Glycosphingolipid Biosynthesis Research Articles

Multi-biobank Mendelian randomization analyses identify opposing pathways in plasma low-density lipoprotein-cholesterol lowering and gallstone disease

Plasma low-density lipoprotein (LDL)-cholesterol is positively associated with coronary artery disease risk while biliary cholesterol promotes gallstone formation. Different plasma LDL-cholesterol lowering pathways may have distinct effects on biliary cholesterol and thereby gallstone disease risk. We conducted a Mendelian randomization (MR) study using data from the UK Biobank (30,547 gallstone disease cases/336,742 controls), FinnGen (34,461 cases/301,383 controls) and Biobank Japan (9,305 cases/168,253 controls). We first performed drug-target MR analyses substantiated by colocalization to investigate the effects of plasma LDL-cholesterol lowering therapies on gallstone disease risk. We then performed clustered MR analyses and pathway analyses to identify distinct mechanisms underlying the association of plasma LDL-cholesterol with gallstone disease risk. For a 1-standard deviation reduction in plasma LDL-cholesterol, genetic mimics of statins were associated with lower gallstone disease risk (odds ratio 0.72 [95% confidence interval 0.62, 0.83]), but genetic mimics of PCSK9 inhibitors and targeting apolipoprotein B were associated with higher risk (1.11 [1.03, 1.19] and 1.23 [1.13, 1.35]). The association for statins was supported by colocalization (posterior probability 98.7%). Clustered MR analyses identified variant clusters showing opposing associations of plasma LDL-cholesterol with gallstone disease risk, with some evidence for ancestry-and sex-specific associations. Among variants lowering plasma LDL-cholesterol, those associated with lower gallstone disease risk were mapped to glycosphingolipid biosynthesis pathway, while those associated with higher risk were mapped to pathways relating to plasma lipoprotein assembly, remodelling, and clearance and ATP-binding cassette transporters. This MR study provides genetic evidence that different plasma LDL-cholesterol lowering pathways have opposing effects on gallstone disease risk.

Age-related and species-specific methylation changes in the protein-coding marmoset sperm epigenome.

The sperm epigenome is thought to affect the developmental programming of the resulting embryo, influencing health and disease in later life. Age-related methylation changes in the sperm of old fathers may mediate the increased risks for reproductive and offspring medical problems. The impact of paternal age on sperm methylation has been extensively studied in humans and, to a lesser extent, in rodents and cattle. Here, we performed a comparative analysis of paternal age effects on protein-coding genes in the human and marmoset sperm methylomes. The marmoset has gained growing importance as a non-human primate model of aging and age-related diseases. Using reduced representation bisulfite sequencing, we identified age-related differentially methylated transcription start site (ageTSS) regions in 204 marmoset and 27 human genes. The direction of methylation changes was the opposite, increasing with age in marmosets and decreasing in humans. None of the identified ageTSS was differentially methylated in both species. Although the average methylation levels of all TSS regions were highly correlated between marmosets and humans, with the majority of TSS being hypomethylated in sperm, more than 300 protein-coding genes were endowed with species-specifically (hypo)methylated TSS. Several genes of the glycosphingolipid (GSL) biosynthesis pathway, which plays a role in embryonic stem cell differentiation and regulation of development, were hypomethylated (<5%) in human and fully methylated (>95%) in marmoset sperm. The expression levels and patterns of defined sets of GSL genes differed considerably between human and marmoset pre-implantation embryo stages and blastocyst tissues, respectively.

Transcriptomic analyses provide new insights into immune response of the Procambarus clarkii intestines to Aeromonas hydrophila challenge

In recent years, the death rate of Procambarus clarkii increased sharply due to pathogenic bacterial infections, leading to huge economic losses. To determine the immune response of the intestinal tissues of P. clarkii to bacterial infections, we compared the immune indexes, histopathological damage, and transcriptome data of the control and Aeromonas hydrophila-infected P. clarkii. With the injection of A. hydrophila, it was found that intestinal mucosal folds increased, intestinal villi showed obvious vacuolation, and the tissue structure changed significantly. The results of non-specific immunoenzyme activity showed that the Total protein (TP) content was significantly lower than that of the control group at 24 h, and the Acid phosphatase (ACP) and Alkaline phosphatase (AKP) activities in the treatment group showed a significant increasing trend (p < 0.05). In terms of the transcriptome, an average of 22,927,523 clean reads and 27,866 genes were found in the control and treatment groups through gene assembly and annotation. Among these genes, 567 were identified as differentially expressed genes (DEGs), of which 501 were up-regulated and 66 were down-regulated. Quantitative-polymerase chain reaction (qPCR) analysis of six randomly selected genes was used to verify the RNA-sequencing results. The Gene Ontology enrichment analysis revealed that the DEGs were primarily involved in cell processes, metabolic processes, and biological regulation, while the Kyoto Encyclopedia of Genes and Genomes enrichment analysis showed that these DEGs were involved in tyrosine metabolism, glycosphingolipid biosynthesis—ganglionic series, and nitrogen metabolism. We studied the tyrosine metabolic pathway and found that it contributes to the immune defense mechanism of P. clarkii. The results of our study provide important insights into the intestinal immune response of P. clarkii to A. hydrophila infection.

Transcriptional analysis of the molecular response of Arabidopsis to manganese stress and recovery

Manganese (Mn) is an essential element for plants and plays a role in various metabolic processes. However, excess manganese can be toxic to plants. This study aimed to analyze the changes in various physiological activities and the transcriptome of Arabidopsis under different treatments: 1 mmol/L MnCl2 treatment for 1 day or 3 days, and 1 day of recovery on MS medium after 3 days of MnCl2 treatment. During the recovery phase, minor yellowing symptoms appeared on the leaves of Arabidopsis, and the content of chlorophyll and carotenoid decreased significantly, but the content of malondialdehyde and soluble sugar increased rapidly. Transcriptome sequencing data shows that the expression patterns of differentially expressed genes exhibit three major models: initial response model, later response model, recovery response model. Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis identified several affected metabolic pathways, including plant hormone signal transduction mitosolysis activates protein kinase (MAPK) phytohormone signaling, phenylpropanoid biosynthesis, ATP binding cassette transporters (ABC transporter), and glycosphingolipid biosynthesis. Differential expressed genes (DEGs) involved in phenylpropanoid biosynthesis, ABC transporter, and glycosphingolipid biosynthesis, were identified. Sixteen randomly selected DEGs were validated through qRT-PCR and showed consistent results with RNA-seq data. Our findings suggest that the phenylpropanoid metabolic pathway is activated to scavenge reactive oxygen species, the regulation of ABC transporter improves Mn transport, and the adjustment of cell membrane lipid composition occurs through glycerophospholipid metabolism to adapt to Mn stress in plants. This study provides new insights into the molecular response of plants to Mn stress and recovery, as well as theoretical cues for cultivating Mn-resistant plant varieties.

Dysregulated lipid metabolism networks modulate T-cell function in people with relapsing-remitting multiple sclerosis.

Altered cholesterol, oxysterol, sphingolipid, and fatty acid concentrations are reported in blood, cerebrospinal fluid, and brain tissue of people with relapsing-remitting multiple sclerosis (RRMS) and are linked to disease progression and treatment responses. CD4 + T cells are pathogenic in RRMS, and defective T-cell function could be mediated in part by liver X receptors (LXRs)-nuclear receptors that regulate lipid homeostasis and immunity. RNA-sequencing and pathway analysis identified that genes within the 'lipid metabolism' and 'signalling of nuclear receptors' pathways were dysregulated in CD4 + T cells isolated from RRMS patients compared with healthy donors. While LXRB and genes associated with cholesterol metabolism were upregulated, other T-cell LXR-target genes, including genes involved in cellular lipid uptake (inducible degrader of the LDL receptor, IDOL), and the rate-limiting enzyme for glycosphingolipid biosynthesis (UDP-glucosylceramide synthase, UGCG) were downregulated in T cells from patients with RRMS compared to healthy donors. Correspondingly, plasma membrane glycosphingolipids were reduced, and cholesterol levels increased in RRMS CD4 + T cells, an effect partially recapitulated in healthy T cells by in vitro culture with T-cell receptor stimulation in the presence of serum from RRMS patients. Notably, stimulation with LXR-agonist GW3965 normalized membrane cholesterol levels, and reduced proliferation and IL17 cytokine production in RRMS CD4 + T-cells. Thus, LXR-mediated lipid metabolism pathways were dysregulated in T cells from patients with RRMS and could contribute to RRMS pathogenesis. Therapies that modify lipid metabolism could help restore immune cell function.

Whole transcriptome mapping reveals the lncRNA regulatory network of TFP5 treatment in diabetic nephropathy.

TFP5 is a Cdk5 inhibitor peptide, which could restore insulin production. However, the role of TFP5 in diabetic nephropathy (DN) is still unclear. This study aims to characterize the transcriptome profiles of mRNA and lncRNA in TFP5-treated DN mice to mine key lncRNAs associated with TFP5 efficacy. We evaluated the role of TFP5 in DN pathology and performed RNA sequencing in C57BL/6Jcontrol mice, C57BL/6J db/db model mice, and TFP5 treatment C57BL/6J db/db model mice. The differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) were analyzed. WGCNA was used to screen hub-gene of TFP5 in treatment of DN. Our results showed that TFP5 therapy ameliorated renal tubular injury in DN mice. In addition, compared with the control group, the expression profile of lncRNAs in the model group was significantly disordered, while TFP5 alleviated the abnormal expression of lncRNAs. A total of 67 DElncRNAs shared among the three groups, 39 DElncRNAs showed a trend of increasing in the DN group and decreasing after TFP treatment, while the remaining 28 showed the opposite trend. DElncRNAs were enriched in glycosphingolipid biosynthesis signaling pathways, NF-κB signaling pathways, and complement activation signaling pathways. There were 1028 up-regulated and 1117 down-regulated DEmRNAs inthe model group compared to control group, and 123 up-regulated and 153 down-regulated DEmRNAs in theTFP5 group compared to themodel group. The DEmRNAs were involved in PPAR and MAPK signaling pathway. We confirmed that MSTRG.28304.1 is a key DElncRNA for TFP5 treatment of DN. TFP5 ameliorated DN maybe by inhibiting MSTRG.28304.1 through regulating the insulin resistance and PPAR signaling pathway. The qRT-PCR results confirmed the reliability of the sequencing data through verifying the expression of ENSMUST00000211209, MSTRG.31814.5, MSTRG.28304.1, and MSTRG.45642.14. Overall, the present study provides novel insights into molecular mechanisms of TFP5 treatment in DN.

Potential targets for the treatment of MI: GRP75-mediated Ca2+ transfer in MAM

After myocardial infarction (MI), there is a notable disruption in cellular calcium ion homeostasis and mitochondrial function, which is believed to be intricately linked to endoplasmic reticulum (ER) stress. This research endeavors to elucidate the involvement of glucose regulated protein 75 (GRP75) in post-MI calcium ion homeostasis and mitochondrial function. In MI rats, symptoms of myocardial injury were accompanied by an increase in the activation of ER stress. Moreover, in oxygen-glucose deprivation (OGD)-induced cardiomyocytes, it was confirmed that inhibiting ER stress exacerbated intracellular Ca2+ disruption and cell apoptosis. Concurrently, the co-localization of GRP75 with IP3R and VDAC1 increased under ER stress in cardiomyocytes. In OGD-induced cardiomyocytes, knockdown of GRP75 not only reduced the Ca2+ levels in both the ER and mitochondria and improved the ultrastructure of cardiomyocytes, but it also increased the number of contact points between the ER and mitochondria, reducing mitochondria associated endoplasmic reticulum membrane (MAM) formation, and decreased cell apoptosis. Significantly, knockdown of GRP75 did not affect the protein expression of PERK and hypoxia-inducible factor 1α (HIF-1α). Transcriptome analysis of cardiomyocytes revealed that knockdown of GRP75 mainly influenced the molecular functions of sialyltransferase and IP3R, as well as the biosynthesis of glycosphingolipids and lactate metabolism. The complex interaction between the ER and mitochondria, driven by the GRP75 and its associated IP3R1-GRP75-VDAC1 complex, is crucial for calcium homeostasis and cardiomyocyte's adaptive response to ER stress. Modulating GRP75 could offer a strategy to regulate calcium dynamics, diminish glycolysis, and thereby mitigate cardiomyocyte apoptosis.

Metatranscriptomic analysis shows functional alterations in subgingival biofilm in young smokers with periodontitis: a pilot study.

This study aimed to assess the influence of smoking on the subgingival metatranscriptomic profile of young patients affected by stage III/IV and generalized periodontal disease. In total, six young patients, both smokers and non-smokers (n=3/group), who were affected by periodontitis were chosen. The STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guidelines for case-control reporting were followed. Periodontal clinical measurements and subgingival biofilm samples were collected. RNA was extracted from the biofilm and sequenced via Illumina HiSeq. Differential expression analysis used Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and differentially expressed genes were identified using the Sleuth package in R, with a statistical cutoff of ≤0.05. This study found 3351 KEGGs in the subgingival biofilm of both groups. Smoking habits altered the functional behavior of subgingival biofilm, resulting in 304 differentially expressed KEGGs between groups. Moreover, seven pathways were modulated: glycan degradation, galactose metabolism, glycosaminoglycan degradation, oxidative phosphorylation, peptidoglycan biosynthesis, butanoate metabolism, and glycosphingolipid biosynthesis. Smoking also altered antibiotic resistance gene levels in subgingival biofilm by significantly overexpressing genes related to beta-lactamase, permeability, antibiotic efflux pumps, and antibiotic-resistant synthetases. Due to the limitations of a small sample size, our data suggest that smoking may influence the functional behavior of subgingival biofilm, modifying pathways that negatively impact the behavior of subgingival biofilm, which may lead to a more virulent community.

Integrative proteomic and metabonomic profiling elucidates amino acid and lipid metabolism disorder in CA-MRSA-infected breast abscesses.

Bacterial culture and drug sensitivity testing have been the gold standard for confirming community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) infection in breast abscess with a long history. However, these tests may delay treatment and increase the risk of nosocomial infections. To handle and improve this critical situation, this study aimed to explore biomarkers that could facilitate the rapid diagnosis of CA-MRSA infection. This study for the first time applied label-free quantitative proteomics and non-targeted metabonomics to identify potential differentially expressed proteins (DEPs) and differentially expressed metabolites (DEMs) in breast abscess infected with CA-MRSA compared to methicillin-susceptible S. aureus (MSSA). The two omics data were integrated and analyzed using bioinformatics, and the results were validated using Parallel Reaction Monitoring (PRM). Receiver operating characteristic (ROC) curves were generated to evaluate the predictive efficiency of the identified biomarkers for diagnosing CA-MRSA infection. After using the above-mentioned strategies, 109 DEPs were identified, out of which 86 were upregulated and 23 were downregulated. Additionally, a total of 61 and 26 DEMs were initially screened in the positive and negative ion modes, respectively. A conjoint analysis indicated that the amino acid metabolism, glycosphingolipid biosynthesis, and glycerophospholipid metabolism pathways were co-enriched by the upstream DEPs and downstream DEMs, which may be involved in structuring the related network of CA-MRSA infection. Furthermore, three significant DEMs, namely, indole-3-acetic acid, L-(-)-methionine, and D-sedoheptulose 7-phosphate, displayed good discriminative abilities in early identification of CA-MRSA infection in ROC analysis. As there is limited high-quality evidence and multiple omics research in this field, the explored candidate biomarkers and pathways may provide new insights into the early diagnosis and drug resistance mechanisms of CA-MRSA infection in Chinese women.

Abstract 14943: Trans-Biobank Mendelian Randomization Analyses Identify Distinct and Opposing Pathways in the Association of Lower Plasma Low-Density Lipoprotein-Cholesterol With Risk of Gallstone Disease

Background: Higher plasma LDL-cholesterol increases coronary artery disease risk, while biliary cholesterol promotes gallstone formation. Trial evidence suggests that lowering plasma LDL-cholesterol with bempedoic acid increases gallstone risk. Hypothesis: Different plasma LDL-cholesterol lowering pathways have distinct effects on biliary cholesterol and thereby gallstone disease risk. Methods: We conducted a Mendelian randomization (MR) study using data from the UK Biobank (30,547 gallstone disease cases/336,742 controls), FinnGen (34,461 cases/301,383 controls) and Biobank Japan (9,305 cases/168,253 controls). We firstly performed drug-target MR analyses substantiated by colocalization to investigate the effects of plasma LDL-cholesterol lowering therapies on gallstone risk. We then performed clustered MR analyses followed by pathway analyses to identify distinct pathways in the association of lower plasma LDL-cholesterol with gallstone risk. Results: Genetic mimics of statins were associated with lower gallstone risk, but genetic mimics of PCSK9 inhibitors, mipomersen and bile acid sequestrants were associated with higher gallstone risk. The association for statins was supported by colocalization (posterior probability 98.7%). Clustered MR analyses consistently identified clusters showing opposing associations of lower plasma LDL-cholesterol with gallstone risk. Among variants predicting lower plasma LDL-cholesterol, those associated with lower gallstone risk were mapped to glycosphingolipid biosynthesis pathway, while those associated with higher gallstone risk were mapped to pathways relating to ATP-binding cassette transporters and plasma lipoprotein assembly, remodelling, and clearance. Conclusions: Genetic evidence suggests that different plasma LDL-cholesterol lowering pathways may have opposing effects on gallstone disease risk. Specifically, statins may reduce gallstone disease risk.

Extracellular vesicle-coupled miRNA profiles of chicken seminal plasma and their potential interaction with recipient cells

The presence of EVs in seminal plasma (SPEVs) suggests their involvement on fertility via transmitting information between the original cells and recipient cells. SPEVs-coupled miRNAs have been shown to affect sperm motility, maturation, and capacitation in mammals, but rarely in poultry species. The present study aims to reveal the profile of SPEVs miRNAs and their potential effect on sperm storage and function in poultry. The SPEVs was successfully isolated from 4 different chicken breeds by ultracentrifugation and verified. Deep sequencing of SPEVs small RNA library of each breed identified 1077 miRNAs in total and 563 shared ones. The top 10 abundant miRNAs (such as miR-10-5p, miR-100-5p, and miR-10a-5p etc.) accounted for around 60% of total SPEVs miRNA reads and are highly conserved across species, predisposing their functional significance. Target genes prediction and functional enrichment analysis indicated that the most abundantly expressed miRNAs may regulate pathways like ubiquitin-mediated proteolysis, endocytosis, mitophagy, glycosphingolipid biosynthesis, fatty acid metabolism, and fatty acid elongation. The high abundant SPEVs-coupled miRNAs were found to target 107 and 64 functionally important mRNAs in the potential recipient cells, sperm and sperm storage tubules (SST) cells, respectively. The pathways that enriched by target mRNAs revealed that the SPEVs-coupled miRNA may rule the fertility by affecting the sperm maturation and regulating the female's immune response and lipid metabolism. In summary, this study presents the distinctive repertoire of SPEVs-coupled miRNAs, and extends our understanding about their potential roles in sperm maturation, capacitation, storage, and fertility, and may help to develop new therapeutic strategies for male infertility and sperm storage.

Neural-specific alterations in glycosphingolipid biosynthesis and cell signaling associated with two human ganglioside GM3 synthase deficiency variants.

GM3 Synthase Deficiency (GM3SD) is a neurodevelopmental disorder resulting from pathogenic variants in the ST3GAL5 gene, which encodes GM3 synthase, a glycosphingolipid (GSL)-specific sialyltransferase. This enzyme adds a sialic acid to the terminal galactose of lactosylceramide (LacCer) to produce the monosialylated ganglioside GM3. In turn, GM3 is extended by other glycosyltransferases to generate nearly all the complex gangliosides enriched in neural tissue. Pathogenic mechanisms underlying the neural phenotypes associated with GM3SD are unknown. To explore how loss of GM3 impacts neural-specific glycolipid glycosylation and cell signaling, GM3SD patient fibroblasts bearing one of two different ST3GAL5 variants were reprogrammed to induced pluripotent stem cells (iPSCs) and then differentiated to neural crest cells (NCCs). GM3 and GM3-derived gangliosides were undetectable in cells carrying either variant, while LacCer precursor levels were elevated compared to wildtype (WT). NCCs of both variants synthesized elevated levels of neutral lacto- and globo-series, as well as minor alternatively sialylated GSLs compared to WT. Ceramide profiles were also shifted in GM3SD variant cells. Altered GSL profiles in GM3SD cells were accompanied by dynamic changes in the cell surface proteome, protein O-GlcNAcylation, and receptor tyrosine kinase abundance. GM3SD cells also exhibited increased apoptosis and sensitivity to erlotinib-induced inhibition of epidermal growth factor receptor signaling. Pharmacologic inhibition of O-GlcNAcase rescued baseline and erlotinib-induced apoptosis. Collectively, these findings indicate aberrant cell signaling during differentiation of GM3SD iPSCs and also underscore the challenge of distinguishing between variant effect and genetic background effect on specific phenotypic consequences.

The Sphingolipids Metabolism Mechanism and Associated Molecular Biomarker Investigation in Keloid.

Sphingolipid metabolism plays important roles in maintaining cell growth and signal transduction. However, this pathway has not been investigated in keloid, a disease characterized by the excessive proliferation of fibroblasts. Based on the expression profiles of three datasets, the differentially expressed genes (DEGs) were explored between keloid fibroblasts and normal fibroblasts. Metabolism-related genes were obtained from a previous study. Then, enrichment analysis and protein-protein interaction (PPI) network analysis were performed for genes. Differences in metabolism-related pathways between keloid fibroblasts and normal fibroblasts were analyzed by the gene set variation analysis (GSVA). Quantitative PCR was used to confirm the expression of key genes in keloid fibroblast. A total of 42 up-regulated co-DEGs and 77 down-regulated co-DEGs were revealed based on three datasets, and were involved in extracellular matrix structural constituent, collagencontaining extracellular matrix and sphingolipid metabolism pathway. A total of 15 metabolism- DEGs were screened, including serine palmitoyltransferase long chain base subunit (SPTLC) 3, UDP-glucose ceramide glucosyltransferase (UGCG) and sphingomyelin synthase 2 (SGMS2). All these three genes were enriched in the sphingolipid pathway. GSVA showed that the biosynthesis of glycosphingolipids (GSLs) in keloid fibroblasts was lower than that in normal fibroblasts. Quantitative PCR suggested SPTLC3, UGCG and SGMS2 were regulated in keloid fibroblasts. Sphingolipids metabolism pathway might take part in the disease progression of keloid by regulating keloid fibroblasts. SPTLC3, UGCG and SGMS2 might be key targets to investigate the underlying mechanism.

Identification and Classification of Long Non-Coding RNAs in the Mammary Gland of the Holstein Cow.

The mammary glands, responsible for milk secretion, are regulated at a local level by various hormones, growth factors, non-coding RNAs, and other elements. Recent research has discovered the presence of lncRNAs in these glands, with suggestions that they may be essential for the maintenance and function of mammary glands. Besides directly controlling the gene and protein expression, lncRNAs are believed to play a significant part in numerous physiological and pathological processes. This study focused on examining the mammary gland tissues of Chinese Holstein cows, to identify and categorize long non-coding RNAs (lncRNAs). The research intended to distinguish lncRNAs in the mammary tissues of Holstein cows and contrast them between lactation and non-lactation periods. In this study, mammary gland tissues were sampled from three Holstein cows in early lactation (n = 3, 30 days postpartum) and non-lactation (n = 3, 315 days postpartum) on a large dairy farm in Jiangsu province. Mammary tissue samples were collected during early lactation and again during non-lactation. In total, we detected 1905 lncRNAs, with 57.3% being 500 bp and 612 intronic lncRNAs. The exon count for lncRNAs varied from 2 to 10. It was observed that 96 lncRNA expressions markedly differed between the two stages, with 83 genes being upregulated and 53 downregulated. Enrichment analysis results revealed that Gene Ontology (GO) analysis was primarily abundant in cellular processes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that target genes were predominantly abundant in metabolic pathways, fatty acid biosynthesis, the immune system, and glycosphingolipid biosynthesis. This study analyzed the expression profile and characteristics of lncRNAs in the mammary gland tissues of Holstein cows during both lactation and non-lactation stages, forming a foundation for further investigation into the functional roles of lncRNAs in Holstein cows throughout lactation.

Genetic Characterization of the Acidic and Neutral Glycosphingolipid Biosynthetic Pathways in Neurospora crassa.

Fungal glycosphingolipids (GSLs) are important membrane components which play a key role in vesicle trafficking. To assess the importance of GSLs in the fungal life cycle, we performed a mutant phenotypic study of the acidic and neutral GSL biosynthetic pathways in Neurospora crassa. GSL biosynthesis begins with two reactions leading up to the formation of dihydrosphingosine. The first of these reactions is catalyzed by serine palmitoyltransferase and generates 3-keto dihydrosphinganine. In N. crassa, this reaction is catalyzed by GSL-1 and GSL-2 and is required for viability. The second reaction is carried out by GSL-3, a 3-keto dihydrosphinoganine reductase to generate dihydrosphingosine, which is used for the synthesis of neutral and acidic GSLs. We found that deletion mutations in the acidic GSL pathway leading up to the formation of mannosylinositol-phosphoceramide are lethal, indicating that acidic GSLs are essential for viability in N. crassa. Once mannosylinositol-phosphoceramide is made, it is further modified by GSL-5, an inositol-phosphoceramide-B C26 hydroxylase, which adds a hydroxyl group to the amide-linked fatty acid. GSL-5 is not required for viability but gives a clear mutant phenotype affecting all stages of the life cycle. Our results show that the synthesis of mannosylinositol-phosphoceramide is required for viability and that the modification of the amide-linked fatty acid is important for acidic GSL functionality. We also examined the neutral GSL biosynthetic pathway and identified the presence of glucosylceramide. The deletion of neutral GSL biosynthetic genes affected hyphal morphology, vegetative growth rate, conidiation, and female development. Our results indicate that the synthesis of neutral GSLs is essential for normal growth and development of N. crassa.

Gaining New Insights into Fundamental Biological Pathways by Bacterial Toxin-Based Genetic Screens.

Genetic screen technology has been applied to study the mechanism of action of bacterial toxins-a special class of virulence factors that contribute to the pathogenesis caused by bacterial infections. These screens aim to identify host factors that directly or indirectly facilitate toxin intoxication. Additionally, specific properties of certain toxins, such as membrane interaction, retrograde trafficking, and carbohydrate binding, provide robust probes to comprehensively investigate the lipid biosynthesis, membrane vesicle transport, and glycosylation pathways, respectively. This review specifically focuses on recent representative toxin-based genetic screens that have identified new players involved in and provided new insights into fundamental biological pathways, such as glycosphingolipid biosynthesis, protein glycosylation, and membrane vesicle trafficking pathways. Functionally characterizing these newly identified factors not only expands our current understanding of toxin biology but also enables a deeper comprehension of fundamental biological questions. Consequently, it stimulates the development of new therapeutic approaches targeting both bacterial infectious diseases and genetic disorders with defects in these factors and pathways.

P-257 Transcriptomic signature of live birth revealed in cumulus cells of women undergoing an ICSI cycle for infertility treatment

Abstract Study question Is the transcriptome of cumulus cells surrounding an oocyte that was fertilised and transferred predictive of live birth? Summary answer Live birth is associated with a distinct transcriptomic signature in cumulus cells isolated from an oocyte that was fertilised and transferred. What is known already The selection of the best embryo for transfer is currently based on standard morphological assessment, which is intrinsically subjective, and time-lapse monitoring, which fails to show benefit in predicting pregnancy. Alternative non-invasive selection methods are in development including analysis of cumulus cells (CCs) isolated from oocytes that resulted in an eligible embryo for transfer. However the majority of the existing studies were conducted using outdated methodologies such as transcriptomic analysis with PCR and microarrays. Moreover, the studies associating CCs’ transcriptome with clinical and embryological outcomes lack consensus due to non-homogenous cohorts while the outcome of live birth is inadequately explored. Study design, size, duration CC samples (n = 17), collected between 2018 and 2021 and biobanked, were retrospectively selected for RNA sequencing on the basis of their embryo transfer outcome. Thus, we analysed CCs associated with an oocyte that resulted in pregnancy (n = 6), no pregnancy (n = 7), live birth (n = 5) and pregnancy without live birth (n = 5). Differential gene expression analysis identified the differentially expressed genes (DEGs) between the groups in relation to pregnancy and live birth. Participants/materials, setting, methods The samples were retrieved from subfertile couples undergoing controlled ovarian stimulation and intracytoplasmic sperm injection with subsequent unbiopsied fresh or frozen embryo transfer at the IVF centre of University Hospital Zurich. Maternal age was below 43 years. Libraries from CCs were prepared using the Smart-seq2 pipeline and the RNA sequencing was performed on the NovaSeq 6000 system. The DESeq2 workflow was conducted and the threshold for the adjusted p-values (FDR) was set to 0.05. Main results and the role of chance No DEGs emerged when comparing the transcriptomic profiles of cumulus cells between the “no pregnancy” and “pregnancy” groups. However, 139 DEGs were identified in the comparison between the “pregnancy without live birth” and “live birth” groups. 28 DEGs were familiar and relevant to competent cumulus-oocyte-complexes: CTGF (fold change: 4.38, FDR: 0.005), SERPINE2 (fc: 3.08, FDR: 0.0002), PCK1 (fc:3.04, FDR: 0.0001), HHIP (fc:2.69, FDR: 0.006), HS3ST (fc:2.45, FDR: 0.009) and BIRC5 (fc: -2.69, FDR 0.0001) among others. Functional enrichment analysis of DEGs revealed that the incidence of live birth is associated with promotion of pathways such as runt-related transcription factor 1 (RUNX1/2) signalling, glycosaminoglycan biosynthesis and sonic hedgehog signalling (SHH), and reduction in pathways such as glycosphingolipid biosynthesis and pyrimidine metabolism in cumulus cells. Limitations, reasons for caution We reveal the transcriptome associated with live birth in CCs analysed with RNAseq on a limited number of samples. Consolidation of these findings via utilisation of a validation prospective cohort is pending. Wider implications of the findings CCs’ transcriptome could inform the development of non-invasive biomarkers to predict the chance of live birth across a group of embryos eligible for transfer. The transcriptomic findings inspire further explorations in the field of cumulus-oocyte bidirectional communication and establishment of oocyte competence. Trial registration number N/A

A BRAF mutation-associated gene risk model for predicting the prognosis of melanoma

BRAF mutation plays an important role in the pathogenesis and progression of melanoma and is correlated to the prognosis of melanoma patients. However, fewer studies have attempted to develop a BRAF mutation-associated gene risk model for predicting the prognosis of melanoma. The current research explores BRAF mutation-related biological features in melanoma and establishes a prognostic signature. First, we identified three significantly enriched KEGG pathways (glycosphingolipid biosynthesis - ganglio series, ether lipid metabolism, and glycosaminoglycan biosynthesis - keratan sulfate) and corresponding genes in the BRAF mutant group by gene set enrichment analysis. We then developed a prognostic signature based on 7 BRAF-associated genes (PLA2G2D, FUT8, PLA2G4E, PLA2G5, PLA2G1B, B3GNT2, and ST3GAL5) and assessed its prediction accuracy using ROC curve analysis. Finally, the nomogram was established according to the prognostic signature and independent clinical characteristics to predict the survival of melanoma patients. Furthermore, we found higher proportions of naive B cells, plasma cells, CD8 T cells, CD4 memory-activated T cells, and regulatory T cells in the low-risk group. Whereas lower proportions of M0, M1, and M2 macrophages and resting NK cells were observed in the high-risk group. The analysis also showed a significantly higher expression of immune checkpoint molecules (PD-1, PD-L1, CTLA4, BTLA, CD28, CD80, CD86, HAVCR2, ICOS, LAG3, and TIGIT) in the low-risk group. Our results provide novel insights into the effect of BRAF mutation on melanoma growth and indicate a promising direction toward immunotherapy and precision medicine in melanoma patients.

Plasma metabolites associated with clinically diagnosed Alzheimer’s Disease and the association is augmented using blood-based biomarkers. (P10-6.003)

<h3>Objective:</h3> Identify metabolites associated with clinical diagnosis and pathology of Alzheimer’s Disease (AD) <h3>Background:</h3> We investigated metabolites in plasma to capture systemic biochemical changes associated with AD diagnosed clinically and then using plasma biomarkers. <h3>Design/Methods:</h3> Exogenous and endogenous metabolites in plasma were measured in 300 clinical AD and 430 healthy non-demented individuals of Caribbean Hispanic ancestry using untargeted liquid-chromatography, performed on HILIC (+ ionization) and C18 (− ionization) columns, coupled to a Thermo Orbitrap HF-X mass spectrometer. Genome-wide SNP data and plasma biomarkers including Ab40, Ab42, P-tau181 and Neurofilament light chain (NfL) were obtained on all participants. Metabolite association with AD and P-tau181 levels were tested after adjustment for age and sex. Metabolites associated with P-tau181 levels (but not with NfL, a non-specific marker of neurodegeneration) were categorized as AD-specific. <h3>Results:</h3> Over 4000 metabolomic features were measured with high accuracy in the sample. 96 metabolites were associated with P-tau181 levels, but no metabolites survived multiple testing correction for association with clinical AD alone. Features putatively annotated as phosphatidylcholine (4.11E-07) and Carotamine (p=1.2e-05) were strongly associated P-tau181 levels. We observed similar results when using a diagnostic cutoff of P-tau181 levels for AD as the outcome. Metabolites associated with P-tau181 levels were enriched in amino acid metabolism, glutathione metabolism, tryrosine metabolism and hexose phosphorylation pathways. Ramipril (p=2.9e-04) and glycerophosphocholine (p=9.9e-04) were among the top associated with AD diagnosis. PLS-DA analysis identified biopterin metabolism, glycosphingolipid biosynthesis and tyrosine metabolism as the most discriminating pathways between AD and healthy controls when augmented by biomarkers. <h3>Conclusions:</h3> Metabolites specifically lipids and those involved in cardiovascular function were associated with AD and P-tau181 levels. Metabolite profiling can identify perturbed pathways in clinical and pre-clinical AD and integration with genome-wide SNP data will identify mechanisms underlying genetic association with disease. <b>Disclosure:</b> Dr. Kalia has nothing to disclose. Mrs. Reyes-Dumeyer has nothing to disclose. Mr. DUBEY has nothing to disclose. Dr. Nandakumar has nothing to disclose. Dr. Lee has nothing to disclose. Rafael Lantigua has nothing to disclose. Dr. Medrano has nothing to disclose. Dr. Rivera Mejia has nothing to disclose. Dr. Honig has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Eisai. Dr. Honig has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Biogen. Dr. Honig has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Medscape. Dr. Honig has received personal compensation in the range of $5,000-$9,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Prevail. Dr. Honig has received personal compensation in the range of $10,000-$49,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Cortexyme. Dr. Honig has received personal compensation in the range of $10,000-$49,999 for serving as an Expert Witness for Legal Firms . The institution of Dr. Honig has received research support from Roche. The institution of Dr. Honig has received research support from Eisau. Dr. Honig has received research support from Alector. The institution of Dr. Honig has received research support from Transposon. Dr. Mayeux has nothing to disclose. Badri Vardarajan has received personal compensation in the range of $10,000-$49,999 for serving as a Consultant for Kodikaz Therapeutics.

Abstract 2042: Graph-based pathway analysis of T cell populations in hepatocellular carcinoma reveals novel metabolic regulators of tumor-infiltration lymphocyte activity

Abstract With advances in single cell RNA sequencing (scRNAseq), accurate detection of perturbed pathways between conditions or cell types of interest becomes a critical analytical step. The ability to model alterations in a set of genes corresponding of a biological function is particularly useful when comparing cells between healthy and tumor tissues. Yet, few tools exist to detect changes in the multivariate distribution of genes corresponding to a given pathway. We developed a novel, graph-based statistical framework based on optimal matching for testing differential distribution of biological pathways in scRNAseq. We applied our method to data generated from &amp;gt;4,000 T cells isolated from six individuals with hepatocellular carcinoma (HCC). The T cell populations were purified from three tissue locations: peripheral blood, tumor-infiltrating immune cells (TIICs), and normal tissue adjacent to the tumor. We examined the distribution of gene sets that belong to a particular metabolic pathway across T cell subtypes to address the following questions: (1) Which pathways have a similar distribution across T cell subtypes in a given tissue? (2) Are there pathways that have a stable distribution across T cell subtypes in a normal/healthy tissue, but a perturbed distribution in HCC? (3) For pathways that have a disparate distribution across the T cell subtypes, which subtypes show the most distinct distribution? Of the 86 metabolic pathways compared, 41, 63 and 76 metabolic pathways were indistinguishable across T cell subtypes in the tumor, peripheral blood and adjacent normal tissues, respectively. We observed that most metabolic pathways do not show evidence for dissimilar distribution across cell types, suggesting that T cell subtypes might be more similar than previously appreciated in terms of how they regulate their basic metabolic machinery. Further, for each pair of tissue locations, we computed the overlap in perturbed pathways, and found that the concordance was substantially higher for blood and adjacent normal, than what either of these tissues had with the tumor tissue. Interestingly, five metabolic pathways were differentially distributed across the T cell subtypes in each tissue examined in HCC: glycolysis, purine metabolism, glycosphingolipid biosynthesis, pyruvate metabolism, and glycerophospholipid metabolism. Our model also found that CD4+ regulatory T cells were the strongest contributors, driving the differential distribution of these pathways between T cell subtypes in HCC. Altogether, our approach allows for a systems level characterization of pathway activity across multiple cell types with a variety of applications in single cell pathway analysis in oncology. Our work here also highlights unexpected regulatory mechanisms of regulatory T cells that might play in role in the immunobiology of HCC. Citation Format: Divyansh Agarwal, Tina Bharani, Somabha Mukherjee. Graph-based pathway analysis of T cell populations in hepatocellular carcinoma reveals novel metabolic regulators of tumor-infiltration lymphocyte activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2042.