Search Results (189)

Background/Objectives: Neisseria gonorrhoeae is a high-priority pathogen for the development of new therapeutic alternatives. Efflux pumps are attractive drug targets because their inactivation influences N. gonorrhoeae susceptibility to multiple antimicrobials. Since most gonococcal efflux systems are energy-dependent, interference with energy metabolism and membrane transport may indirectly compromise efflux activity. Efflux inhibitors may increase intracellular antibiotic concentration, although this requires validation in resistant strains. The most effective efflux inhibitors interfere with energy metabolism, affecting several physiological processes, including efflux. In this work, we used an in silico drug repurposing strategy targeting proteins involved in membrane transport and energy metabolism in N. gonorrhoeae. A subset of candidate drugs were subsequently evaluated in vitro using only the reference strain N. gonorrhoeae ATCC 49226. Methods: Predicted drug–target interactions were identified using publicly available databases such as DrugBank and STITCH. Minimum inhibitory concentrations (MICs) of selected drugs against N. gonorrhoeae were determined by microdilution. Changes in intracellular ethidium bromide accumulation were assessed by real-time fluorometry as an indirect indicator of possible efflux-related interference. Results: In silico analysis identified 32 predicted targets associated with 57 approved drugs. Triclabendazole and dequalinium showed the lowest MIC values of the tested compounds (2 and 4 mg/L, respectively). Ketotifen and verapamil demonstrated activity consistent with possible efflux interference, as indicated by increased ethidium bromide accumulation. Atovaquone showed adjuvant-like effects in combination assays, suggesting that mechanisms other than efflux-related interference may contribute to its activity. Conclusions: Overall, this preliminary study identifies approved drugs with antimicrobial or adjuvant activity against a single N. gonorrhoeae reference strain, supporting further investigation in clinically relevant and efflux-variant strains. Full article

Probiotic lactic acid bacteria are widely recognized for their health-promoting effects. However, the use of live microorganisms may pose safety concerns and stability limitations. Consequently, postbiotics, defined as inactivated microbial cells and/or their components, have emerged as a promising alternative. This study integrates genome-guided evaluation of probiotic potential, experimental validation of in silico predictions and process optimization for the production of inactivated Lactiplantibacillus plantarum DM1 and KK1 cells as postbiotics. Genome mining identified genes and gene clusters associated with metabolic versatility, antimicrobial activity, gastrointestinal stress tolerance, adhesion and prebiotic substrate utilization. Building on these findings, to generate postbiotics, the efficiency of thermal, enzymatic, mechanical and radiation-based inactivation methods was evaluated in bacterial suspensions prepared in three dairy by-product matrices: milk permeate, sweet whey and sour whey. Complete inactivation of both strain cells was achieved by thermal treatment (3 min pasteurization), γ-irradiation (3 kGy), and combined lysozyme–pasteurization treatment, whereas other treatments showed partial and matrix-dependent effects. Matrix composition significantly influenced treatment efficacy, suggesting a protective role of food components used. These findings highlight the importance of combining genome mining for potential probiotic strain characterization with robust, matrix-adapted inactivation strategies for the development of stable postbiotic formulations. Full article

Atypical Salmonella enterica strains that evade conventional detection pose significant challenges to food safety surveillance. A hydrogen sulfide (H₂S)-negative and serologically untypable S. enterica strain (SF1060) was detected by qPCR from cooked farmed mussels in Galicia, Spain, and characterized using phenotypic and genomic approaches. Despite typical biochemical profiles, SF1060 failed to produce black colonies on Xylose Lysine Deoxycholate (XLD) agar and lacked detectable somatic antigens by conventional serotyping. Hybrid genome assembly using nanopore and illumina sequencing yielded a closed chromosome and five plasmids. In silico analyses identified the strain as S. Senftenberg ST14. Comparative genomics revealed a chromosomal inversion at the rfb operon (encoding enzymes needed to synthesize deoxysugars and O antigens) mediated by IS5-family transposase ISEc68, which truncated the rfbD gene and separated the remaining rfb genes at rfbD, disrupting O-antigen biosynthesis, explaining the inconclusive phenotypic serotyping results. The phs operon responsible for H₂S production lacked premature stop codons, suggesting the H₂S-negative phenotype may result from an alternative mechanism. This study demonstrates how whole-genome sequencing resolves identification of atypical strains that fail culture-based detection and emphasizes the critical need for molecular surveillance methods in seafood safety programs, particularly in regions where atypical S. enterica variants may be endemic. Full article

Background/Objectives: Alzheimer’s disease (AD) is characterized by beta-amyloid (Aβ) plaque deposition, which impairs several cellular processes, including autophagy. Considering the multifactorial nature of AD, the development of therapies acting on alternative molecular targets is necessary. In this study, we evaluated the neuroprotective effect of a molecule from the hydrozoan Eudendrium carneum and investigated its impact on autophagy-related pathways. Methods: The secretion of E. carneum was fractionated by RP-HPLC according to its neuroprotective activity in SH-SY5Y cells exposed to oAβ42, evaluated using LDH and MTT assays. The purified molecule (named EC5), characterized by mass spectrometry, was evaluated regarding in silico toxicity and calcium dynamics. Neuronal lysosomal morphology was assessed using the LysoTracker probe, and cathepsin D activity was determined using a synthetic substrate. The expression of autophagy-related proteins (mTOR, LAMP-1, and LC3B) was evaluated by dot blotting, and amyloid plaque clearance was quantified using Thioflavin-T staining. Results: The steroid glycoside putatively identified as Sarmentoside B (EC5) exhibited neuroprotective effects and showed no toxicity or alterations in neuronal calcium or sodium channel dynamics. EC5 restored lysosomal morphology and cathepsin D activity, reversing the impairment induced by oAβ42. Furthermore, EC5 reduced mTOR expression, and this interaction was supported by molecular docking analysis. Lysosomal restoration promoted the clearance of oAβ42 aggregates, as evidenced by Thioflavin-T staining, resulting in reduced neuronal death. Conclusions: EC5, putatively identified as Sarmentoside B, exerts neuroprotective effects against oAβ42-induced toxicity by promoting autophagy-related amyloid clearance, highlighting its therapeutic potential for AD. Full article

Imprinted proteins (IPs) are promising materials for producing artificial alternatives to natural recognition systems (antibodies, aptamers, etc.) due to their high sorption properties and specificity. However, contemporary understanding of the imprinting process at the atomic level is rather limited, which hinders the rational design of more efficient IPs. In this paper, we use computational modeling to provide a description of fundamental principles of protein imprinting at the atomic level. We have modeled several potential associates between the protein matrix and template molecules that form during the imprinting process up to the addition of the cross-linking agent. We used bovine serum albumin (BSA) as the protein matrix and 4-hydroxycoumarin (4–HC) as a molecular template. In combination with computational modeling, extensive experimental analyses including isothermal titration calorimetry (ITC) and NMR spectroscopic methods (¹H NMR and diffusion-ordered NMR spectroscopy (DOSY)) were used to evaluate the potential efficiency of imprinted BSA. This study represents a step toward the future rational in silico design of IPs. Full article

Background/Objectives: The increasing resistance of Candida species to conventional antifungals, particularly azoles, poses a critical public health challenge due to high mortality rates associated with candidemia. This study aimed to describe the chemical composition of the essential oil from Haplopappus foliosus (EO-BAI) and evaluate its antifungal properties, along with its nanoemulsion (NE-BAI) and major constituents, against a panel of clinical Candida isolates. Methods: EO-BAI was extracted via steam distillation and analyzed using GC-MS. A nanoemulsion was synthesized through ultrasonic emulsification and characterized by DLS and microscopy (SEM/STEM). Antifungal activity (MIC/MFC) was determined following CLSI M27-A3 guidelines. Time–kill kinetic studies were conducted on C. albicans, and an in silico approach was used to describe interactions with Als9-2 and CYP51 targets. Results: The EO-BAI profile was dominated by terpinen-4-ol (27.27%) and α-bisabolol (10.40%). The NE-BAI exhibited a droplet size of approximately 22 nm with an encapsulation efficiency of 88.72%. Among the tested samples, α-bisabolol emerged as the core bioactive driver (MIC = 16 µg/mL against C. albicans). While NE-BAI showed reduced initial activity at 24 h, it demonstrated enhanced efficacy by 48 h, matching fluconazole’s potency and maintaining a definitive fungicidal effect. Docking analysis confirmed that α-bisabolol establishes stabilizing interactions with key virulence and membrane homeostasis targets. Conclusions: The NE-BAI provides a sustained delivery of its bioactive terpenes, preserving their fungicidal nature and positioning them as robust therapeutic alternatives to conventional treatments. Full article

Objectives: Visceral leishmaniasis (VL), caused by Leishmania donovani and Leishmania infantum, is a life-threatening neglected tropical disease, particularly in endemic regions such as Djibouti. Current therapies are constrained by toxicity, high cost, and limited availability, highlighting the urgent need for safe, effective, and affordable alternatives. This study aimed to identify novel antileishmanial candidates from Djiboutian medicinal plants using an integrated in silico approach. Methods: A total of 136 phytoconstituents isolated from local medicinal plants were screened via molecular docking against validated protein targets (6UAK and 2JK6). Promising candidates were further analyzed for interaction patterns, drug-likeness according to Lipinski’s Rule of Five, and ADMET properties. Molecular dynamics (MD) simulations over 100 ns were performed to assess the structural stability of selected protein–ligand complexes. Results: Compound C41 emerged as a leading candidate, showing binding affinities of −8.3 kcal/mol and −7.5 kcal/mol toward 6UAK and 2JK6, respectively, comparable to reference drugs. Interaction analysis revealed stable hydrogen bonds and hydrophobic contacts within the catalytic sites. Drug-likeness assessment confirmed compliance with Lipinski’s Rule, while ADMET predictions indicated high intestinal absorption and favorable safety profiles for several candidates. MD simulations corroborated the structural stability of the 2JK6-C41 complex throughout the 100 ns trajectory. Conclusions: These findings underscore Djiboutian medicinal plants as a valuable source of potential antileishmanial leads. Among them, Compound C41 represents a promising candidate for future experimental validation, supporting the development of innovative, safe, and cost-effective therapies against visceral leishmaniasis. Full article

Background/Objectives: Due to the widespread problem of antimicrobial resistance (AMR), there is an urgent need to identify new antibacterial targets that act through mechanisms distinct from those of existing antibiotics. One of these targets is the essential cell division protein FtsQ, which is a central hub of the Gram-negative divisome, but the druggability of its extensive protein–protein interaction (PPI) interfaces remains poorly defined. Here, we present a comprehensive structure-based in silico characterization of Escherichia coli FtsQ aimed at identifying and prioritizing druggable regions for PPI modulation. Methods: We analyzed E. coli FtsQ in both apo and complexed states (FtsQB, FtsQL, and FtsQBL) using a combination of pocket-mapping tools (FTMap and SiteMap), evolutionary conservation analysis (ConSurf), and structure property assessment (BLAST, ProBiS) to map and evaluate potential binding pockets of FtsQ protein. Results: Eight potential binding sites were predicted across the β and POTRA domains of FtsQ. One previously unreported site within the POTRA domain was prioritized as a candidate site, characterized by favorable druggability scores, strong evolutionary conservation, and a putative role in the FtsQ–FtsW/FtsN/FtsI interaction network. In contrast, two highly conserved sites at the FtsQ–FtsB/FtsL interaction interface were structurally flat, indicating limited suitability for classical small-molecule binding and greater compatibility with alternative modalities such as macrocycles or peptidomimetics. Conclusions: Although FtsQ lacks deep canonical binding pockets, this study proposes several conserved and potentially tractable regions as candidate sites, supporting its potential as a non-classical but promising antibacterial target for disrupting bacterial cytokinesis. Full article

Background/Objectives: Paracetamol is a widely analgesic and antipyretic drug; however, its limited anti-inflammatory efficacy and safety concerns motivate the search for novel non-opioid alternatives. In this study, a series of 4-hydroxyphenylamino-naphthoquinones were designed as paracetamol-inspired analogs and synthesized via a solvent-free, silica-assisted Michael addition, providing a sustainable and efficient synthetic route. Methods: The compounds were evaluated using an integrated strategy combining in silico prediction, density functional theory calculations, molecular docking, ADMET profiling, and in vivo phenotypic pharmacological assays. Results: In vivo evaluation revealed pronounced peripheral antinociceptive activity in the acetic acid-induced writhing model and robust anti-inflammatory effects in carrageenan-induced paw edema, comparable to those of naproxen. These findings suggest a predominantly peripheral mechanism consistent with anti-inflammatory and antinociceptive profiles linked to cyclooxygenase inhibition. A normalization-based multi-criteria analysis integrating peripheral, anti-inflammatory, central, and antipyretic endpoints enabled transparent phenotypic prioritization within the series. Under this framework, compound 7 emerged as the most balanced peripheral–anti-inflammatory candidate, whereas compound 8, evaluated experimentally as a regioisomeric mixture, showed comparatively stronger central antinociceptive activity in the hot plate test. Antipyretic activity in an LPS-induced fever model was limited and not sustained. Conclusions: Overall, these findings indicated that the 4-hydroxyphenylamino-naphthoquinone scaffold emerges as a promising non-opioid platform for peripheral inflammatory pain, supporting further investigation of its pharmacological and mechanistic properties. Full article

Background/Objectives: Leishmaniasis is a group of diseases caused by obligate intracellular parasites of the Leishmania genus and is classified by the World Health Organization as a category I neglected tropical disease. Leishmania infantum predominantly affects children under five years of age and shows an increasing incidence of cutaneous and visceral forms. The development of new therapeutic alternatives remains challenging, making in silico approaches valuable for accelerating antileishmanial drug discovery. This study aimed to identify new compounds with potential activity against Leishmania infantum amastigotes using artificial intelligence-based classification models. Methods: A curated database of compounds with reported biological activity was constructed. Molecular representation employed zero- to two-dimensional descriptors calculated with Dragon software (v 7.0.10). Unsupervised k-means cluster analysis was applied to define training and external prediction sets. Supervised models were developed on the WEKA platform using IBk, J48, multilayer perceptron, and sequential minimal optimization algorithms. Model performance was assessed through internal cross-validation and external validation procedures. Results: All models achieved classification accuracies above eighty percent for both training and prediction sets, indicating consistent predictive performance and good generalization ability. The validated models were applied to virtual screening of the DrugBank database and a collection of synthetic compounds. This screening campaign enabled the identification of one hundred twenty compounds with potential activity against the amastigote form of Leishmania infantum. Conclusions: Artificial intelligence-based QSAR models proved to be useful tools for prioritizing antileishmanial candidates. The integration of molecular descriptors, machine learning, and virtual screening offers an efficient strategy for drug discovery. Full article

The common metabolic disorder, lactose intolerance, is often treated with oral lactase enzyme supplements, which can frequently cause gastrointestinal instability. This work utilizes Malbranchea cinnamomea’s AA9 lytic polysaccharide monooxygenase (LPMO) to target β-lactose (β-lactose) in an investigation of a new enzymatic approach for lactose breakdown. Potential possibilities for lactose breakdown are AA9 LPMOs, copper-dependent enzymes that oxidatively cleave glycosidic bonds in polysaccharides. We employed a combined in silico method that incorporated molecular docking, density functional theory (DFT) calculations, and molecular dynamics (MD) simulations. Docking studies revealed that β-lactose formed hydrogen bonds with key residues SER100, ASN54, and ARG56, exhibiting a greater binding affinity (−5.4 kcal/mol) toward LPMO compared to the control citric acid (−4.9 kcal/mol). Upon DFT analysis, (LPMO) showed excellent stability and appropriate reactivity for enzyme interaction. The higher stability of the LPMO-β-lactose complex was highlighted by MD simulation over 100 ns, which showed lower root mean square deviation (RMSD) and root mean square fluctuation (RMSF) values, greater structural compactness, and reduced solvent accessibility when compared to the control. These collective findings suggest that β-lactose interacts efficiently with the AA9 LPMO active site, supporting its potential as a novel enzymatic target for lactose degradation. This computational study provides a theoretical foundation for developing alternative therapeutic strategies for lactose intolerance, though further in vitro and in vivo investigations are required to validate these findings. Full article

Background/Objectives: Severe and recurrent infections due to multidrug-resistant (MDR) Pseudomonas aeruginosa necessitate alternative antimicrobial strategies. Fermented cacao beans represent a niche microbial ecosystem with the potential to harbor beneficial lactic acid bacteria (LAB). This study aimed to isolate and characterize LAB strains from fermented cacao beans in southern Thailand and to evaluate their probiotic potential and antimicrobial activity against MDR P. aeruginosa. Methods and Results: Five Lactiplantibacillus plantarum isolates were identified via MALDI-TOF MS and whole-genome sequencing (WGS). All strains demonstrated antimicrobial activity against 17 clinical MDR P. aeruginosa isolates and CR14 exhibited the largest inhibition zone. The isolates displayed robust probiotic traits, including survival under simulated gastrointestinal conditions. Acid tolerance (pH 2.0) reached 61.15 ± 7.75%, while resistance to pepsin, pancreatin, and bile salts exceeded 88%, 91%, and 92%, respectively. Strong adhesion was confirmed via auto-aggregation (55.02 ± 1.75%), hydrophobicity (45.58 ± 0.96%) and Caco-2 cell attachment (up to 98.11 ± 3.28%). WGS revealed multiple plantaricin-encoding clusters. Coarse-grained molecular dynamic simulations showed that two-peptide plantaricins (plnJ/K and plnNC8-αβ) self-assembled and formed stable pores in bacterial membrane models, confirming a pore-forming antimicrobial mechanism. The strains lacked acquired resistance genes and virulence factors, confirmed by in silico safety assessments. Conclusions: Thus, these L. plantarum strains are promising probiotics for managing MDR P. aeruginosa via functional foods or adjunct therapies. Full article

Background: Avian pathogenic Escherichia coli (APEC) contributes substantially to colibacillosis outbreaks in chickens. Because APEC cells readily attach to surfaces and develop biofilms, they pose a notable hazard to poultry production and food safety. This study investigated the antibiofilm and anti-adhesion activities of deep eutectic solvent-based emulsion containing Piper betle L. extract (DEPE) and hydroxychavicol, a pure compound isolated from P. betle leaves against APEC. Methods: Antibiofilm and anti-adhesion activities of DEPE and hydroxychavicol against APEC were investigated. Molecular docking and dynamics simulation of DEPE and hydroxychavicol was conducted. In addition, anti-adhesion activity of DEPE on chicken meat during storage was evaluated. Results: DEPE and hydroxychavicol significantly inhibited biofilm formation at sub-MIC, with DEPE achieving up to 80% inhibition and hydroxychavicol up to 69%. At 8 × MIC, DEPE and hydroxychavicol diminished the viability of both early and established biofilms. Furthermore, DEPE and hydroxychavicol reduced APEC adhesion on the surface as observed by SEM. In silico analyses demonstrated the stable binding of hydroxychavicol to adhesion-related proteins, particularly EcpA and FimH, suggesting a possible mechanism for its anti-adhesion activity. At day 5, DEPE at 4 × MIC significantly reduced 63% bacterial adhesion to chicken meat surfaces during storage, while maintaining the meat’s color. Conclusions: These findings indicate that DEPE and hydroxychavicol are promising candidates for limiting APEC biofilm formation and surface attachment and may serve as alternative antibacterial agents in poultry-related food safety applications. Full article

Spotted fever group (SFG) rickettsioses are tick-borne infectious diseases caused by more than 30 Rickettsia species. As ticks may harbor and transmit multiple pathogens during a single blood meal, sensitive and specific molecular detection methods are essential for early diagnosis. Conventional nested PCR is commonly used but is time-consuming and prone to cross-contamination due to multiple amplification steps. This study evaluated a dual-target one-step nested PCR assay developed as a rapid alternative to conventional nested PCR for SFG Rickettsia detection. Gene-specific primers targeting the Rickettsia outer membrane protein A (ompA) gene and the 17 kDa antigen gene were designed, with a Plasmodium falciparum thrombospondin-related anonymous protein (TRAP) gene included as an internal amplification control. Primer specificity was verified in silico, and assay performance was assessed using synthetic DNA templates. The dual-target one-step nested PCR achieved detection limits of 10 gene copies for the 17 kDa gene and 1000 gene copies for ompA, compared with 10 and 100,000 gene copies, respectively, using conventional nested PCR. Screening of 184 tick specimens identified one positive sample (0.54%) for the Rickettsia 17 kDa gene. Overall, the dual-target one-step nested PCR demonstrated comparable sensitivity to conventional nested PCR while reducing assay time and contamination risk, indicating its potential as a reliable tool for SFG Rickettsia detection. Full article

Background/Objectives: Citrus limon leaf essential oil (EO) is traditionally used for its calming and cognitive-enhancing properties. Although the chemical composition of C. limon leaf essential oils (EOs) obtained by means of hydrodistillation (HD) and solvent-free microwave extraction (SFME) has been previously characterized, the influence of the extraction method on their neuroprotective efficacy and dose–response effects remains insufficiently explored. In the present study, EOs obtained by means of HD (CEH) and SFME (CEM) were compared for their behavioral, biochemical, and in silico neuroprotective effects against scopolamine (SCOP)-induced cognitive and anxiety-like impairments in adult zebrafish. Methods: Adult Tübingen zebrafish were exposed to CEH or CEM via immersion at 10, 100, and 150 µL/L for 19 days prior to SCOP challenge (100 µM). Cognitive performance was evaluated using the Y-maze and novel object recognition (NOR) tests, while anxiety-like behavior was assessed using the novel tank test (NTT) and novel approach test (NAT). Brain acetylcholinesterase (AChE) activity and oxidative stress markers were quantified. Molecular docking analyses were conducted to investigate interactions between major EO constituents and AChE and monoamine oxidase A (MAO A). Results: Both CEH and CEM significantly attenuated SCOP-induced memory deficits, improved spontaneous alternation and NOR discrimination, and reduced anxiety-like behaviors. These effects were associated with AChE inhibition and restoration of redox balance. Notably, CEM generally exhibited stronger neurobehavioral and biochemical effects at comparable doses. In silico analyses supported these findings, revealing favorable binding affinities of key EO constituents toward cholinergic and monoaminergic targets. Conclusions: This study demonstrates that the extraction method influences the neuroprotective efficacy of C. limon leaf EOs. While both CEH and CEM exert antioxidant and cholinergic modulatory effects, CEM shows enhanced neuroprotective potential in a zebrafish model of SCOP-induced cognitive impairment, supporting the relevance of extraction-dependent biological profiling in EO-based neurotherapeutic research. Full article