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Background: CA19.9 is the unique marker recommended for the preoperative staging and the follow-up of patients suffering from pancreatic ductal adenocarcinoma (PDAC) but up to 30% of PDAC patients maintain normal CA19.9 values and cannot be monitored in this way. Lewis a (Lea Galβ1,3[Fucα1,4]GlcNAc) and b (Leb, Fucα1,2Galβ1,3[Fucα1,4]GlcNAc) are antigens which are structurally similar to sialyl-Lewis a (Siaα2,3Galβ1,3[Fucα1,4]GlcNAc), the epitope of CA19.9., Methods: We set an ELISA procedure determining the levels of Lea, Leb, and CA19.9 in the blood of healthy individuals or PDAC patients. Moreover, such antigens were also detected in cancer resections by immunofluorescence microscopy, and the levels of glycosyltransferase transcripts involved in Lewis antigen biosynthesis were determined by RT-qPCR., Results: In our cohort of 116 healthy individuals, the distribution of circulating Lea and Leb was similar to that of CA19.9, allowing us to set putative cutoff values for both antigens. In a cohort of 115 PDAC patients, the differential distribution with respect to the controls was statistically significant for both antigens (p < 0.001). Out of 37 patients presenting normal CA19.9 values, 15 patients presented Lea or Leb above the cutoffs. By immunofluorescence, Lea, Leb and CA19.9 were all detected in cancer resections and expression levels were heterogeneous among patients in terms of intensity, localization and diffusion. The levels of relevant glycosyltransferase transcripts were found to be heterogeneous between cancers of different patients and no association was detectable with the levels of any circulating antigen., Conclusions: The concurrent quantification of Lea and Leb together with CA19.9 improves the management of PDAC patients., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

We evaluated the performance of three different single-phase extraction methods to be used before untargeted lipidomics analysis by Liquid Chromatography High-Resolution Mass Spectrometry. Lipids were extracted from a pool of healthy human donors' plasma in triplicates and run in both positive and negative ESI. The most satisfactory results were attained using methanol/chloroform (2:1, v/v) mixture. Eventually, we evaluated whether a filtration of the samples could be beneficial to yield cleaner and more mass-friendly extracts. Instead of using syringes, we set up a method we called tip-tip filtration, which requires the usage of a filtrating pipette tip. This way of purification led to superior results than the solvent extraction method alone. This additional procedure not only increased reproducibility but also allowed the same lipid coverage. In addition, it permitted to spare time and money, as tip-tip filtration is not particularly expensive nor time-consuming and hopefully it may be useful to increase analytical column lifetime., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background and Aims: CA.ME.LI.A (CArdiovascular risks, MEtabolic syndrome, LIver and Autoimmune disease) is a cross-sectional, epidemiological study performed between 2009-2011 in Abbiategrasso (Milan, Italy) to estimate the prevalence of cardiovascular risk factors, metabolic syndrome, liver and autoimmune diseases in the general adult population. This report focuses on the description and presentation of baseline characteristics of the population., Methods and Results: Citizens were randomly selected from the city electoral registers (n = 30903), yielding a sample of 2554 subjects (M = 1257, F = 1297; age, 47 ± 15 yrs; range 18-77 yrs). Men had higher prevalence of overweight or obesity (60.8% vs 41.6%; p < 0.0001) and greater thickness of visceral adipose tissue (40 ± 19 vs 27 ± 17 mm; p < 0.0001); no gender difference was found in subcutaneous adipose tissue thickness. Men also showed higher levels of serum triglycerides, γ-GT, fasting blood glucose, insulin and Homa-IR Index, while HDL, CRP, and prevalence of elevated (>5.0 mg/L) CRP were lower. Compared to normal weight men, risk-ratio (RR) of CRP elevation was 1.32 (ns) in overweight and 2.68 (p < 0.0001) in obese subjects. The corresponding figures in females were 2.68 (p < 0.0001) and 5.18 (p < 0.0001). Metabolic syndrome was more frequent in men (32.7% vs 14.5%; RR: 2.24, p < 0.0001). Interadventitia common carotid artery diameter was higher in men and increased with age and BMI., Conclusions: The present study reports on the overall characteristics of a large population from Northern Italy. It aims to identify the associations among cardiovascular risk factors to prevent their development and progression, improve healthy lifestyle and identify subjects liable to pharmacological interventions., Competing Interests: Declaration of competing interest The authors have nothing to disclose., (Copyright © 2021 The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

Short and medium fatty acids derived from either dietary sources, gut microbiota, and liver production might play a role in the modulation of metabolism and inflammation. The outcome of different autoimmune or inflammatory diseases could be related to microbiota composition and consequently fatty acids production. Their analytical detection, historically completed by GC, was herein investigated using a sensitive approach of LC-MS/MS with straightforward chemical derivatization, using 3-NPH, to the respective acylhydrazines. An isopropanol protein precipitation coupled to LC-MS/MS analysis allowed to separate and quantify butyric, valeric, hexanoic acid and their branched forms. The serum physiological ranges of short and medium chain fatty acids were determined in a heterogeneous healthy population (n = 54) from 18 to 85 years finding a concentration of 935.6 ± 246.5 (butyric), 698.8 ± 204.7 (isobutyric), 62.9 ± 15.3 (valeric), 1155.0 ± 490.4 (isovaleric) and 468.7 ± 377.5 (hexanoic) ng/mL respectively (mean ± SD). As expected, the biological levels in human serum are reasonably wide-ranging depending on several factors such as body-weight, gut microbiome dysbiosis, gut permeability, cardiometabolic dysregulation, and diet., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Ceramide is emerging as one of the players of inflammation in lung diseases. However, data on its inflammatory role in Cystic Fibrosis (CF) as part of the extracellular machinery driven by lung mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) are missing. We obtained an in vitro model of CF-MSC by treating control human lung MSCs with a specific CFTR inhibitor. We characterized EVs populations derived from MSCs (ctr EVs) and CF-MSCs (CF-EVs) and analyzed their sphingolipid profile by LC-MS/MS. To evaluate their immunomodulatory function, we treated an in vitro human model of CF, with both EVs populations. Our data show that the two EVs populations differ for the average size, amount, and rate of uptake. CF-EVs display higher ceramide and dihydroceramide accumulation as compared to control EVs, suggesting the involvement of the de novo biosynthesis pathway in the parental CF-MSCs. Higher sphingomyelinase activity in CF-MSCs, driven by inflammation-induced ceramide accumulation, sustains the exocytosis of vesicles that export new formed pro-inflammatory ceramide. Our results suggest that CFTR dysfunction associates with an enhanced sphingolipid metabolism leading to the release of EVs that export the excess of pro-inflammatory Cer to the recipient cells, thus contributing to maintain the unresolved inflammatory status of CF., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

During the lactation, the choice of a proper antibiotic is crucial since the drug can cross into breast milk causing toxicity to the infant. Therefore, an extraction protocol and LC/MS-MS method for the determination of daptomycin in human milk and plasma were developed, validated and applied to a case of a breastfeeding mother affected by a purulent acute soft skin infection treated with daptomycin. Because of daptomycin high protein binding and its high molecular weight, the optimisation of the extraction protocol and analytical conditions were deeply investigated, and several parameters were taken into account: in particular the type of extraction, internal standard, the type of organic modifier, pH of the aqueous solution, and gradient. The use of a protein precipitation protocol coupled to a C8-reverse phase LC-MS/MS allows for a reliable quantification of daptomycin in both plasma (in the range of 19-199 μg/mL) and breast milk (in the range of 0.12-0.32 μg/mL). The determination of milk/plasma (M/P) ratio, which ranged from 0.002 to 0.006, allowed to assess that daptomycin, effective for the mother, was contemporarily safe for the breastfed newborn., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Accumulating experimental and clinical evidences over the last decade indicate that GLP-1 analogues have a series of central nervous system and peripheral target tissues actions which are able to significantly influence the liver metabolism. GLP-1 analogues pleiotropic effects proved to be efficacious in T2DM subjects not only reducing liver steatosis and ameliorating NAFLD and NASH, but also in lowering plasma glucose and liver inflammation, improving cardiac function and protecting from kidney dysfunction. While the experimental and clinical data are robust, the precise mechanisms of action potentially involved in these protective multi-target effects need further investigation. Here we present a systematic review of the most recent literature data on the multi-target effects of GLP-1 analogues on the liver, on adipose and muscular tissue and on the nervous system, all capable of influencing significant aspects of the fatty liver disease physiopathology. From this analysis, we can conclude that the multi-target beneficial action of the GLP-1 analogues could explain the positive effects observed in animal and human models on progression of NAFLD to NASH., (Copyright © 2018 Elsevier Ltd. All rights reserved.)