Search Results (178)

Cataracts are the leading cause of reversible blindness worldwide; this condition results from the aggregation of lens proteins. Currently, surgery remains the only treatment; however, there is growing interest in non-surgical approaches, including the use of bioactive compounds incorporated into nanostructured systems designed to enhance solubility, enable controlled release, and improve bioavailability and bioactivity. Among the bioactive compounds investigated, curcumin has attracted considerable attention due to its antioxidant and anti-inflammatory properties, positioning it as a potential anticataractogenic agent. In the present study, curcumin-loaded nanoemulsion was developed via ultrasonication and characterized by average particle size, D90 percentile, ζ potential, and rheological behavior. In addition, its anti-cataract efficacy was evaluated both using an in vivo model in rats and an ex vivo model employing human cataract samples. The resulting curcumin-loaded nanoemulsion exhibited an average particle size of 152 ± 19.79 nm with a monomodal distribution, along with good physical stability over time. The nanoemulsion exhibited apparent viscosity between 30 and 25 mPa·s, at shear rate values (100 to 0 s⁻¹), indicating slight shear-thinning behavior. Regarding the effect on cataracts, in the in vivo model, cataract reversal was observed. Furthermore, ex vivo isothermal titration calorimetry (ITC) analyses indicated exothermic heat exchange between the curcumin nanoemulsions and cataract fragments, consistent with binding interactions occurring within lens components, likely involving crystallin proteins. These findings provide biophysical and in vivo evidence that intravitreally administered curcumin-loaded nanoemulsions not only prevent but actively reverse lens opacity, positioning them as a promising non-surgical therapeutic approach for cataract treatment. Full article

α-Glucosidase inhibitors (AGIs) significantly regulate blood glucose by delaying carbohydrate digestion and slowing glucose absorption, thus playing a critical role in glycemic control. Structurally, dihydromyricetin (Unless otherwise stated, the term dihydromyricetin used throughout this manuscript refers to trans-(2R,3R)-(+)-dihydromyricetin.) differs from myricetin in that the C2=C3 double bond in the C-ring is saturated, resulting in a dihydroflavonol instead of a flavonol. This study investigated the inhibition mechanism of α-glucosidase by the C2=C3 double bond structure using a set of integrated and multi-perspective approaches combining enzyme kinetics, multi-spectroscopic methods, molecular docking, isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR). Myricetin (IC₅₀ = 13.648 ± 0.157 μM) was found to be a more potent α-glucosidase inhibitor than dihydromyricetin (IC₅₀ = 453.922 ± 1.643 μM). Enzyme kinetics indicated that myricetin acted as a competitive inhibitor, whereas dihydromyricetin functioned as a non-competitive inhibitor. To further examine these interactions, multi-spectroscopic analysis demonstrated that binding of myricetin caused significant changes in the microenvironment around fluorescent amino acids (such as tyrosine and tryptophan) in α-glucosidase, resulting in slight unfolding of the enzyme structure. Additionally, molecular docking provided a detailed molecular perspective, identifying hydrogen bonding and hydrophobic interactions as the primary forces driving the binding of two flavonoids to α-glucosidase. Delving deeper into the binding mechanism, ITC analysis provided thermodynamic evidence that myricetin (K_D = 6.215 ± 0.022 μM) exhibited a stronger binding affinity to α-glucosidase than dihydromyricetin (K_D = 232.648 ± 1.236 μM), with both interactions being enthalpy-driven and primarily mediated by hydrogen bonds. Building on this, SPR analysis offered additional insights into the binding process, showing that myricetin not only had a higher binding affinity (K_D = 3.416 ± 0.015 μM) but also a faster association rate (k_a = 1668 ± 23 M⁻¹ s⁻¹) compared to dihydromyricetin (K_D = 11.539 ± 0.056 μM, k_a = 339.7 ± 17.1 M⁻¹ s⁻¹). In conclusion, this study demonstrated that the C2=C3 double bond plays a key role in enhancing α-glucosidase/inhibitor interactions, providing a theoretical basis for the design of novel AGIs and proposing a new set of multi-perspective methods for elucidating these inhibition mechanisms. Full article

Pseudomonas aeruginosa is a predominant spoilage organism in meat products. The extensive use of antimicrobial preservatives has, however, led to the emergence of resistant strains. Mitigating the resistance of foodborne P. aeruginosa to conventional preservatives has become a critical challenge in food safety. Piperonylpiperazine (Pip), a piperazine derivative sourced from Piper nigrum, was first examined in this work as a novel agent capable of both suppressing virulence and enhancing preservative efficacy against P. aeruginosa, with its mode of action elucidated. At sub-inhibitory concentrations, Pip strongly suppressed the production of virulence factors and potentiated the susceptibility of P. aeruginosa to the common preservative EDTA. Mechanistically, a multi-pronged approach involving pull-down assay, transcriptomic profiling, isothermal titration calorimetry (ITC) analysis, and gene knockout models demonstrated that Pip binds specifically to the LYS-164 and ASP-35 residues of the RhlI synthase, blocking the quorum sensing (QS) signaling cascade. This QS disruption led to reduced virulence factor production and attenuated pathogenicity in a Caenorhabditis elegans model. The compromised QS system subsequently induced oxidative stress, which disrupted cell membrane integrity and permeability, thereby potentiating EDTA’s antibacterial action. These findings suggest that Pip is a promising natural additive that can be used in combination with existing preservatives to enhance food safety. Full article

Oxybaphus himalaicus Edgew. is a traditional Tibetan medicinal plant used to treat nephritis and edema; however, its active constituents and the molecular mechanisms underlying its renoprotective properties remain poorly elucidated. This study investigated the pharmacological activity and mechanism of Boeravinone A (BA), a major constituent of O. himalaicus, in lipopolysaccharide (LPS)-induced acute kidney injury (AKI). A mouse model of LPS-induced AKI and LPS-stimulated RAW264.7 macrophages were used to evaluate the anti-inflammatory and renoprotective effects of BA in vivo and in vitro. Activity-based protein profiling (ABPP) was performed to identify potential molecular targets, followed by validation using isothermal titration calorimetry (ITC), cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) assays. The functional role of the identified target was further examined using shRNA-mediated knockdown and virtual knockout analysis. BA dose-dependently attenuate LPS-induced renal injury and reduced inflammatory responses. Phosphoglycerate kinase 1 (PGK1) was identified as a direct target of BA. Mechanistically, BA activated the Kelch-like ECH-associated protein 1–nuclear factor erythroid 2-related factor 2 (Keap1–Nrf2) pathway through PGK1, enhanced the expression of antioxidant enzymes such as Nqo1, and reduced the production of pro-inflammatory cytokines, including IL-1β and IL-6. Virtual knockout of PGK1 in macrophages further supported its regulatory role in this pathway. These findings suggest that BA exerts renoprotective effects by targeting PGK1 and activating the Keap1-Nrf2 pathway, thereby reducing oxidative stress and inflammation. This study provides a pharmacological basis for the traditional use of O. himalaicus and supports BA as a potential candidate for mechanism-based intervention in AKI. Full article

This study investigates the thermodynamics of binding between the affibody proteins ZTaq and anti-ZTaq across a broad temperature range, aiming to deepen the understanding of the underlying mechanisms governing their interaction. Affibodies are small, engineered proteins of notable stability and practical utility, serving as robust models for molecular recognition processes. Here, the anti-idiotypic binders ZTaq and anti-ZTaq were expressed and purified, and their interaction was characterized using isothermal titration calorimetry (ITC). The analysis revealed that the formation of the ZTaq:anti-ZTaq complex is marked by a large negative free energy of binding ΔG_b that is virtually unaffected by changes in salt concentration, in contrast to typical protein–polyelectrolyte systems where ionic strength plays a major role. Furthermore, the thermodynamic data indicated a large, negative heat capacity change ΔC_p, which is primarily attributed to conformational transitions, especially the disruption of the molten-globule-like (MG) state of anti-ZTaq above 303 K. By comparing thermodynamic and structural properties with related affibody systems, the study aims to clarify how specific sequence features contribute to the exceptional binding properties of these proteins, providing new insights into protein engineering for high-affinity molecular recognition. Full article

Adeno-associated viruses (AAVs) have emerged as promising vectors for gene therapy due to their non-pathogenic nature and ability to transduce various cell types efficiently. In recent years, there has been an increasing effort to optimize the production and purification of AAV to support clinical applications; however, challenges exist in affinity ligand design, synthesis, and characterization. Understanding the binding interactions of these viruses with functional molecules is pivotal for the development of affinity-based separation methods of AAVs. Classical methods to measure thermodynamic parameters such as Isothermal Titration Calorimetry (ITC) are challenging to employ in these scenarios, as the concentrations of the viral titers are significantly lower than those used in binding experiments with small biomolecules. Here, we present design principles that enable ITC-based determination of binding interactions between AAV2 and heparin. We observe increasing binding affinity with increasing molecular weight of heparin. We also elucidate the binding stoichiometry between AAV2 and heparins of varying molecular weights. Additionally, we report on the impact of buffer conditions and pH values on AAV2–heparin binding properties. Lastly, we also present the binding affinities and thermodynamic properties of interactions between the two species with heparin immobilized onto surfaces, namely, silica nanoparticles, as surface immobilization of the ligand is a common pathway for affinity-based separations. Overall, our results may provide key information for optimization of AAV-ligand binding protocols that are an essential step toward optimizing AAV capture and immobilization methods. 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

Glyphosate is a highly polar herbicide, the reliable molecular recognition of which is complicated by co-occurring structural analogues, metabolites, and derivatives in real-world samples. Rather than reporting new aptamer discovery, this study establishes a standardized, solution-phase isothermal titration calorimetry (ITC) workflow to thermodynamically reassess two literature-reported glyphosate DNA aptamers, Seq03 and Seq05, under matched buffer composition and instrument settings. After verification of baseline stability and evaluation of heat-of-dilution contributions, ligand-to-aptamer titrations yielded apparent dissociation constants of approximately 8.14 μM for Seq03 and 40.2 μM for Seq05, enabling affinity-based prioritization of these reported candidates within the tested concentration window. To define an application-relevant selectivity boundary, we further constructed a counter-screen panel restricted to glyphosate-related chemicals, including structural analogues, metabolites, and derivatives, and evaluated all candidates using an identical ITC protocol with explicit background handling. None of the counter-screen compounds produced binding-consistent, saturable isotherms after integration and control-based interpretation; instead, their responses remained close to background heat and were therefore operationally classified as having no detectable binding under the tested conditions, including a reverse-titration format check with Glufosinate-N-acetyl. Collectively, these results position ITC as a label-free, platform-independent validation step for small-molecule aptamer benchmarking prior to analytical translation, while also highlighting that the present conclusions are bounded by the tested PBS-based conditions and the sensitivity window of the current ITC configuration. Full article

Background/Objectives: PDE4 is a key regulator of cAMP signaling and a clinically validated anti-inflammatory target; however, the use of PDE4 inhibitors is often limited by adverse effects such as nausea, vomiting, and diarrhea. The natural compound braylin was previously identified as a novel PDE4 inhibitor scaffold, exhibiting an IC₅₀ of 0.96 µM. Using the PDE4–braylin co-crystal structure, we conducted structure-based design and optimization to enhance its potency. Methods: A series of novel braylin derivatives was synthesized and characterized. Their inhibitory activities against PDE4D were evaluated via enzymatic assays, and binding thermodynamics were analyzed by isothermal titration calorimetry (ITC). Molecular modeling was used to predict binding modes, and anti-inflammatory effects were assessed in LPS-stimulated macrophages. Results: Structure-guided optimization yielded lead compound L27, which showed significantly improved PDE4D inhibition (IC₅₀ = 67 nM) and high-affinity binding (K_d = 45 nM) as confirmed by ITC. L27 also exhibited remarkable selectivity against PDE isoforms. Molecular simulations highlighted key interactions with Gln369 and hydrophobic residues in the PDE4 active site. In cellular assays, L27 dose-dependently suppressed LPS-induced inflammation in macrophages at non-cytotoxic concentrations with efficacy comparable to roflumilast. Conclusions: We developed L27, a potent and selective PDE4 inhibitor derived from natural braylin. It demonstrated promising in vitro anti-inflammatory activity and represents a valuable lead for further therapeutic development. Full article

The rapid and accurate detection of SARS-CoV-2 biomarkers remains a critical requirement for effective outbreak control and decentralized diagnostics. Although RT-PCR is the current gold standard, its reliance on centralized laboratories and long processing times limits its applicability in point-of-care settings. In this context, optical biosensing platforms based on surface plasmon resonance (SPR) offer attractive features, including label-free, real-time, and quantitative detection. This study explores the use of synthetic receptors for the highly sensitive detection of the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. Specifically, soft molecularly imprinted polymer nanoparticles (nanoMIPs) were employed as synthetic receptors and integrated into a high-sensitivity, portable plasmonic platform based on a D-shaped plastic optical fiber (POF) SPR sensor. The nanoMIPs were selectively imprinted against the RBD, characterized by Dynamic Light Scattering (DLS), Isothermal Titration Calorimetry (ITC), and Scanning Electron Microscopy (SEM) to confirm nanoMIPs size, binding properties, and surface morphology. Next, the nanoMIPs were immobilized onto a gold-coated sensing surface, enabling enhanced specificity, affinity, and signal amplification compared to conventional biological recognition elements. The resulting RBD-SPR-nanoMIPs sensor demonstrated promising analytical performance, exhibiting high selectivity against potentially interfering proteins and an anticipated sensitivity suitable for RBD detection at femtomolar concentrations. The inherent stability of nanoMIPs suggests the potential for reusable SPR sensing platforms, paving the way for next-generation synthetic receptor-based plasmonic biosensors. Full article

This article investigates the thermodynamic driving force of the interaction between lysozyme (Lys) and sulfated β-cyclodextrin (β-CDS), with a particular emphasis on the elusive role of hydration during polyelectrolyte–protein binding. Using isothermal titration calorimetry (ITC), the binding affinity was quantified across varying temperatures and salt concentrations, employing a recently developed thermodynamic framework that explicitly separates the contributions from counterion release and hydration effects. The study reveals that while counterion release is minimal in the Lys/β-CDS system, hydration effects become a dominant factor influencing the binding free energy ΔG_b, especially as experimental temperature deviates from the characteristic temperature T₀. It demonstrates that hydration contributions can substantially weaken binding at increased salt concentration c_s. The high characteristic temperature T₀ and the salt-dependent heat capacity change indicate a complex interplay of water structure and ion association—significantly departing from commonly linear interpretations of ΔG_b vs. log c_s based solely on counterion release effects. This work advances the understanding of polyelectrolyte–protein interactions by providing the first direct quantification of the hydration effect in such complexes and may have an impact on the rational design of biomolecular assemblies and therapeutic carriers. Full article

Histatins are histidine-rich antimicrobial peptides present in human saliva, with histatin 5 (Hst5) demonstrating the most potent antifungal activity. Previous studies have linked the antifungal properties of histatins, particularly those against Candida species, to their ability to bind metal ions such as Cu(II) and Zn(II). While the antimicrobial activity of some histatins is well established, the impact of metal ion coordination on this activity remains an area of ongoing investigation. This study focuses on histatin 8 (Hst8), a less-explored member of the histatin family, and compares its metal-binding and antimicrobial properties to those of Hst5. Using isothermal titration microcalorimetry (ITC), we examined the interactions of Hst8 with Cu(II), Zn(II), and Ni(II) ions and evaluated its antimicrobial activity against Escherichia coli, Staphylococcus aureus and two Candida albicans strains. Our findings revealed significant differences in copper and zinc binding between Hst5 and Hst8, with both peptides exhibiting distinct antifungal profiles. Interestingly, it has been shown that copper ions bind to Hst5 in a distinctly different manner than to Hst8. Hst5 exhibits two binding sites with dissociation constants (K_DITC) of 0.2 µM and 14.8 µM, whereas Hst8 has only one set of binding sites with a K_DITC of 12.3 µM. These results highlight the potential role of metal ion coordination in modulating the antimicrobial efficacy of histatins, providing further insight into their therapeutic potential. Full article

Isothermal titration calorimetry (ITC) provides direct insight into the energetics of DNA polymerase function, including binding, catalysis, and exonuclease activity. We characterized a Phi29 mutant polymerase (SS_01) engineered to incorporate non-natural nucleotides in the presence of Mg²⁺, a function absent in the wild-type enzyme. ITC analyses revealed that SS_01 binding to the primed template was strongly influenced by metal ions. In the presence of Mg²⁺, the polymerase displayed tight binding (KD = 243 nM) and a clear exothermic signal, indicating activation of a large fraction of catalytically competent molecules. By contrast, in the presence of Ca²⁺, binding produced weaker exothermic signals (KD = 317 nM), suggesting less efficient binding complex formation. During dNTP- or oligonucleotide-tagged dNTP-driven polymerization, ITC profiles with Mg²⁺ exhibited pronounced endothermic heat changes, whereas with Ca²⁺, only minimal heat changes were observed. When binding only oligonucleotide-tagged dNTPs, the polymerases showed distinct thermodynamic behavior: in the presence of Mg²⁺, high substrate concentrations induced endothermic responses, while in the absence of catalytic ions, binding remained exothermic. Exonuclease activity monitored using unmodified oligonucleotides yielded strong exothermic signals in the presence of Mg²⁺ but weak responses in the presence of Ca²⁺, confirming strict ion dependence. Together, these data demonstrate that ITC directly captures the metal ion-dependent energetics of SS_01, providing mechanistic insight into its polymerization and exonuclease functions. Full article

Proteins play an important role in living organisms, and, for most of them, the function depends on their structure. There are some proteins that have similar properties but different structures. An example of this is ice-binding proteins (IBPs), which have different structures but share the ability to bind to ice. Many organisms have evolved such proteins to help them survive in cold environments. Therefore, it is important to study the oligomeric state of the active form in solutions. The activity of IBP is related to the area of their ice-binding site. We have demonstrated the presence of oligomeric forms of protein in solution using multiple techniques, such as mass spectrometry, native gel electrophoresis, atomic force microscopy (AFM), isothermal titration calorimetry (ITC) and small-angle X-ray scattering (SAXS). It is noteworthy that, to date, there have been no reports of the oligomerization of ice-binding protein from Longhorn sculpin. Additionally, our findings suggest that larger molecules may influence the ability of proteins to bind to ice. In our study, the ice-binding protein forms elongated assemblies with limited intermonomer interfaces. The combination of SAXS and AFM data indicates a structure that combines compactness and flexibility and probably consists of four monomeric units. The employment of molecular modelling methodologies resulted in the attainment of a tetrameric complex that is in alignment with AFM data. Details of oligomers observed using the methods in our study emphasize the importance of different techniques that complement each other in resolving structural features. Additionally, we suggest that the protein particles, which were dispersed on the surface, exhibit softness or the form planar complexes with loose quaternary structures. It is conceivable that, depending on ionic strength and/or temperature, the various oligomeric forms of the ice-binding protein form thermodynamically more favorable complexes than their monomeric forms. Full article

Background: Cancer therapeutics development has been a challenge in medical and scientific areas due to their toxicity, limited biocompatibility, and unfortunate side effects. However, despite advances in early detection and the study of novel treatments, the mortality rate for breast cancer remains high, making it a significant global health concern. Objectives: In this study, poly(methyl methacrylate) (PMMA) nanoparticles functionalized with acrylic acid (AA), fumaramide (FA), and curcumin (CUR) as chelating and inhibitor agents were synthesized by emulsion polymerization techniques. Methods and Results: Comprehensive physiochemical characterization studies based on gravimetry, dynamic light scattering (DLS), electrophoresis, Fourier transform infrared (FT-IR), ultraviolet–visible (UV–Vis) and photoluminescence (PL) spectroscopy, X-ray diffraction (XRD), and scanning electron microscopy (SEM) revealed a pH dependence of nanoparticles that exhibit structural changes upon interaction with calcium (Ca²⁺) and magnesium (Mg²⁺) ions. Calorimetric thermodynamic properties measured by isothermal titration calorimetry (ITC) confirmed chelating coordination and positive cooperativity between the nanoparticles and metal ions. In vitro studies showed the low cytotoxicity of nanoparticles by fibroblast proliferation, and their chelation process was observed by fluorescence microscopy, with the loss of interaction between cells. Conclusions: These results suggest that the functionalized nanoparticles have potential in drug delivery systems (DDS) for targeted breast cancer therapies, providing a promising polymer material for more efficient and less toxic treatments. Full article