Pesticides play a crucial role in protecting crops from pests, ensuring food security and higher agricultural yields. However, excessive pesticide use can harm human health, disrupt ecosystems, and contaminate soil and water sources. Endosulfan, a potent organochlorine pesticide, presents severe toxicological risks, manifesting as nausea, emesis, vertigo, cephalalgia, cognitive impairment, convulsions, and, in extreme cases, mortality. Owing to its extensive utilization in agriculture, it demonstrates a high propensity for vegetable contamination. The present investigation elucidates the molecular interplay between endosulfan and lysozyme, a protein particularly susceptible to its perturbative effects. Notably, an ADMET analysis confirms the facile permeation of endosulfan across the blood-brain barrier (BBB), underscoring its neurotoxic potential. Our findings reveal that while the overarching secondary structural integrity of lysozyme remains ostensibly preserved, its active site undergoes substantial perturbation upon endosulfan exposure. Despite its initial interaction at an allosteric site, the pesticide induces destabilization of key active-site residues, leading to a partial attenuation of enzymatic activity. Spectroscopic analyses indicate that endosulfan directly associates with Trp63, eliciting a subtle quenching effect in the fluorescence spectrum. However, this quenching is unequivocally attributable to static mechanisms, as substantiated by an increase in absorbance spectra coupled with an unaltered fluorescence lifetime. Moreover, this allosteric interaction profoundly modulates the protein's functional dynamics through allosteric perturbation propagation. Allosteric coupling intensity (ACI) analysis further delineates that His15 and Val92 experience significant perturbation, with ACI values exceeding 0.9, a phenomenon consistently observed in both the bound and unbound states. This underscores the pervasive influence of endosulfan on lysozyme's conformational stability and functional integrity.

The therapeutic efficacy of growth factor-mediated bone regeneration is frequently compromised by structural destabilization and loss of bioactivity following adsorption onto biomaterial surfaces. Addressing this challenge requires biomaterials capable of not only delivering osteogenic cues but also preserving protein functionality at the nanobiointerface. Herein, a hyaluronic acid-conjugated Zn, Se codoped hydroxyapatite nanobiocomposite (HA-Zn, Se-HAP) was engineered to investigate the molecular determinants governing growth factor stabilization and cellular response. Zn and Se incorporation modulated the apatite lattice and surface reactivity, while hyaluronic acid established a hydrated extracellular matrix-mimetic interface conducive to protein interaction. Using BMP-2 as a model osteogenic growth factor, spectroscopic, calorimetric, and computational analyses revealed a thermodynamically favorable and reversible adsorption process dominated by hydrogen bonding, electrostatic interactions, and desolvation effects, while preserving the native conformational architecture of the protein. Density functional theory (DFT) and molecular docking further elucidated the electronic redistribution, binding orientation, and intermolecular interaction motifs responsible for BMP-2 stabilization. The nanobiocomposite exhibited excellent cytocompatibility toward both L929 fibroblasts and MG-63 osteoblast-like cells, promoting cellular metabolic activity, proliferation, and collagen-rich extracellular matrix formation. The findings presented herein reveal the critical role of nanobiointerface engineering in governing growth factor adsorption, structural preservation, and downstream cellular behavior. By bridging interfacial physicochemistry with biological function, this work provides fundamental mechanistic insight into growth factor-biomaterial interactions and advances the design of bioactive osteoinductive platforms that extend beyond conventional carrier systems toward the active modulation of protein fate and regenerative outcomes.

Liraglutide, a glucagon-like peptide-1 receptor agonist, is widely used as a therapeutic macromolecule for the treatment of type 2 diabetes; however, its large-scale production is limited by high manufacturing costs and the frequent occurrence of closely related deletion impurities during synthesis. In the present study, we describe a novel, impurity-controlled, and industrially feasible synthetic strategy for liraglutide and its sequence-modified analogs. This method utilizes solution-phase incorporation of Pal-γ-Glu-OtBu in combination with a preassembled Boc-His (Boc)-Ala-Glu (OtBu)-OH tripeptide fragment, effectively minimizing the formation of des-His, des-Ala, and des-Glu impurities. The optimized protocol enabled the production of liraglutide and its analogs with consistent isolated yields and high chromatographic purity (> 95% by RP-HPLC), suitable for subsequent biological evaluation. The antidiabetic potential of liraglutide and three sequence-modified analogs, Lira (Trp-O25), Lira (desGly31), and Lira (Glu17), was evaluated using a Drosophila melanogaster model of high-sucrose diet induced diabetes. Among these, Lira (Glu17) exhibited the most pronounced metabolic improvements, significantly reducing free glucose (p < 0.001), trehalose (p < 0.001), and triglyceride levels (p < 0.001) when compared to diabetic controls. Furthermore, this analog effectively decreased lipid accumulation and reactive oxygen species in larval gut tissues and enhanced locomotor performance in both larva and adult flies. While Lira (Trp-O25) also demonstrated beneficial effects, Lira (desGly31) showed comparatively limited efficacy. Collectively, this study presents a cost-effective and impurity-controlled synthetic platform for liraglutide production and identifies Lira (Glu17) as a promising analog with enhanced antidiabetic activity, offering valuable insights for peptide manufacturing and GLP-1-based therapeutic development.

The development of IL-N8 and its magnetic micelle-forming derivative MIL-N8 offers a promising advancement in nanostructured carriers for efficient hydrophobic drug delivery. The structural validation for MIL-N8 was obtained through Raman, EPR, and NMR spectroscopy, which suggested successful synthesis and molecular integrity of both IL-N8 and MIL-N8 compounds. Furthermore, the thermogravimetric analysis demonstrated that MIL-N8 exhibits better stability than its precursor. Due to the structural attributes of MIL-N8 (containing both hydrophilic and hydrophobic moieties), it readily self-assembles into uniform nanostructures, such as micelles, at the critical aggregation concentration (CAC). This was further characterized through confocal microscopy using ANS as a fluorescent probe and visualized using TEM and FESEM imaging. These nanomicellar structures enabled the efficient encapsulation of the hydrophobic anticancer drug, quercetin (QCT), with high loading and sustained release behavior adhering to the Korsmeyer-Peppas model, which suggests diffusion-controlled transport through aggregated micellar layers. Furthermore, biological evaluation using SW-480 colon cancer cells demonstrated a remarkable enhancement in the anticancer activity of QCT-encapsulated and delivered via MIL-N8, moreover, in comparison to free QCT, the QCT-MIL-N8 formulation produced substantially lower IC50 values and pronounced dose and time-dependent effects. QCT-MIL-N8 formulation demonstrated increased apoptotic features, including nuclear condensation, fragmentation, and an increase in AO/EtBr-positive cells, as well as elevated intracellular ROS levels, that further supported oxidative stress-driven cell death as the predominant mechanism. Collectively, MIL-N8 nanomicelles emerge as a highly effective, low-toxicity delivery platform that significantly improves the therapeutic potential and controlled release of QCT for anticancer applications.

The aberrant aggregation of human islet amyloid polypeptide (hIAPP) or Amylin into toxic oligomers and fibrils leads to pancreatic β-cell dysfunction and progressive cell death, which is a key pathological feature of Type II diabetes mellitus (T2DM). In this study, we adopted a hybrid approach combining virtual screening and molecular dynamics (MD) simulation, with experimental validation, to identify inhibitors of hIAPP aggregation. Herein, we screened 2000 phytoconstituents from natural products using molecular docking, followed by in silico ADMET predictions. Withaferin A and Withanolide A (phytoconstituents of Ashwagandha) were found to be lead molecules with suitable drug-like properties. Next, we performed MD simulation to assess the stability and interaction dynamics of hIAPP-ligand complexes, and the effect of ligands on hIAPP fibrils. Building on the computational screening, we further carried out a comprehensive experimental analysis to validate the inhibitory effects of lead molecules. The collective experimental results from the Thioflavin T (ThT) assays, FTIR experiment, combined with Confocal and Transmission Electron Microscopy (TEM), suggest that the ligands (preferably Withanolide A) have a potent inhibitory effect against hIAPP aggregation by increasing the lag phase, reducing β-sheet content, and inhibiting fibril formation of hIAPP. For in vivo validation using Drosophila model, Withanolide A was found to mitigate hIAPP oligomer-induced toxicity by reducing apoptosis, necrosis, and oxidative stress in the Drosophila gut, as confirmed by multiple cell death staining assays and reactive oxygen species (ROS) analysis. Besides, in diabetic flies, Withanolide A lowered glucose levels, demonstrating anti-diabetic activity. Thus, this work, for the first time, suggests that Withanolide A may be a potential candidate for inhibiting hIAPP aggregation and as a T2DM drug.

Hydrogels have emerged as highly versatile biomaterials in biomedical science owing to their intrinsic biocompatibility, biodegradability, and tunable mechanical properties, enabling their widespread application in drug delivery, wound healing, tissue engineering, and corneal prostheses. In the present study, we have developed a composite hydrogel by incorporating hyaluronic acid (HA) into an unfolded, aggregation-driven bone morphogenetic protein-2 (BMP-2) network to enhance its functional performance for biomedical use. The inclusion of HA induced significant modifications in both physicochemical and biological properties of the system. Notably, the composite hydrogel exhibited a markedly improved pore architecture, with an average pore size of ∼15 µm, compared to the denser BMP-2-only hydrogel, thereby creating a more favorable microenvironment for cellular infiltration. In parallel, a substantial increase in water retention capacity was observed, attributable to the hydrophilic nature of HA and its influence on gelation behavior, resulting in a hydrated, extracellular matrix (ECM)-mimetic scaffold. Furthermore, the BMP-2/HA hydrogel demonstrated enhanced hemocompatibility and reduced cytotoxicity relative to the BMP-2 system, alongside sustained antibiotic release, which is critical for infection control during wound healing. Importantly, in vivo wound healing studies using Drosophila melanogaster revealed that the composite hydrogel accelerates wound closure by approximately 40-45% compared to BMP-2 alone. This improvement highlights the synergistic interplay between HA-driven biological activity and the structural support provided by the aggregated BMP-2 network, underscoring the potential of this composite hydrogel as a promising candidate for advanced biomedical and translational applications.

Protein refolding and unfolding assays are essential for understanding of protein conformational transitions, assessing the stability of proteins, particularly their acclimatization into various environmental conditions, and for deeper insights into the mechanisms of diseases and therapeutic treatment. Ionic liquids (ILs) are a common adduct in protein folding assays as they help solubilize and stabilize proteins. Here, we describe the "IL-supramolecular host-guest complexation" with β-cyclodextrin (β-CD) and the effect of β-CD on protein unfolding and refolding using the assistance of ILs. Isothermal titration calorimetry (ITC) was employed to demonstrate IL binding, whereas fluorescence titrations and NMR experiments were used to determine the host-guest complex conformations. Thermal denaturation and renaturation experiments showed that β-CD reversed the IL-stabilizing influence in these experiments providing a new technique for demonstrating a supramolecular strategy to regulate protein folding through controlled modulation of ionic liquid-protein interactions.

Macromolecular crowding significantly influences protein folding, stability, and aggregation within cellular environments. Here, Cellular Retinoic Acid Binding Protein I (CRABP I), a highly stable β-barrel protein with low intrinsic aggregation propensity, was employed to investigate the concentration-dependent effects of PEG 1000 on temperature-assisted refolding. Structural, functional, and biological consequences of refolding were examined using spectroscopic, microscopic, rheological, ligand-binding, zeta potential, and in vivo analyses. Refolding in the absence of PEG produced soluble but structurally heterogeneous intermediates. In contrast, low PEG concentration promoted intermolecular protein-protein interactions, leading to amorphous aggregation, altered structural properties, reduced retinoic acid-binding activity, and enhanced oxidative stress and cellular damage in Drosophila melanogaster. Higher PEG concentrations suppressed aggregation and favoured structurally stabilized conformations with preserved ligand-binding function. These findings demonstrate that PEG 1000 acts as a concentration-dependent molecular switch governing the balance between aggregation and productive refolding of CRABP I.

The present study introduces the development of a sustainable ZnO/ACPS composite derived from waste pistachio shells that combines adsorption and photocatalytic degradation for efficient methylene blue removal. The synergistic interaction between ZnO nanoparticles and activated carbon enhanced charge separation and dye removal efficiency. Comprehensive characterization by Fourier Transform Infrared Spectroscopy, X-ray diffraction, Scanning Electron Microscopy, Energy-Dispersive X-Ray Spectroscopy, Brunauer–Emmett–Teller, and Thermogravimetric analysis confirmed the functional groups, crystalline structure, morphology, elemental composition, surface area, and thermal stability of the composite. Parameters, including “catalyst dosage, initial dye concentration, stirring speed, contact time, and pH, were systematically evaluated” to improve adsorption and photocatalytic degradation performance. Under optimized conditions, the material exhibited nearly 90

Macromolecular crowding plays a pivotal role in shaping protein stability and bridges insights from in vitro studies to the cellular environment. We investigated the stability of CRABP I through urea melt studies in the presence of PEG 2000 and PEG 4000, monitoring fluorescence wavelength shifts as sensitive indicators of structural transitions. In the absence of crowding agents, CRABP I unfolded with Cm at 4.39 M urea, whereas both PEG variants shifted the unfolding transition to higher concentrations, indicating an enhanced stability. This stabilization reflects crowding-induced reshaping of the free energy landscape, where excluded-volume effects entropically favor the native compact state. PEG 4000, with its larger size, imposed stronger steric constraints and augmented preferential hydration, thereby reinforcing intramolecular interactions and restricting the access of urea to the hydrophobic core. Complementary molecular dynamics simulations corroborated these mechanisms, highlighting how macromolecular crowding governs protein folding pathways and stability under physiologically relevant conditions.

In the present study, the effects of three structurally distinct imidazolium-based ionic liquids on the model protein CRABP I were investigated using a combination of spectroscopic and computational approaches. The spectroscopic results reveal that the ionic liquids interact primarily with surface-exposed regions of CRABP I, leading to concentration-dependent fluorescence quenching and minor perturbations in the local environment of tryptophan residues while largely preserving the β-sheet-dominated secondary structure of the protein. Spectroscopic observations suggest that the quenching process involves contributions from both ground-state association and excited-state interactions, indicating a mixed quenching mechanism. Circular dichroism measurements further confirm that the global secondary structure of CRABP I remains largely intact in the presence of the ionic liquids. To complement the experimental observations, molecular docking and molecular dynamics simulations were carried out. The combined results demonstrate that variations in ionic liquid cation architecture influence the strength and mode of interaction with CRABP I, with each system showing distinct interaction patterns and stabilization effects. This study provides insight into how structural differences in ionic liquids govern their interactions with proteins and highlights the importance of ionic liquid design in developing biocompatible media for protein stabilization and related biotechnological applications.

Designing multifunctional nanocarriers that integrate controlled drug delivery, favorable protein interactions, and accelerated tissue repair remains a key challenge in nanomedicine. In this study, we have engineered a magnetically responsive, biocompatible Fe3O4@SiO2 nanoparticle functionalized with a bromocholine-based ionic liquid. This surface design enabled efficient encapsulation and sustained release of the hydrophobic anticancer drug quercetin over 72 h under physiological conditions. Given the critical role of plasma protein adsorption in determining nanomaterial fate, systematic interaction studies with human serum albumin (HSA) were investigated using spectroscopic analyses, molecular docking, and esterase-like activity assays. The results showed that the nanocarriers exhibited stable protein binding with minimal conformational perturbation and preserved enzymatic activity, indicating excellent biocompatibility. Further, in an in vivo wound healing model, topical application of quercetin-loaded nanocarriers at 500 ppm led to rapid wound closure, with significant wound reduction observed within 3 h. The magnetic core further offers potential for external guidance to wound sites. Overall, this bromocholine-functionalized platform combines tunable drug release, biointerfacial compatibility, and rapid healing efficacy, making it a promising candidate for advanced wound therapy.

Human serum albumin (HSA) plays a key role in regulating the transport and bioavailability of therapeutic molecules, making protein-ligand interaction studies essential for assessing biomedical compatibility. In this work, the interaction of cotarnine and its derivatives with HSA was systematically investigated using spectroscopic, thermodynamic, and molecular docking approaches, along with evaluation of antibacterial, wound healing, and hemostatic activities. Spectroscopic analyses confirmed efficient binding of the derivatives to HSA without disrupting its native secondary structure, while thermodynamic studies revealed spontaneous, enthalpy-driven interactions dominated by noncovalent forces. Docking studies identified favorable binding orientations within HSA binding pockets, complementing the experimental results. All derivatives of cotarnine show greater binding and biological activity, such as wound healing and hemostatic, than cotarnine. Among the derivatives, L2 exhibited the strongest binding affinity and superior biological performance, which can be attributed to enhanced hydrophobic interactions arising from its chloro-substituted aromatic moiety. Overall, the combined biophysical, computational, and biological findings highlight cotarnine derivatives, particularly L2, as promising multifunctional candidates for wound-healing and related biomedical applications.

Glycosaminoglycans (GAGs), a class of carbohydrates integral to the extracellular matrix in biological systems, such as the connective tissue, cornea, and synovial fluid, significantly influence protein dynamics. This study investigates the unfolding kinetics and dynamics of Bone Morphogenetic Protein-2 (BMP-2) in the presence of glycosaminoglycans, specifically, hyaluronic acid (HA) and sulfated hyaluronic acid (SHA). The findings reveal that BMP-2 undergoes faster unfolding in the presence of SHA compared with HA in chemical denaturation. This accelerated unfolding can be attributed to a complex interplay between viscosity and the binding or nonbinding interactions between the glycosaminoglycans and the protein. Despite HA exhibiting a higher viscosity than SHA, the anisotropy of the intrinsic fluorophore of the protein demonstrates a significantly higher fluorescence anisotropy and anisotropy decay time in the SHA environment. Fluorescence lifetime measurements and rotational correlation times further substantiate this observation, with anisotropy kinetics indicating a binding interaction of BMP-2 with SHA. Additionally, the protein's unfolding mechanism in the presence of HA and SHA follows a mixed pathway, involving both direct and indirect processes. Energetically, the unfolding of BMP-2 in the SHA environment is considerably more stable than that in HA, suggesting a more robust interaction between the protein and sulfated glycosaminoglycans.

Amyloid formation and protein aggregation are essential mechanisms in a number of physiologic and pathological circumstances. This study explores the influence of ammonium-based ionic liquids with varying hydrophobic alkyl chain...

The present article examined the impact of ammonium-based dicationic ionic liquids (ILs) as co-solvent on the biophysical behaviour of CRABP I protein using various spectroscopic techniques along with molecular docking to support the experimental outcomes. Fluorescence study revealed significant alternations in the microenvironments of aromatic fluorescent amino acid residues. Fluorescence intensity indicated a decline upon increase in concentration of co-solvents, with a red shift. A similar trend also observed with the synchronous study. Stern-Volmer parameters revealed the static quenching behaviour whereas, existence of electrostatic interaction between protein and ILs is revealed from binding study using temperature-dependent fluorescence measurements. Conformational and structural changes of the protein were analysed using Circular Dichroism spectrometer. Molecular docking study supported the spectroscopic outcomes at the molecular level.

Bone fractures remain a formidable clinical challenge, particularly in oxidative and inflammatory microenvironments where conventional hydroxyapatite (HAP)-based scaffolds exhibit poor redox buffering, limited biointeractivity, and suboptimal integration with native extracellular matrix (ECM) signaling pathways. In order to address these multifactorial limitations, we report the rational design and one-pot synthesis of bioinspired nanocomposites comprising chondroitin sulfate (CS)-functionalized HAP, which is further engineered with surface-exposed thiol (-SH) or amine (-NH₂) group. This aqueous-phase synthesis simultaneously orchestrates Ca2-templated HAP nucleation and CS conjugation, enabling hierarchical assembly with molecular level interface control. Comprehensive spectroscopic and microscopic analyses validate the formation of structurally coherent, chemically stable CS-HAP nanocomposites with post-synthetic -SH and -NH₂ functionalization. The developed nanobiocomposites exhibit enhanced cytocompatibility in the MG-63 osteoblast-like cell line and effectively attenuate ROS induced apoptosis in the Drosophila melanogaster model. This one-pot synthesis strategy presents a unified platform for engineering biomimetic and osteoinductive nanoscaffolds with dual functionality, supporting bone regeneration while concurrently serving as potent antioxidant candidates. The approach holds significant translational potential for therapeutic application in oxidative stress associated bone fracture microenvironments.

Protein aggregation and disaggregation are critical in determining biomolecular stability and function. This study explores the influence of ammonium-based ionic liquids with varying hydrophobic chain lengths on the aggregation and disaggregation of bone morphogenetic protein-2 (BMP-2). Thioflavin T (ThT) fluorescence spectroscopy showed a fivefold increase in fluorescence upon aggregation, which further intensified in the presence of N8, indicating enhanced fibrillation. Fourier transform infrared (FTIR) spectroscopy analysis revealed that heat-aggregated BMP-2 exhibited a β-sheet content of 63% ± 9%, which increased to 68% ± 9% upon treatment with N8, whereas shorter-chain ionic liquids (N2 and N4) reduced the β-sheet content to 37% ± 4% and 32% ± 5%, respectively. Dynamic light scattering (DLS) measurements confirmed that the native protein had a hydrodynamic radius of 12 ± 5 nm, which increased to 390 ± 60 nm upon aggregation. Treatment with N8 and N6 further increased the radius to 680 ± 150 nm and 530 ± 100 nm, respectively, whereas N2 and N4 reduced it to 37 ± 5 nm and 60 ± 11 nm, respectively. Cytotoxicity assays indicated increased nuclear fragmentation and reactive oxygen species (ROS) generation with longer-chain ionic liquids. These findings suggest that shorter-chain ammonium-based ionic liquids exhibit greater disaggregating potential owing to the interruption of hydrogen bonds and π-π stacking interactions, which stabilize the fibril structure. Longer chains stabilize fibrils and promote fibrillation owing to the hydrophobic interaction between the alkyl chain of the cation and the hydrophobic surface of aggregates, providing insights into protein stability modulation.