Summary: Dictyostelium discoideum is one of eight non‐mammalian model organisms recognized by the National Institute of Health for the study of human pathology. The use of this slime mould is possible owing to similarities in cell structure, behaviour and intracellular signalling with mammalian cells. Its haploid set of chromosomes completely sequenced amenable to genetic manipulation, its unique and short life cycle with unicellular and multicellular stages, and phenotypic richness encoding many human orthologues, make Dictyostelium a representative and simple model organism to unveil cellular processes in human disease. Dictyostelium studies within the biomedical field have provided fundamental knowledge in the areas of bacterial infection, immune cell chemotaxis, autophagy/phagocytosis and mitochondrial and neurological disorders. Consequently, Dictyostelium has been used to the development of related pharmacological treatments. Herein, we review the utilization of Dictyostelium as a model organism in biomedicine.

Nitro-fatty acids (NO2-FA) undergo reversible Michael adduction reactions with cysteine and histidine residues leading to the post-translational modification (PTM) of proteins. This electrophilic character of NO2-FA is strictly related to their biological roles. The NO2-FA-induced PTM of signaling proteins can lead to modifications in protein structure, function, and subcellular localization. The nitro lipid-protein adducts trigger a series of downstream signaling events that culminates with anti-inflammatory, anti-hypertensive, and cytoprotective effects mediated by NO2-FA. These lipoxidation adducts have been detected and characterized both in model systems and in biological samples by using mass spectrometry (MS)-based approaches. These MS approaches allow to unequivocally identify the adduct together with the targeted residue of modification. The identification of the modified proteins allows inferring on the possible impact of the NO2-FA-induced modification. This review will focus on MS-based approaches as valuable tools to identify NO2-FA-protein adducts and to unveil the biological effect of this lipoxidation adducts.

Cancer associated body wasting is the cause of physical disability, reduced tolerance to anticancer therapy and reduced survival of cancer patients and, similarly to cancer, its incidence is increasing. There is no cure for this clinical condition, and the pathophysiological process involved is largely unknown. Exercise training appears as the gold standard non-pharmacological therapy for the management of this wasting syndrome. Herein we used a lipidomics approach based on liquid chromatography coupled with high-resolution mass spectrometry (LC-HR-MS) to study the effect of exercise in the modulation of phospholipids profile of mitochondria isolated from gastrocnemius muscle of a pre-clinical model of urothelial carcinoma-related body wasting (BBN induced), submitted to 13 weeks of treadmill exercise after diagnosis. Multivariate analysis showed a close relationship between the BBN exercise group and both control groups (control sedentary and control exercise), while the BBN sedentary group was significantly separated from the control groups and the BBN exercise group. Univariate statistical analysis revealed differences mainly in phosphatidylserine (PS) and cardiolipin (CL), although some differences were also observed in phosphatidylinositol (PI, LPI) and phosphatidylcholine (PC) phospholipids. PS with shorter fatty acyl chains were up-regulated in the BBN sedentary group, while the other species of PS with longer FA and a higher degree of unsaturation were down-regulated, but the BBN exercise group was mostly similar to control groups. Remarkably, exercise training prevented these alterations and had a positive impact on the ability of mitochondria to produce ATP, restoring the healthy phospholipid profile. The remodelling of mitochondria phospholipid profile in rats with urothelial carcinoma allowed confirming the importance of the lipid metabolism in mitochondria dysfunction in cancer-induced skeletal muscle remodelling. The regulation of phospholipid biosynthetic pathways observed in the BBN exercise group supported the current perspective that exercise is an adequate therapeutic approach for the management of cancer-related muscle remodeling.

Ashbya gossypii is a filamentous fungus that is currently exploited for the industrial production of riboflavin. In addition, metabolically engineered strains of A. gossypii have also been described as valuable biocatalysts for the production of different metabolites such as folic acid, nucleosides, and biolipids. Hence, bioproduction in A. gossypii relies on the availability of well-performing gene expression systems both for endogenous and heterologous genes. In this regard, the identification of novel promoters, which are critical elements for gene expression, decisively helps to expand the A. gossypii molecular toolbox. In this work, we present an adaptation of the Dual Luciferase Reporter (DLR) Assay for promoter analysis in A. gossypii using integrative cassettes. We demonstrate the efficiency of the analysis through the identification of 10 new promoters with different features, including carbon source-regulatable abilities, that will highly improve the gene expression platforms used in A. gossypii. Three novel strong promoters (P_CCW12, P_SED1, and P_TSA1) and seven medium/weak promoters (P_HSP26, P_AGL366C, P_TMA10, P_CWP1, P_AFR038W, P_PFS1, and P_CDA2) are presented. The functionality of the promoters was further evaluated both for the overexpression and for the underexpression of the A. gossypii MSN2 gene, which induced significant changes in the sporulation ability of the mutant strains.

Lipids are well‐known targets of reactive nitrogen species and this reaction leads to the formation of nitrated lipids that have been associated with anti‐inflammatory and cytoprotective effects. Nitro‐fatty acids (NO₂‐FA) are highly electrophilic compounds that can form covalent adducts with proteins, leading to the formation of lipoxidation adducts, which modulate the protein structure and function. Nitrated phospholipids (NO₂‐PL) have been detected recently in biological samples, but their biological effects are unknown, although similarly to what has been described for nitrated lipids, it has been hypothesized that they may react with peptides and proteins. In this study, in vitro biomimetic assays are used to synthetize adducts of nitrated POPC (NO₂POPC), already detected in biological samples, and GSH peptide. The formation of NO₂POPC‐GSH adducts is studied by ESI‐MS and MS², using both low and high energy CID in different MS platforms: a LXQ linear ion trap, a Q‐TOF 2, and a Q‐Exactive Hybrid Quadrupole‐Orbitrap. Typical product ions observed under MS² conditions are modified b, y, and C ions bearing NO₂POPC covalently linked that unequivocally confirms the presence of the lipid‐peptide adduct. Typical loss of HNO₂ is only observed in the MS² of the mono‐charged precursor ions, [M+H]⁺. Product ions at m/z 184 or neutral loss of 183 Da are assigned as typical fragmentations that confirm the presence of the phosphatidylcholine. In summary, the characterization of nitro PL‐peptide adducts by MS and MS² allows the identification of the structure and specific MS² reporter ions to be used to pinpoint these adductions in biological systems.Practical Applications: The covalent interaction between nitro phospholipids and peptides suggests a new pathway in cellular transduction of nitroxidative stress signal. This adduction can be considered a post‐translational modification (PTM) of lipoxidation type, similarly as it has been described for nitro‐fatty acids and with important physiological implications. The identification and characterization of the nitro phospholipids and peptides adducts are possible by MS and tandem MS. This analytical technique also represents a robust and sensitive approach for detection of nitro‐lipids adduction to peptides or proteins in biological samples, allowing to disclose their physiological and clinical implications. Tandem MS fingerprinting is an essential feature for this purpose, so in this work, the identification of the reporter ions typical for this type of lipoxidation adducts is provided. These reporter ions can be used to design target (NL, PIS, or MRM) approaches to detect these type of PTM in biological environments.Nitrated phospholipids can adduct covalently to nucleophilic peptide gluthathione, as demonstrated by characterization using tandem MS.