Glycosphingolipid Biosynthesis Research Articles

The Link between Gaucher Disease and Parkinson's Disease Sheds Light on Old and Novel Disorders of Sphingolipid Metabolism.

Sphingolipid metabolism starts with the biosynthesis of ceramide, a bioactive lipid and the backbone for the biosynthesis of complex sphingolipids such as sphingomyelin and glycosphingolipids. These are degraded back to ceramide and then to sphingosine, which enters the ceramide–sphingosine-1-phosphate signaling pathway or is further degraded. Several enzymes with multiple catalytic properties and subcellular localizations are thus involved in such metabolism. Hereditary defects of lysosomal hydrolases have been known for several years to be the cause of lysosomal storage diseases such as gangliosidoses, Gaucher disease, Niemann–Pick disease, Krabbe disease, Fabry disease, and Farber disease. More recently, many other inborn errors of sphingolipid metabolism have been recognized, involving enzymes responsible for the biosynthesis of ceramide, sphingomyelin, and glycosphingolipids. Concurrently, epidemiologic and biochemical evidence has established a link between Gaucher disease and Parkinson’s disease, showing that glucocerebrosidase variants predispose individuals to α-synuclein accumulation and neurodegeneration even in the heterozygous status. This appears to be due not only to lysosomal overload of non-degraded glucosylceramide, but to the derangement of vesicle traffic and autophagy, including mitochondrial autophagy, triggered by both sphingolipid intermediates and misfolded proteins. In this review, old and novel disorders of sphingolipid metabolism, in particular those of ganglioside biosynthesis, are evaluated in light of recent investigations of the link between Gaucher disease and Parkinson’s disease, with the aim of better understanding their pathogenic mechanisms and addressing new potential therapeutic strategies.

Biochemical diversity of glycosphingolipid biosynthesis as a driver of Coccolithovirus competitive ecology.

Coccolithoviruses (EhVs) are large, double-stranded DNA-containing viruses that infect the single-celled, marine coccolithophore Emiliania huxleyi. Given the cosmopolitan nature and global importance of E. huxleyi as a bloom-forming, calcifying, photoautotroph, E. huxleyi-EhV interactions play a key role in oceanic carbon biogeochemistry. Virally-encoded glycosphingolipids (vGSLs) are virulence factors that are produced by the activity of virus-encoded serine palmitoyltransferase (SPT). Here, we characterize the dynamics, diversity and catalytic production of vGSLs in an array of EhV strains in relation to their SPT sequence composition and explore the hypothesis that they are a determinant of infectivity and host demise. vGSL production and diversity was positively correlated with increased virulence, virus replication rate and lytic infection dynamics in laboratory experiments, but they do not explain the success of less-virulent EhVs in natural EhV communities. The majority of EhV-derived SPT amplicon sequences associated with infected cells in the North Atlantic derived from slower infecting, less virulent EhVs. Our lab-, field- and mathematical model-based data and simulations support ecological scenarios whereby slow-infecting, less-virulent EhVs successfully compete in North Atlantic populations of E. huxleyi, through either the preferential removal of fast-infecting, virulent EhVs during active infection or by having access to a broader host range.

Genome-Wide Signatures of Selection Detection in Three South China Indigenous Pigs.

South China indigenous pigs are famous for their superior meat quality and crude feed tolerance. Saba and Baoshan pigs without saddleback were located in the high-altitude area of Yunnan Province, while Tunchang and Ding’an pigs with saddleback were located in the low-altitude area of Hainan Province. Although these pigs are different in appearance, the underlying genetic differences have not been investigated. In this study, based on the single-nucleotide polymorphism (SNP) genotypes of 124 samples, both the cross-population extended haplotype homozygosity (XP-EHH) and the fixation index (FST) statistic were used to identify potential signatures of selection in these pig breeds. We found nine potential signatures of selection detected simultaneously by two methods, annotated 22 genes in Hainan pigs, when Baoshan pigs were used as the reference group. In addition, eleven potential signatures of selection detected simultaneously by two methods, annotated 24 genes in Hainan pigs compared with Saba pigs. These candidate genes were most enriched in GO: 0048015~phosphatidylinositol-mediated signaling and ssc00604: Glycosphingolipid biosynthesis—ganglio series. These selection signatures were likely to overlap with quantitative trait loci associated with meat quality traits. Furthermore, one potential selection signature, which was associated with different coat color, was detected in Hainan pigs. These results contribute to a better understanding of the underlying genetic architecture of South China indigenous pigs.

Proteomic analysis of oxidized proteins in the brain and liver of the Nile tilapia (Oreochromis niloticus) exposed to a water-accommodated fraction of Maya crude oil

Crude oil (CO) is a super mixture of chemical compounds whose toxic effects are reported in fish species according to international guidelines. In the current study a proteomic analysis of oxidized proteins (ox) was performed on the brain and liver of Nile tilapia exposed to WAF obtained from relevant environmental loads (0.01, 0.1 and 1.0 g/L) of Maya CO. Results have shown that oxidation of specific proteins was a newly discovered organ-dependent process able to disrupt key functions in Nile tilapia. In control fish, enzymes involved on aerobic metabolism (liver aldehyde dehydrogenase and brain dihydrofolate reductase) and liver tryptophan--tRNA ligase were oxidized. In WAF-treated liver specimens, fructose-bisphosphate aldolase (FBA), β-galactosidase (β-GAL) and dipeptidyl peptidase 9 (DPP-9) were detected in oxidized form. oxDPP-9 could be favorable by reducing the risk associated with altered glucose metabolism, the opposite effects elicited by oxFBA and oxβ-GAL. oxTrypsin showed a clear adverse effect by reducing probably the hepatocyte capacity to achieve proteolysis of oxidized proteins as well as for performing the proper digestive function. Additionally, enzyme implicated in purine metabolism adenosine (deaminase) was oxidized. Cerebral enzymes of mitochondrial respiratory chain complex (COX IV, COX5B), of glycosphingolipid biosynthesis (β-N-acetylhexosaminidase), involved in catecholamines degradation (catechol O-methyltransferase), and microtubule cytoskeleton (stathmin) were oxidized in WAF-treated specimens. This response suggests, in the brain, an adverse scenario for the mitochondrial respiration process and for ATP provision as for ischemia/reoxygenation challenges. Proteomic analysis of oxidized proteins is a promising tool for monitoring environmental quality influenced by hydrocarbons dissolved in water.

The AGC Kinase YpkA Regulates Sphingolipids Biosynthesis and Physically Interacts With SakA MAP Kinase in Aspergillus fumigatus.

Sphingolipids (SL) are complex lipids and components of the plasma membrane which are involved in numerous cellular processes, as well as important for virulence of different fungal pathogens. In yeast, SL biosynthesis is regulated by the “AGC kinases” Ypk1 and Ypk2, which also seem to connect the SL biosynthesis with the cell wall integrity (CWI) and the High Osmolarity Glycerol (HOG) pathways. Here, we investigate the role of ypkAY PK1 in SL biosynthesis and its relationship with the CWI and the HOG pathways in the opportunistic human pathogen Aspergillus fumigatus. We found that ypkA is important for fungal viability, since the ΔypkA strain presented a drastically sick phenotype and complete absence of conidiation. We observed that under repressive condition, the conditional mutant niiA::ypkA exhibited vegetative growth defects, impaired germination and thermosensitivity. In addition, the ypkA loss of function caused a decrease in glycosphingolipid (GSL) levels, especially the metabolic intermediates belonging to the neutral GSL branch including dihydroceramide (DHC), ceramide (Cer), and glucosylceramide (GlcCer), but interestingly a small increase in ergosterol content. Genetic analyzes showed that ypkA genetically interacts with the MAP kinases of CWI and HOG pathways, mpkA and sakA, respectively, while only SakA physically interacts with YpkA. Our results suggest that YpkA is important for fungal survival through the regulation of GSL biosynthesis and cross talks with A. fumigatus MAP kinase pathways.

Comparison of the Gut Microbiota of Centenarians in Longevity Villages of South Korea with Those of Other Age Groups.

Several studies have attempted to identify factors associated with longevity and maintenance of health in centenarians. In this study, we analyzed and compared the gut microbiota of centenarians in longevity villages with the elderly and adults in the same region and urbanized towns. Fecal samples were collected from centenarians, elderly, and young adults in longevity villages, and the gut microbiota sequences of elderly and young adults in urbanized towns of Korea were obtained from public databases. The relative abundance of Firmicutes was found to be considerably higher in subjects from longevity villages than those from urbanized towns, whereas Bacteroidetes was lower. Age-related rearrangement of gut microbiota was observed in centenarians, such as reduced proportions of Faecalibacterium and Prevotella, and increased proportion of Escherichia, along with higher abundances of Akkermansia, Clostridium, Collinsella, and uncultured Christensenellaceae. Gut microbiota of centenarians in rehabilitation hospital were also different to those residing at home. These differences could be due to differences in diet patterns and living environments. In addition, phosphatidylinositol signaling system, glycosphingolipid biosynthesis, and various types of N-glycan biosynthesis were predicted to be higher in the gut microbiota of centenarians (corrected p < 0.05). These three metabolic pathways of gut microbiota can be associated with the immune status and healthy gut environment of centenarians. Although further studies are necessary to validate the function of microbiota between groups, this study provides valuable information on centenarians' gut microbiota.

Total loss of GM3 synthase activity by a normally processed enzyme in a novel variant and in all ST3GAL5 variants reported to cause a distinct congenital disorder of glycosylation.

ST3GAL5-CDG is a rare syndrome which is caused by variant GM3 synthases, the enzyme involved in the biosynthesis of a-b-c-series gangliosides. Here we report a novel homozygous ST3GAL5 variant, p.Gly342Ser, in a patient suffering from failure to thrive, severe hearing, visual, motor, and cognitive impairment, and respiratory chain dysfunction. A GM3 synthase assay towards the natural acceptor substrate lactosylceramide was performed upon transfection in HEK-293T cells of expression plasmids carrying wild type and mutated ST3GAL5 cDNAs. The assay revealed a complete loss of enzyme activity. Identical results were obtained with the other four ST3GAL5 variants which have been reported to be pathogenic. HEK-293T clones permanently expressing HaloTag-ST3GAL5 carrying each of the five variants were assessed by quantitative PCR, flow cytometry, western blotting and confocal microscopy. The results indicated that transcription, translation, stability and intracellular localization of the tagged protein were identical to those of the wild type construct. Compared with the very mild phenotype of st3gal5 KO mouse models, the results suggest that unknown mechanisms, in addition to the lack of a-b-c-series gangliosides, contribute to the syndrome. Direct enzyme assay upon transfection in model cells appears to be an effective tool for characterizing variants of glycosyltransferases involved in glycosphingolipid biosynthesis.

The allodynia induced by intrathecal injection of sulfatides.

Glycosphingolipids (GSLs) play many important roles in cellular interaction, proliferation, vesicular transport and intracellular signal transduction. Our previous study revealed that the gene expressions of six glycosyltransferases in the GSLs biosynthesis pathway were altered in the spinal cord and dorsal root ganglion one day or 15 days after inflammation caused by complete Freund's adjuvant (CFA). We focused on CFA-induced upregulation of gal3st1 glycosyltransferase that catalyzes the synthesis of sulfatides, the sulfated GSLs in myelin structures, because the roles of sulfatides in nociceptive behavior are unclear. In this study, we intrathecally injected sulfatides into naïve mice and measured mechanical thresholds using von Frey test. Indeed, sulfatides induced mechanical allodynia within 40 min.

Metabolomic study of natrin-induced apoptosis in SMMC-7721 hepatocellular carcinoma cells by ultra-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry

Natrin, a new member of the cysteine-rich secretory protein (CRISP) family purified from the snake venom of Naja naja atra, has been demonstrated to have anticancer activity. However, the underlying molecular mechanisms need further elucidation. In this study, MTT was used to evaluate cell viability. Apoptotic cells were analyzed by employing a transmission electron microscope (TEM). Metabolomic study of the metabolic perturbations caused by natrin-induced apoptosis in differentiated SMMC-7721 cells was performed for the first time by using integrative ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF MS). To investigate the possible mechanism in the mitochondrial pathway of natrin-induced apoptosis, we measured apoptosis-related mRNA changes using real-time fluorescent quantitative PCR (FQ-PCR). Cell proliferation was significantly inhibited after treatment with natrin in a dose-dependent manner. Principal component analysis (PCA) and partial least squares-discriminate analysis (PLS-DA) clearly demonstrated that metabolic profiles were affected by natrin. The results of multivariate statistical analysis showed that a total of 13 metabolites were characterized as potential biomarkers highly implicated in natrin-induced apoptosis, which corresponded to fluctuations of five pathways, including sphingolipid metabolism, fatty acid biosynthesis, fatty acid metabolism, glycerophospholipid metabolism and glycosphingolipid biosynthesis. Furthermore, natrin-induced apoptosis showed an increase in the Bax/Bcl-2 ratio in the mitochondrial pathway compared with controls. This study illustrated that rapid and holistic cell metabolomics combining molecular biological approaches might be a powerful tool for evaluating the underlying mechanisms of natrin-induced apoptosis, which would help to deepen specific insights into the anti-hepatoma mechanisms of natrin and facilitate the clinical application of natrin in the future.

Comprehensive analysis of coexpressed long noncoding RNAs and genes in breast cancer.

To identify the differentially expressed long noncoding RNAs (lncRNAs) in breast cancer and analyze the potential roles. RNA-sequencing data (ID: GSE52194) of breast cancer and normal breast samples were downloaded from Gene Expression Omnibus. Clean reads were aligned against the human genome hg19 using TopHat2 and assembled into transcripts using cufflinks. Protein-coding potential of transcripts with length 200 bp or more were assessed using Coding Potential Calculator, followed by alignment against human lncRNAs using Blastn. Differentially expressed lncRNAs and genes were identified using NOISeq, followed by coexpression analysis with the criterion of Pearson correlation coefficient greater than 0.99. Enrichment analysis was performed using Database for Annotation, Visualization and Integrated Discovery. Totally 181 differentially expressed lncRNAs and 3967 differentially expressed genes (DEG) were obtained. Besides, coexpression analysis revealed ST6GALNAC3, ST6GALNAC4, ST8SIA5 and DEDD were coexpressed with FAM9B, LINC00320, LOC100169752 and RFX5. H2AFY2 and NOL11 were coexpressed with LOC286002. ADORA2B was coexpressed with GRHPR, LOC100652909 and TEX26-AS1. CD226, FCER1A and ADORA3 were coexpressed with LINC00323 and LOC100505540. PVRL2 and CD300LB were coexpressed with C1QTNF1, CENPA, FAM22F and SNX2. The DEG were predominantly enriched in biological processes of regulation of transcription from RNA polymerase I (e.g. H2AFY2, NOL11 and DEDD) and regulation of mast cell activation (e.g. ADORA3, CD226, FCER1A, MS4A2, ADORA2B and PVRL2), as well as pathways of glycosphingolipid biosynthesis (e.g. ST6GALNAC3, ST6GALNAC4 and ST8SIA5) and peroxisome. This study revealed the potential involvement of lncRNAs in the progression of breast cancer via participating in glycosphingolipid biosynthesis pathway and regulation of transcription and mast cell activation biological processes.

Chromosomal-level assembly of yellow catfish genome using third-generation DNA sequencing and Hi-C analysis.

BackgroundThe yellow catfish, Pelteobagrus fulvidraco, belonging to the Siluriformes order, is an economically important freshwater aquaculture fish species in Asia, especially in Southern China. The aquaculture industry has recently been facing tremendous challenges in germplasm degeneration and poor disease resistance. As the yellow catfish exhibits notable sex dimorphism in growth, with adult males about two- to three-fold bigger than females, the way in which the aquaculture industry takes advantage of such sex dimorphism is another challenge. To address these issues, a high-quality reference genome of the yellow catfish would be a very useful resource.FindingsTo construct a high-quality reference genome for the yellow catfish, we generated 51.2 Gb short reads and 38.9 Gb long reads using Illumina and Pacific Biosciences (PacBio) sequencing platforms, respectively. The sequencing data were assembled into a 732.8 Mb genome assembly with a contig N50 length of 1.1 Mb. Additionally, we applied Hi-C technology to identify contacts among contigs, which were then used to assemble contigs into scaffolds, resulting in a genome assembly with 26 chromosomes and a scaffold N50 length of 25.8 Mb. Using 24,552 protein-coding genes annotated in the yellow catfish genome, the phylogenetic relationships of the yellow catfish with other teleosts showed that yellow catfish separated from the common ancestor of channel catfish ∼81.9 million years ago. We identified 1,717 gene families to be expanded in the yellow catfish, and those gene families are mainly enriched in the immune system, signal transduction, glycosphingolipid biosynthesis, and fatty acid biosynthesis.ConclusionsTaking advantage of Illumina, PacBio, and Hi-C technologies, we constructed the first high-quality chromosome-level genome assembly for the yellow catfish P. fulvidraco. The genomic resources generated in this work not only offer a valuable reference genome for functional genomics studies of yellow catfish to decipher the economic traits and sex determination but also provide important chromosome information for genome comparisons in the wider evolutionary research community.

Follicular dynamics of glycerophospholipid and sphingolipid metabolisms in polycystic ovary syndrome patients

Polycystic ovary syndrome (PCOS) is a common heterogeneous disease, affecting up to 5–10% women at reproductive age. Although PCOS patients could produce morphologically normal metaphase II oocytes undergoing assisted reproductive techniques (ART), oocyte developmental competence and embryo development have been impaired in following in-vitro fertilization (IVF) steps. Follicular fluid (FF) provides a variety of information in oocyte environment when oocytes grow. In the present work, based on ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS), the metabolic signatures of PCOS FF have been compared with healthy women using untargeted metabolomics approach. Significant abundance differences of a series of glycerolipid, glycerophospholipids, sphingolipids, and carboxylic acids have been discovered. Among them, reduced levels of phosphatidylglycerolphosphate (PGP) and a triglyceride (TG) were highly related to the lower fertilization rate in PCOS; increased abundance of lysoPE and decreased amount of PC were significantly correlated with LH/FSH (ratio of luteinizing hormone to follicle stimulating hormone). Some metabolites, including decreased sphingolipids, glycerophospholipids, and fluctuated fatty acyls, also performed close relationship with other ART and clinical results. We concluded that dysfunctions in the metabolism of glycerolipid, glycerophospholipid, sphingolipid, and glycosphingolipid biosynthesis in PCOS patients’ follicles play a non-ignorable role in declining the 2 pronuclei (PN) fertilization rate during IVF procedure.

‘Concord’ grapevine nutritional status and chlorosis rank associated with fungal and bacterial root zone microbiomes

Leaf chlorosis in vineyards is associated with reduced crop yields and quality. While iron (Fe) is understood to play a crucial role in chlorosis, total plant and soil Fe are not always indicative of chlorosis in grapevines. Physiology of chlorosis in vineyards has been well-studied, but the soil microbial consequences of and contributions to chlorosis have received little attention. We used next-generation sequencing (NGS) to examine the bacterial and fungal communities associated with grapevines demonstrating varying degrees of visual chlorosis symptoms. Additionally, chemical analyses of soils and grape leaves were used to explore the influence of plant nutritional status and soil chemistry on microbial community composition. Finally, factors influencing bacterial community composition were correlated with predicted bacterial community function. Leaf tissue magnesium (leaf Mg) concentrations and chlorosis rank were correlated with bacterial community composition as determined via dbRDA (distance-based Redundancy Analysis) using Bray-Curtis dissimilarities. Non-metric multidimensional scaling (NMDS) revealed a significant correlation between fungal community composition and soil Fe and pH, along with leaf N, Mg, and Ca (mg.kg−1). Chlorosis rank was moderately correlated with KEGG Orthology (KO) terms associated with nitrogen (N) and carbon (C) metabolism in soils, while leaf Mg was associated with a spectrum of KO terms including glycosphingolipid biosynthesis, glycan degradation, transporters, and porphyrin and chlorophyll metabolism. Additionally, abundance of many bacterial operational taxonomic units was significantly correlated with leaf Mg, including those from the following orders: Rhodobacterales, Acidobacteriales, Opitutales, Sphingomonadales, Burkholderiales, Saprospirales, and Flavobacteriales. Our findings suggest grapevine chlorosis is interrelated with soil microbial community structure and function, plant nutrition, and soil chemistry.

Lipidomic Profiling Links the Fanconi Anemia Pathway to Glycosphingolipid Metabolism in Head and Neck Cancer Cells.

Purpose: Mutations in Fanconi anemia (FA) genes are common in sporadic squamous cell carcinoma of the head and neck (HNSCC), and we have previously demonstrated that FA pathway depletion in HNSCC cell lines stimulates invasion. The goal of our studies was to use a systems approach in order to define FA pathway-dependent lipid metabolism and to extract lipid-based signatures and effectors of invasion in FA-deficient cells.Experimental Design: We subjected FA-isogenic HNSCC keratinocyte cell lines to untargeted and targeted lipidomics analyses to discover novel biomarkers and candidate therapeutic targets in FA-deficient cells. Cellular invasion assays were carried out in the presence and absence of N-butyldeoxynojirimycin (NB-DNJ), a biosynthetic inhibitor of the newly identified class of gangliosides, to investigate the requirement of ganglioside upregulation in FA-deficient HNSCC cells.Results: The most notable element of the lipid profiling results was a consistent elevation of glycosphingolipids, and particularly the accumulation of gangliosides. Conversely, repression of this same class of lipids was observed upon genetic correction of FA patient-derived HNSCC cells. Functional studies demonstrate that ganglioside upregulation is required for HNSCC cell invasion driven by FA pathway loss. The motility of nontransformed keratinocytes in response to FA loss displayed a similar dependence, thus supporting early and late roles for the FA pathway in controlling keratinocyte invasion through lipid regulation.Conclusions: Elevation of glycosphingolipids including the ganglioside GM3 in response to FA loss stimulates invasive characteristics of immortalized and transformed keratinocytes. An inhibitor of glycosphingolipid biosynthesis NB-DNJ attenuates invasive characteristics of FA-deficient HNSCC cells. Clin Cancer Res; 24(11); 2700-9. ©2018 AACR.

Effect of tamoxifen on the sphingolipid biosynthetic pathway in the different intraerythrocytic stages of the apicomplexa Plasmodium falciparum

Parasites of the genus Plasmodium responsible for Malaria are obligate intracellular pathogens residing in mammalian red blood cells, hepatocytes, or mosquito midgut epithelial cells. Regarding that detailed knowledge on the sphingolipid biosynthetic pathway of the apicomplexan protozoan parasites is scarce, different stages of Plasmodium falciparum were treated with tamoxifen in order to evaluate the effects of this drug on the glycosphingolipid biosynthesis. Thin layer chromatography, High performance reverse phase chromatography and UV-MALDI-TOF mass spectrometry were the tools used for the analysis. In the ring forms, the increase of NBD-phosphatidyl inositol biosynthesis was notorious but differences at NBD-GlcCer levels were undetectable. In trophozoite forms, an abrupt decrease of NBD-acylated GlcDHCer and NBD-GlcDHCer in addition to an increase of NBD-PC biosynthesis was observed. On the contrary, in schizonts, tamoxifen seems not to be producing substantial changes in lipid biosynthesis. Our findings indicate that in this parasite, tamoxifen is exerting an inhibitory action on Glucosylceramidesynthase and sphingomyelin synthase levels. Moreover, regarding that Plasmodium does not biosynthesize inositolphosphoceramides, the accumulation of phosphatidylinositol should indicate an inhibitory action on glycosylinositol phospholipid synthesis.

The bilateral change of the spinal cord and dorsal root ganglion glycosyltransferase expression involved in glycosphingolipids biosynthesis during inflammatory pain

The bilateral change of the spinal cord and dorsal root ganglion glycosyltransferase expression involved in glycosphingolipids biosynthesis during inflammatory pain

Ganglioside Metabolism in Health and Disease.

Gangliosides (GGs) are cell type-specific sialic acid-containing glycosphingolipids (GSLs), which are enriched in mammalian brain. Defects in GSL metabolism cause fatal human diseases. GSLs are composed of a hydrophilic oligosaccharide linked in 1-O-position to a hydrophobic ceramide anchor, which itself is composed of a long-chain amino alcohol, the sphingoid base, and an amide-bound acyl chain. Biosynthesis of mammalian GGs and other GSLs starts with the formation of their hydrophobic ceramide anchor in the endoplasmic reticulum, followed by sequential glycosylation reactions along the secretory pathway, mainly at the luminal surface of Golgi and trans-Golgi network (TGN) membranes. Few membrane-anchored and often promiscuous glycosyltransferases allow the formation of cell type-specific glycolipid patterns in a combinatorial process. Inherited defects of these transferases therefore affect not only single structures but defined glycolipid series. GGs and other GSLs are thereafter transported by an exocytotic membrane flow to the plasma membrane where they are expressed in cell type-specific patterns, which can be modified by metabolic reactions at or near the cellular surface. Endocytosed (glyco)sphingolipids are degraded, together with other membrane lipids in a stepwise fashion by endolysosomal enzymes with the help of small lipid-binding proteins, the sphingolipid activator proteins (SAPs), at the surface of intraluminal lysosomal vesicles. Inherited defects in a sphingolipid-degrading enzyme or SAP cause the accumulation of the corresponding lipid substrates. Endolysosomal GSL degradation is strongly modified by the lipid components of the organelle microenvironments.

Pharmacological Modulation of Glycosphingolipid Metabolism.

The experimental approach to deplete cellular glycosphingolipids (GSLs) with the specific inhibitors of glycosphingolipid biosynthesis has the potential to identify functions of endogenous GSLs. Most GSLs are derived from glucosylceramide (GlcCer). D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) inhibits GIcCer synthase and has been used extensively to study the biological functions of living cells. D-PDMP inhibits mTORC1 activity, which is independent of its inhibitory activity on GlcCer synthase. We also developed an analog of D-PDMP, D-threo-1-phenyl-2-benzyloxycarbonylamino-3-pyrrolidino-1-propanol (D-PBPP) lacking the effect on mTORC1. Here, we summarize the effects of D-PDMP and D-PBPP on the metabolism of GSLs and cell growth.

RNA-Seq reveals seven promising candidate genes affecting the proportion of thick egg albumen in layer-type chickens

Eggs with a much higher proportion of thick albumen are preferred in the layer industry, as they are favoured by consumers. However, the genetic factors affecting the thick egg albumen trait have not been elucidated. Using RNA sequencing, we explored the magnum transcriptome in 9 Rhode Island white layers: four layers with phenotypes of extremely high ratios of thick to thin albumen (high thick albumen, HTA) and five with extremely low ratios (low thick albumen, LTA). A total of 220 genes were differentially expressed, among which 150 genes were up-regulated and 70 were down-regulated in the HTA group compared with the LTA group. Gene Ontology (GO) analysis revealed that the up-regulated genes in HTA were mainly involved in a wide range of regulatory functions. In addition, a large number of these genes were related to glycosphingolipid biosynthesis, focal adhesion, ECM-receptor interactions and cytokine-cytokine receptor interactions. Based on functional analysis, ST3GAL4, FUT4, ITGA2, SDC3, PRLR, CDH4 and GALNT9 were identified as promising candidate genes for thick albumen synthesis and metabolism during egg formation. These results provide new insights into the molecular mechanisms of egg albumen traits and may contribute to future breeding strategies that optimise the proportion of thick egg albumen.

Transcriptome Analysis Reveals the Mechanism of Fungicidal of Thymol Against Fusarium oxysporum f. sp. niveum.

Fusarium oxysporum: f. sp. niveum (Fusarium oxysporum) causes watermelon wilt, drastically reducing its yield. Thymol (5-methyl-2-(1-methylethyl) phenol, PubChem CID: 6989) has been extensively reported to have antimicrobial activity against pathogenic microorganisms. In this study, the growth of F. oxysporum was significantly inhibited by thymol in vitro. The dry weight of F. oxysporum was decreased significantly. Thymol-induced cell membrane damage was observed at 24 hpi (hours post-incubation). Therefore, the changes in the gene expression level of F. oxysporum after treatment with 80μg/mL (average EC50 value) of thymol were analyzed using RNA-Seq to reveal the underlying fungicidal mechanisms of thymol. Among the 5057 differentially expressed genes (DEGs), 2440 and 2617 were up-regulated and down-regulated, respectively. Bioinformatics analysis demonstrated that the expression levels of most genes, including glycosphingolipid biosynthesis and sphingolipid metabolism, were down-regulated. However, the genes involved in antioxidant activity, chitin biosynthesis, and cell wall modification were up-regulated. These results were verified by quantitative reverse transcription PCR (qRT-PCR). According to these results, thymol produces reactive oxygen species (ROS) accumulation and destroys the integrity of the cell wall and cell membrane by inhibiting the genes involved in cell wall and cell membrane synthesis.