout. This study provides evidence that GM1 use for 24weeks was superior to placebo for improving motor symptoms and that extended GM1 use (up to 120weeks) resulted in a lower than expected rate of symptom progression. The data from this small study suggest that GM1 may have symptomatic and potentially disease modifying effects on PD. [Copyright &y& Elsevier]

Background Measurement of anti-GM1 IgM antibodies in multifocal motor neuropathy (MMN) sera is confounded by relatively low sensitivity that limits clinical usefulness. Combinatorial assay methods, in which antibodies react to heteromeric complexes of two or more glycolipids, are being increasingly applied to this area of diagnostic testing. Methods A newly developed combinatorial glycoarray able to identify antibodies to 45 different heteromeric glycolipid complexes and their 10 individual glycolipid components was applied to a randomly selected population of 33 MMN cases and 57 normal or disease controls. Comparison with an enzyme-linked immunosorbent assay (ELISA) was conducted for selected single glycolipids and their complexes. Results By ELISA, 22/33 MMN cases had detectable anti-GM1 IgM antibodies, whereas 19/33 MMN samples were positive for anti-GM1 antibodies by glycoarray. Analysis of variance (ANOVA) revealed that of the 55 possible single glycolipids and their 1:1 complexes, antibodies to the GM1:galactocerebroside (GM1:GalC) complex were most significantly associated with MMN, returning 33/33 MMN samples as positive by glycoarray and 29/33 positive by ELISA. Regression analysis revealed a high correlation in absolute values between ELISA and glycoarray. Receiver operator characteristic analysis revealed insignificantly different diagnostic performance between the two methods. However, the glycoarray appeared to offer slightly improved sensitivity by identifying antibodies in four ELISA-negative samples. Conclusions The use of combinatorial glycoarray or ELISA increased the diagnostic sensitivity of anti-glycolipid antibody testing in this cohort of MMN cases, without significantly affecting specificity, and may be a useful assay modification for routine clinical screening. [ABSTRACT FROM AUTHOR]

Abstract: The functioning and metabolic pathway of the amyloid β-precursor protein (APP) have not been fully elucidated. To fill this research gap, this study immunocytochemically investigated the intracellular localization of APP in the neuroblastoma cell line SK-N-SH and in normal primary cells. Using antibodies against the amino-terminal portion of the APP molecule, immunoreactivity was detected not only in the cytoplasm but also in the nucleus and nucleolus. Further analysis revealed the co-localization of amino acids 44–63 of the APP molecule with fibrillarin, a nucleolus marker. These findings indicate that a fraction of APP, including its amino-terminal portion, may be localized in the nucleus as well as in the nucleolus, suggesting an important role of APP in RNA metabolism and other intra-nucleolus functions. [Copyright &y& Elsevier]

Abstract: Neurotensin (NT) is an endogenous tridecapeptide neurotransmitter that shows multiple biological function in central and peripheral nervous systems. Gangliosides are glycosphingolipids, most abundant in the plasma membrane of nerve cells. Here we investigate the change of neurotensin solution structure induced by isotropic CHAPS-PC bicelles with and without ganglioside GM1 using solution state NMR spectroscopy. In aqueous solution the peptide is predominately unstructured. In the presence of bicelle overall structure of the peptide is stabilized. In CHAPS-PC bicelle neurotensin adopts 310 helical structure. In the presence of GM1 containing bicelle, the peptide adopts predominately 310 helical structures with small amount of α-helical structure. These results are consistent with the CD spectroscopic results. Neurotensin interacts better with GM1 containing bicelle than that of the CHAPS-PC bicelle. Docking studies between the Neurotensin Receptor3 (NTS3) and different NT conformations also indicated better binding of the NT conformation obtained in presence of GM1-containing bicelles. [Copyright &y& Elsevier]

Alzheimer's disease (AD) is the most common cause of dementia in elderly people, and age is the major nongenetic risk factor for sporadic AD. A hallmark of AD is the accumulation of amyloid in the brain, which is composed mainly of the amyloid beta-peptide (Aβ) in the form of oligomers and fibrils. However, how aging induces Aβ aggregation is not yet fully determined. Some residues in the Aβ sequence seem to promote Aβ-induced toxicity in association with age-dependent risk factors for AD, such as (i) increased GM1 brain membrane content, (ii) altered lipid domain in brain membrane, (iii) oxidative stress. However, the role of Aβ sequence in promoting aggregation following interaction with the plasma membrane is not yet demonstrated. As Tyr10 is implicated in the induction of oxidative stress and stabilization of Aβ aggregation, we substituted Tyr 10 with a synthetic amino acid that abolishes Aβ-induced oxidative stress and shows an accelerated interaction with GM1. This variant peptide shows impaired aggregation properties and increased affinity for GM1. It has a dominant negative effect on amyloidogenesis in vitro, in cellulo, and in isolated synaptosomes. The present study shed new light in the understanding of Aβ-membrane interactions in Aβ-induced neurotoxicity. It demonstrates the relevance of Aβ sequence in (i) Aβ-membrane interaction, underlining the role of age-dependent enhanced GM1 content in promoting Aβ aggregation, (ii) Aβ aggregation, and (iii) Aβ-induced oxidative stress. Our results open the way for the design of peptides aimed to inhibit Aβ aggregation and neurotoxicity. [ABSTRACT FROM AUTHOR]

Abstract: In order to analyze the mechanisms for cancer metastasis, high metastatic sublines (H7-A, H7-Lu, H7-O, C4-sc, and C4-ly) were obtained by repeated injection of mouse Lewis lung cancer sublines H7 and C4 into C57BL/6 mice. These sublines exhibited increased proliferation and invasion activity in vitro. Ganglioside profiles exhibited lower expression of GM1 in high metastatic sublines than the parent lines. Then, we established GM1-Si-1 and GM1-Si-2 by stable silencing of GM1 synthase in H7 cells. These GM1-knockdown clones exhibited increased proliferation and invasion. Then, we explored genes that markedly altered in the expression levels by DNA microarray in the combination of C4 vs. C4-ly or H7 vs. H7 (GM1-Si). Consequently, pp-GalNAc-T13 gene was identified as up-regulated genes in the high metastatic sublines. Stable transfection of pp-GalNAc-T13 cDNA into C4 (T13-TF) resulted in increased invasion and motility. Then, immunoblotting and flow cytometry using various antibodies and lectins were performed. Only anti-trimeric Tn antibody (mAb MLS128), showed increased expression levels of trimeric Tn antigen in T13-TF clones. Moreover, immunoprecipitation/immunoblotting was performed by mAb MLS128, leading to the identification of an 80kDa band carrying trimeric Tn antigen, i.e. Syndecan-1. Stable silencing of endogenous pp-GalNAc-T13 in C4-sc (T13-KD) revealed that primary tumors generated by subcutaneous injection of T13-KD clones showed lower coalescence to fascia and peritoneum, and significantly reduced lung metastasis than control clones. These data suggested that high expression of pp-GalNAc-T13 gene generated trimeric Tn antigen on Syndecan-1, leading to the enhanced metastasis. [Copyright &y& Elsevier]

Abstract: Repression of human papillomavirus (HPV) E6 and E7 oncogenes in established cervical carcinoma cell lines causes senescence due to reactivation of cellular tumor suppressor pathways. Here, we determined whether ongoing expression of HPV16 or HPV18 oncogenes is required for the proliferation of primary human cervical carcinoma cells in serum-free conditions at low passage number after isolation from patients. We used an SV40 viral vector expressing the bovine papillomavirus E2 protein to repress E6 and E7 in these cells. To enable efficient SV40 infection and E2 gene delivery, we first incubated the primary cervical cancer cells with the ganglioside GM1, a cell-surface receptor for SV40 that is limiting in these cells. Repression of HPV in primary cervical carcinoma cells caused them to undergo senescence, but the E2 protein had little effect on HPV-negative primary cells. These data suggest that E6 and E7 dependence is an inherent property of human cervical cancer cells. [Copyright &y& Elsevier]

Abstract: Glycolipids are important biological molecules that modulate cellular recognitions and pathogen adhesions. In this paper, we report a sensitive glycolipid microarray for non-covalently immobilizing glycolipids on a microarray substrate and we perform a set of immunoassays to explore glycolipid–protein interactions. This substrate utilizes a three-dimensional hydrazide-functionalized dendrimer monolayer attached onto a microscopic glass surface, which possesses the characteristics to adsorb glycoliplids non-covalently and facilitates multivalent attributes on the substrate surface. In the proof-of-concept experiments, gangliosides such as GM1, FucGM1, GM3, GD1b, GT1b, and GQ1b, and a lipoarabinomannan were tested on the substrate and interrogated with toxins and antibodies. The resulting glycolipid microarrays exhibited hypersensitivity and specificity for detection of glycolipid–protein interactions. In particular, a robust and specific binding of a pentameric cholera toxin B subunit to the GM1 glycolipid spotted on the array has demonstrated its superiority in sensitivity and specificity. In addition, this glycolipid microarray substrate was used to detect lipoarabinomannan in buffer within a limit-of-detection of 125ng/mL. Furthermore, Mycobacterium tuberculosis (Mtb) Lipoarabinomannan was tested in human urine specimens on this platform, which can effectively identify urine samples either infected or not infected with Mtb. The results of this work suggest the possibility of using this glycolipid microarray platform to fabricate glycoconjugate microarrays, which includes free glycans and glycolipids and potential application in detection of pathogen and toxin. [Copyright &y& Elsevier]

Abstract: The effect of Aβ25–35 peptide, in its fibrillar and non-fibrillar forms, on ganglioside expression in organotypic hippocampal slice cultures was investigated. Gangliosides were endogenously labeled with D-[1-C14] galactose and results showed that Aβ25–35 affected ganglioside expression, depending on the peptide aggregation state, that is, fibrillar Aβ25–35 caused an increase in GM3 labeling and a reduction in GD1b labeling, whereas the non-fibrillar form was able to enhance GM1 expression. Interestingly, GM1 exhibited a neuroprotective effect in this organotypic model, since pre-treatment of the hippocampal slices with GM1 10μM was able to prevent the toxicity triggered by the fibrillar Aβ25–35, when measured by propidium iodide uptake protocol. With the purpose of further investigating a possible mechanism of action, we analyzed the effect of GM1 treatment (1, 6, 12 and 24h) upon the Aβ-induced alterations on GSK3β dephosphorylation/activation state. Results demonstrated an important effect after 24-h incubation, with GM1 preventing the Aβ-induced dephosphorylation (activation) of GSK3β, a signaling pathway involved in apoptosis triggering and neuronal death in models of Alzheimer’s disease. Taken together, present results provide a new and important support for ganglioside participation in development of Alzheimer’s disease experimental models and suggest a protective role for GM1 in Aβ-induced toxicity. This may be useful for designing new therapeutic strategies for Alzheimer’s treatment. [Copyright &y& Elsevier]

Abstract: Pharmacological regulation of gene expression was one of the top professional interests of Dr. Costa. He promoted the idea that drugs can improve the endogenous mechanisms of synaptic plasticity by modulating gene expression. In this article I reflect upon Dr. Costa''s leadership in projects undertaken at FGIN that were aimed at elucidating how neurotransmitter receptor activation could affect brain function by modulating genes and their products. I will be presenting examples of how pharmacological tools can change gene expression. These include the ability of drugs of abuse to alter the synthesis of opioid peptides or an endogenous ligand for GABAA receptor. I will conclude with a brief summary of intriguing discoveries about the regulation of nerve growth factor (NGF) and its receptors by beta-receptor agonists, adrenal steroids and cytokines. [Copyright &y& Elsevier]

Cell therapy is a promising approach for the treatment of neurodegenerative conditions such as Alzheimer's and Parkinson's diseases. However, the presence of toxic aggregates in tissue raises the question of whether grafted stem cells are susceptible to amyloid toxicity before they differentiate into mature neurons. To address this question, we investigated the relative vulnerability of human mesenchymal stromal cells and their neuronally differentiated counterparts to Aβ42 oligomers and whether susceptibility correlates with membrane GM1 content, a key player in oligomer toxicity. We found that our cell model was highly susceptible to aggregate toxicity, whereas neuronal differentiation induced resistance to amyloid species. This data correlated well with the content of membrane GM1, levels of which decreased considerably in differentiated cells. These findings extend our knowledge of stem cell vulnerability to amyloid species, which remains a controversial issue, and confirm that amyloid-GM1 interactions play an important role in cell impairment. [ABSTRACT FROM AUTHOR]

Abstract: Cholera toxin B-subunit (CTB) treatment of K562 erythroleukemia cells increased their sensitivity to be killed by NK-92 cells with more than 10%, compared to untreated cells. A similar treatment of non-T, non-B acute lymphoblastic REH leukemia cells, known to be unsensitive to NK cell mediated cytotoxicity, did not have any impact at all. Visualization of the cross-linked gangliosideM1 (GM1) using fluorescent labeled CTB, indicated accumulation of the fluorescence to one cap and a few smaller patches in both type of cells. Additional cross-linking using anti-CTB antibodies further accentuated capping and increased lysis in the case of K562 cells. Blocking experiments performed with anti-MICA/B, ULBP-2 and/or CD59 antibodies could not inhibit the increased sensitivity mediated by CTB. [Copyright &y& Elsevier]

Abstract: Gangliosides are lipophilic compounds found in cell plasma membranes throughout the brain that play a role in neuronal plasticity and regeneration. Indeed, absence or abnormal accumulation of gangliosides has been shown to lead to neurological disorders. Experimental data have shown that exogenous gangliosides exhibit properties similar to the neurotrophins, a family of neurotrophic factors that are important in the survival and maintenance of neurons and prevention of neurological diseases. Brain-derived neurotrophic factor (BDNF) is the most abundant of the neurotrophins. This work was done to reveal the neurotrophic mechanism of exogenous gangliosides. In particular, we examined whether gangliosides promote the release of BDNF. Rat hippocampal neurons or human neuroblastoma cells were transduced with a recombinant adenovirus expressing BDNF-flag to facilitate detection of BDNF. Release of BDNF was then determined by Western blot analysis and a two-site immunoassay of culture medium. The depolarizing agent KCl was used as a comparison. In hippocampal neurons, both GM1 ganglioside and KCl evoked within minutes the release of mature BDNF. In human cells, GM1 and other gangliosides released both mature BDNF and pro-BDNF. The effect of gangliosides was structure-dependent. In fact, GT1b preferentially released mature BDNF whereas GM1 released both mature and pro-BDNF. Ceramide and sphingosine did not modify the release of BDNF. This work provides additional experimental evidence that exogenous gangliosides can be used to enhance the neurotrophic factor environment and promote neuronal survival in neurological diseases. This article is part of a Special Issue entitled ‘Trends in Neuropharmacology: In Memory of Erminio Costa’. [Copyright &y& Elsevier]