Search Results (43)

Parkinson’s disease is the second leading neurodegenerative disease of aging. For over five decades, oral levodopa has been used to manage the progressive motor deficits that are the hallmark of the disease. However, individual dose requirements are highly variable, and patients typically require increased levodopa dosage as the disease progresses, which can cause undesirable side effects. It has become increasingly apparent that the gut microbiome can have a major impact on the metabolism and efficacy of therapeutic drugs. In this Perspective, we examine recent studies highlighting the impact of metabolism by Enterococcus faecalis, a common commensal gut bacterium, on levodopa bioavailability. E. faecalis expresses a highly conserved tyrosine decarboxylase that promiscuously converts levodopa to dopamine in the gut, resulting in decreased neuronal uptake of levodopa and reduced dopamine formation in the brain. Mitochondria-targeted antioxidants conjugated to a triphenylphosphonium moiety have shown promise in transiently suppressing the growth of E. faecalis and decreasing microbial levodopa metabolism, providing an approach to modulating the microbiome that is less perturbing than conventional antibiotics. Thus, mitigating metabolism by the gut microbiota is an attractive therapeutic target to preserve and potentiate the efficacy of oral levodopa therapy in Parkinson’s disease. Full article

Introduction: Mpox emerged as a multi-country outbreak in 2022 and disproportionately affected gay, bisexual, and other men who have sex with men (GBMSM). Stigma is known to exacerbate health crises by discouraging testing, treatment, and vaccination. This review aimed to explore stigma associated with Mpox among GBMSM from July 2022, when mpox was declared a public health emergency of international concern. Methods: The PICO framework guided this narrative review. A search was conducted across the following databases from inception to June 2025: PubMed/MEDLINE, Embase, CINAHL, and Web of Science. The literature had to be empirical, peer-reviewed research that focused on mpox-related stigma in GBMSM. Results: Forty-seven studies were included in this review. The following themes were derived: (1) healthcare experiences, (2) media influence, (3) internalised and anticipated stigma, (4) public health messaging, (5) community responses, and (6) psychosocial impact. Healthcare experiences were marked by anticipated discrimination; many GBMSM delayed testing or vaccination for fear of being disclosed or labelled promiscuous. This was especially apparent in contexts where same-sex relationships are criminalised, leading some men to self-medicate or seek clandestine services. Media analyses revealed that social and traditional platforms often amplified blame and homophobia, though community-led counter-messaging helped shift narratives. Internalised and anticipated stigma resulted in shame, concealment of symptoms, avoidance of care, and heightened anxiety. Public health messaging that framed mpox as a behaviour-linked rather than identity-linked risk was more acceptable, and flexible vaccination strategies (e.g., offering less conspicuous injection sites) increased uptake. Stigma contributed to psychosocial distress and may have impeded outbreak control. Conclusions: Mpox-related stigma among GBMSM operates at individual, community, and structural levels, echoing patterns from the HIV era. Effective mitigation requires rights-based, destigmatising communication, culturally competent care, and collaboration. Addressing stigma is vital to controlling future outbreaks and ensuring equitable healthcare access. Full article

Cholinergic neuron impairment is a significant cause of cognitive decline in Alzheimer’s disease (AD), making acetylcholinesterase (AChE) a key therapeutic target. AChE inhibitors are principal drugs prescribed to alleviate symptoms in AD patients, while up to 50% of these individuals also suffer from depression, frequently treated with selective serotonin reuptake inhibitors (SSRIs). Due to the multisymptomatic nature of AD, there is a growing interest in developing multitargeted ligands that simultaneously enhance cholinergic and serotonergic tone. This study presents the synthesis of novel ligands based on functionalized piperidines, evaluated through radioligand binding assays at the serotonin transporter (SERT) and AChE and butyrylcholinesterase (BuChE) inhibition. The pharmacological results showed that some compounds exhibited moderate inhibitory activity against AChE, with one compound 19 standing out as the most potent, also displaying a moderate BuChE inhibitory activity, while showing low affinity for SERT. On the other hand, compound 21 displayed an interesting polypharmacological profile, with good and selective activity against BuChE and SERT. The results underscore the difficulty of designing promiscuous ligands for these targets and suggest that future structural modifications could optimize their therapeutic potential in AD. Full article

Enzymes of the cytochrome P450 monooxygenase family 4 (CYP4) in mammals are generally involved either in endobiotic metabolism (e.g., acting on fatty acids or eicosanoids), or the modification of xenobiotics including therapeutic drugs. CYP4B1 is special, as it is an enigmatic enzyme acting at the interface between xenobiotic and endobiotic metabolism. However, a systematic analysis of CYP4B1’s substrate scope has not yet been reported. Herein, a three-step approach to identify novel substrates for three CYP4B1 orthologs (namely from rabbits, green monkeys, and mouse lemurs) is described. First, screening of a library containing 502 natural products revealed potential novel substrate groups for CYP4B1. Second, based on these results, a systematic library was defined consisting of 63 compounds representing 10 compound groups. Third, in vitro conversion of these compounds by CYP4B1 and identification of conversion products were conducted, supported by in silico docking, allowing the prediction of binding probabilities and potential oxidation sites. We report five new substrate groups (acyclic, monocyclic and bicyclic terpenoids, stilbenoids, and vanilloids), twenty-eight new substrates (inter alia capsaicin, gingerol, genistein, stilbene, myristicin, thioanisole), and two new reaction types for CYP4B1 (S-oxidation, O-demethylation). Consequently, CYP4B1 is a far more promiscuous enzyme than previously thought. Full article

Background: Whole genome sequencing of clinical bacterial isolates holds promise in predicting their susceptibility to antibiotic therapy, based on a detailed understanding of the phenotypic manifestation of genotypic variation. The aac(6′) aminoglycoside acetyltransferase gene family is the most abundant aminoglycoside resistance determinant encountered in clinical practice. A variety of AAC(6′) isozymes have been described, suggesting a phenotypic distinction between subtype I, conferring resistance to amikacin (AMK), and subtype II, conferring resistance to gentamicin (GEN) instead. However, the epidemiology and thus clinical relevance of the various and diverse isozymes and their phenotypic distinction demand systematic and contemporary re-assessment to reliably predict bacterial susceptibility to aminoglycoside antibiotics. Methods: We analyzed the resistance gene annotations of 657,603 clinical bacterial isolates to assess the prevalence and diversity of aac(6′) genes. Seventeen unique aac(6′) amino acid sequences were cloned and expressed under defined promoter control in otherwise isogenic E. coli cells for phenotypic analysis with twenty distinct aminoglycoside antibiotics. A panel of clinical isolates was analyzed for the genotype–phenotype correlation of aac(6′). Results: An aac(6′) resistance gene annotation was found in 139,236 (21.2%) of the clinical isolates analyzed. AMK resistance-conferring aac(6′)-I genes dominated in Enterobacterales (28.5%). In Pseudomonas aeruginosa and Acinetobacter baumannii, a gene conferring the aac(6′)-II phenotype but annotated as aac(6′)-Ib₄ was the most prevalent. None of the aac(6′) genes were annotated as subtype III, but gene aac(6′)-Ii identified in Gram-positive isolates displayed a subtype III phenotype. Genes that were annotated as aac(6′)-Ib₁₁ in Enterobacterales conferred resistance to both AMK and GEN, which we propose constitutes a novel subtype IV when applying established nomenclature. A phenotypic assessment facilitated structural re-assessment of the substrate promiscuity of AAC(6′) enzymes. Conclusions: Our study provides the most comprehensive analysis of clinically relevant aac(6′) gene sequence variations to date, providing new insights into a differentiated substrate promiscuity across the genotypic spectrum of this gene family, thus translating into a critical contribution towards the development of amino acid sequence-based in silico antimicrobial susceptibility testing (AST). Full article

Recently, the use of click chemistry for localization of chemically modified cyanopeptides has been introduced, i.e., taking advantage of promiscuous adenylation (A) domains in non-ribosomal peptide synthesis (NRPS), allowing for the incorporation of clickable non-natural amino acids (non-AAs) into their peptide products. In this study, time-lapse experiments have been performed using pulsed feeding of three different non-AAs in order to observe the synthesis or decline of azide- or alkyne-modified microcystins (MCs) or anabaenopeptins (APs). The cyanobacteria Microcystis aeruginosa and Planktothrix agardhii were grown under maximum growth rate conditions (r = 0.35–0.6 and 0.2–0.4 (day⁻¹), respectively) in the presence of non-AAs for 12–168 h. The decline of the azide- or alkyne-modified MC or AP was observed via pulse-feeding. In general, the increase in clickable MC/AP in peptide content reached a plateau after 24–48 h and was related to growth rate, i.e., faster-growing cells also produced more clickable MC/AP. Overall, the proportion of clickable MC/AP in the intracellular fraction correlated with the proportion observed in the dissolved fraction. Conversely, the overall linear decrease in clickable MC/AP points to a rather constant decline via dilution by growth instead of a regulated or induced release in the course of the synthesis process. Full article

The comparative analysis of homologous enzymes is a valuable approach for elucidating enzymes’ structure–function relationships. Glutathione transferases (GSTs, EC. 2.5.1.18) are crucial enzymes in maintaining the homeostatic stability of plant cells by performing various metabolic, regulatory, and detoxifying functions. They are promiscuous enzymes that catalyze a broad range of reactions that involve the nucleophilic attack of the activated thiolate of glutathione (GSH) to electrophilic compounds. In the present work, three highly homologous (96–98%) GSTs from ryegrass Lolium perenne (LpGSTs) were identified by in silico homology searches and their full-length cDNAs were isolated, cloned, and expressed in E. coli cells. The recombinant enzymes were purified by affinity chromatography and their substrate specificity and kinetic parameters were determined. LpGSTs belong to the tau class of the GST superfamily, and despite their high sequence homology, their substrate specificity displays remarkable differences. High catalytic activity was determined towards hydroxyperoxides and alkenals, suggesting a detoxification role towards oxidative stress metabolites. The prediction of the structure of the most active LpGST by molecular modeling allowed the identification of a non-conserved residue (Phe215) with key structural and functional roles. Site-saturation mutagenesis at position 215 and the characterization of eight mutant enzymes revealed that this site plays pleiotropic roles, affecting the affinity of the enzyme for the substrates, catalytic constant, and structural stability. The results of the work have improved our understanding of the GST family in L. perenne, a significant threat to agriculture, sustainable food production, and safety worldwide. Full article

This paper analyzes themes of male insecurities and distrust of the exclusive culture of female sexuality and reproduction in E. T. A. Hoffmann’s Vampirism, one of the earliest psychologically sophisticated female vampires in Western literature. The doomed heroine, Aurelia, escapes a life of maternal abuse and sexual trauma by marrying the wealthy Count Hippolytus, but his attraction warps into suspicion when she becomes pregnant and loses her appetite for his food. Worried that losing her virginity has activated promiscuity inherited from her late mother, he begins following her and thinks he sees her conspiring with a coven of female ghouls who train her to satisfy her pregnancy cravings by feeding on a male corpse. Real or imagined, this vision confirms his suspicions and leads to their mutual destruction. In my analysis, I explore vampire literature’s early history, its place within Gothic literature, the prominent role of female vampires, their relationship to gender anxieties exacerbated by the Romantic Era’s subversive political movements, and the way in which Hoffmann’s cynical story operates as a misogynistic conspiracy theory aimed at the secret female space of reproduction, symbolized by Aurelia’s cannibalistic pregnancy cravings. As such, it contributes to the destructive folklore of social distrust. Full article

PSD95-disc large-zonula occludens (PDZ) domains are globular modules of 80–90 amino acids that co-evolved with multicellularity. They commonly bind to carboxy-terminal sequences of a plethora of membrane-associated proteins and influence their trafficking and signaling. We previously built a PDZ resource (PDZome) allowing us to unveil human PDZ interactions by Yeast two-hybrid. Yet, this resource is incomplete according to the current knowledge on the human PDZ proteome. Here we built the PDZome 2.0 library for Yeast two-hybrid, based on a PDZ library manually curated from online resources. The PDZome2.0 contains 305 individual clones (266 PDZ domains in isolation and 39 tandems), for which all boundaries were designed based on available PDZ structures. Using as bait the E6 oncoprotein from HPV16, a known promiscuous PDZ interactor, we show that PDZome 2.0 outperforms the previous resource. Full article

Extracellular synthesis of functional cyclodextrins (CDs) as intermediates of starch assimilation is a convenient microbial adaptation to sequester substrates, increase the half-life of the carbon source, carry bioactive compounds, and alleviate chemical toxicity through the formation of CD-guest complexes. Bacteria encoding the four steps of the carbohydrate metabolism pathway via cyclodextrins (CM-CD) actively internalize CDs across the microbial membrane via a putative type I ATP-dependent ABC sugar importer system, MdxEFG-(X/MsmX). While the first step of the CM-CD pathway encompasses extracellular starch-active cyclomaltodextrin glucanotransferases (CGTases) to synthesize linear dextrins and CDs, it is the ABC importer system in the second step that is the critical factor in determining which molecules from the CGTase activity will be internalized by the cell. Here, structure-function relationship studies of the cyclo⁄maltodextrin-binding protein MdxE of the MdxEFG-MsmX importer system from Thermoanaerobacter mathranii subsp. mathranii A3 are presented. Calorimetric and fluorescence studies of recombinant MdxE using linear dextrins and CDs showed that although MdxE binds linear dextrins and CDs with high affinity, the open-to-closed conformational change is solely observed after α- and β-CD binding, suggesting that the CM-CD pathway from Thermoanaerobacterales is exclusive for cellular internalization of these molecules. Structural analysis of MdxE coupled with docking simulations showed an overall architecture typically found in sugar-binding proteins (SBPs) that comprised two N- and C-domains linked by three small hinge regions, including the conserved aromatic triad Tyr193/Trp269/Trp378 in the C-domain and Phe87 in the N-domain involved in CD recognition and stabilization. Structural bioinformatic analysis of the entire MdxFG-MsmX importer system provided further insights into the binding, internalization, and delivery mechanisms of CDs. Hence, while the MdxE-CD complex couples to the permease subunits MdxFG to deliver the CD into the transmembrane channel, the dimerization of the cytoplasmatic promiscuous ATPase MsmX triggers active transport into the cytoplasm. This research provides the first results on a novel thermofunctional SBP and its role in the internalization of CDs in extremely thermophilic bacteria. Full article

Corynebacterium glutamicum has been regarded as a food-grade microorganism. In recent years, the research to improve the activities of beneficial therapeutics and pharmaceutical substances has resulted in the engineering of the therapeutically favorable cell factory system of C. glutamicum. In this study, we successfully glucosylated isoeugenol and other monoterpene derivatives in C. glutamicum using a promiscuous YdhE, which is a glycosyltransferase from Bacillus lichenformis. For efficient glucosylation, cultivation conditions such as the production time, substrate concentration, carbon source, and culture medium were optimized. Our system successfully converted about 93% of the isoeugenol to glucosylated compounds in the culture. The glucoside compounds were then purified, analyzed, and identified as isoeugenol-1-O-β-d-glucoside and isoeugenol-1-O-β-d-(2″-acetyl)-glucoside. Full article

The objective of the present study was to evaluate the synergistic effect of two important pharmacophores, coumarin and α-amino dimethyl phosphonate moieties, on antimicrobial activity toward selected LPS-varied E. coli strains. Studied antimicrobial agents were prepared via a Kabachnik–Fields reaction promoted by lipases. The products were provided with an excellent yield (up to 92%) under mild, solvent- and metal-free conditions. A preliminary exploration of coumarin α-amino dimethyl phosphonate analogs as novel antimicrobial agents was carried out to determine the basic features of the structure responsible for the observed biological activity. The structure–activity relationship revealed that an inhibitory activity of the synthesized compounds is strongly related to the type of the substituents located in the phenyl ring. The collected data demonstrated that coumarin-based α-aminophosphonates can be potential antimicrobial drug candidates, which is particularly crucial due to the constantly increasing resistance of bacteria to commonly used antibiotics. Full article

Hydroxytyrosol, a valuable plant-derived phenolic compound, is increasingly produced from microbial fermentation. However, the promiscuity of the key enzyme HpaBC, the two-component flavin-dependent monooxygenase from Escherichia coli, often leads to low yields. To address this limitation, we developed a novel strategy utilizing microbial consortia catalysis for hydroxytyrosol production. We designed a biosynthetic pathway using tyrosine as the substrate and selected enzymes and overexpressing glutamate dehydrogenase GdhA to realize the cofactor cycling by coupling reactions catalyzed by the transaminase and the reductase. Additionally, the biosynthetic pathway was divided into two parts and performed by separate E. coli strains. Furthermore, we optimized the inoculation time, strain ratio, and pH to maximize the hydroxytyrosol yield. Glycerol and ascorbic acid were added to the co-culture, resulting in a 92% increase in hydroxytyrosol yield. Using this approach, the production of 9.2 mM hydroxytyrosol was achieved from 10 mM tyrosine. This study presents a practical approach for the microbial production of hydroxytyrosol that can be promoted to produce other value-added compounds. Full article

Glutathione transferases (GSTs) are promiscuous enzymes whose main function is the detoxification of electrophilic compounds. These enzymes are characterized by structural modularity that underpins their exploitation as dynamic scaffolds for engineering enzyme variants, with customized catalytic and structural properties. In the present work, multiple sequence alignment of the alpha class GSTs allowed the identification of three conserved residues (E137, K141, and S142) at α-helix 5 (H5). A motif-directed redesign of the human glutathione transferase A1-1 (hGSTA1-1) was performed through site-directed mutagenesis at these sites, creating two single- and two double-point mutants (E137H, K141H, K141H/S142H, and E137H/K141H). The results showed that all the enzyme variants displayed enhanced catalytic activity compared to the wild-type enzyme hGSTA1-1, while the double mutant hGSTA1-K141H/S142H also showed improved thermal stability. X-ray crystallographic analysis revealed the molecular basis of the effects of double mutations on enzyme stability and catalysis. The biochemical and structural analysis presented here will contribute to a deeper understanding of the structure and function of alpha class GSTs. Full article

Viral hepatitis is a significant cause of morbidity and mortality worldwide. In Croatia, hepatitis B virus (HBV) and hepatitis C virus (HCV) are widely distributed, especially in some high-risk groups such as people who inject drugs (PWID), prisoners, and highly promiscuous groups. The seroprevalence of HBV ranges from 7.0% in the general population to 38.8% in PWID, depending on the region. The seroprevalence of HCV is highest among PWID (29–75.5%) as compared to 0.9% in the general population. Analyzing the distribution of HCV genotypes, no substantial changes in the molecular epidemiology of the two most frequent HCV genotypes (1 and 3) in the past 20 years were observed. However, the predominance of subtype 1b compared to subtype 1a as detected in 1996–2005 was not confirmed in 2008–2015. Hepatitis A virus (HAV) incidence was high in the past with a decreasing trend since the 2000s, except for an outbreak in 2017–2018 as part of the large European outbreak, which was mainly among men who have sex with men. Hepatitis E virus (HEV) is an emerging virus detected for the first time in Croatia in 2012. The seroprevalence of HEV is high among hemodialysis patients (27.9%) and liver transplant recipients (19.3–24.4%). In addition, higher seroprevalence rates were observed in animal-related professions (e.g., veterinarians, 15.2%; hunters, 14.9%). All detected HEV strains belonged to genotype 3. Full article