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.
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 1H1H cross relaxation rate, and the H2O-assisted 1H13C cross polarization rate to lipid alkyl 13Cs, 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.
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.
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 13C13C 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.
Neurodegenerative diseases such as Alzheimer's, Parkinson's, frontotemporal dementia, and ALS are characterized by amyloid protein aggregation involving intrinsically disordered proteins that are also capable of liquid-liquid phase separation (LLPS). LLPS, known to drive the formation of dynamic membraneless organelles essential for cellular functions, can play a role in limiting fibrillation process or aberrantly transition into solid aggregates under pathological conditions. Here we review how mutations, post-translational modifications, and environmental factors can modulate LLPS of proteins like Tau, TDP-43, FUS, and α-synuclein, potentially regulating amyloid aggregation. We also examine the interplay of these proteins exploring how LLPS and condensate maturation could impinge on the emergence of co-pathologies contributing to disease progression. Finally we discuss emerging therapeutic strategies, aimed at modulating phase separation dynamics.
The phosphatidylinositol 3-kinase (PI3K) pathway is a vital intracellular signaling cascade that plays a key role in cancer cell survival, angiogenesis, and metastasis. Consequently, PI3K has been a primary target for therapeutic inhibition in the treatment of various malignancies. Accumulating studies indicate that targeting multiple isoforms of PI3K may enhance antitumor activity. The advancement of pan-PI3K inhibitors for cancer treatment offers a promising research and development opportunity. This study introduces a machine learning-based virtual screening (VS) method that combines a Naïve Bayesian classification model employing molecular fingerprints and descriptors, alongside a pharmacophore model and consensus scoring-based molecular docking, to discover new pan-PI3K inhibitors. The VS method was validated for its strong predictive accuracy by successfully identifying the commercially available pan-PI3K inhibitor copanlisib. The hybrid VS strategy was applied to the SPECS database, leading to the discovery of several promising PI3K inhibitor compounds. The machine learning-based VS approach is expected to offer meaningful insights and a practical framework for discovering novel PI3K inhibitors.
Transmission electron microscopy (TEM), together with Thioflavin T (ThT) fluorescence assays, is widely used to visualize amyloid fibrils and to characterize the kinetics of amyloid formation. However, discrepancies between ThT fluorescence data and TEM observations are sometimes reported, which may arise from limitations in fibril visualization by TEM. In particular, TEM imaging can be strongly influenced by the sample loading procedure on the grid, which governs fibril deposition and retention. In this work, five different grid preparation methods were compared to evaluate their efficiency in detecting and visualizing human islet amyloid polypeptide (hIAPP) fibrils, which are present in 95% of patients with type 2 diabetes mellitus. The methods were assessed based on detection speed, morphological representation, fibril abundance and grid contamination. The two best-performing methods were further evaluated for detecting early hIAPP aggregates and subsequently applied to another amyloid forming protein, namely amyloid-β 42 (Aβ42), which is involved in Alzheimer's disease. Among the tested approaches, method 2 (a droplet-deposition protocol) and method 3 (a centrifugation-based loading protocol) provided the most efficient fibril detection and morphological representation. Method 2 was identified as the best compromise between rapid detection, experimental simplicity, and low grid contamination, and was further tested under different buffer conditions. Overall, this comparative study demonstrated that variations in grid preparation protocols can significantly influence TEM observations and provide practical guidance for selecting optimal conditions for amyloid fibril imaging depending on experimental objectives.
Antimicrobial resistance (AMR) has emerged as a major global health challenge, contributing to nearly 5 million deaths annually, according to recent WHO reports. Nanotechnology offers an alternative, with biologically produced nanoparticles being regarded as sustainable and potent antimicrobial agents. Iron oxide nanoparticles (IONPs), particularly Fe3O4 and Fe2O3, are of particular interest due to their unique magnetic, physicochemical, and bio-functional properties. The review discusses the reduction mechanism that explains the biological production of IONPs using microbial biomolecules and phytochemicals as natural capping, stabilizing, and reducing agents. Particular attention is given to biomolecules-mediated electron transfer, Fe2+ and Fe3+ redox cycling during the nucleation process and how surface functionalization determines nanoparticles' stability and activity. Additionally, this review discussed how these reduction mechanisms directly affect antimicrobial action through the generation of reactive oxygen species (ROS), membrane rupture, DNA/protein damage, and biofilm inhibition. A comparative analysis of plant and microbe-mediated synthesis is presented, along with structure-activity correlates that regulate antibacterial activity. Applications of biogenic IONPs are examined in the realms of biomedicine, industry, and the environment, with particular focus on their potential use in combination therapy to prevent antibiotic resistance. The positive factors of eco-friendliness, increased biocompatibility and multifunctionality are offset by such factors as variability in synthesis, scaling and long-term safety. Finally, in the future, the combination of clinical translation regulatory frameworks, omics-based mapping of bacterial responses, and molecular-level mechanistic investigations will be of relative importance. Altogether, biologically synthesized IONPs are an eco-friendly and effective approach to prevent microbial resistance that can be applied to the interface between green chemistry and more sophisticated nanomedicine. This review uniquely integrates comparative plant- and microbial-mediated synthesis, mechanistic Fe3+/Fe2+ reduction pathways, physicochemical optimization parameters, and molecular antimicrobial mechanisms involved in combating multidrug-resistant pathogens.
This study introduces a novel high-performance biochar adsorbent derived from Pennisetum Alopecuroides, a previously unexplored biomass source for water remediation, through phosphoric acid-activated chemical carbonization. Employing a systematic Box-Behnken experimental design, we optimized the adsorption process for tartrazine dye removal, achieving an exceptional adsorption capacity of 71.75 mg/g (20 mg adsorbent dose, pH 6.4, 328.15 K, 40 mg/L initial concentration). The adsorption mechanism was comprehensively elucidated through an integrated approach combining experimental characterization (FTIR, SEM-EDX, XRD) with theoretical DFT calculations (B3LYP/6-311G), revealing specific interaction sites and confirming monolayer adsorption following pseudo-second-order kinetics and Langmuir isotherm models. Thermodynamic analysis demonstrated spontaneous, endothermic adsorption (positive ΔH°, negative ΔG°). Notably, this work represents the first reported application of Pennisetum Alopecuroides-derived activated carbon for dye remediation, offering a sustainable and cost-effective alternative to conventional adsorbents. The demonstrated high efficiency, coupled with the renewable nature of the precursor material, positions this adsorbent as a promising candidate for industrial-scale implementation in textile wastewater treatment systems.
Coronavirus disease 2019 (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), was first identified in the People's Republic of China and is characterized by infection of airway epithelial cells, frequently leading to severe acute respiratory syndrome and clinically diagnosed as severe human pneumonia. The primary targets of SARS-CoV-2 are lung cells, where viral-host interactions trigger extensive molecular and structural changes. In this work, we developed fully atomistic reactive molecular dynamics simulations to investigate the binding energies, binding atoms, and dynamic behavior of biological assemblies of viral protein-ligand fragments associated with SARS-CoV-2. The studied protein structures correspond to the PDB entries (6Y84), (5REH), (5REJ), (5REK), (5REN), (5REO), (5RF0), (5RGG), and (6LU7). All simulations were performed using the reactive force field ReaxFF implemented in the Large-scale Atomic/Molecular Massively Parallel Simulator (LAMMPS), enabling explicit treatment of bond formation and bond breaking processes. The molecular dynamics simulations were conducted at room temperature to analyze the dynamical behavior of viral protein ligands under realistic external conditions. Our results reveal that all classes of viral ligand proteins exhibit pronounced chemical reactivity, characterized by the spontaneous formation of new chemical bonds during thermal equilibration. Our reactive classical molecular dynamics (ReaxFF) simulations show that SARS-CoV-2 viral protein assemblies undergo significant chemical reactivity, forming both covalent (mainly C-C, C-N, and C-O) and non-covalent interactions. Newly formed covalent bonds exhibit lengths of 1.2-1.4 Å and average angles around 120°, consistent with sp2-like geometries that contribute to local nanostructural stabilization. Nanometric solvation analysis further reveals distinct hydration profiles, where 6Y84 exhibits a compact shell at 3.04 Å suggesting a "dry" catalytic pocket, while 6LU7 displays the most extended hydration influence and the highest hydrogen bond stability (avg. 78.88). Detailed analysis of the ReaxFF energy contributions, including bond, angle, and nonbonded interaction terms, demonstrates that the observed reactivities are energetically consistent and thermodynamically stable. The potential energy curves further confirm both the validity of the ReaxFF parameter set and the nanostructural stability of the viral protein-ligand fragments under ambient temperature conditions. These findings highlight the intrinsic mutability and reactivity of SARS-CoV-2 binding proteins at the nanoscale. The present theoretical results complement previous crystallographic and experimental studies and provide novel insights into nanostructural activity-reactivity relationships of viral fragments that remain inaccessible to current experimental techniques, thereby offering valuable perspectives for future antiviral research.
The clinical application of berberine is limited by its poor bioavailability. To overcome this, berberine-saturated fatty acid salts have been developed, yet their interaction mechanism with hemoglobin, a key transport protein, remains unclear. This study aims to comprehensively investigate the interactions between bovine hemoglobin (BHb) and three berberine mid-chain saturated fatty acid salts ([BBR][FAs], specifically berberine caproate ([BBR][CAP]), berberine heptylate ([BBR][HEP]), berberine octanoate ([BBR][OCT])) by various experimental and molecular docking methodologies. UV-visible and fluorescence spectroscopic analyses demonstrate that [BBR][FAs] can bind to BHb with 1:1 stoichiometry and quench its fluorescence intensity through a static quenching mechanism. According to the evidence of synchronous fluorescence, three-dimensional fluorescence and FT-IR spectrometry, it was found that the microenvironment and secondary structure of BHb undergo alterations upon binding with [BBR][FAs]. Based on Förster resonance energy transfer analysis, the binding distance between [BBR][FAs] and BHb was determined to be within the range of 2.98 to 3.15 nm. This binding is associated with an increase in the esterase-like activity of BHb. The aggregation behavior of [BBR][FAs] in BHb aqueous solution was investigated from conductivity measurement. The critical aggregation concentration of [BBR][FAs] exhibited a positive correlation with increasing concentrations of BHb. Molecular docking data confirmed that the binding of [BBR][FAs] to BHb was principally mediated by hydrophobic interaction, hydrogen bonding and van der Waals force. Furthermore, this work systematically compared the chain length-dependent binding affinity between [BBR][FAs] and BHb, and observed the order of binding strength as: [BBR][CAP] ˂ [BBR][HEP] ˂ [BBR][OCT]. These findings provide quantitative structure-affinity relationship data.
Acute respiratory distress syndrome (ARDS), a life-threatening pulmonary dysfunctional condition marked by hypoxemia, is a leading cause of high mortality in critically ill patients. Despite its clinical significance, the metabolic disruptions underlying ARDS, particularly in the context of severe acute pancreatitis (SAP), need further understanding. This study employed Nuclear Magnetic Resonance (NMR)-based metabolomics to investigate serum samples from 92 intensive care unit (ICU) patients, 27 with SAP-induced ARDS and 65 patients with ARDS due to other aetiologies. Multivariate and univariate statistical analyses were utilized to distinguish the metabolic profile between these groups. The study identified delineation in 13 significant metabolites and metabolic pathways corresponding to severity in ARDS patients due to SAP. Decreased levels of proline and glucose, along with increased levels of 3-Hydroxybutyrate (3-HB), citrate, lactate and branched-chain amino acids (BCAAs) were identified. The findings of this study may provide potential biomarkers to facilitate early diagnosis, guide prognosis, and support the development of novel therapeutic interventions, which, aligned with established ICU guidelines, could contribute to improved patient outcomes in ARDS induced by SAP.
Electrostatic Force Microscopy (EFM), an extension of Atomic Force Microscopy (AFM), characterizes electrical properties at the nanoscale by detecting long range electrostatic force gradients. It enables simultaneous acquisition of topography and electrostatic information from biological samples. Dynamic electrical changes are fundamental across all life processes, from biomolecular charge transport to cellular electrophysiology. EFM's high resolution and nondestructive nature make it essential for revealing the physical mechanisms at biological interfaces. This review systematically elaborates the evolution of EFM principles, including optimized working modes tailored for biology, and analyzes its hierarchical applications, from biomolecules and subcellular structures to cells and pathological diagnosis. Cross scale dielectric correlations and current technical challenges are discussed, alongside advanced solutions such as heterodyne high harmonic detection and multimodal integration, offering a systematic perspective for EFM's translational development in biomedicine.
A yeast cell template technology is described to biomimetically synthesize FeTi oxides. The resulting samples, yeast Fe2O3 and yeast Fe2O3/TiO2, are then employed as active materials for the electrochemical detection of cancer-associated miRNAs. For the perfect matched cancer miR-141, electrochemical analysis shows a main oxidation peak at about +0.4 V, along with a reduction peak at about +0.30 V. For the detection of cancer miR-29c, the introduction of the Ti element is found to stabilize the structure of the resulting materials. These results suggest a potential of the biomimetic FeTi oxides as promising sensing materials for miRNA electrochemical biosensors.
The development of nanotechnology mainly encompasses with establishment of a technical evolution in every phase of science along with physical, chemical, biological and material science. Nanoparticles synthesized from chemical pathway engendering serious biological risk and toxicity to living organisms. The toxicity of nanoparticles to the natural world mainly caused by synthetic process and small size rang which can easily pass across physiological barrier. Toxicity because of nanoparticle dimensions can't be neglected however, nullification of toxicity due to synthetic pathway can be done by biosynthesis pathway using plants, bacteria, algae, fungus, yeast and viruses as a valuable alternative to non-biological physicochemical method. Among all plant extract-based synthesis is more famous. The phytochemicals present in plant extracts are acted as bio-reductant as well as capping agent. This makes it ecologically effective and toxicity free. In this review, the synthesis method using plant extract is discussed. We also summarized data of some plant mediated nanocrystals with description on synthetic techniques, and characterization techniques. Further, it contains their role and their applications in terms of removal of dye, antimicrobial activity and anticancer activity. This review involves critical evaluation of near about 60 already discussed research papers on plant base nanoparticle synthesis to create fundamental theoretical framework that allows knowledge development for young researchers. We summarized the study of various nanoparticles that will definitely help new researchers to grasp trends and patterns in previously studied nanoparticles. Overall, our review will beneficial for readers to create general interest regarding this emerging field and will encourage to investigate those plants which have not been used in the past.
