Tommaso Beccari

The journal retracts the article titled “Neutral Sphingomyelinase Modulation in the Protective/Preventive Role of rMnSOD from Radiation-Induced Damage in the Brain”

Background/Objectives: Extracellular Vesicles (EVs) have shown great promise as diagnostic and therapeutic tools, as well as pharmacological nanocarriers. Various strategies are being explored to develop EVs for monitoring, imaging, loading with pharmacological agents, and surface decoration with tissue-specific ligands. EVs derived from Mesenchymal Stromal Cells (MSC-EVs) are of particular interest both as therapeutics per se and as natural nanocarriers for the targeted delivery of biotherapeutics. Methods: In this study, we investigated the ability of different tags to deliver a reporter protein into canine MSC-EVs with the aim of identifying the most effective endogenous loading mechanism. To this aim, canine MSCs were engineered to express the Green Fluorescent Protein (GFP) fused to CD63, Syntenin-1, TSG101, and the palmitoylation signal of Lck, which were expected to promote GFP incorporation into EVs. Overexpression of tagged GFP in canine MSCs was confirmed by Western blotting and examined by confocal microscopy and transmission electron microscopy to map intracellular localization. Results: All tags were able to deliver GFP into EVs. Syntenin-1 showed relatively high loading efficiency and secretion index but exhibited a diffuse localization pattern in the transfected cells. The palmitoylation signal showed low loading efficiency and localization specificity. TSG101 displayed a morphological pattern consistent with specific localization in endosomal structures, but its low expression level prevented further evaluations. Finally, CD63 showed the highest expression efficiency, as GFP-CD63 levels were approximately 5-fold higher than untagged GFP. Conclusions: In conclusion, CD63 emerged as the most suitable tag for canine MSC-EV engineering. Indeed, even if the secretion index favours Syntenin-1, CD63’s higher abundance in the lysate suggests its substantial post-secretion uptake. Further studies aimed at elucidating CD63’s specific contribution and identifying the domains involved in vesicle trafficking could provide valuable insights into EV bioengineering.

Background/Objectives: Extracellular Vesicles (EVs) have shown promise as diagnostic and therapeutic tools as well as pharmacological nanocarriers. Strategies are being explored to develop EVs to enable their monitoring, imaging, loading with pharmacological agents and surface decoration with tissue-specific ligands. EVs derived from mesenchymal stromal cells (MSC-EVs) are of interest both as therapeutics per se and as natural nanocarriers for the targeted delivery of biotherapeutics. Methods: In this study, we investigated the ability of different tags to deliver a reporter protein into canine MSC-EVs with the aim of identifying the most effective endogenous loading mechanism. To this end, canine MSCs were engineered to express the green fluorescent protein (GFP) associated with CD63, syntenin-1, TSG101 and the palmitoylation signal of Lck, which were expected to introduce GFP into EVs. Overexpression of tagged GFP in canine MSCs was detected by Western blotting and examined by confocal and transmission electron microscopy to map intracellular localization. Results: All tags were able to deliver GFP into EVs. Syntenin-1 showed high efficiency but exhibited a diffuse localization pattern in the transfected cells. Palmitoylation signal showed low efficiency and low specificity in terms of localization. TSG101 showed a morphological pattern consistent with a specific localization in endosomal structures, but its low expression did not allow further considerations. Finally, CD63 showed the highest expression efficiency, as GFP-CD63 was 5-fold higher than untagged GFP. Conclusions: In conclusion, CD63 is the most effective tag for canine MSC-EVs engineering. Further studies aimed at better deciphering its contribution and clarifying which part of the molecule is involved in vesicle trafficking could provide insights for EV bioengineering.

Sphingomyelin is a crucial molecule in the sphingolipid metabolic pathway, and its action is closely related to that of vitamin D3. Both molecules are recognized for their involvement in brain pathophysiology. In this study, the effect of a sphingomyelin + vitamin D3-enriched diet was investigated in rabbits. The results showed a strong immunopositive GFAP staining in the brain’s white matter. Furthermore, a remodeling of the balance between glycero-phospholipids and ether-phospholipids was observed in the brain, along with an increase in ceramides and hexose ceramides, molecules relevant for the structure, function, and stability of myelin. Taken together, these findings provide clues as to how the combination sphingomyelin + vitamin D3 may play a vital role in normal brain physiology and could potentially be leveraged in the context of neurodegenerative diseases.

Ceramide is a critical molecule in both the physiology and pathology of the central nervous system. The most studied aspect is its effect on embryonic/stem cells. A salient question is whether low doses of ceramide induce neuronal differentiation without interfering with sphingolipid metabolism and whether high doses can be used in glioblastoma for their cytotoxic effect. Here, we examined the effect of a high dose of ceramide (13 µM) on HN9.10e cells. Interestingly, 13 µM ceramide induced an immediate increase in cell viability, followed by an increase in the number of mitochondria. Microscopic and morphometric analysis revealed a decrease in the number of differentiated cells with 13 µM compared to 0.1 µM but with longer neurites. Furthermore, the lipidomic study demonstrated an increase in the formation of medium–long-chain ceramide and sphingomyelin species and sphingosine 1 phosphate. Sphingolipid modification correlated with SMPD3, ASAH2, and SPHK2 gene expression coding for neutral sphingomyenase 2, ceramidase 2, and sphingosine kinase 2, respectively. Overall, our data show that the variety of responses to ceramide of the same cell type is dependent on the concentration used. Low doses do not affect sphingolipid metabolism, and high doses do so with a different cellular response.

Background: Persistent alterations in taste and smell affect a significant proportion of individuals following COVID-19, representing a component of post-acute COVID-19 syndrome, commonly referred to as long COVID. The degradation of sphingomyelin by acid sphingomyelinase is regarded as a biomarker for acquired demyelinating neuropathies. Objectives: This study was aimed to enroll women who contracted COVID-19 during pregnancy and experienced persistent alterations in taste and/or smell for more than 1 year post-infection, in comparison to pregnant women without any disturbances in these senses. Methods: The patients were subjected to a questionnaire investigating smell and taste disorders more than 1 year after the infection. Then, the levels of acid sphingomyelinase in the plasma of the participants were assessed. Results: The results showed that in women who had been pregnant and who had been infected with SARS Cov-2 during the COVID period and who still had taste and smell disorders 1 year later, plasma acid sphingomyelinase levels were double that of pregnant women who had contracted the infection during the COVID period but had not reported taste and smell disorders and that of pregnant women analyzed after the COVID period. Conclusions: The results suggest a hypothesis that the persistence of sensory disturbances in long COVID was probably due to a failure to utilize brain circuitry with demyelination resulting from chemosensory dysfunction of the olfactory epithelium.

The role of sphingomyelin metabolism and vitamin C in cancer has been widely described with conflicting results ranging from a total absence of effect to possible preventive and/or protective effects. The aim of this study was to establish the possible involvement of sphingomyelin metabolism in the changes induced by vitamin C in breast cancer cells. The MCF7 cell line reproducing luminal A breast cancer and the MDA-MB-231 cell line reproducing triple-negative breast cancer were used. Cell phenotype was tested by estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2 expression, and proliferation index percentage. Sphingomyelin was localized by an EGFP-NT-Lys fluorescent probe. Sphingomyelin metabolism was analyzed by RT-PCR, Western blotting and UFLC-MS/MS. The results showed that a high dose of vitamin C produced reduced cell viability, modulated cell cycle related genes, and changed the cell phenotype with estrogen receptor downregulation in MCF7 cell. In these cells, the catabolism of sphingomyelin was promoted with a large increase in ceramide content. No changes in viability and molecular expression were observed in MB231 cells. In conclusion, a high dose of vitamin C induces changes in the luminal A cell line involving sphingomyelin metabolism.