Theoretical Studies of Cyanophycin Dipeptides as Inhibitors of Tyrosinases

The three-dimensional structure of tyrosinase has been crystallized from many species but not from Homo sapiens. Tyrosinase is a key enzyme in melanin biosynthesis, being an important target for melanoma and skin-whitening cosmetics. Several studies employed the structure of tyrosinase from Agaricus bisporus as a model enzyme. Recently, 98% of human genome proteins were elucidated by AlphaFold. Herein, the AlphaFold structure of human tyrosinase and the previous model were compared. Moreover, tyrosinase-related proteins 1 and 2 were included, along with inhibition studies employing kojic and cinnamic acids. Peptides are widely studied for their inhibitory activity of skin-related enzymes. Cyanophycin is an amino acid polymer produced by cyanobacteria and is built of aspartic acid and arginine; arginine can be also replaced by other amino acids. A new set of cyanophycin-derived dipeptides was evaluated as potential inhibitors. Aspartate–glutamate showed the strongest interaction and was chosen as a leading compound for future studies.     

Detection of Serum-Specific IgE by Fluoro-Enzyme Immunoassay for Diagnosing Type I Hypersensitivity Reactions to Penicillins

Diagnosis of type I hypersensitivity reactions (IgE-mediated reactions) to penicillins is based on clinical history, skin tests (STs), and drug provocation tests (DPTs). Among in vitro complementary tests, the fluoro-enzyme immunoassay (FEIA) ImmunoCAP^® (Thermo-Fisher, Waltham, MA, USA) is the most widely used commercial method for detecting drug-specific IgE (sIgE). In this study, we aimed to analyze the utility of ImmunoCAP^® for detecting sIgE to penicillin G (PG) and amoxicillin (AX) in patients with confirmed penicillin allergy. The study includes 139 and 250 patients evaluated in Spain and Italy, respectively. All had experienced type I hypersensitivity reactions to penicillins confirmed by positive STs. Additionally, selective or cross-reactive reactions were confirmed by DPTs in a subgroup of patients for further analysis. Positive ImmunoCAP^® results were 39.6% for PG and/or AX in Spanish subjects and 52.4% in Italian subjects. When only PG or AX sIgE where analyzed, the percentages were 15.1% and 30.4%, respectively, in Spanish patients; and 38.9% and 46% in Italian ones. The analysis of positive STs showed a statistically significant higher percentage of positive STs to PG determinants in Italian patients. False-positive results to PG (16%) were detected in selective AX patients with confirmed PG tolerance. Low and variable sensitivity values observed in a well-defined population with confirmed allergy diagnosis, as well as false-positive results to PG, suggest that ImmunoCAP^® is a diagnostic tool with relevant limitations in the evaluation of subjects with type I hypersensitivity reactions to penicillins.     

Noncatalytic Domains in DNA Glycosylases

Many proteins consist of two or more structural domains: separate parts that have a defined structure and function. For example, in enzymes, the catalytic activity is often localized in a core fragment, while other domains or disordered parts of the same protein participate in a number of regulatory processes. This situation is often observed in many DNA glycosylases, the proteins that remove damaged nucleobases thus initiating base excision DNA repair. This review covers the present knowledge about the functions and evolution of such noncatalytic parts in DNA glycosylases, mostly concerned with the human enzymes but also considering some unique members of this group coming from plants and prokaryotes.     

Increased Acetylcholine Levels and Other Brain Effects in 5XFAD Mice after Treatment with 8,14-Dihydroxy Metabolite of Efavirenz

Efavirenz (EFV), an FDA-approved anti-HIV drug, has off-target binding to CYP46A1, the CNS enzyme which converts cholesterol to 24-hydroxycholesterol. At small doses, EFV allosterically activates CYP46A1 in mice and humans and mitigates some of the Alzheimer’s disease manifestations in 5XFAD mice, an animal model. Notably, in vitro, all phase 1 EFV hydroxymetabolites activate CYP46A1 as well and bind either to the allosteric site for EFV, neurotransmitters or both. Herein, we treated 5XFAD mice with 8,14-dihydroxyEFV, the binder to the neurotransmitter allosteric site, which elicits the highest CYP46A1 activation in vitro. We found that treated animals of both sexes had activation of CYP46A1 and cholesterol turnover in the brain, decreased content of the amyloid beta 42 peptide, increased levels of acetyl-CoA and acetylcholine, and altered expression of the brain marker proteins. In addition, male mice had improved performance in the Barnes Maze test and increased expression of the acetylcholine-related genes. This work expands our knowledge of the beneficial CYP46A1 activation effects and demonstrates that 8,14-dihydroxyEFV crosses the blood–brain barrier and has therapeutic potential as a CYP46A1 activator.     

Lipophilic Bioactive Compounds Transported in Triglyceride-Rich Lipoproteins Modulate Microglial Inflammatory Response

Microglial cells can contribute to Alzheimer’s disease by triggering an inflammatory response that leads to neuronal death. In addition, the presence of amyloid-β in the brain is consistent with alterations in the blood–brain barrier integrity and triglyceride-rich lipoproteins (TRL) permeation. In the present work, we used lab-made TRL as carriers of lipophilic bioactive compounds that are commonly present in dietary oils, namely oleanolic acid (OA), α-tocopherol (AT) and β-sitosterol (BS), to assess their ability to modulate the inflammatory response of microglial BV-2 cells. We show that treatment with lab-made TRL increases the release and gene-expression of IL-1β, IL-6, and TNF-α, as well as NO and iNOS in microglia. On the other hand, TRL revealed bioactive compounds α-tocopherol and β-sitosterol as suitable carriers for oleanolic acid. The inclusion of these biomolecules in TRL reduced the release of proinflammatory cytokines. The inclusion of these biomolecules in TRL reduced the release of proinflammatory cytokines. AT reduced IL-6 release by 72%, OA reduced TNF-α release by approximately 50%, and all three biomolecules together (M) reduced IL-1β release by 35% and TNF-α release by more than 70%. In addition, NO generation was reduced, with the inclusion of OA by 45%, BS by 80% and the presence of M by 88%. Finally, a recovery of the basal glutathione content was observed with the inclusion of OA and M in the TRL. Our results open the way to exploiting the neuro-pharmacological potential of these lipophilic bioactive compounds through their delivery to the brain as part of TRL.     

Interplay between Mast Cells and Regulatory T Cells in Immune-Mediated Cholangiopathies

Immune-mediated cholangiopathies are characterised by the destruction of small and large bile ducts causing bile acid stasis, which leads to subsequent inflammation, fibrosis, and eventual cirrhosis of the liver tissue. A breakdown of peripheral hepatic immune tolerance is a key feature of these diseases. Regulatory T cells (Tregs) are a major anti-inflammatory immune cell subset, and their quantities and functional capacity are impaired in autoimmune liver diseases. Tregs can undergo phenotypic reprogramming towards pro-inflammatory Th1 and Th17 profiles. The inflamed hepatic microenvironment influences and can impede normal Treg suppressive functions. Mast cell (MC) infiltration increases during liver inflammation, and active MCs have been shown to be an important source of pro-inflammatory mediators, thus driving pathogenesis. By influencing the microenvironment, MCs can indirectly manipulate Treg functions and inhibit their suppressive and proliferative activity. In addition, direct cell-to-cell interactions have been identified between MCs and Tregs. It is critical to consider the effects of MCs on the inflammatory milieu of the liver and their influence on Treg functions. This review will focus on the roles and crosstalk of Tregs and MCs during autoimmune cholangiopathy pathogenesis progression.     

The Beneficial Effects of Triterpenic Acid and Acteoside in an In Vitro Model of Nonalcoholic Steatohepatitis (NASH)

Triterpenic acid (TA) and acteoside (ACT), the major components of APPLIVER and ACTEOS, respectively, have been reported to exert hepatoprotective effects, but the molecular mechanisms remain elusive, particularly in the NAFLD/NASH context. We assessed their effects in our well-established in vitro model resembling the pathophysiological mechanisms involved in NASH. Human hepatocytes and hepatic stellate cells were exposed to free fatty acids (FFA) alone or in combination with APPLIVER and ACTEOS as a mono- or co-culture. Steatosis, inflammation, generation of reactive oxygen species (ROS), and collagen deposition were determined. ACTEOS reduced both the TNF-α and ROS production, and, most importantly, attenuated collagen deposition elicited by the excess of FFA in the co-culture model. APPLIVER also showed inhibition of both TNF-α production and collagen deposition caused by FFA accumulation. The compounds alone did not induce any cellular effects. The present study showed the efficacy of APPLIVER and ACTEOS on pathophysiological mechanisms related to NASH. These in vitro data suggest that these compounds deserve further investigation for possible use in NASH treatment.     

The Therapeutic Role of Exercise and Probiotics in Stressful Brain Conditions

Oxidative stress has been recognized as a contributing factor in aging and in the progression of multiple neurological disorders such as Parkinson’s disease, Alzheimer’s dementia, ischemic stroke, and head and spinal cord injury. The increased production of reactive oxygen species (ROS) has been associated with mitochondrial dysfunction, altered metal homeostasis, and compromised brain antioxidant defence. All these changes have been reported to directly affect synaptic activity and neurotransmission in neurons, leading to cognitive dysfunction. In this context two non-invasive strategies could be employed in an attempt to improve the aforementioned stressful brain status. In this regard, it has been shown that exercise could increase the resistance against oxidative stress, thus providing enhanced neuroprotection. Indeed, there is evidence suggesting that regular physical exercise diminishes BBB permeability as it reinforces antioxidative capacity, reduces oxidative stress, and has anti-inflammatory effects. However, the differential effects of different types of exercise (aerobic exhausted exercise, anaerobic exercise, or the combination of both types) and the duration of physical activity will be also addressed in this review as likely determinants of therapeutic efficacy. The second proposed strategy is related to the use of probiotics, which can also reduce some biomarkers of oxidative stress and inflammatory cytokines, although their underlying mechanisms of action remain unclear. Moreover, various probiotics produce neuroactive molecules that directly or indirectly impact signalling in the brain. In this review, we will discuss how physical activity can be incorporated as a component of therapeutic strategies in oxidative stress-based neurological disorders along with the augmentation of probiotics intake.     

Amorphous Inclusion Complexes: Molecular Interactions of Hesperidin and Hesperetin with HP-Β-CD and Their Biological Effects

This study aimed at obtaining hesperidin (Hed) and hesperetin (Het) systems with HP-β-CD by means of the solvent evaporation method. The produced systems were identified using infrared spectroscopy (FT-IR), X-ray powder diffraction (XRPD), and differential scanning calorimetry (DSC). Moreover, in silico docking and molecular dynamics studies were performed to assess the most preferable site of interactions between tested compounds and HP-β-CD. The changes of physicochemical properties (solubility, dissolution rate, and permeability) were determined chromatographically. The impact of modification on biological activity was tested in an antioxidant study as well as with regards to inhibition of enzymes important in pathogenesis of neurodegenerative diseases. The results indicated improvement in solubility over 1000 and 2000 times for Hed and Het, respectively. Permeability studies revealed that Hed has difficulties in crossing biological membranes, in contrast with Het, which can be considered to be well absorbed. The improved physicochemical properties influenced the biological activity in a positive manner by the increase in inhibitory activity on the DPPH radical and cholinoesterases. To conclude the use of HP-β-CD as a carrier in the formation of an amorphous inclusion complex seems to be a promising approach to improve the biological activity and bioavailability of Hed and Het.