Lactosylceramide is necessary for the biosynthesis of almost all classes of glycosphingolipids and plays a relevant role in pathways involved in neuroinflammation. It is synthesized by the action of galactosyltransferases B4GALT5 and B4GALT6, which transfer galactose from UDP-galactose to glucosylceramide. Lactosylceramide synthase activity was classically determined in vitro by a method based on the incorporation of radiolabeled galactose followed by the chromatographic separation and quantitation of the product by liquid scintillation counting. Here, we used deuterated glucosylceramide as the acceptor substrate and quantitated the deuterated lactosylceramide product by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). We compared this method with the classical radiochemical method and found that the reactions have similar requirements and provide comparable results in the presence of high synthase activity. Conversely, when the biological source lacked lactosylceramide synthase activity, as in the case of a crude homogenate of human dermal fibroblasts, the radiochemical method failed, while the other provided a reliable measurement. In addition to being very accurate and sensitive, the proposed use of deuterated glucosylceramide and LC-MS/MS for the detection of lactosylceramide synthase in vitro has the relevant advantage of avoiding the costs and discomforts of managing radiochemicals.

The last step of ex novo ceramide biosynthesis consists of the conversion of dihydroceramide into ceramide catalyzed by sphingolipid Δ4-desaturase DEGS1. DEGS1 variants were found to be responsible for heterogeneous clinical pictures belonging to the family of hypomyelinating leukodystrophies. To investigate the mechanisms making such variants pathogenic, we designed a procedure for the efficient detection of desaturase activity in vitro using LC-MS/MS and prepared a suitable cell model knocking out DEGS1 in HEK-293T cells through CRISPR-Cas9 genome editing (KO-DES-HEK). Transfecting KO-DES-HEK cells with DEGS1 variants, we found that their transcripts were all overexpressed as much as the WT transcripts, while the levels of cognate protein were 40%-80% lower. In vitro desaturase activity was lost by many variants except L175Q and N255S, which maintain a catalytic efficiency close to 12% of the WT enzyme. Metabolic labeling of KO-DES-HEK with deuterated palmitate followed by LC-MS/MS analysis of the formed sphingolipids revealed that the ceramide/dihydroceramide and sphingomyelin/dihydrosphingomyelin ratios were low and could be reverted by the overexpression of WT DEGS1 as well as of L175Q and N255S variants, but not by the overexpression of all other variants. Similar analyses performed on fibroblasts from a patient heterozygous for the N255S variant showed very low variant DEGS1 levels and a low ratio between the same unsaturated and saturated sphingolipids formed upon metabolic labeling, notwithstanding the residual activity measured at high substrate and homogenate protein concentrations. We conclude that loss of function and reduced protein levels are both relevant in disease pathogenesis.

Background and Aims: Cholangiocarcinoma (CCA) is a globally rare, increasingly incident cancer. Metabolic reprogramming is common in cancer cells, and altered lipid homeostasis favors tumor development and progression. Previous studies have described lipid deregulation in HCC cells, while in CCA, the lipidome profile is still poorly characterized. Methods: We used liquid chromatography–tandem mass spectrometry to examine the lipid level profile of intrahepatic CCA (iCCA) and non-tumor surrounding tissue from patients, as well as in patients’ and healthy controls’ sera. Results: All lipid classes were upregulated in tumor specimens and iCCA-derived sera. Newly synthesized fatty acids (FAs) accumulated in iCCA and were only marginally directed to mitochondrial β-oxidation and scarcely folded in lipid droplets as neutral species. Metabolic flux assay showed that FAs were instead redirected toward plasma membrane formation and remodeling, being incorporated into phospholipids and sphingomyelin. A distinct lipid droplet and macrophage distribution was revealed by immunohistochemistry and Imaging Mass Cytometry. Lipid droplets were fewer in iCCA than in normal tissue and present mainly in the intratumoral fibrous septa and in M2 macrophages. Monocytes modified their lipid content and phenotype in the presence of iCCA cells, and the same effect could be recapitulated by FA supplementation. Conclusions: Our results reveal a profound alteration in the lipid content of iCCA tissues and demonstrate that FA accumulation prompts iCCA aggressiveness by supporting membrane biogenesis, generating bioactive lipids that boost proliferation, and by modifying macrophage phenotype.

Angiopoietin-like protein 3 (ANGPTL3) is a plasmatic protein that plays a crucial role in lipoprotein metabolism by inhibiting the lipoprotein lipase (LPL) and the endothelial lipase (EL) responsible for the hydrolysis of phospholipids on high-density lipoprotein (HDL). Interest in developing new pharmacological therapies aimed at inhibiting ANGPTL3 has been growing due to the hypolipidemic and antiatherogenic profile observed in its absence. The goal of this study was the in silico characterization of the interaction between ANGPTL3 and EL. Because of the lack of any structural information on both the trimeric coiled-coil N-terminal domain of ANGPTL3 and the EL homodimer as well as data regarding their interactions, the first step was to obtain the three-dimensional model of these two proteins. The models were then refined via molecular dynamics (MD) simulations and used to investigate the interaction mechanism. The analysis of interactions in different docking poses and their refinement via MD allowed the identification of three specific glutamates of ANGPTL3 that recognize a positively charged patch on the surface of EL. These ANGPTL3 key residues, i.e., Glu154, Glu157, and Glu160, could form a putative molecular recognition site for EL. This study paves the way for future investigations aimed at confirming the recognition site and at designing novel inhibitors of ANGPTL3.

Background/Objectives: Chronic graft-versus-host disease (cGVHD) and oral lichen planus (LPO) are chronic inflammatory conditions with similar oral manifestations. This study aimed to assess whether serum and salivary cytokines (IL-1α, IL-6, IL-17) could serve as reliable biomarkers for cGVHD. Methods: A prospective cohort study was conducted involving cGVHD patients, LPO patients, and healthy controls. Cytokine levels in serum and saliva were measured by ELISA and compared across the groups using the Kruskal–Wallis test. Results: IL-17 levels were significantly elevated in the serum of cGVHD patients compared to LPO patients and controls (p < 0.05). However, IL-6 and IL-1α did not show significant differences among the groups. A comparison of salivary samples between the three groups did not reach statistical significance (p > 0.05). Conclusions: This study suggests that IL-17 could be a potential biomarker for cGVHD-related inflammation, warranting further investigation. Salivary samples do not seem to be a reliable biological marker for the diagnosis of cGVHD. The findings underline the need for larger studies to validate these preliminary results.

Background: Pathogenic ST3GAL3 variants cause neurological and cognitive impairment, defining a distinct congenital disorder of glycosylation (ST3GAL3-CDG). Nonetheless, limited enzyme characterization exists due to the lack of a non-radiochemical assay. Methods: Here, we developed an LC-MS/MS-based method using the artificial substrate para-nitrophenyl-lacto-N-biose (LNB-pNP; Galβ1,3GlcNAcβ1-O-C6H4NO2) to measure ST3GAL3 activity in vitro. Results: A peak corresponding to sialyl-LNB-pNP was detected in reactions with homogenate from HEK-293T cells transfected with pCDNA3 ST3GAL3 plasmid, but was virtually absent in mock-transfected cells. A substrate dependence curve provided an apparent Km value for the substrate (0.40 mM) and closely matched values from prior radiochemical methods. No activity was detected with homogenates from cells expressing pathogenic ST3GAL3 variants, except p.A13D, which is known to retain about 10% of residual activity. Compared to ST3GAL4 and ST3GAL6, ST3GAL3 showed markedly higher specificity toward LNB-pNP, lactotetraosylceramide (Lc4) and asialo-GM1, which are rather specific substrates. Instead, neo-lactotetraosylceramide (neoLc4) was processed by all three ST3GALs. Conclusions: These findings suggest that ST3GAL4 or ST3GAL6 cannot compensate for ST3GAL3 loss in the biosynthesis of gangliosides sialyl-Lc4 and GM1b, but may do so for sialyl-neoLc4. This non-radiochemical assay enables screening and diagnostic evaluation of novel ST3GAL3 variants potentially associated with ST3GAL3-CDG.