Cholesterol is the most considerable member of a family of polycyclic compounds understood as sterols, and represents an amphipathic molecule, such as phospholipids, with the polar hydroxyl group located in position 3 and the rest of the molecule is completely hydrophobic. In cells, it is usually present as free, unesterified cholesterol, or as esterified cholesterol, in which the hydroxyl group binds to a carboxylic acid and thus generates an apolar molecule. Filipin is a naturally fluorescent antibiotic that exerts a primary antifungal effect with low antibacterial activity, interfering with the sterol stabilization of the phospholipid layers and favoring membrane leakage. This polyene macrolide antibiotic does not bind to esterified sterols, but only to non-esterified cholesterol, and it is commonly used as a marker to label and quantify free cholesterol in cells and tissues. Several lines of evidence have indicated that filipin staining could be a good diagnostic tool for the cholesterol alterations present in neurodegenerative (e.g., Alzheimer’s Disease and Huntington Disease) and lysosomal storage diseases (e.g., Niemann Pick type C Disease and GM1 gangliosidosis). Here, we have discussed the uses and applications of this fluorescent molecule in lipid storage diseases and neurodegenerative disorders, exploring not only the diagnostic strength of filipin staining, but also its limitations, which over the years have led to the development of new diagnostic tools to combine with filipin approach.

The number of antibiotic-resistant microbial infections is dramatically increasing, while the discovery of new antibiotics is significantly declining. Furthermore, the activity of antibiotics is negatively influenced by the ability of bacteria to form sessile communities, called biofilms, and by the microenvironment of the infection, characterized by an acidic pH, especially in the lungs of patients suffering from cystic fibrosis (CF). Antimicrobial peptides represent interesting alternatives to conventional antibiotics, and with expanding properties. Here, we explored the effects of an acidic pH on the antimicrobial and antibiofilm activities of the AMP Esc(1-21) and we found that it slightly lost activity (from 2- to 4-fold) against the planktonic form of a panel of Gram-negative bacteria, with respect to a ≥ 32-fold of traditional antibiotics. Furthermore, it retained its activity against the sessile form of these bacteria grown in media with a neutral pH, and showed similar or higher effectiveness against the biofilm form of bacteria grown in acidic media, simulating a CF-like acidic microenvironment, compared to physiological conditions.

l-Homocysteine, formed from S-adenosyl methionine following demethylation and adenosine release, accumulates when the methionine recycling pathway and other pathways become impaired, thus leading to hyperhomocysteinemia, a biomarker in cardiovascular diseases, neurological/psychiatric disorders, and cancer. The partial oxidation of the l-homocysteine thiol group and its decarboxylation on C-alpha lead to the formation of l-homocysteinesulfinic acid (l-HCSA) and homohypotaurine (HHT), respectively. Both compounds are not readily available from commercial suppliers, which hinders the investigation of their biological activities. Herein, the chemical synthesis of l-HCSA, from l-homocystine, was the starting point for establishing the bio-based synthesis of HHT using recombinant Escherichia coli glutamate decarboxylase (EcGadB), an enzyme already successfully employed for the bio-based synthesis of GABA and its phosphinic analog. Prior to HHT synthesis, k_cat (33.92 ± 1.07) and K_M (38.24 ± 3.45 mM) kinetic constants were determined for l-HCSA on EcGadB. The results of our study show that the EcGadB-mediated synthesis of HHT can be achieved with good yields (i.e., 40% following enzymatic synthesis and column chromatography). Purified HHT was tested in vitro on primary human umbilical vein endothelial cells and rat cardiomyoblasts and compared to the fully oxidized analog, homotaurine (OT, also known as tramiprosate), in widespread pharmaceutical use. The results show that both cell lines display statistically significant recovery from the cytotoxic effects induced by H₂O₂ in the presence of HHT.