Myrosinase isogenes in wasabi (Wasabia japonica Matsum) and their putative roles in glucosinolate metabolism.

BACKGROUND: Wasabi, a Brassicaceae member, is well-known for its unique pungent and hot flavor which is produced from glucosinolate (GSL) degradation. Myrosinase (MYR) is a principle enzyme catalyzing the primary conversion of GSLs to GSL hydrolysis products (GHPs) which is responsible for plant defense system and food quality. Due to the limited information in relation to MYRs present in wasabi (Wasabia japonica M.), this study aimed to identify the MYR isogenes in W. japonica and analyze their roles in relation to GSL metabolism. RESULTS: In results, WjMYRI-1 was abundantly expressed in all organs, whereas WjMYRI-2 showed only trace expression levels. WjMYRII was highly expressed in the aboveground tissues. Interestingly, WjMYRII expression was significantly upregulated by certain abiotic factors, such as methyl jasmonate (more than 40-fold in petioles and 15-fold in leaves) and salt (tenfold in leaves). Young leaves and roots contained 97.89 and 91.17 µmol‧g-1 of GSL, whereas less GSL was produced in mature leaves and petioles (38.36 and 44.79 µmol‧g-1, respectively). Similar pattern was observed in the accumulation of GHPs in various plant organs. Notably, despite the non-significant changes in GSL production, abiotic factors treated samples enhanced significantly GHP content. Pearson's correlation analysis revealed that WjMYRI-1 expression significantly correlated with GSL accumulation and GHP formation, suggesting the primary role of WjMYRI-1-encoding putative protein in GSL degradation. In contrast, WjMYRII expression level showed no correlation with GSL or GHP content, suggesting another physiological role of WjMYRII in stress-induced response. CONCLUSIONS: In conclusions, three potential isogenes (WjMYRI-1, WjMYRI-2, and WjMYRII) encoding for different MYR isoforms in W. japonica were identified. Our results provided new insights related to MYR and GSL metabolism which are important for the implications of wasabi in agriculture, food and pharmaceutical industry. Particularly, WjMYRI-1 may be primarily responsible for GSL degradation, whereas WjMYRII (clade II) may be involved in other regulatory pathways induced by abiotic factors.

Mass Spectrometric and Glycan Microarray-Based Characterization of the Filarial Nematode Brugia malayi Glycome Reveals Anionic and Zwitterionic Glycan Antigens.

Millions of people worldwide are infected with filarial nematodes, responsible for lymphatic filariasis (LF) and other diseases causing chronic disablement. Elimination programs have resulted in a substantial reduction of the rate of infection in certain areas creating a need for improved diagnostic tools to establish robust population surveillance and avoid LF resurgence. Glycans from parasitic helminths are emerging as potential antigens for use in diagnostic assays. However, despite its crucial role in host-parasite interactions, filarial glycosylation is still largely, structurally, and functionally uncharacterized. Therefore, we investigated the glycan repertoire of the filarial nematode Brugia malayi. Glycosphingolipid and N-linked glycans were extracted from several life-stages using enzymatic release and characterized using a combination of MALDI-TOF-MS and glycan sequencing techniques. Next, glycans were purified by HPLC and printed onto microarrays to assess the host anti-glycan antibody response. Comprehensive glycomic analysis of B. malayi revealed the presence of several putative antigenic motifs such as phosphorylcholine and terminal glucuronic acid. Glycan microarray screening showed a recognition of most B. malayi glycans by immunoglobulins from rhesus macaques at different time points after infection, which permitted the characterization of the dynamics of anti-glycan immunoglobulin G and M during the establishment of brugian filariasis. A significant level of IgG binding to the parasite glycans was also detected in infected human plasma, while IgG binding to glycans decreased after anthelmintic treatment. Altogether, our work identifies B. malayi glycan antigens and reveals antibody responses from the host that could be exploited as potential markers for LF.

Glucosinolates and Biotic Stress Tolerance in Brassicaceae with Emphasis on Cabbage: A Review.

Glucosinolates (GSLs) and GSL-associated genes are receiving increasing attention from molecular biologists due to their multifunctional properties. GSLs are secondary metabolites considered to be highly active in most Brassica species. Their importance has motivated the discovery and functional analysis of the GSLs and GSL hydrolysis products involved in disease development in brassicas and other plants. Comprehensive knowledge of the GSL content of Brassica species and the molecular details of GSL-related genes will help elucidate the molecular control of this plant defense system. This report provides an overview of the current status of knowledge on GSLs, GSL biosynthesis, as well as hydrolysis related genes, and GSL hydrolysis products that regulate fungal, bacterial, and insect resistance in cabbage and other brassicas.

Arabidopsis Glucan Synthase-Like1 (GSL1) Is Required for Tolerance to Low-Calcium Conditions and Exhibits a Function Comparable to GSL10.

Crops that exhibit symptoms of calcium (Ca) deficiency constitute a major agricultural problem. Molecular breeding of resistant cultivars is a promising method for overcoming this problem. However, the involved genes must first be identified. Here, we show that the glucan synthase-like (GSL) 1 gene is essential for low-Ca tolerance in Arabidopsis thaliana. GSL1 is homologous to GSL10, which we previously showed was essential for low-Ca tolerance. Under low-Ca conditions, gsl1 mutants exhibit reduced growth and the onset of necrosis in new leaves. These symptoms are typical of Ca-deficient crops. A grafting experiment suggested that the shoot genotype, but not the root genotype, was important for the suppression of shoot necrosis. The ectopic accumulation of callose under low-Ca conditions was significantly reduced in gsl1 mutants compared with wild-type plants. Because the corresponding single-mutant phenotypes are similar, we investigated the interaction between GSL1 and GSL10 by testing the gsl1 gsl10 double mutant for sensitivity to low-Ca conditions. The double mutant exhibited a more severe phenotype than did the single mutants, indicating that the effects of GSL1 and GSL10 on low-Ca tolerance are additive. Because GSL genes are highly conserved within the plant kingdom, the GSL loci may be useful for breeding low-Ca tolerant crops.

A genome-scale TF-DNA interaction network of transcriptional regulation of Arabidopsis primary and specialized metabolism.

Plant metabolism is more complex relative to individual microbes. In single-celled microbes, transcriptional regulation by single transcription factors (TFs) is sufficient to shift primary metabolism. Corresponding genome-level transcriptional regulatory maps of metabolism reveal the underlying design principles responsible for these shifts as a model in which master regulators largely coordinate specific metabolic pathways. Plant primary and specialized metabolism occur within innumerable cell types, and their reactions shift depending on internal and external cues. Given the importance of plants and their metabolites in providing humanity with food, fiber, and medicine, we set out to develop a genome-scale transcriptional regulatory map of Arabidopsis metabolic genes. A comprehensive set of protein-DNA interactions between Arabidopsis thaliana TFs and gene promoters in primary and specialized metabolic pathways were mapped. To demonstrate the utility of this resource, we identified and functionally validated regulators of the tricarboxylic acid (TCA) cycle. The resulting network suggests that plant metabolic design principles are distinct from those of microbes. Instead, metabolism appears to be transcriptionally coordinated via developmental- and stress-conditional processes that can coordinate across primary and specialized metabolism. These data represent the most comprehensive resource of interactions between TFs and metabolic genes in plants.

Glycosphingolipid metabolism and its role in ageing and Parkinson's disease.

It is well established that lysosomal glucocerebrosidase gene (GBA) variants are a risk factor for Parkinson's disease (PD), with increasing evidence suggesting a loss of function mechanism. One question raised by this genetic association is whether variants of genes involved in other aspects of sphingolipid metabolism are also associated with PD. Recent studies in sporadic PD have identified variants in multiple genes linked to diseases of glycosphingolipid (GSL) metabolism to be associated with PD. GSL biosynthesis is a complex pathway involving the coordinated action of multiple enzymes in the Golgi apparatus. GSL catabolism takes place in the lysosome and is dependent on the action of multiple acid hydrolases specific for certain substrates and glycan linkages. The finding that variants in multiple GSL catabolic genes are over-represented in PD in a heterozygous state highlights the importance of GSLs in the healthy brain and how lipid imbalances and lysosomal dysfunction are associated with normal ageing and neurodegenerative diseases. In this article we will explore the link between lysosomal storage disorders and PD, the GSL changes seen in both normal ageing, lysosomal storage disorders (LSDs) and PD and the mechanisms by which these changes can affect neurodegeneration.

Reasons for choice of glaucoma surgery in eyes not treated with anti-glaucoma medications.

BACKGROUND: In the real world, some glaucoma patients can undergo an incisional glaucoma surgery without using medication. The rate of cases with no medication treatment at the time of surgery among those that underwent incisional glaucoma surgeries performed in our department was reported. METHODS: The department database of Shimane University Hospital for eyes that underwent incisional surgeries to manage glaucoma at the hospital between April 2018 and September 2020 were searched. By reviewing the medical charts of 1,417 consecutive eyes listed, 90 (6.4%) eyes of 67 subjects (mean age of 72 ± 16 years; 22 men, 29 eyes; 45 women, 61 eyes) who underwent a surgery without use of antiglaucoma medication were identified. The types of glaucoma, glaucoma procedures, and reasons for choosing the glaucoma surgeries rather than medical therapy were collected for the 90 eyes. RESULTS: Among the 90 eyes, primary angle-closure disease (PACD) (60%) was the most frequent type of glaucoma followed by EXG (17%), POAG (16%), and others (8%). Among the reasons for the choice of incisional surgery, relief of angle closure (64%) was the most frequent, the second most frequent was the incidental diagnosis of glaucoma during the ocular examinations both for that eye's cataract surgery or the contralateral glaucoma surgery (13%). Other reasons included poor medication adherence (10%), dementia (6%), multiple medication allergy (3%), and acute IOP elevation other than PACD (3%). Cataract extraction (CE) alone (33%) was the most frequent glaucoma procedures performed in these eyes, followed by CE combined with goniosynechialysis (27%), CE + iStent (16%), CE + goniotomy by Tanito microhook ab interno trabeculotomy or using the Kahook Dual Blade (11%), Ahmed Glaucoma valve implantation (11%), and trabeculectomy (2%). CONCLUSION: In the real-world, 6.4% of incisional glaucoma surgeries were performed in the absence of medication use; of them, 32 eyes (2.3%) were with open angle glaucoma. In open angle glaucoma, the reasons can be classified into; 1) patients' inability to instill the medication, 2) incidental diagnosis of glaucoma during the pre-surgical examinations, and 3) the eyes with acute IOP rise.

ATP-binding cassette transporters mediate differential biosynthesis of glycosphingolipid species.

The cytosolic-oriented glucosylceramide (GlcCer) synthase is enigmatic, requiring nascent GlcCer translocation to the luminal Golgi membrane to access glycosphingolipid (GSL) anabolic glycosyltransferases. The mechanism by which GlcCer is flipped remains unclear. To investigate the role of GlcCer-binding partners in this process, we previously made cleavable, biotinylated, photoreactive GlcCer analogs in which the reactive nitrene was closely apposed to the GlcCer head group, while maintaining a C16-acyl chain. GlcCer-binding protein specificity was validated for both photoprobes. Using one probe, XLB, here we identified ATP-binding cassette (ABC) transporters ABCA3, ABCB4, and ABCB10 as unfractionated microsomal GlcCer-binding proteins in DU-145 prostate tumor cells. siRNA knockdown (KD) of these transporters differentially blocked GSL synthesis assessed in toto and via metabolic labeling. KD of ABCA3 reduced acid/neutral GSL levels, but increased those of LacCer, while KD of ABCB4 preferentially reduced neutral GSL levels, and KD of ABCB10 reduced levels of both neutral and acidic GSLs. Depletion of ABCA12, implicated in GlcCer transport, preferentially decreased neutral GSL levels, while ABCB1 KD preferentially reduced gangliosides, but increased neutral GSL Gb3. These results imply that multiple ABC transporters may provide distinct but overlapping GlcCer and LacCer pools within the Golgi lumen for anabolism of different GSL series by metabolic channeling. Differential ABC family member usage may fine-tune GSL biosynthesis depending on cell/tissue type. We conclude that ABC transporters provide a new tool for the regulation of GSL biosynthesis and serve as potential targets to reduce selected GSL species/subsets in diseases in which GSLs are dysregulated.

The case for free and open source software in research and scholarship.

Free and open source software (FOSS) is any computer program released under a licence that grants users rights to run the program for any purpose, to study it, to modify it, and to redistribute it in original or modified form. Our aim is to explore the intersection between FOSS and computational reproducibility. We begin by situating FOSS in relation to other 'open' initiatives, and specifically open science, open research, and open scholarship. In this context, we argue that anyone who actively contributes to the research process today is a computational researcher, in that they use computers to manage and store information. We then provide a primer to FOSS suitable for anyone concerned with research quality and sustainability-including researchers in any field, as well as support staff, administrators, publishers, funders, and so on. Next, we illustrate how the notions introduced in the primer apply to resources for scientific computing, with reference to the GNU Scientific Library as a case study. We conclude by discussing why the common interpretation of 'open source' as 'open code' is misplaced, and we use this example to articulate the role of FOSS in research and scholarship today. This article is part of the theme issue 'Reliability and reproducibility in computational science: implementing verification, validation and uncertainty quantification in silico'.

Anterior vitrectomy, phacoemulsification cataract extraction and irido-zonulo-hyaloid-vitrectomy in protracted acute angle closure crisis.

PURPOSE: To describe a modified surgical approach with anterior vitrectomy, phacoemulsification (phaco) cataract extraction and irido-zonulo-hyaloid-vitrectomy (IZHV) in protracted acute angle closure crisis (AACC). PATIENTS AND METHODS: Non-comparative, retrospective case series including 21 eyes in 19 consecutive cases of protracted AACC, which persists for at least 7 days despite maximal medical and laser therapies, were included in this study. All patients underwent a modified surgical procedure with anterior vitrectomy, phaco cataract extraction, IOL implantation, goniosynechialysis (GSL) and IZHV, using modest phaco dynamic parameters with intraocular pressure (IOP) set at 30 mmHg through the procedure using Centurion® Vision System equipped with active fluidics while the anterior vitrectomy was set at 4000 or 5000 rpm. IOP and anterior chamber space were maintained through the procedure using ophthalmic viscosurgical device (OVD) injected through paracentesis whenever the Phaco or I/A probe was withdrawn from within the anterior chamber. Medical history, visual acuity (VA), IOP and anterior and posterior segment findings were recorded and compared before and after surgical treatment. RESULTS: The average age of all patients was 60.05 years old, while the average period of persistent AACC was 20.05 days. Preoperatively, the average IOP of all included eyes was 44.40 ± 8.42 mmHg despite maximal topical and systemic anti-glaucoma medications and/or laser surgeries, while the average VA was 1.46 ± 0.88 (log MAR). Postoperatively, IOP was well controlled in all patients with an average IOP at 12.06 ± 3.07 mmHg without any anti-glaucoma medications at follow-ups, which was decreased significantly from that in preoperative measurements (P < 0.001). Visual acuity was improved significantly at final follow-up with an average postoperative VA at 0.74 ± 0.77 (log MAR, P < 0.001). Anterior segment inflammation was surprisingly mild with no or minimal inflammatory cells or exudates. Anterior segment configuration was resolved in all the cases. There was no recurrent IOP spike, anterior chamber shallowing or severe complications during an average follow-up of 5.38 months (ranging from 3 to 6 months). CONCLUSIONS: Protracted AACC is a complex situation while a modified surgical strategy of anterior vitrectomy, phaco cataract extraction and IZHV provides a safe and efficient solution.

PapG subtype-specific binding characteristics of Escherichia coli towards globo-series glycosphingolipids of human kidney and bladder uroepithelial cells.

Uropathogenic Escherichia coli (UPEC) are the primary cause of urinary tract infections (UTIs) in humans. P-fimbriae are key players for bacterial adherence to the uroepithelium through the Galα1-4Gal-binding PapG adhesin. The three identified classes I, II and III of PapG are supposed to adhere differently to host cell glycosphingolipids (GSLs) of the uroepithelial tract harboring a distal or internal Galα1-4Gal sequence. In this study, GSL binding characteristics were obtained in a nonradioactive adhesion assay using biotinylated E. coli UTI and urine isolates combined with enzyme-linked NeutrAvidin for detection. Initial experiments with reference globotriaosylceramide (Gb3Cer, Galα1-4Galß1-4Glcß1-1Cer), globotetraosylceramide (Gb4Cer, GalNAcß1-3Galα1-4Galß1-4Glcß1-1Cer) and Forssman GSL (GalNAcα1-3GalNAcß1-3Galα1-4Galß1-4Glcß1-1Cer) revealed balanced adhesion toward the three GSLs for PapG I-mediated attachment. In contrast, E. coli carrying PapG II or PapG III increasingly adhered to growing oligosaccharide chain lengths of Gb3Cer, Gb4Cer and Forssman GSL. Binding studies with GSLs from human A498 kidney and human T24 bladder epithelial cells, both being negative for the Forssman GSL, revealed the less abundant Gb4Cer vs. Gb3Cer as the prevalent receptor in A498 cells of E. coli expressing PapG II or PapG III. On the other hand, T24 cells exhibited a higher relative content of Gb4Cer vs. Gb3Cer alongside dominant binding of PapG II- or PapG III-harboring E. coli toward Gb4Cer and vastly lowered attachment to minor Gb3Cer. Further studies on PapG-mediated interaction with cell surface-exposed GSLs will improve our knowledge on the molecular mechanisms of P-fimbriae-mediated adhesion and may contribute to the development of antiadhesion therapeutics to combat UTIs.

High-sensitivity quantification of glycosphingolipid glycans by ESI-MS utilizing ozonolysis-based release and isotopic Girard's reagent labeling.

Quantitative analysis of glycosphingolipids (GSLs) has been hindered by the lack of chromogenic groups for spectral detection or active functional groups for specific derivatization. In this study, a highly sensitive method based on ozonolysis-induced release and isotopic Girard's reagent P labeling of GSL glycans coupled with mass spectrometric detection for the quantification of animal tissue GSLs is developed. First, different approaches for the release of glycans from GSLs were compared with each other and the ozonolysis-based method was found to have the highest glycan yield under relative mild reaction conditions. Then a relative quantification method of ozonolysis-released GSL glycans based on stable isotope labeling using nondeuterated (d0-) and 2,3,4,5,6-pentadeuterated (d5-) Girard's reagent P (GP) was established, and its good linearity, accuracy and reproducibility were statistically verified. Finally, the new method was successfully applied to revealing the difference between porcine brain and liver as well as between the brain of normal and aging rats in GSL glycome by online hydrophilic interaction liquid chromatography coupling with ultraviolet detection and tandem mass spectrometry (HILIC-UV-MS/MS). This novel method is versatile and sensitive, enabling accurate quantitative analysis of tissue GSLs and showing great significance for glycomic studies.

Evaluation of Residual Human-Induced Pluripotent Stem Cells in Human Chondrocytes by Cell Type-Specific Glycosphingolipid Glycome Analysis Based on the Aminolysis-SALSA Technique.

Cartilage damage may eventually lead to osteoarthritis because it is difficult to repair. Human-induced pluripotent stem cell (iPSC)-derived chondrocytes may potentially be used to treat cartilage damage, but the tumorigenicity of iPSCs is a major concern for their application in regenerative medicine. Many glycoconjugates serve as stem cell markers, and glycosphingolipids (GSLs) including H type 1 antigen (Fucα1-2Galß1-3GlcNAc) have been expressed on the surface of iPSCs. The purpose of the present study was to investigate whether GSL-glycome analysis is useful for quality control of residual iPSCs in chondrocytes. We performed GSL-glycome analysis of undifferentiated iPSCs in chondrocytes by combining glycoblotting and aminolysis-sialic acid linkage-specific alkylamidation (SALSA) method, enabling the detection of small quantities of iPSC-specific GSL-glycans from 5 × 104 cells. Furthermore, we estimated the residual amount of iPSCs using R-17F antibody, which possesses cytotoxic activity toward iPSCs that is dependent on the Lacto-N-fucopentaose I (LNFP I) of GSL. Moreover, we could detect a small number of LNFP I during mesenchymal stem cells (MSCs) differentiation from iPSCs. This is the first demonstration that GSL-glycome analysis is useful for detecting undifferentiated iPSCs, and can thereby support safe regenerative medicine.

Excessive interferon-α signaling in autoimmunity alters glycosphingolipid processing in B cells.

Excessive interferon-α (IFN-α) production by innate immune cells is a hallmark of autoimmune diseases. What other cell type secretes IFN-α and how IFN-α affects immune cell metabolism and homeostasis in autoimmunity are largely unclear. Here, we report that autoimmune B cells, arising from two different B cell-specific genetic lesions in mice, secrete IFN-α. In addition, IFN-α, found in abundance in autoimmunity, elicited profound changes in the B cell lipidome, increasing their expression of glycosphingolipids (GSLs) and leading to their CD1d-mediated depletion of iNKT cells in vitro and in vivo. IFN-α receptor blockade could reverse the loss of iNKT cells. Excessive stimulation of B cells with IFN-α altered the expression of enzymes that catalyze critical steps in GSL processing, increasing the expressions of glucosylceramide synthase (GCS) and globotrihexosylceramide synthase (Gb3S) but decreasing that of α-galactosidase A (α-galA). Inhibiting GCS or restoring α-galA expression prevented iNKT depletion by IFN-α-activated B cells. Taken together, our work indicated that excessive IFN-α perturbs GSL metabolism in B cells which in turn adversely affects iNKT homeostasis.

OXI1 kinase plays a key role in resistance of Arabidopsis towards aphids (Myzus persicae).

Plants have co-evolved with a diverse array of pathogens and insect herbivores and so have evolved an extensive repertoire of constitutive and induced defence mechanisms activated through complex signalling pathways. OXI1 kinase is required for activation of mitogen-activated protein kinases (MAPKs) and is an essential part of the signal transduction pathway linking oxidative burst signals to diverse downstream responses. Furthermore, changes in the levels of OXI1 appear to be crucial for appropriate signalling. Callose deposition also plays a key role in defence. Here we demonstrate, for the first time, that OXI1 plays an important role in defence against aphids. The Arabidopsis mutant, oxi1-2, showed significant resistance both in terms of population build-up (p < 0.001) and the rate of build-up (p < 0.001). Arabidopsis mutants for ß-1,3-glucanase, gns2 and gns3, showed partial aphid resistance, significantly delaying developmental rate, taking two-fold longer to reach adulthood. Whilst ß-1,3-glucanase genes GNS1, GNS2, GNS3 and GNS5 were not induced in oxi1-2 in response to aphid feeding, GNS2 was expressed to high levels in the corresponding WT (Col-0) in response to aphid feeding. Callose synthase GSL5 was up-regulated in oxi1-2 in response to aphids. The results suggest that resistance in oxi1-2 mutants is through induction of callose deposition via MAPKs resulting in ROS induction as an early response. Furthermore, the results suggest that the ß-1,3-glucanase genes, especially GNS2, play an important role in host plant susceptibility to aphids. Better understanding of signalling cascades underpinning tolerance to biotic stress will help inform future breeding programmes for enhancing crop resilience.

Glucosinolate and isothiocyanate intakes are inversely associated with breast cancer risk: a case-control study in China.

Although previous studies have investigated the association of cruciferous vegetable consumption with breast cancer risk, few studies focused on the association between bioactive components in cruciferous vegetables, glucosinolates (GSL) and isothiocyanates (ITC), and breast cancer risk. This study aimed to examine the association between consumption of cruciferous vegetables and breast cancer risk according to GSL and ITC contents in a Chinese population. A total of 1485 cases and 1506 controls were recruited into this case-control study from June 2007 to March 2017. Consumption of cruciferous vegetables was assessed using a validated FFQ. Dietary GSL and ITC were computed by using two food composition databases linking GSL and ITC contents in cruciferous vegetables with responses to the FFQ. The OR and 95 % CI were assessed by unconditional logistic regression after adjusting for the potential confounders. Significant inverse associations were found between consumption of cruciferous vegetables, GSL and ITC and breast cancer risk. The adjusted OR comparing the highest with the lowest quartile were 0·51 (95 % CI 0·41, 0·63) for cruciferous vegetables, 0·54 (95 % CI 0·44, 0·67) for GSL and 0·62 (95 % CI 0·50, 0·76) for ITC, respectively. These inverse associations were also observed in both premenopausal and postmenopausal women. Subgroup analysis by hormone receptor status found inverse associations between cruciferous vegetables, GSL and ITC and both hormone-receptor-positive or hormone-receptor-negative breast cancer. This study indicated that consumption of cruciferous vegetables, GSL and ITC was inversely associated with breast cancer risk among Chinese women.

Down-regulation of the glucan synthase-like 6 gene (HvGsl6) in barley leads to decreased callose accumulation and increased cell wall penetration by Blumeria graminis f. sp. hordei.

The recent characterization of the polysaccharide composition of papillae deposited at the barley cell wall during infection by the powdery mildew pathogen, Blumeria graminis f. sp. hordei (Bgh), has provided new targets for the generation of enhanced disease resistance. The role of callose in papilla-based penetration resistance of crop species is largely unknown because the genes involved in the observed callose accumulation have not been identified unequivocally. We have employed both comparative and functional genomics approaches to identify the functional orthologue of AtGsl5 in the barley genome. HvGsl6 (the barley glucan synthase-like 6 gene), which has the highest sequence identity to AtGsl5, is the only Bgh-induced gene among the HvGsls examined in this study. Through double-stranded RNA interference (dsRNAi)-mediated silencing of HvGsl6, we have shown that the down-regulation of HvGsl6 is associated with a lower accumulation of papillary and wound callose and a higher susceptibility to penetration of the papillae by Bgh, compared with control lines. The results indicate that the HvGsl6 gene is a functional orthologue of AtGsl5 and is involved in papillary callose accumulation in barley. The increased susceptibility of HvGsl6 dsRNAi transgenic lines to infection indicates that callose positively contributes to the barley fungal penetration resistance mechanism.

Glucose availability and glycolytic metabolism dictate glycosphingolipid levels.

Cancer therapeutics has seen an emergence and re-emergence of two metabolic fields in recent years, those of bioactive sphingolipids and glycolytic metabolism. Anaerobic glycolysis and its implications in cancer have been at the forefront of cancer research for over 90 years. More recently, the role of sphingolipids in cancer cell metabolism has gained recognition, notably ceramide's essential role in programmed cell death and the role of the glucosylceramide synthase (GCS) in chemotherapeutic resistance. Despite this knowledge, a direct link between these two fields has yet to be definitively drawn. Herein, we show that in a model of highly glycolytic cells, generation of the glycosphingolipid (GSL) glucosylceramide (GlcCer) by GCS was elevated in response to increased glucose availability, while glucose deprivation diminished GSL levels. This effect was likely substrate dependent, independent of both GCS levels and activity. Conversely, leukemia cells with elevated GSLs showed a significant change in GCS activity, but no change in glucose uptake or GCS expression. In a leukemia cell line with elevated GlcCer, treatment with inhibitors of glycolysis or the pentose phosphate pathway (PPP) significantly decreased GlcCer levels. When combined with pre-clinical inhibitor ABT-263, this effect was augmented and production of pro-apoptotic sphingolipid ceramide increased. Taken together, we have shown that there exists a definitive link between glucose metabolism and GSL production, laying the groundwork for connecting two distinct yet essential metabolic fields in cancer research. Furthermore, we have proposed a novel combination therapeutic option targeting two metabolic vulnerabilities for the treatment of leukemia.

Surfactins modulate the lateral organization of fluorescent membrane polar lipids: a new tool to study drug:membrane interaction and assessment of the role of cholesterol and drug acyl chain length.

The lipopeptide surfactin exhibits promising antimicrobial activities which are hampered by haemolytic toxicity. Rational design of new surfactin molecules, based on a better understanding of membrane:surfactin interaction, is thus crucial. We here performed bioimaging of lateral membrane lipid heterogeneity in adherent living human red blood cells (RBCs), as a new relevant bioassay, and explored its potential to better understand membrane:surfactin interactions. RBCs show (sub)micrometric membrane domains upon insertion of BODIPY analogs of glucosylceramide (GlcCer), sphingomyelin (SM) and phosphatidylcholine (PC). These domains exhibit increasing sensitivity to cholesterol depletion by methyl-ß-cyclodextrin. At concentrations well below critical micellar concentration, natural cyclic surfactin increased the formation of PC and SM, but not GlcCer, domains, suggesting preferential interaction with lipid assemblies with the highest vulnerability to methyl-ß-cyclodextrin. Surfactin not only reversed disappearance of SM domains upon cholesterol depletion but further increased PC domain abundance over control RBCs, indicating that surfactin can substitute cholesterol to promote micrometric domains. Surfactin sensitized excimer formation from PC and SM domains, suggesting increased lipid recruitment and/or diffusion within domains. Comparison of surfactin congeners differing by geometry, charge and acyl chain length indicated a strong dependence on acyl chain length. Thus, bioimaging of micrometric lipid domains is a visual powerful tool, revealing that intrinsic lipid domain organization, cholesterol abundance and drug acyl chain length are key parameters for membrane:surfactin interaction. Implications for surfactin preferential location in domains or at their boundaries are discussed and may be useful for rational design of better surfactin molecules.

New insights on glucosylated lipids: metabolism and functions.

Ceramide, cholesterol, and phosphatidic acid are major basic structures for cell membrane lipids. These lipids are modified with glucose to generate glucosylceramide (GlcCer), cholesterylglucoside (ChlGlc), and phosphatidylglucoside (PtdGlc), respectively. Glucosylation dramatically changes the functional properties of lipids. For instance, ceramide acts as a strong tumor suppressor that causes apoptosis and cell cycle arrest, while GlcCer has an opposite effect, downregulating ceramide activities. All glucosylated lipids are enriched in lipid rafts or microdomains and play fundamental roles in a variety of cellular processes. In this review, we discuss the biological functions and metabolism of these three glucosylated lipids.