Loss of function and reduced levels of sphingolipid desaturase DEGS1 variants are both relevant in disease mechanism.

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.

Medicinal Cannabis: Extended Stability of Cannabis Extracts Produced Using a New Ethanol-based Extraction Method.

Cannabis as a therapeutic agent is increasing in popularity all around the globe, particularly in Western countries, and its potential is now well assessed. On the other hand, each country has its own regulation for the preparation of cannabis macerated oils; in Italy, there are only a few preparation methods allowed. With this work, we aim to perform a stability study of cannabis oils produced with a novel method for the extraction of cannabinoids from cannabis inflorescence. Three different varieties of cannabis were used, with and without the adding of tocopherol acetate as an antioxidant. Cannabinoids were extracted using ethanol at room temperature; then, the solvent was evaporated under reduced pressure and the preparations reconstituted with olive oil. In this work, we assessed the stability of both cannabinoids and terpenes in these formulas over 8 months. Cannabinoid stability was assessed by monitoring the concentrations of THC and CBD, while terpene stability was assessed by monitoring ß-Caryophyllene and α-Humulene concentrations. Stability of the extracts was not influenced by the presence of tocopherol acetate, though refrigeration seems to be detrimental for a long storage of products, especially regarding THC concentrations. The improvements offered by this method reside in the flexibility in controlling the concentration of the extract and the ability to produce highly concentrated oils, alongside the possibility to produce standardized oils despite the variability of the starting plant material.