Treatment options for hepatocellular carcinoma (HCC) remain unsatisfactory, and JAK2 continues to attract interest as a point of pharmacological intervention in this malignancy. The present work asked whether Neoruscogenin, a spirostanol sapogenin, and its predicted human metabolites can engage that node. Intersecting predicted compound targets with HCC-associated genes returned 97 shared proteins. Degree-based ranking placed IL2, SRC, STAT3, MTOR, PIK3CB, MAPK1, JAK2, MDM2, KIT and MAPK14 at the center of the resulting interaction network, and functional enrichment pointed toward cancer-related, MAPK, PI3K-Akt, apoptotic and immune-associated signaling. Four biotransformation products retaining the steroidal core were carried forward. Across ten prioritized receptors, the parent compound scored best against JAK2 (PDB 5AEP, -10.48 kcal/mol), among the metabolites, the 3-O-glucuronide BTM00003 came within 0.17 kcal/mol of it (-10.31 kcal/mol), and both exceeded the reference inhibitor QUP (-8.87 kcal/mol). All three complexes survived 200 ns of unrestrained dynamics, but the routes to stability differed: free energy landscape, principal component and cross-correlation analyses described a narrow, dynamically restrained basin for QUP, whereas Neoruscogenin and BTM00003 stayed bound while exploring wider conformational space. MMGBSA returned favorable binding free energies for all three, with QUP most negative. Predicted ADMET combined near-complete intestinal absorption for Neoruscogenin with poor aqueous solubility and a hepatotoxicity alert, and DFT descriptors described a hard, electronically stable scaffold. Neoruscogenin and its glucuronide therefore merit experimental follow-up in HCC as starting points rather than as optimized candidates.

Traditional Chinese medicine (TCM) supramolecular assemblies are commonly found in TCM decoctions. As a form of the material basis for pharmacological efficacy, TCM supramolecular assemblies not only enhance therapeutic effects and reduce toxicity but also represent an important manifestation of the "integration of medicine and adjuvant" characteristic of TCM components. However, current research rarely focuses on the structural features of TCM components capable of forming supramolecular assemblies, and there is a lack of guiding principles for screening self-assembling components. Evaluating the self-assembly ability of various components solely through classical experiments is time-consuming and labor-intensive. Therefore, in this study, we employed a computational simulation method (N-GAFF-CM) to assess the self-assembly capabilities of 55 structurally representative TCM components in a simulated decoction environment. Furthermore, quantitative structure-property relationship (QSPR) models correlating structure and self-assembly binding energy were constructed using multiple machine learning (ML) algorithms. Isothermal titration calorimetry (ITC) was utilized to construct an independent external test set, strictly validating the external predictive performance of the established QSPR models on unseen compounds. Finally, cluster analysis was applied to systematically explore the structural characteristics of components capable of self-assembly. Kinetic and thermodynamic evaluations revealed that at an optimal concentration of 10 mM, 46 components show a favorable assembly trend, while exactly 28 components could spontaneously form stable supramolecular structures (ΔG < 0) primarily driven by non-covalent interactions (e.g., π-π stacking and hydrogen bonding) closely related to their specific structural features. Among the ML algorithms tested, the model built using the ExtraTrees algorithm performed best (R[2] = 0.963, RMSEP = 3.075), proving suitable for predicting the self-assembly binding energy of TCM components. Analysis of descriptor importance in the model and clustering results revealed that molecules with smaller molecular weight (Mw < 326 Da), a higher number of carbonyl groups connected to aromatic rings (nArCO ≥2), and greater molecular planarity were more prone to form supramolecular assemblies in the simulated decoction environment. The present work provides a rapid and reliable computational approach for screening monomer components capable of forming supramolecular assemblies in TCM decoctions and lays a methodological foundation for the efficient development of supramolecular nanodrugs and nano-delivery systems based on TCM decoctions system.

The widespread use of antibiotics in human medicine and veterinary practices has raised significant public health concerns, particularly regarding the development of antibiotic resistance and the presence of antibiotic residues in food products. To address these challenges, we present aptamers-single-stranded oligonucleotides that can bind specific targets with high affinity-as promising tools for the detection of antibiotics. Aptamers offer advantages over traditional antibodies, including cost-effectiveness, rapid synthesis, and ease of modification. The integration of aptamers into biosensors, particularly fluorescent aptasensors, has emerged as an innovative approach for the sensitive and selective detection of antibiotic residues in various matrices, including food and environmental samples. This review examines the application of graphene-based materials, particularly graphene oxide, in enhancing the performance of aptasensors. The unique properties of graphene oxide facilitate the development of highly sensitive biosensors capable of real-time monitoring of antibiotic contamination. We summarize recent advancements in aptamer-based detection methods, highlighting their potential to mitigate the health risks associated with antibiotic residues in food products. Ultimately, this review underscores the necessity for the development of rapid, economical, and precise detection methods to ensure food safety and public health.

Cognitive decline (CD) and dementia are major age-associated neurodegenerative conditions lacking minimally invasive biomarkers that reflect underlying disease biology. Systemic metabolic alterations closely mirror brain bioenergetic dysfunction and may provide accessible indicators of disease progression. This study applied quantitative [1]H NMR-based serum metabolomics to identify metabolic signatures associated with cognitive decline and to evaluate their diagnostic relevance. The serum samples from individuals with cognitive decline (CD, n = 40) and cognitively normal controls (NCD, n = 54) were analyzed using high-field 800 MHz [1]H NMR spectroscopy. Multivariate (PCA, PLS-DA, OPLS-DA, Random Forest) and univariate statistical analyses were performed to explore their discriminatory performance. Receiver operating characteristic (ROC) analysis evaluated diagnostic performance, and correlations with Mini-Mental State Examination (MMSE) scores assessed clinical relevance. Thirty-three metabolites and key metabolic ratios were quantified. CD subjects showed significantly increased glucose, pyruvate, acetate, creatine, and mannose, while lactate, Leucine, Methanol, Valine, and Histidine were decreased. Metabolic ratios including glutamine-to-glucose (QGR), alanine-to-glucose (AGR), and histidine-to-tyrosine (HTR) were markedly reduced. AGR and QGR further declined with increasing disease severity and AGR positively correlated with MMSE scores. ROC analysis showed promising discriminatory performance within the study cohort for QGR (AUC = 0.876), AGR (AUC = 0.840), and HTR (AUC = 0.822). The observed metabolic pattern is consistent with alterations in systemic energy metabolism, amino-acid homeostasis and metabolic flexibility. These findings support the concept of dementia as a disorder of metabolic network dysregulation and highlight NMR-based serum metabolomics as a promising approach for objective disease assessment and progression monitoring.

Practically all the publications on properties of spread and adsorbed plant protein fibrils deal with the aggregates obtained from protein isolates containing the admixtures of other proteins. On the contrary, this work is devoted to layers of amyloid fibrils of recombinant vicilin, storage 7S globulin from garden pea, at an aqueous surface. The application of surface tensiometry, dilational surface rheology and ellipsometry allowed discovering that, unlike the layers of non-fibrillar protein, the spreading occurred only if the ionic strength of the spreading dispersion or of the subphase exceeded a certain value. The dynamic surface elasticity of the layers of vicilin fibrils proved to be a non-monotonic function of the surface pressure but reached much lower values at high surface compressions than in the case of layers of the non-fibrillar protein. The observed distinction can be explained by different structures of the surface aggregates in the two systems. Relatively rigid and long fibrils cannot form dense and rigid surface aggregates unlike the non-fibrillar state of the vicilin protein. At the same time, atomic force microscopy shows that the layers of fibrils still have some holes at high surface pressures. These finding highlight how fibril rigidity and salt screening control interfacial organization and may guide the design of plant protein-based stabilizers for foams and emulsions.

A new metal complex of ethylenediamine and sulfosalicylic acid, incorporating Pd[2+] as metal ion, was synthesized and analyzed using spectroscopic, electrochemical, physicochemical and quantum mechanical methods. Quantum mechanical calculations identified the lowest-energy structure for the new compound. The metal center adopts a four-coordinate, slightly distorted square planar geometry. Molecular electrostatic potential surface and HOMO - LUMO analysis were accomplished for the obtained structure. The compound demonstrated promising cytotoxic activity against K562 cancer cells, with an IC50 value of 36 μM. The biological activity was assessed by investigating interactions with calf-thymus DNA (CT-DNA) and bovine serum albumin (BSA) using UV-Vis and fluorescence spectroscopy. Hydrogen bonds/van der Waals interaction with Kb (CT-DNA) = 1.71 × 10[5] and Kb (BSA) = 1.00 × 10[5] were determined. Viscosity and electrophoresis experiments for CT-DNA and circular dichroism (CD) for BSA were also applied to further investigate the binding of the title complex with the mentioned biomolecules. Viscosity assay confirmed the groove binding with CT-DNA, and CD suggested the refolding of BSA in the presence of Pd(II) complex. Molecular docking modeling predicted the groove binding with CT-DNA and positioning in site I of BSA through some hydrogen bonds.

Adenosine-5'-triphosphate (ATP) can accumulate to near-molar concentrations within specific biological compartments, influencing a diverse range of cellular processes. However, its structural organization and molecular dynamics under these crowded conditions remain poorly understood. In this study, ATP gel systems containing ATP at near-molar concentrations were prepared and investigated using solid-state nuclear magnetic resonance (ssNMR) spectroscopy and all-atom molecular dynamics (MD) simulations. The ssNMR results reveal that ATP undergoes spontaneous hydrolysis and yields adenosine diphosphate (ADP) and inorganic phosphate (Pi). Combined ssNMR and MD analyses further suggest local purine-stacked and ion-mediated "tail-to-tail" arrangements of ATP/ADP molecules, in which cationic clusters attract anionic phosphate groups and restrict terminal phosphate mobility. The suppression of ATP β-phosphate signals is attributed to reduced hydration arising from steric hindrance and limited solvent accessibility. A comparison of ATP-HCl and ATP-HAc gels demonstrates that both ATP hydrolysis level and molecular dynamics are strongly modulated by anion identity. MD simulations further reveal a modest increase in Mg[2+]-mediated intermolecular bridging and a slightly higher local mass density in ATP-HCl gels. These results provide molecular-level insight into ATP behavior at high concentrations and highlight the critical role of anion species in regulating ATP stability and organization.

The interaction between c-Myb and the CBP/p300 KIX domain is a critical transcriptional regulatory event and an attractive target for the development of candidate disruptors of the recombinant c-Myb-KIX interaction. In this study, we used an integrated computational and experimental strategy to identify new small molecules capable of disrupting this protein-protein interaction. A focused Umbelliferyl phosphate scaffold library was subjected to stepwise virtual screening via drug-likeness assessment and docking to the c-Myb-binding region of the KIX domain and short molecular dynamics refinement. Selected compounds were then evaluated by 500 ns molecular dynamics simulations, MM/PBSA analysis, free energy landscape (FEL) mapping, and finally by microscale thermophoresis (MST) assay. Computational analyses showed that stable ligand binding did not necessarily translate into disruption of the c-Myb-KIX interface, allowing separation of compounds that stabilized the complex from those predicted to weaken it. Consistent with this distinction, ΔΔGPPIanalysis identified only MUP and Naphthol AS-BI phosphate as protein-protein interaction-weakening ligands, with Naphthol AS-BI phosphate showing the strongest predicted disruptive effect (ΔΔGPPI=+3.25 kcal/mol), whereas DiFMUP and Naphthol AS-D phosphate were predicted to stabilize the complex. Among the tested molecules, Naphthol AS-BI phosphate showed the clearest disruption-like behavior in silico and was the most potent inhibitor in vitro, with an IC50 of 18.9 ± 0.6 μM. Importantly, MUP emerged as the most promising umbelliferyl phosphate-derived hit, displaying measurable inhibitory activity (IC50 = 33.5 ± 0.3 μM) comparable to the reference Naphthol AS-E phosphate (IC50 = 31.2 ± 1.3 μM) and a more favorable predicted ADMET profile. Overall, this work identifies new chemical starting points for targeting the c-Myb-CBP/p300 KIX interaction and supports MUP as an attractive scaffold for further optimization.

Cathepsins are predominantly cysteine proteases that function in lysosomes and the extracellular matrix, where they regulate essential proteolytic processes. They are synthesised as inactive zymogens (procathepsins), in which an N-terminal propeptide blocks access to the active site and is removed during maturation. Glycosaminoglycans (GAGs), a class of linear, sulfated polysaccharides composed of repeating disaccharide units, are known to modulate both cathepsin activity and proenzyme processing. Here, we use molecular modelling to elucidate the role of GAGs in the maturation of procathepsin K. Molecular docking, molecular dynamics (MD) simulations, and binding free energy calculations identify putative allosteric sites on the procathepsin surface that mediate GAG recognition. Microsecond-scale MD simulations of the most stable complexes, analysed via principal component analysis (PCA), reveal GAG-dependent shifts in the conformational landscape of the proenzyme. To probe environmental effects, we further simulate apo and GAG-bound procathepsin K under lysosomal conditions (pH 4). These simulations demonstrate a synergistic interplay between acidification and GAG binding in promoting activation. While low pH destabilises propeptide secondary structure, GAG binding at a distinct allosteric hotspot amplifies this effect, promoting helix unwinding and facilitating propeptide dissociation. Overall, our results provide a molecular-level framework for GAG-assisted maturation of procathepsin K, highlighting cooperative environmental and allosteric regulation of zymogen activation.

Melittin, an anticancer peptide, is the main component of bee venom. However, bee venom also contains phospholipase A2 and hyaluronidase-toxic enzymes that induce inflammatory responses. To facilitate therapeutic applications of melittin, these toxic components must be isolated and removed. This study explored optimal conditions for separating phospholipase A2 and hyaluronidase from bee venom using molecular dynamics simulations. pH = 10 was identified as the optimal condition for melittin separation, as significant conformational changes in melittin were observed at pH = 12. Additionally, a temperature of 30 °C was found to be ideal for melittin separation, whereas a temperature of 40 °C altered its secondary structure and reduced its hydrophilicity. These findings provide a foundational framework for protein separation processes, ensuring that melittin maintains its therapeutic integrity during purification.

The emergence of methicillin-resistant Staphylococcus aureus (MRSA) calls for novel antibiotic adjuvants. Carvacrol (Car), a natural monoterpene phenol derived from the essential oils of aromatic plants, exhibits membrane-active properties and has potential as an antibacterial adjuvant. Here, we found that Car markedly enhanced the bactericidal activities of Gentamicin (Gen) against MRSA. Mechanistically, Car dissipated membrane potential, inhibited respiratory chain dehydrogenase activity, and depleted intracellular ATP. Moreover, Car promoted membrane lipid peroxidation, ultimately leading to structural and functional membrane damage and bacterial death. Notably, pharmacological perturbation of respiratory and metabolic pathways further supported that this synergistic effect depends on bacterial respiratory bioenergetics. In addition, the combination showed favorable in vivo antibacterial efficacy in both Galleria mellonella and murine infection models, without apparent toxicity under the tested conditions. Collectively, these findings indicate that Car may serve as a natural antibacterial adjuvant for combination therapy against drug-resistant infections.

Membrane disruption along the amyloidogenic aggregation of β-amyloid (Aβ) peptides is considered a molecular mechanism for the Aβ-induced cell toxicity and death. Yet, the underlying structural basis for the harmful Aβ-membrane interactions that lead to disruption remains poorly understood. We have been utilizing solid-state nuclear magnetic resonance (ssNMR) spectroscopy to explore the intermediate states of membrane-associated Aβ aggregation, as well as their roles in membrane disruption process. Aligning with this general objective, complementary quantitative ssNMR spectroscopy focusing on the modulation of phospholipid dynamics in membrane bilayers will provide useful insight about how these intermediate states influence the physicochemical properties and architecture of membranes. In the current work, we systematically investigate how specific molecular motions of phospholipids change in the presence of membrane-associated 40- and 42-residue Aβ isoforms, within the time frame of nucleation processes. Physicochemical parameters, including the lipid headgroup and lateral diffusive motion correlation time, the lipid alkyl chain to headgroup [1]H[1]H cross relaxation rate, and the H2O-assisted [1]H[13]C cross polarization rate to lipid alkyl [13]Cs, were monitored by various ssNMR spectroscopic approaches. The outcomes suggest a general rigidification of bilayers upon instant Aβ-bilayer interactions across different bilayer phospholipid compositions and Aβ isoforms, accompanied by increase of water accessibility to bilayer interiors. These observations, together with the knowledge of molecular structural evolution of Aβ within the same time frame, help to establish a molecular-level, schematic explanation of the membrane-associated Aβ nucleation process.

The formation of biomolecular condensates has been linked to a broad range of essential cellular processes, including immune signaling cascades, mRNA transport, and autophagy. Conversely, phase-separated condensates have also been associated with aberrant protein misfolding and subsequent aggregation in neurodegenerative disease-related processes. Protein phase separation and phase transitions involve the formation of heterogeneous and dynamic assemblies that can evolve into gel-like or semi-crystalline states, which are challenging to characterize using high-resolution structural biology techniques. Solid-state nuclear magnetic resonance (NMR) spectroscopy now offers a broad arsenal of methods to probe the structural and dynamic features of viscous condensates, elastic solids, coacervates and rigid protein assemblies. This review provides an overview of solid-state NMR approaches that are readily applicable and discusses potential methodological developments to investigate protein condensates and coacervates as well as to study protein phase separation and phase transitions.

Hydrogen sulfide (H₂S) has emerged as a crucial endogenous gaseous signaling molecule involved in diverse physiological and pathological processes, including vascular regulation, neuronal transmission, inflammation, and cellular redox homeostasis. The transient and highly reactive nature of endogenous H₂S necessitates the development of highly sensitive and selective analytical techniques capable of real-time detection in living systems. Among various sensing strategies, ratiometric fluorescent probes have attracted considerable attention due to their inherent ability to provide self-calibrated signals, thereby minimizing environmental interference and enabling quantitative imaging of H₂S in complex biological environments. Recent advances in probe design have introduced novel molecular scaffolds, organelle-targeted fluorophores, near-infrared emitters, and reaction-based sensing mechanisms that significantly enhance sensitivity, selectivity, and spatiotemporal resolution. These probes exploit diverse photophysical mechanisms including intramolecular charge transfer (ICT), fluorescence resonance energy transfer (FRET), excited-state intramolecular proton transfer (ESIPT), and photoinduced electron transfer (PET). In addition, nanomaterial-integrated probes and reversible sensing platforms have further improved quantitative detection and long-term monitoring of endogenous H₂S dynamics. Recent studies have demonstrated successful application of these probes in cellular systems, zebrafish, and mammalian disease models such as oxidative stress and acute liver injury. This review comprehensively summarizes the recent progress in ratiometric fluorescent probes for quantitative imaging of endogenous H₂S, focusing on molecular design strategies, sensing mechanisms, biological applications, and emerging technological trends. Furthermore, current challenges and future research directions are discussed to guide the development of next-generation H₂S imaging tools for biomedical and translational research.

Solid-state NMR spectroscopy is increasingly used to investigate the structure and dynamics of a wide range of chemical, material, and biological systems. Although limited sensitivity has long posed a major challenge, the recently developed MAS cryoprobe substantially alleviates this limitation. By enhancing the signal-to-noise (S/N) ratio without requiring sample freezing, the MAS cryoprobe is particularly well suited for studies of non-isotropic systems, including rigid solids (e.g., amyloid fibrils), semi-solids (e.g., membrane mimetics), and soft materials (e.g., nanodiscs and hydrogels). In this study, we demonstrate the enhanced sensitivity of solid-state NMR experiments on α-synuclein fibrils using a MAS cryoprobe. Compared with a conventional MAS probe, substantial improvements in S/N were observed in CPMAS, refocused INEPT, and 2D [13]C[13]C chemical-shift correlation spectra. The increased sensitivity enables the detection of slowly decaying signals in the indirect dimension, thereby accelerating the acquisition of high-resolution multidimensional solid-state NMR data. These results highlight the potential of MAS cryoprobes for structural studies of samples that are scarce, unstable, or transient, such as amyloid intermediaries.