Thrombin is the key serine proteinase of the coagulation cascade and therefore a suitable target for inhibition of blood coagulation. A number of pharmacologically active secondary metabolites from mushrooms have already been isolated, thus providing the rationale for screening for new thrombin inhibitors in mushrooms. In this study, inhibitory activities of mushroom extracts on thrombin and trypsin were measured using the chromogenic substrates H-d-phenylalanine-l-pipecolyl-l-arginine-paranitroaniline dihydrochloride (S-2238) for thrombin and N-benzoyl-d,l-Arg-p-nitroanilide (BAPNA) for trypsin. The inhibitory activities of extracts from 95 Basidiomycete species have been determined. The majority of samples inhibited trypsin and thrombin with various potencies; however, some extracts showed no activity against one or both of the enzymes. An aqueous extract of Gleophyllum odoratum exhibited high inhibitory activity on both thrombin and trypsin (72 and 60%, respectively), while extracts of Clitocybe gibba, Amanita virosa, Cantharellus lutescens, Suillus tridentinus, Hypoloma fasciculare and Lactarius badiosanguineus considerably inhibited thrombin (49, 48, 36, 34, 32 and 31%, respectively) and showed no inhibitory activity on trypsin. The results at this point are promising for further research with the objective of finding an effective and safe thrombin inhibitor.

Congenital heart disease is one of the most common congenital malformations and thus represents a considerable public health burden. Hence, the identification of individuals and families with an increased genetic predisposition to congenital heart disease (CHD) and its possible prevention is important. Even though CHD is associated with the lack of folate during early pregnancy, the genetic background of folate and methionine metabolism perturbations and their influence on CHD risk is not clear. While some genes, such as those coding for cytosolic enzymes of folate/methionine cycles, have been extensively studied, genetic studies of folate transporters (de)glutamation enzymes and mitochondrial enzymes of the folate cycle are lacking. Among genes coding for cytoplasmic enzymes of the folate cycle, MTHFR, MTHFD1, MTR, and MTRR have the strongest association with CHD, while among genes for enzymes of the methionine cycle BHMT and BHMT2 are the most prominent. Among mitochondrial folate cycle enzymes, MTHFD2 plays the most important role in CHD formation, while FPGS was identified as important in the group of (de)glutamation enzymes. Among transporters, the strongest association with CHD was demonstrated for SLC19A1.

Neuronal differentiation into specific subtypes is crucial for nervous system development and function, guided by neurotrophic factors. γ-Enolase, a neuron-specific glycolytic enzyme, exhibits neurotrophic-like properties and supports neuronal differentiation; however, its role in specific neuronal subtypes remains unknown. Here, we investigate the role of γ-enolase in differentiation dopaminergic-, cholinergic-, and adrenergic-like neuronal cells. Our results demonstrate that γ-enolase expression is significantly upregulated in differentiated cells, with the highest expression observed in cholinergic-like neurons. Full-length γ-enolase, compared to its truncated form, promoted enhanced neurite outgrowth and increased β-tubulin, a cytoskeletal marker. Conversely, silencing endogenous γ-enolase significantly reduced neurite length, confirming its essential role in driving neuronal morphological maturation. Furthermore, a γ-enolase-derived peptide corresponding to the active C-terminus of γ-enolase significantly promoted neurite outgrowth and increased β-tubulin expression, particularly in cholinergic-like neuronal cells. Notably, γ-enolase activity is regulated by cathepsin X, a lysosomal peptidase that cleaves γ-enolase at its C-terminus, reducing its neurotrophic effects. Confocal microscopy revealed increased co-localization of γ-enolase and cathepsin X in differentiated neuronal cells, emphasizing their interaction in cholinergic-like neurons. Inhibiting cathepsin X preserved active γ-enolase, promoted neuronal differentiation, and altered cytoskeletal marker expression. These findings suggest an important role for γ-enolase in cholinergic-like neuronal cells and propose cathepsin X as a regulatory modulator of γ-enolase activity, suggesting novel therapeutic strategies for neuroregeneration.