S. Plasma lipids in circulation are integral to the physiopathological processes of the ovary and may impact the development of various ovarian conditions, including ovarian cancer (OC). This study utilized a two-sample Mendelian randomization method to examine the causal link between changes in 179 plasma lipid groups and ovarian cancer (OC) to gain deeper insights into this association. We used the inverse variance weighted (IVW) method as the main tool for analysis. We utilized statistical data from plasma lipidomics involving 7,174 Finnish individuals and OC data from the FinnGen consortium, including 2,339 European OC patients and 222,078 European healthy controls. Our analysis revealed that elevated levels of four plasma lipids-Phosphatidylcholine (14:0_16:0, O-18:2_18:2, 16:0_20:4)-are linked to an increased risk of OC, while Sphingomyelin (d34:2) seems to act as a protective factor(all P < 0.05). We also conducted tests for heterogeneity and pleiotropy in the MR results. Additionally, reverse MR analysis indicated that OC does not affect plasma levels of these lipids. To determine whether the observed significant plasma lipids influence OC through common risk factors, we selected BMI as a confounder for multivariable Mendelian randomization (MVMR) analysis. The results showed that Sphingomyelin (d34:2) levels remained significantly associated with OC even after including BMI as an exposure factor. Furthermore, we investigated whether these four lipids mediated the effect of BMI on OC but found no evidence supporting their mediating role. In summary, our findings confirm a causal link between certain plasma lipid species and OC, providing fresh perspectives for risk evaluation and potential therapeutic strategies.

Lactate metabolism and signaling intricately intertwine in the context of cancer and immunity. Basigin, working alongside monocarboxylate transporters MCT1 and MCT4, orchestrates the movement of lactate across cell membranes. Despite their potential in treating formidable tumors, the mechanisms by which basigin antibodies affect basigin and MCTs remain unclear. Our research demonstrated that basigin positively modulates MCT activity. We subsequently developed a basigin antibody that converts basigin into a negative modulator, thereby suppressing lactate transport and enhancing anti-tumor immunity. Additionally, the antibody alters metabolic profiles in NSCLC-PDOs and T cells. Cryo-EM structural analysis and molecular dynamics simulations reveal that the extracellular Ig2 domain and transmembrane domain of basigin regulate MCT1 activity through an allosteric mechanism. The antibody decreases MCT1 transition rate by reducing the flexibility of basigin's Ig2 domain and diminishing interactions between basigin's transmembrane domain and MCT1. These findings underscore the promise of basigin antibodies in combating tumors by modulating metabolism and immunity, and the value of a common therapeutic subunit shared by multiple transporter targets.

Elevated de novo lipid synthesis is a remarkable adaptation of cancer cells that can be exploited for therapy. However, the role of altered lipid metabolism in the regulation of apoptosis is still poorly understood. Using thermal proteome profiling, we identified Manidipine-2HCl, targeting UGT8, a key enzyme in the synthesis of sulfatides. In agreement, lipidomic analysis indicated that sulfatides are strongly reduced in colorectal cancer cells upon treatment with Manidipine-2HCl. Intriguingly, this reduction led to severe mitochondrial swelling and a strong synergism with BH3 mimetics targeting BCL-XL, leading to the activation of mitochondria-dependent apoptosis. Mechanistically, Manidipine-2HCl enhanced mitochondrial BAX localization in a sulfatide-dependent fashion, facilitating its activation by BH3 mimetics. In conclusion, our data indicates that UGT8 mediated synthesis of sulfatides controls mitochondrial homeostasis and BAX localization, dictating apoptosis sensitivity of colorectal cancer cells.

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, causes significant global morbidity, including fever, rash, and persistent arthralgia. Utilizing untargeted lipidomics, we investigated how CHIKV infection alters host cell lipid metabolism in Vero cells. CHIKV infection induced marked catabolism of hexosylceramides, reducing their levels while increasing ceramide byproducts. Functional studies revealed a reliance on fatty acid synthesis, β-oxidation, and glycosphingolipid biosynthesis. Notably, inhibition of uridine diphosphate glycosyltransferase 8 (UGT8), essential for galactosylceramide production, significantly impaired CHIKV replication and entry in Vero cells. Sensitivity of CHIKV to UGT8 inhibition was reproduced in a disease-relevant cell line, mouse hepatocytes (Hepa1-6). CHIKV was also sensitive to evacetrapib, a cholesterol ester transfer protein (CETP) inhibitor, though the mechanism of inhibition appeared independent of CETP itself, suggesting an off-target effect. These findings highlight specific lipid pathways, particularly glycosphingolipid metabolism, as critical for CHIKV replication and further refine our understanding of how CHIKV exploits host lipid networks. This study provides new insights into CHIKV biology and suggests that targeted investigation of host lipid pathways may inform future therapeutic strategies.

The efficient identification of microbial strains capable of producing rare sphingoid bases, such as sphingosine and sphinganine, is critical for advancing microbial fermentation processes and addressing increasing industrial demands. Wickerhamomyces ciferrii, a non-conventional yeast, naturally overproduces tetraacetyl phytosphingosine (TAPS); however, the production of other valuable sphingoid bases, including sphingosine, sphinganine, and triacetyl sphingosine, remains a key target. In this study, we developed a novel screening method utilizing fluorescein sodium, a selective fluorescent dye that specifically reacts with non-acetylated sphingoid bases-sphinganine, sphingosine, and phytosphingosine-while exhibiting no reactivity with TAPS. A mutant library of W. ciferrii was generated via gamma-ray mutagenesis and screened using fluorescence-activated cell sorting (FACS). Mutants exhibiting high fluorescence intensity, indicative of non-acetylated or partially acetylated sphingoid base production, were isolated through three rounds of sorting and further validated via HPLC analysis. This approach successfully identified three mutant strains: P41C3 (sphingosine-producing), M01_5 (sphinganine-producing), and P41E7 (triacetyl sphingosine-producing). Among them, the P41C3 mutant achieved a sphingosine titer of 36.7 mg/L during shake-flask cultivation, accompanied by a significant reduction in TAPS production, indicating a redirection of metabolic flux. This study demonstrates the utility of fluorescein sodium as a selective screening dye for sphingoid base-producing strains and establishes an effective platform for the metabolic engineering of W. ciferrii to enhance the production of industrially significant sphingolipids.

While increasing evidence suggests that alterations in the gut microbiota and metabolites are associated with ovarian cancer (OC) risk, whether these associations imply causation remains to be identified.

We conducted a two-sample Mendelian randomization (MR) study utilizing a large-scale genome-wide association study (GWAS) to explore the causal effects of the gut microbiota of 196/220 individuals and 1,400 plasma metabolites on OC and epithelial ovarian cancer (EOC) subtypes. Data on the gut microbiota were obtained from the MiBioGen consortium of 18,340 subjects and the Dutch Microbiome Project of 7,738 volunteers. Data on plasma metabolites were derived from a GWAS of plasma metabolites in 8,299 participants. Ovarian cancer (n = 25,509) and EOC subtypes were obtained from the Ovarian Cancer Association Consortium (OCAC). Metabolites and associated targets were analyzed via network pharmacology and molecular docking.

At the genus and species levels, we identified seven risk factors for the gut microbiota: the genus Dialister (P = 0.024), genus Ruminiclostridium5 (P = 0.0004), genus Phascolarctobacterium (P = 0.0217), species Bacteroides massiliensis (P = 0.011), species Phascolarctobacterium succinatutens (P = 0.0212), species Paraprevotella clara (P = 0.0247) and species Bacteroides dorei (P = 0.0054). In addition, five gut microbes at the genus and species levels were found to be protective: genus Family XIII AD3011 group (P = 0.006), genus Butyrivibrio (P = 0.0095), genus Oscillibacter (P = 0.0206), species Roseburia hominis (P = 0.0241), and species Bifidobacterium bifidum (P = 0.0224). For plasma metabolites, we revealed five positive and four negative correlations with OC. Among these, caffeic acid and caffeine metabolites and sphingomyelin and ceramide metabolites were identified as risk factors, whereas phenylalanine metabolites, butyric acid metabolites, and some lipid metabolites were recognized as protective factors. A series of sensitivity analyses revealed no abnormalities, including pleiotropy and heterogeneity analyses.

Our MR analysis demonstrated that the gut microbiota and metabolites are causally associated with OC, which has significant potential for the early detection and diagnosis of OC and EOC subtypes, providing valuable insights into this area of research.

Cancer biology revolves around understanding how cells undergo uncontrolled proliferation leading to the formation of malignant tumors. Key aspects include self-sufficiency in growth signals, the lack of response to signals of growth inhibition, the evasion of apoptosis, sustained angiogenesis, the evasion of immune response, the capacity to invade and metastasize, and alterations in cellular metabolism. A vast amount of research, which is exponentially growing, over the past few decades highlights the role of sphingolipids in cancer. They act not only as structural membrane components but also as bioactive molecules that regulate cell fate in different physio-pathological conditions. In cancer, sphingolipid metabolism is dysregulated, contributing to tumor progression, metastasis, and drug resistance. In this review, we outline the impact of sphingosine-1-phosphate (S1P) as a key bioactive sphingolipid in cancer. We give an overview of its metabolism summarizing the role of S1P as an intracellular and extracellular mediator through specific plasma membrane receptors in different cancers. We also describe previous findings on how the disruption in the balance between S1P and ceramide (Cer) is common in cancer cells and can contribute to tumorigenesis and resistance to chemotherapy. We finally consider the potential of targeting the metabolic pathways of S1P as well as its receptors and transporters as a promising therapeutic approach in cancer treatments.

Spingosine-1-phosphate (S1P) and ceramides are bioactive sphingolipids that influence cancer cell fate. Anti-ceramide antibodies might inhibit the effects of ceramide. The aim of this study was to assess the potential role of circulating S1P and anti-ceramide antibody as biomarkers in non-small cell lung cancer (NSCLC).

We recruited 66 subjects (34 controls and 32 patients with NSCLC). Patient history and clinical variables were taken from all participants. Venous blood samples were collected to evaluate plasma biomarkers. If bronchoscopy was performed, bronchial washing fluid (BWF) was also analyzed. We measured the levels of S1P and anti-ceramide antibody with ELISA.

S1P levels were significantly higher in the NSCLC group (3770.99 ± 762.29 ng/mL vs. 366.53 ± 249.38 ng/mL, patients with NSCLC vs. controls, respectively, p < 0.001). Anti-ceramide antibody levels were significantly elevated in the NSCLC group (278.70 ± 19.26 ng/mL vs. 178.60 ± 18 ng/mL, patients with NSCLC vs. controls, respectively, p = 0.007). Age or BMI had no significant effect on anti-ceramide antibody or S1P levels. BWF samples had higher levels of anti-ceramide antibody (155.29 ± 27.58 ng/mL vs. 105.87 ± 9.99 ng/mL, patients with NSCLC vs. controls, respectively, p < 0.001). Overall survival (OS) was 13.36 months. OS was not affected by anti-ceramide antibody or S1P levels.

Higher levels of S1P and anti-ceramide antibody were associated with active cancer. These results suggest that sphingolipid alterations might be important features of NSCLC.

Canine mammary carcinomas (CMCs) represent the most prevalent form of cancer in female dogs, characterized by a high incidence and mortality rate. C6 ceramide is recognized for its multifaceted anti-cancer properties, yet its specific influence on CMCs remains to be elucidated. Long noncoding RNAs (lncRNAs), now recognized as functional "dark matter" in precision oncology, are particularly intriguing, with 44% of canine lncRNAs exhibiting tissue-specific expression. In this study, we performed a thorough analysis of lncRNA expression profiles to uncover the mechanisms behind C6 ceramide's anti-cancer activity in CHMp cells. Our findings reveal that C6 ceramide notably inhibits the proliferation of CHMp cells. RNA sequencing identified 4522 lncRNAs with expression changes following C6 ceramide treatment, of which 2936 were upregulated and 1586 were downregulated. Further investigation into Lnc_025370 showed that it is predominantly nuclear-localized and is significantly downregulated by C6 ceramide treatment. Functional studies discovered that overexpression of Lnc_025370 enhances the growth and metastatic capabilities of CHMp cells, which is associated with an increase in NRG1, and concurrently diminishes the anti-cancer effectiveness of C6 ceramide in vitro. Mouse xenograft models also showed that Lnc_025370 overexpression promotes tumor growth and Ki67 expression. Together, our results suggest that Lnc_025370 acts as a pivotal target mediator of C6 ceramide's anti-cancer effects, facilitating the malignant progression of CHMp cells.

Traumatic brain injury (TBI) is associated with disturbances in energy metabolism. This study aimed to construct a lncRNA-miRNA-mRNA network through bioinformatics methods to explore energy metabolism-related genes in the pathogenesis of TBI.

Data from datasets GSE171718, GSE131695, and GSE223245 obtained from the Gene Expression Omnibus, were analyzed to identify differentially expressed (DE) genes. Regulatory relationships were investigated through miRDB, miRTarBase, and TargetScan, thereby forming a lncRNA-miRNA-mRNA network. The Molecular Signatures Database (MSigDB) was utilized to identify energy metabolism-related genes, and a protein-protein interaction (PPI) network was established through the STRING database. Functional annotation and enrichment analysis were conducted using GO and KEGG. The TBI mouse model was established to detect the expression levels of GOLGA8B, ZNF367, and SMPD3 in brain tissues.

SMPD3 emerged as the key DE gene linked to energy metabolism in TBI, demonstrating a negative correlation with miR-218-5p and being associated with moderate unconsciousness and female patients. The PPI network revealed SMPD3 interactions with proteins associated with cell death, sphingolipid metabolism, and neurodegenerative diseases such as Alzheimer's disease. In vivo, GOLGA8B, ZNF367, and SMPD3 mRNA levels were significantly lower in TBI mice.

In summary, SMPD3 represents a crucial metabolic gene in the progression of TBI. It potentially provides a new therapeutic target for metabolic disorders caused by traumatic brain injury (TBI) and holds significant theoretical value for further research.

Patients with high-grade serous carcinoma of tubo-ovarian origin (HGSC) often experience significant side effects related to their disease and treatments, such as pain, discomfort, nausea, and vomiting. Over the last two decades, the use of cannabinoids (CBD) to manage pain and anxiety has become more mainstream. However, there is limited data on how CBD interacts with HGSC tumor cells or whether CBD impacts the effect of chemotherapy. Prior preclinical data has suggested the antitumor benefits of cannabinoids; however, the mechanism and data in ovarian cancer are limited. The objectives of this proposed research are to define the endocannabinoid system milieu in ovarian cancer, determine if CBD influences the growth of ovarian cancer cells, measure the cell viability when cannabinoids such as CBD are combined with standard-of-care therapies, and identify potential molecular pathways in which cannabinoids have a therapeutic effect. We conducted publicly available database searches, in vitro proliferation and apoptotic assays, functional protein signaling via reverse phase protein array analysis of CBD-treated cells using 2D cultured cells, and immunohistological analysis of ex vivo cultured patient-derived tumor slices treated with CBD. Our data suggests that CBD is unlikely to affect the growth of cancer cells at physiologic doses but promotes apoptosis and can have growth inhibitory effects at higher concentrations. The inhibitory effects seen at high dose concentrations are likely from the upregulation of apoptotic pathways and inhibition of oncogenic pathways. Overall, physiologic CBD levels have minimal impact on cancer cell growth or chemotherapy efficacy.

Sphingosine kinase-1 (SPHK1), the enzyme that catalyzes the synthesis of the pro-oncogenic molecule sphingosine-1-phosphate, is commonly upregulated in breast cancer cells and has been linked with poor prognosis and progression by promoting cell transformation, proliferation, angiogenesis, and metastasis. Therefore, SPHK1-targeting drugs have been proposed for breast cancer treatment, with better antitumor results when they are combined with chemotherapy. Previously, we demonstrated that the synthetic flavonoid 2'-nitroflavone (2'NF) exerted a potent and selective antiproliferative effect in murine HER2-positive LM3 mammary tumor cells. As we found that these cells overexpress SPHK1, we decided to explore the antitumor action of the combination of SPHK inhibitors (safingol or SKI-II) with 2'NF. In vitro assays showed that the combination induced a synergistic antiproliferative effect in LM3 cells. Similar results were obtained when human HER2-positive MDA-MB-453 breast cancer cells were treated with the combination of 2'NF/safingol. We also found that safingol potentiated the 2'NF apoptotic effect in both cell lines. The synergistic antitumor effect was confirmed in vivo in an LM3 syngeneic breast cancer model. Moreover, western blot analysis of tumor lysates revealed that combined treatment increased PARP cleavage and Bax protein levels and decreased anti-apoptotic Bcl-xL and Bcl-2 protein levels. Additionally, mice treated with both compounds showed no histopathological effects on different organ tissues. In summary, these findings suggest that the combination safingol/2'NF can be proposed as a potential therapeutic strategy for HER2-positive breast cancer treatment. KEY MESSAGES: The combination safingol/2'-nitroflavone exerts a synergic antitumor action in vitro. Safingol potentiates 2'-nitroflavone apoptotic effect in breast cancer cells. Safingol enhances the 2'-nitroflavone antitumor activity in vivo in breast cancer.

Long non-coding RNAs (lncRNAs) have emerged as pivotal regulators of cancer pathogenesis, influencing various cellular processes and contributing to tumorigenesis. Sphingolipid metabolism has garnered interest as a potential target for cancer therapy owing to its considerable diagnostic and prognostic value. Recent studies have demonstrated that lncRNAs regulate tumor-associated metabolic reprogramming via sphingolipid metabolism. However, the precise nature of the interactions between lncRNAs and sphingolipid metabolism remains unclear. This review summarizes the key roles of lncRNAs and sphingolipid metabolism in tumorigenesis. We emphasize that the interaction between lncRNAs and sphingolipid metabolism influences their impact on both cancer prognosis and drug resistance. These findings suggest that lncRNA-sphingolipid metabolism interaction holds great potential as a newl target for cancer treatment.

Sphingolipids, including ceramides, are an important component of high-fat diets. These molecules can regulate fatty acid oxidation and intestinal stem cell proliferation, predisposing the gut to tumorigenesis. However, the molecular mechanisms involved in ceramide metabolism-mediated intestinal stem cell (ISC) proliferation and tumorigenesis are poorly understood. To understand how changes in sphingolipid metabolite flux affect intestinal stem cells, we manipulated the activities of each of the enzymes of the ceramide synthetic pathway using cell type-specific over-expression or depletion of the corresponding mRNAs in each intestinal cell type of the Drosophila midgut. We documented cell-autonomous and non-cell-autonomous effects, including alterations in cell size, number, differentiation, and proliferation. In our screen, the altered expression of several ceramide metabolism enzymes led to changes in ISC proliferation, cell sizes, and overall cellularity. Among other genes, over-expression of ceramidase homolog, Brain washing (bwa) in gut enteroblasts (EB) increased EB cell size and caused a non-cell-autonomous, 7-8-fold increase in ISC proliferation. Our analysis confirmed previous reports that bwa does not have ceramidase activity, and lipidomic studies indicated that bwa increases the saturation status of sphingolipids, free fatty acids, and other lipids. The pro-proliferative effects of bwa could be counter-acted by depleting a serine palmitoyltransferase, Lace , or a sphingosine acyltransferase, Schlank , which are needed for ceramide synthesis, or by co-expressing a ceramide desaturase enzyme, ifc , indicating that increased saturated ceramides were causal for ISC proliferation and the disruption of gut homeostasis. Accumulating saturated sphingolipids and fatty acids induced inflammatory signaling in the gut, and activated ISC proliferation through the pro-inflammatory cytokines, Upd3 and Upd2. We propose that saturated sphingolipids promote ISC proliferation through pro-inflammatory pathways.

Sphingolipids, particularly sphingosine-1-phosphate (S1P), are bioactive lipids involved in regulating cellular processes such as proliferation, apoptosis, inflammation, and tumor progression. Alkaline ceramidase 2 (ACER2) plays a critical role in sphingolipid metabolism by catalyzing the hydrolysis of ceramide to sphingosine, which is subsequently converted to S1P. Dysregulation of ACER2 has been implicated in various gastrointestinal cancers, including colorectal cancer, gastric cancer, and hepatocellular carcinoma. ACER2-mediated sphingolipid signaling, particularly through the SphK/S1P pathway, influences cancer development by modulating immune responses, inflammation, and the balance between cell survival and death. This review examines the physiological functions of ACER2, and its role in sphingolipid metabolism, and its contribution to the pathogenesis of gastrointestinal cancers. Understanding the mechanisms by which ACER2 regulates tumor progression and immune modulation may open new avenues for targeted therapies in gastrointestinal malignancies.

Abnormal sphingolipid metabolism (SM) is closely linked to the incidence of cancers. However, the role of SM in pancreatic cancer (PC) remains unclear. This study aims to explore the significance of SM in the prognosis, immune microenvironment, and treatment of PC.

Single-cell and bulk transcriptome data of PC were acquired via TCGA and GEO databases. SM-related genes (SMRGs) were obtained via MSigDB database. Consensus clustering was utilized to construct SM-related molecular subtypes. LASSO and Cox regression were utilized to build SM-related prognostic signature. ESTIMATE and CIBERSORT algorithms were employed to assess the tumour immune microenvironment. OncoPredict package was used to predict drug sensitivity. CCK-8, scratch, and transwell experiments were performed to analyze the function of ANKRD22 in PC cell line PANC-1 and BxPC-3.

A total of 153 SMRGs were acquired, of which 48 were linked to PC patients' prognosis. Two SM-related subtypes (SMRGcluster A and B) were identified in PC. SMRGcluster A had a poorer outcome and more active SM process compared to SMRGcluster B. Immune analysis revealed that SMRGcluster B had higher immune and stromal scores and CD8 + T cell abundance, while SMRGcluster A had a higher tumour purity score and M0 macrophages and activated dendritic cell abundance. PC with SMRGcluster B was more susceptible to gemcitabine, paclitaxel, and oxaliplatin. Then SM-related prognostic model (including ANLN, ANKRD22, and DKK1) was built, which had a very good predictive performance. Single-cell analysis revealed that in PC microenvironment, macrophages, epithelial cells, and endothelial cells had relatively higher SM activity. ANKRD22, DKK1, and ANLN have relatively higher expression levels in epithelial cells. Cell subpopulations with high expression of ANKRD22, DKK1, and ANLN had more active SM activity. In vitro experiments showed that ANKRD22 knockdown can inhibit the proliferation, migration, and invasion of PC cells.

This study revealed the important significance of SM in PC and identified SM-associated molecular subtypes and prognostic model, which provided novel perspectives on the stratification, prognostic prediction, and precision treatment of PC patients.

Ceramides have recently been identified as novel biomarkers associated with diabetes mellitus (DM) and major adverse cardiac and cerebrovascular events (MACCE). This study aims to explore their utility in diagnosing microvascular disease.

This study prospectively enrolled 309 patients from 2018 to 2020 into three groups: healthy controls (Group 1, N = 51), DM patients without acute myocardial infarction (AMI) (Group 2, N = 150), and DM patients with AMI (Group 3, N = 108). We assessed outcomes using stress perfusion cardiac magnetic resonance (CMR) imaging for coronary microvascular disease (CMD) (Outcome 1), retinography for retinal microvascular disease (RMD) (Outcome 2), both CMD and RMD (Outcome 3), and absence of microvascular disease (w/o MD) (outcome 4). We evaluated the classification performance of ceramides using receiver operating characteristic (ROC) analysis and multiple logistic regression. 11-ceramide panel previously identified by our research group as related to macrovascular disease were used.

Average glycated hemoglobin (HbA1c) values were 5.1% in Group 1, 8.3% in Group 2, and 7.6% in Group 3. Within the cohort, CMD was present in 59.5% of patients, RMD in 25.8%, both CMD and RMD in 18.8%, and w/o MD in 38.5%. The AUC values for the reference ceramide ratios were as follows: CMD at 0.66 (p = 0.012), RMD at 0.61 (p = 0.248), CMD & RMD at 0.64 (p = 0.282), and w/o MD at 0.67 (p = 0.010). In contrast, the AUC values using 11-ceramide panel showed significant improvement in the outcomes prediction: CMD at 0.81 (p = 0.001), RMD at 0.73 (p = 0.010), CMD & RMD at 0.73 (p = 0.04), and w/o MD at 0.83 (p = 0.010). Additionally, the plasma concentration of C14.0 was notably higher in the w/o MD group (p < 0.001).

Plasma ceramides serve as potential predictors for health status and microvascular disease phenotypes in diabetic patients.

Sphingolipids are a major lipid species found in all eukaryotes. Among structurally complex and diversified lipids, sphingoid bases have been heavily linked to various metabolic diseases. However, most current LC-MS-based methods lack the sensitivity to detect low-abundant sphingoid bases. The 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization reagent, which efficiently forms covalent bonds with amino groups, has been widely used for amino acid detection. Nevertheless, the commonly used reverse-phase HPLC method for amino acid analysis is not suitable for amphipathic sphingolipids. To address this issue, we report a robust reverse-phase HPLC-MS/MS method capable of separating and detecting hydrophilic amino acids and sphingoid bases in a single run with high sensitivity. This method is also inclusive of other amino metabolites with an expandable target list. We tested this method under various conditions and samples, demonstrating its high reproducibility and sensitivity. Using this approach, we systematically analyzed human serum samples from healthy individuals, dyslipidemia, and type II diabetes mellitus (T2DM) patients, respectively. Two sphingolipids and five amino acids were identified with significant differences between the control and T2DM groups, highlighting the potential of this method in clinical studies.

Regulatory T cells (Treg cells) hold promise for sustainable therapy of immune disorders. Recent advancements in chimeric antigen receptor development and genome editing aim to enhance the specificity and function of Treg cells. However, impurities and functional instability pose challenges for the development of safe gene-edited Treg cell products. Here, we examined different Treg cell subsets regarding their fate, epigenomic stability, transcriptomes, T cell receptor repertoires, and function ex vivo and after manufacturing. Each Treg cell subset displayed distinct features, including lineage stability, epigenomics, surface markers, T cell receptor diversity, and transcriptomics. Earlier-differentiated memory Treg cell populations, including a hitherto unidentified naïve-like memory Treg cell subset, outperformed late-differentiated effector memory-like Treg cells in regulatory function, proliferative capacity, and epigenomic stability. High yields of stable, functional Treg cell products could be achieved by depleting the small effector memory-like Treg cell subset before manufacturing. Considering Treg cell subset composition appears critical to maintain lineage stability in the final cell product.

Programmed death ligand 1, PD-L1 (CD274), facilitates immune evasion and exerts pro-survival functions in cancer cells. Here, we report a mechanism whereby internalization of PD-L1 in response to alterations of bioactive lipid/ceramide metabolism by ceramide synthase 4 (CerS4) induces sonic hedgehog (Shh) and transforming growth factor β receptor signaling to enhance tumor metastasis in triple-negative breast cancers (TNBCs), exhibiting immunotherapy resistance. Mechanistically, data showed that internalized PD-L1 interacts with an RNA-binding protein, caprin-1, to stabilize Shh/TGFBR1/Wnt mRNAs to induce β-catenin signaling and TNBC growth/metastasis, consistent with increased infiltration of FoxP3+ regulatory T cells and resistance to immunotherapy. While mammary tumors developed in MMTV-PyMT/CerS4-/- were highly metastatic, targeting the Shh/PD-L1 axis using sonidegib and anti-PD-L1 antibody vastly decreased tumor growth and metastasis, consistent with the inhibition of PD-L1 internalization and Shh/Wnt signaling, restoring anti-tumor immune response. These data, validated in clinical samples and databases, provide a mechanism-based therapeutic strategy to improve immunotherapy responses in metastatic TNBCs.

Alteration of cell metabolism is one of the essential characteristics of tumor growth. Cancer stem cells (CSCs) are the initiating cells of tumorigenesis, proliferation, recurrence, and other processes, and play an important role in therapeutic resistance and metastasis. Thus, identification of the metabolic profiles in prostate cancer stem cells (PCSCs) is critical to understanding prostate cancer progression. Using untargeted metabolomics and lipidomics methods, we show distinct metabolic differences between prostate cancer cells and PCSCs. Urea cycle is the most significantly altered metabolic pathway in PCSCs, the key metabolites arginine and proline are evidently elevated. Proline promotes cancer stem-like characteristics via the JAK2/STAT3 signaling pathway. Meanwhile, the enzyme pyrroline-5-carboxylate reductase 1 (PYCR1), which catalyzes the conversion of pyrroline-5-carboxylic acid to proline, is highly expressed in PCSCs, and the inhibition of PYCR1 suppresses the stem-like characteristics of prostate cancer cells and tumor growth. In addition, carnitine and free fatty acid levels are significantly increased, indicating reprogramming of fatty acid metabolism in PCSCs. Reduced sphingolipid levels and increased triglyceride levels are also observed. Collectively, our data illustrate the comprehensive landscape of the metabolic reprogramming of PCSCs and provide potential therapeutic strategies for prostate cancer.

Sphingosine-1-phosphate (S1P) formed via catalytic actions of sphingosine kinase 1 (SphK1) behaves as a pro-survival substance and activates downstream target molecules associated with various pathologies, including initiation, inflammation, and progression of cancer. Here, we aimed to investigate the SphK1 inhibitory potentials of thymoquinone (TQ), Artemisinin (AR), and Thymol (TM) for the therapeutic management of lung cancer. We implemented docking, molecular dynamics (MD) simulations, enzyme inhibition assay, and fluorescence measurement studies to estimate binding affinity and SphK1 inhibitory potential of TQ, AR, and TM. We further investigated the anti-cancer potential of these compounds on non-small cell lung cancer (NSCLC) cell lines (H1299 and A549), followed by estimation of mitochondrial ROS, mitochondrial membrane potential depolarization, and cleavage of DNA by comet assay. Enzyme activity and fluorescence binding studies suggest that TQ, AR, and TM significantly inhibit the activity of SphK1 with IC50 values of 35.52 µM, 42.81 µM, and 53.68 µM, respectively, and have an excellent binding affinity. TQ shows cytotoxic effect and anti-proliferative potentials on H1299 and A549 with an IC50 value of 27.96 µM and 54.43 µM, respectively. Detection of mitochondrial ROS and mitochondrial membrane potential depolarization shows promising TQ-induced oxidative stress on H1299 and A549 cell lines. Comet assay shows promising TQ-induced oxidative DNA damage. In conclusion, TQ, AR, and TM act as potential inhibitors for SphK1, with a strong binding affinity. In addition, the cytotoxicity of TQ is linked to oxidative stress due to mitochondrial ROS generation. Overall, our study suggests that TQ is a promising inhibitor of SphK1 targeting lung cancer therapy.

Neutral sphingomyelinase-2 (nSMase2), gene name sphingomyelin phosphodiesterase-3 (Smpd3), is a key regulatory enzyme responsible for generating the sphingolipid ceramide. The function of nSMase2 in the brain is still controversial. To better understand the functional roles of nSMase2 in the aging mouse brain, we applied RNA-seq analysis, which identified a total of 1462 differentially abundant mRNAs between +/fro and fro/fro, of which 891 were increased and 571 were decreased in nSMase2-deficient mouse brains. The most strongly enriched GO and KEGG annotation terms among transcripts increased in fro/fro mice included synaptogenesis, synapse development, synaptic signaling, axon development, and axonogenesis. Among decreased transcripts, enriched annotations included ribosome assembly and mitochondrial protein complex functions. KEGG analysis of decreased transcripts also revealed overrepresentation of annotations for Alzheimer's disease (AD), Parkinson's disease (PD), and Huntington disease (HD). Ingenuity Pathway Analysis (IPA) tools predicted lower susceptibility to these neurodegenerative disorders, as well as predictions agreeing with stronger synaptic function, learning, and memory in fro/fro mice. The IPA tools identified signaling proteins, epigenetic regulators, and microRNAs as likely upstream regulators of the broader set of genes encoding the affected transcripts. It also revealed 16 gene networks, each linked to biological processes identified as overrepresented annotations among the affected transcripts by multiple analysis methods. Therefore, the analysis of these RNA-seq data indicates that nSMase2 impacts synaptic function and neural development, and may contribute to the onset and development of neurodegenerative diseases in middle-aged mice.

Glioblastoma multiforme is the most common and fatal brain tumor among human cancers. Ceramide (Cer) and Sphingosine 1-phosphate (S1P) have emerged as bioeffector molecules that control several biological processes involved in both cancer development and resistance. Cer acts as a tumor suppressor, inhibiting cancer progression, promoting apoptosis, enhancing immunotherapy and sensitizing cells to chemotherapy. In contrast, S1P functions as an onco-promoter molecule, increasing proliferation, survival, invasiveness, and resistance to drug-induced apoptosis. The pro-survival PI3K/Akt pathway is a recognized downstream target of S1P, and we have previously demonstrated that in glioma cells it also improves Cer transport and metabolism towards complex sphingolipids in glioma cells. Here, we first examined the possibility that, in T98G glioma cells, S1P may regulate Cer metabolism through PI3K/Akt signaling. Our research showed that exogenous S1P increases the rate of vesicular trafficking of Cer from the endoplasmic reticulum (ER) to the Golgi apparatus through S1P receptor-mediated activation of the PI3K/Akt pathway. Interestingly, the effect of S1P results in cell protection against toxicity arising from Cer accumulation in the ER, highlighting the role of S1P as a survival factor to escape from the Cer-generating cell death response.

Globally, metabolic dysfunction-associated steatotic liver disease (MASLD), previously termed nonalcoholic fatty liver disease (NAFLD), is one of the most common liver disorders and is strongly associated with copper deficiency. To explore the potential effects and mechanisms of Lactiplantibacillus plantarum LPJZ-658, copper deficiency combined with a high-sugar diet-induced MASLD mouse model was utilized in this study. We fed 40-week-old (middle-aged) male C57BL/6 mice a copper-deficient and high-sugar diet for 16 weeks (CuDS), with supplementary LPJZ-658 for the last 6 weeks (CuDS + LPJZ-658). In this study, we measured body weight, liver weight, and serum biochemical markers. Lipid accumulation, histology, lipidomics, and sphingolipid metabolism-related enzyme expression were investigated to analyze liver function. Untargeted metabolomics was used to analyze the serum and the composition and abundance of intestinal flora. In addition, the correlation between differential liver lipid profiles, serum metabolites, and gut flora at the genus level was measured. The results show that LPJZ-658 significantly improves abnormal liver function and hepatic steatosis. The lipidomics analyses and metabolic pathway analysis identified sphingolipid, retinol, and glycerophospholipid metabolism as the most relevant metabolic pathways that characterized liver lipid dysregulation in the CuDS group. Consistently, RT-qPCR analyses revealed that the enzymes catalyzing sphingolipid metabolism that were significantly upregulated in the CuDS group were downregulated by the LPJZ-658 treatment. In addition, the serum metabolomics results indicated that the linoleic acid, taurine and hypotaurine, and ascorbate and aldarate metabolism pathways were associated with CuDS-induced MASLD. Notably, we found that treatment with LPJZ-658 partially reversed the changes in the differential serum metabolites. Finally, LPJZ-658 effectively regulated intestinal flora abnormalities and was significantly correlated with differential hepatic lipid species and serum metabolites. In conclusion, we elucidated the function and potential mechanisms of LPJZ-658 in alleviating copper deficiency combined with sugar-induced middle-aged MASLD and hope this will provide possible treatment strategies for improving MASLD.

Glioblastoma is the most common and aggressive type of malignant brain tumor with a poor prognosis due to the lack of effective treatment options. Therefore, new treatment options are required. Sphingolipids are essential components of the cell membrane, while complement components are integral to innate immunity, and both play a critical role in regulating glioblastoma survival signaling. This review focuses on recent studies investigating the functional roles of sphingolipid metabolism and complement activation signaling in glioblastoma. It also discusses how targeting these two systems together may emerge as a novel therapeutic approach.

Tumour cells secrete various proangiogenic factors like VEGF, PDGF, and EGF that result in the formation of highly vascularized tumours with an immunosuppressive tumour microenvironment. As tumour growth and metastasis are highly dependent on angiogenesis, targeting tumour vasculature along with rapidly dividing tumour cells is a potential approach for cancer treatment. Here, we specifically engineered sub-100 sized nanomicelles (DTX-CA4 NMs) targeting proliferation and angiogenesis using an esterase-sensitive phosphocholine-tethered docetaxel conjugate of lithocholic acid (LCA) (PC-LCA-DTX) and a poly(ethylene glycol) (PEG) derivative of an LCA-combretastatin A4 conjugate (PEG-LCA-CA4). DTX-CA4 NMs effectively inhibit the tumour growth in syngeneic (CT26) and xenograft (HCT116) colorectal cancer models, inhibit tumour recurrence, and enhance the percentage survival in comparison with individual drug-loaded NMs. DTX-CA4 NMs enhance the T cell-mediated anti-tumour immune response and DTX-CA4 NMs in combination with an immune checkpoint inhibitor, anti-PDL1 antibody, enhance the anti-tumour response. We additionally showed that DTX-CA4 NMs effectively attenuate the production of ceramide-1-phosphate, a key metabolite of the sphingolipid pathway, by downregulating the expression of ceramide kinase at both transcriptional and translational levels. Therefore, this study presents the engineering of effective DTX-CA4 NMs for targeting the tumour microenvironment that can be explored further for clinical applications.

Diabetic kidney disease (DKD) is one of the chronic microvascular complications caused by diabetes, which is characterized by persistent albuminuria and/or progressive decline of estimated glomerular filtration rate (eGFR), and has been the major cause of dialysis around the world. At present, although the treatments for DKD including lifestyle modification, glycemic control and even using of Sodium-glucose cotransporter 2 (SGLT2) inhibitors can relieve kidney damage caused to a certain extent, there is still a lack of effective treatment schemes that can prevent DKD progressing to ESRD. It is urgent to find new complementary and effective therapeutic agents. Growing animal researches have shown that mitophagy makes a great difference to the pathogenesis of DKD, therefore, exploration of new drugs that target the restoration of mitophagy maybe a potential perspective treatment for DKD. The use of Chinese botanical drugs (CBD) has been identified to be an effective treatment option for DKD. There is growing concern on the molecular mechanism of CBD for treatment of DKD by regulating mitophagy. In this review, we highlight the current findings regarding the function of mitophagy in the pathological damages and progression of DKD and summarize the contributions of CBD that ameliorate renal injuries in DKD by interfering with mitophagy, which will help us further explain the mechanism of CBD in treatment for DKD and explore potential therapeutic strategies for DKD.

Alzheimer's disease (AD) is associated with disturbed metabolism, prompting investigations into specific metabolic pathways that may contribute to its pathogenesis and pathology. Sphingolipids have garnered attention due to their known physiological impact on various diseases.

We conducted comprehensive profiling of sphingolipids to understand their possible role in AD. Sphingolipid levels were measured in AD brains, Cerad score B brains, and controls, as well as in induced pluripotent stem (iPS) cells (AD, PS, and control), using liquid chromatography mass spectrometry.

AD brains exhibited higher levels of sphingosine (Sph), total ceramide 1-phosphate (Cer1P), and total ceramide (Cer) compared to control and Cerad-B brains. Deoxy-ceramide (Deoxy-Cer) was elevated in Cerad-B and AD brains compared to controls, with increased sphingomyelin (SM) levels exclusively in Cerad-B brains. Analysis of cell lysates revealed elevated dihydroceramide (dhSph), total Cer1P, and total SM in AD and PS cells versus controls. Multivariate analysis highlighted the relevance of Sph, Cer, Cer1P, and SM in AD pathology. Machine learning identified Sph, Cer, and Cer1P as key contributors to AD.

Our findings suggest the potential importance of Sph, Cer1P, Cer, and SM in the context of AD pathology. This underscores the significance of sphingolipid metabolism in understanding and potentially targeting mechanisms underlying AD.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer resistant to current therapies, including oxaliplatin (Oxa). Growing evidence supports the ability of cancers to harness sphingolipid metabolism for survival. Sphingosine-1-phosphate (S1P) is an anti-apoptotic, pro-survival mediator that can influence cellular functions such as endoplasmic reticulum (ER) stress. We hypothesize that PDAC drives dysregulated sphingolipid metabolism and that S1P inhibition can enhance ER stress to improve therapeutic response to Oxa in PDAC.

RNA sequencing data of sphingolipid mediators from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression Project (GTEx) datasets were analyzed. Murine and human PDAC cell lines were treated with small interfering RNA (siRNA) against sphingosine kinase-2 (SPHK2) or ABC294640 (ABC) and incubated with combinations of vehicle control or Oxa. In an orthotopic syngeneic KPC PDAC model, tumors were treated with either vehicle control, Oxa, ABC, or combination therapy.

RNA sequencing analysis revealed multiple significantly differentially expressed sphingolipid mediators (P < 0.05). In vitro, both siRNA knockdown of SPHK2 and ABC sensitized cells to Oxa therapy (P < 0.05), and induced eukaryotic initiation factor 2α (eIF2α) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) phosphorylation, hallmarks of ER stress. In vitro therapy also increased extracellular high mobility group box 1 (HMGB1) release (P < 0.05), necessary for immunogenic cell death (ICD). In vivo combination therapy increased apoptotic markers as well as the intensity of HMGB1 staining compared to control (P < 0.05).

Our evidence suggests that sphingolipid metabolism is dysregulated in PDAC. Furthermore, S1P inhibition can sensitize PDAC to Oxa therapy through increasing ER stress and can potentiate ICD induction. This highlights a potential therapeutic target for chemosensitizing PDAC as well as an adjunct for future chemoimmunotherapy strategies.

The evaluation of toxicity and environmental behavior of bioactive lead molecules is helpful in providing theoretical support for the development of agrochemicals, in line with the sustainable development of the ecological environment. In previous work, some acethydrazide structures have been demonstrated to exhibit excellent and broad-spectrum fungicidal activity; however, its environmental compatibility needs to be further elucidated if it is to be identified as a potential fungicide. In this project, the toxicity of fungicidal acethydrazide lead compounds F51, F58, F72, and F75 to zebrafish was determined at 10 μg mL-1 and 1 μg mL-1. Subsequently, the toxic mechanism of compound F58 was preliminarily explored by histologic section and TEM observations, which revealed that the gallbladder volume of common carp treated with compound F58 increased, accompanied by a deepened bile color, damaged plasma membrane, and atrophied mitochondria in gallbladder cells. Approximately, F58-treated hepatocytes exhibited cytoplasmic heterogeneity, with partial cellular vacuolation and mitochondrial membrane rupture. Metabolomics analysis further indicated that differential metabolites were enriched in the bile formation-associated steroid biosynthesis, primary bile acid biosynthesis, and taurine and hypotaurine metabolism pathways, as well as in the membrane function-related glycerophospholipid metabolism, linolenic acid metabolism, α-linolenic acid metabolism, and arachidonic acid metabolism pathways, suggesting that the acethydrazide F58 may have acute liver toxicity to common carp. Finally, the hydrolysis dynamics of F58 was investigated, with the obtained half-life of 5.82 days. The above results provide important guiding significance for the development of new green fungicides.

Pain is a clinical condition that is currently of great concern and is often caused by tissue or nerve damage or occurs as a concomitant symptom of a variety of diseases such as cancer. Severe pain seriously affects the functional status of the body. However, existing pain management programs are not fully satisfactory. Therefore, there is a need to delve deeper into the pathological mechanisms underlying pain generation and to find new targets for drug therapy. Sphingolipids (SLs), as a major component of the bilayer structure of eukaryotic cell membranes, also have powerful signal transduction functions. Sphingolipids are abundant, and their intracellular metabolism constitutes a huge network. Sphingolipids and their various metabolites play significant roles in cell proliferation, differentiation, apoptosis, etc., and have powerful biological activities. The molecules related to sphingolipid metabolism, mainly the core molecule ceramide and the downstream metabolism molecule sphingosine-1-phosphate (S1P), are involved in the specific mechanisms of neurological disorders as well as the onset and progression of various types of pain, and are closely related to a variety of pain-related diseases. Therefore, sphingolipid metabolism can be the focus of research on pain regulation and provide new drug targets and ideas for pain.

Traumatic brain injury (TBI) is a significant source of disability in the United States and around the world and may lead to long-lasting cognitive deficits and a decreased quality of life for patients across injury severities. Following the primary injury phase, TBI is characterized by complex secondary cascades that involve altered homeostasis and metabolism, faulty signaling, neuroinflammation, and lipid dysfunction. The objectives of the present study were to (1) assess potential correlations between lipidome and cytokine changes after closed-head mild TBI (mTBI), and (2) examine the reproducibility of our acute lipidomic profiles following TBI. Cortices from 54 Sprague Dawley male and female rats were analyzed by ultra-high-performance liquid chromatography mass spectrometry (LC-MS) in both positive and negative ionization modes and multiplex cytokine analysis after single (smTBI) or repetitive (rmTBI) closed-head impacts, or sham conditions. Tissue age was a variable, given that two cohorts (n = 26 and n = 28) were initially run a year-and-a-half apart, creating inter-batch variations. We annotated the lipidome datasets using an in-house data dictionary based on exact masses of precursor and fragment ions and removed features with statistically significant differences between sham control batches. Our results indicate that lipids with high-fold change between injury groups moderately correlate with the cytokines eotaxin, IP-10, and TNF-α. Additionally, we show a significant decrease in the pro-inflammatory markers IL-1β and IP-10, TNF-α, and RANTES in the rmTBI samples relative to the sham control. We discuss the major challenges in correlating high dimensional lipidomic data with functional cytokine profiles and the implications for understanding the biological significance of two related but disparate analysis modes in the study of TBI, an inherently heterogeneous neurological disorder.

Autophagy, a self-degradative process vital for cellular homeostasis, plays a significant role in adipose tissue metabolism and tumorigenesis. This review aims to elucidate the complex interplay between autophagy, obesity, and cancer development, with a specific emphasis on how obesity-driven changes affect the regulation of autophagy and subsequent implications for cancer risk. The burgeoning epidemic of obesity underscores the relevance of this research, particularly given the established links between obesity, autophagy, and various cancers. Our exploration delves into hormonal influence, notably INS (insulin) and LEP (leptin), on obesity and autophagy interactions. Further, we draw attention to the latest findings on molecular factors linking obesity to cancer, including hormonal changes, altered metabolism, and secretory autophagy. We posit that targeting autophagy modulation may offer a potent therapeutic approach for obesity-associated cancer, pointing to promising advancements in nanocarrier-based targeted therapies for autophagy modulation. However, we also recognize the challenges inherent to these approaches, particularly concerning their precision, control, and the dual roles autophagy can play in cancer. Future research directions include identifying novel biomarkers, refining targeted therapies, and harmonizing these approaches with precision medicine principles, thereby contributing to a more personalized, effective treatment paradigm for obesity-mediated cancer.

Myoblast proliferation and differentiation are highly dynamic and regulated processes in skeletal muscle development. Given that proteins serve as the executors for the majority of biological processes, exploring key regulatory factors and mechanisms at the protein level offers substantial opportunities for understanding the skeletal muscle development. In this study, a total of 607 differentially expressed proteins between proliferation and differentiation in myoblasts were screened out using our chicken muscle antibody array. Biological function analysis revealed the importance of energy production processes and compound metabolic processes in myogenesis. Our antibody array specifically identified an upregulation of LDHA during differentiation, which was associated with the energy metabolism. Subsequent investigation demonstrated that LDHA promoted the glycolysis and TCA cycle, thereby enhancing myoblasts differentiation. Mechanistically, LDHA promotes the glycolysis and TCA cycle but inhibits the ETC oxidative phosphorylation through enhancing the NADH cycle, providing the intermediate metabolites that improve the myoblasts differentiation. Additionally, increased glycolytic ATP by LDHA induces Akt phosphorylation and activate the PI3K-Akt pathway, which might also contribute to the promotion of myoblasts differentiation. Our studies not only present a powerful tool for exploring myogenic regulatory factors in chicken muscle, but also identify a novel role for LDHA in modulating myoblast differentiation through its regulation of cellular NAD+ levels and subsequent downstream effects on mitochondrial function.

Sphingosine (Sph) plays important roles in various complex biological processes. Abnormalities in Sph metabolism can result in various diseases, including neurodegenerative disorders. However, due to the lack of rapid and accurate detection methods, understanding sph metabolic in related diseases is limited. Herein, a series of near-infrared fluorogenic probes DMS-X (X = 2F, F, Cl, Br, and I) are designed and synthesized. The fast oxazolidinone ring formation enables the DMS-2F to detect Sph selectively and ultrasensitively, and the detection limit reaches 9.33 ± 0.41 nm. Moreover, it is demonstrated that DMS-2F exhibited a dose- and time-dependent response to Sph and can detect sph in living cells. Importantly, for the first time, the changes in Sph levels induced by Aβ42 oligomers and H2 O2 are assessed through a fluorescent imaging approach, and further validated the physiological processes by which Aβ42 oligomers and reactive oxygen species (ROS)-induce changes in intracellular Sph levels. Additionally, the distribution of Sph in living zebrafish is successfully mapped by in vivo imaging of a zebrafish model. This work provides a simple and efficient method for probing Sph in living cells and in vivo, which will facilitate investigation into the metabolic process of Sph and the connection between Sph and disease pathologies.

Ceramide has a key role in the regulation of cellular senescence and apoptosis. As Ceramide levels are lowered by the action of acid ceramidase (AC), abnormally expressed in various cancers, the identification of AC inhibitors has attracted increasing interest. However, this finding has been mainly hampered by the lack of formats suitable for the screening of large libraries. We have overcome this drawback by adapting a fluorogenic assay to a 384-well plate format. The performance of this optimised platform has been proven by the screening a library of 4100 compounds. Our results show that the miniaturised platform is well suited for screening purposes and it led to the identification of several hits, that belong to different chemical classes and display potency ranges of 2-25 µM. The inhibitors also show selectivity over neutral ceramidase and retain activity in cells and can therefore serve as a basis for further chemical optimisation.