Search Results (1,427)

Galectins are β-galactosyl-binding lectins with key roles in immune regulation and as pattern recognition receptors. To address their potential role(s) in viral infection of mucosal epithelia we currently investigate adhesion and entry mechanisms of the infectious hematopoietic necrosis virus (IHNV) using the zebrafish (Danio rerio) model system. We previously reported the recognition of IHNV envelope glycoprotein by the zebrafish galectin Drgal1-L2 and its inhibitory activity for viral adhesion to epithelial cells. Subsequently, we determined the structure of Drgal1-L2 and proposed a mechanism for Drgal1-mediated inhibition of IHNV spike fusion to the host epithelial cell. We now show that Drgal1 can also hinder viral adhesion and infection by binding to glycans on the host cell surface and epidermal mucus. We identified fibronectin, the reported IHNV receptor, as the cell surface glycoprotein recognized by Drgal1-L2. Surprisingly, IHNV also adhered in vitro to purified β1integrin, and pre-exposure of either IHNV or the immobilized β1integrin to Drgal1-L2 hindered IHNV adhesion. Binding of either anti-fibronectin or anti-β1integrin antibodies to the cell surface partially inhibited IHNV adherence. Drgal1-L2 also hindered IHNV adhesion by binding to mucus glycans. Taken together, our results suggest complementary mechanisms by which Drgal1-L2 may protect mucosal epithelial cells against IHNV infection and tentatively identify β1integrin as a novel receptor for IHNV. Full article

This study reports the plant-mediated synthesis of copper-oxide-decorated magnetic iron oxide composite (CuO@Fe₃O₄) nanoparticles using Dolomiaea costus extract and their application as nanocarriers for β-galactosidase immobilization. The fabricated nanocomposite exhibited favorable physicochemical properties, achieving an immobilization efficiency of 83%, with enhanced thermal and pH tolerance compared to the free enzyme. Kinetic analysis revealed a modest increase in Km and a 31% decrease in Vmax after immobilization, while maintaining 69% of the catalytic activity, confirming the system’s suitability for industrial lactose hydrolysis. Reusability and storage tests showed 79% retained activity after five cycles and 77% after 60 days at 4 °C. In milk hydrolysis, the immobilized enzyme achieved 77% conversion within 3 h, following pseudo-first-order kinetics. Biocompatibility was evaluated using HepG2 cells via the MTT assay. BDH, MDH, and ABC maintained high cell viability across the tested dilution range of 25–100% (v/v), indicating no detectable cytotoxic effect under the experimental conditions, whereas cisplatin showed marked cytotoxicity with an IC₅₀ of 14.98 µg/mL. These findings demonstrate that the green-synthesized CuO@Fe₃O₄ support provides a safe, reusable, and magnetically recoverable platform for β-galactosidase immobilization, offering a promising sustainable strategy for producing lactose-free dairy products. Full article

Single-molecule Förster resonance energy transfer (smFRET) enables direct measurement of nanoscale conformational dynamics and heterogeneity in biomolecules, but quantitative interpretation of smFRET data critically depends on well-controlled excitation geometry, low background fluorescence, robust calibration, and reproducible data-analysis workflows. Prism-based total internal reflection fluorescence (pTIRF) microscopy provides important advantages for such measurements by physically separating excitation and emission paths and generating a highly confined evanescent field, yet practical guidance for implementing reproducible, quantitative pTIRF systems remains fragmented. Here we present a comprehensive, standardized framework for the design, alignment, calibration, validation, and operation of a prism-based TIRF microscope optimized for single-molecule fluorescence measurements. We describe the complete optical architecture for dual-color excitation and detection, establish alignment invariants that ensure reproducible evanescent excitation and stable donor–acceptor channel registration, and detail surface preparation, flow control, and photostabilization strategies required for reliable long-term imaging. Quantitative benchmarking protocols are introduced to evaluate signal-to-noise ratio, photobleaching kinetics, and spectral crosstalk, providing objective criteria for defining optimal operating conditions and instrument performance limits. Finally, we integrate these experimental procedures with an end-to-end single-molecule data-analysis workflow encompassing channel registration, automated and manual trajectory selection, FRET calculation, and kinetic analysis using hidden Markov modeling. The utility of the platform is demonstrated through smFRET measurements of conformational dynamics in a model nucleic acid system. Together, this work provides a reproducible and accessible methodology for implementing prism-based TIRF microscopy as a robust quantitative platform for single-molecule fluorescence studies across a wide range of biomolecular systems. Full article

A composite hydrogel scaffold comprising chitosan, silk fibroin, Aloe vera extract, and Mimosa complex was fabricated and thoroughly characterized. Upon freeze-drying, the scaffolds displayed a uniform cylindrical geometry with a highly porous, interconnected polymeric network. Quantitative image analysis revealed a mean pore diameter of 43.09 ± 2.27 µm alongside an overall porosity of 61.4 ± 6.2%. ATR-FTIR and XRD analyses confirmed successful inclusion of the complex formation and the incorporation of all constituents into the final formulation. The scaffold exhibited a compressive modulus of 46.63 ± 22.71 kPa (dry) and 5.40 ± 3.73 kPa (hydrated), with a swelling ratio of 756.62 ± 114.08%, supporting its suitability for physiological applications. TGF-β3 loading via adsorption yielded an entrapment efficiency of approximately 79.18%, reflecting effective physical immobilization throughout the polymer matrix. Cytocompatibility was subsequently assessed using an indirect contact model combined with an MTT assay, both of which confirmed that TGF-β3-loaded scaffolds exerted no cytotoxic effects on chondrocytes. After 28 days in culture, scanning electron microscopy revealed pronounced cell adhesion, preservation of rounded cell morphology, and ECM deposition along pore walls and throughout interconnected channels. Immunofluorescence analysis further demonstrated a time-dependent accumulation of aggrecan and collagen type II within the three-dimensional scaffold architecture. Collectively, these findings suggest that the developed composite hydrogel scaffold is well-suited for cartilage-related in vitro culture applications. Full article

Bone repair is a complex and dynamic process that demands implanted scaffolds to provide temporal-specific functions: antibacterial activity in the early stage, followed by angiogenic and osteogenic stimulation in later stages. This study introduces a biomimetic scaffold composed of a filled Gel-OSA hydrogel and a 3D-printed PLA framework, enabling sequential multi-drug release for bone regeneration. Zero-dimensional arginine-derived carbon dots were incorporated into the hydrogel to achieve rapid release after implantation, conferring potent antibacterial activity and ROS regulation. Meanwhile, chondroitin sulfate (CS)-loaded mesoporous bioactive glass nanoparticles were immobilized onto the 3D-printed PLA surface via a polydopamine coating, allowing sustained release of CS and Ca/P ions to enhance the scaffold’s long-term osteoinductive capability. The composite scaffold further demonstrated combined effects in promoting cell proliferation and osteogenic differentiation in vitro. Collectively, these findings suggest that this biomimetic scaffold, designed for temporally controlled multi-drug release, represents a promising therapeutic strategy for the reconstruction of bone tissue. 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

The emerging threat of antibiotic-resistant pathogens and the limitations that conventional cancer chemotherapies display have created an urgent need for the development of innovative therapeutic strategies. Combining the pleiotropic biological roles of antimicrobial peptides (AMPs) and nanomaterials through their conjugation presents a promising possibility of targeting both microbial membranes and malignant cells. In the present study, we engineered a novel bioactive material by immobilizing the insect-derived AMP Papiliocin onto multi-walled—decorated with polyethylene–glycol—carbon nanotubes (PEG-MWCNTs) to prevent proteolytic degradation of the peptide and enhance its cellular delivery. Recombinant Papiliocin was cloned, heterologously expressed, purified and conjugated onto the PEG-MWCNT carrier. Successful expression and conjugation were validated via immunoblotting and Fourier transform infrared (FT-IR) spectroscopy, respectively. Further physicochemical characterization of the bionanocomposites was conducted using Dynamic Light Scattering (DLS) and Zeta potential measurements. Biologically, the biofunctionalized material exhibited potent, broad-spectrum antimicrobial activity both on Staphylococcus aureus and Escherichia coli, inhibiting almost 90% of the latter’s growth, highlighting the bioconjugate’s specific interactions with the Gram-negative pathogens’ membranes. Furthermore, it significantly reduced biofilm formation in Candida albicans, as indicated by the TCP assay. In parallel with its antimicrobial effects, CNTs-PEG–Papiliocin significantly reduced cancer cell viability and induced apoptosis via the extrinsic apoptosis pathway in HeLa cells, a response assisted by efficient intracellular delivery. Notably, cytotoxicity assays demonstrated lesser cytotoxic effect against non-tumorigenic HaCaT cells relative to the cancerous cell line. Collectively, these findings indicate the Papiliocin–biofunctionalized CNTs as a versatile, dual-action therapeutic agent with potential for antimicrobial activity and anticancer mode of action. Full article

Background. The highly mutable influenza virus causes severe annual infections worldwide and results in substantial socioeconomic losses. The spread of infection could be effectively controlled by cross-protective vaccines and universal diagnostic test systems based on the nucleoprotein (NP) as one of the most conserved viral antigens. However, NP also undergoes slow evolutionary changes, and little is known about the influence of these mutations on its antigenicity and immunogenicity. Methods. We expressed the full-length recombinant 6xHis-tagged NPs of ten evolutionary distant influenza A strains of different subtypes in E. coli BL21(DE3) cells and purified these proteins by immobilized metal affinity chromatography. The obtained antigens were identified by mass spectrometry and serological methods. NPs served as antigens for three immunizations of BALB/c mice (15 µg/animal at 14-day interval) and as capturing proteins in ELISA at 2 µg/mL, in order to study the effect of adaptive mutations on the antigenic and immunogenic properties of NPs. Results. A pronounced cross-reactivity of anti-NP antibodies induced in mice by immunization with different NPs was revealed. At the same time, we observed the differences in the humoral immunogenicity of NP, which are in line with the accumulation of evolutionarily driven NP mutations. In general, antibody affinity to heterologous NPs was reduced, indicating the differences in the specificity of anti-NP immunoglobulins, which may be caused by evolutionarily determined variability of immunogenic epitopes leading to the emergence of escape mutations. Conclusions. Overall, our results reflect the slightly evolving nature of the NP antigen, which influences the specificity spectrum of anti-NP antibodies and should be considered as a limitation for the development of NP-based cross-protective vaccines and test systems. Full article

Yeast surface display is a powerful strategy for enzyme immobilization and whole-cell biocatalysis; however, the intracellular processing of heterologous enzymes during secretion and anchoring remains poorly understood. In this study, a GH5 endoglucanase gene (eglS, 1.4 kb) from Bacillus subtilis, originally isolated from a paper mill effluent, was cloned into the pYD1 vector and expressed in Saccharomyces cerevisiae EBY100 using the Aga1–Aga2 surface display system. The recombinant strain produced clear degradation halos on carboxymethyl cellulose (CMC) plates, confirming cellulolytic activity at the whole-cell level. Zymographic analysis revealed multiple active enzyme forms depending on the cellular fraction analyzed. Intracellular extracts displayed active bands ranging from 70 to 57 kDa, consistent with immature or partially processed Aga2 fusion proteins, whereas cell wall-associated fractions showed active bands between 55 and 35 kDa, suggesting proteolytic processing during secretion and surface anchoring. The apparent specific activity of the cytoplasmic fraction was 5.33 ± 0.31 U mg⁻¹, while the cell wall-associated fraction exhibited a higher apparent specific activity (58.4 ± 10.1 U mg⁻¹). Although these values were obtained from non-purified fractions and therefore do not represent intrinsic enzymatic constants, they indicate a relative enrichment of catalytically active enzyme in the cell wall-associated fraction, consistent with functional surface display. The presence of multiple active enzyme forms and the enhanced catalytic efficiency observed in the cell wall-associated fraction suggest that the engineered yeast strain may serve as a promising whole-cell biocatalyst, with potential applications in consolidated bioprocessing (CBP) strategies for lignocellulosic biomass conversion. Full article

Mercury ion, a highly toxic and bioaccumulative heavy metal pollutant, poses significant risks to human health and ecosystems even at trace concentrations. Therefore, the development of highly sensitive and selective analytical methods for mercury ions is critically important to safeguard environmental integrity and human health. In this work, 4-mercaptopyridine-functionalized gold nanoparticles (4-MPY-AuNPs) were synthesized and subsequently immobilized onto quartz slides to fabricate a localized surface plasmon resonance (LSPR) sensor. Exploiting the selective coordination interaction between the pyridyl nitrogen atoms of 4-MPY and Hg²⁺, this LSPR sensor enables highly specific detection of Hg²⁺. Moreover, injecting a trace amount of 4-mercaptopyridine-functionalized AuNPs into the flow cell triggers the in situ formation of a surface-confined AuNP–Hg²⁺–AuNP sandwich architecture, thereby enhancing the sensor’s sensitivity. Under the optimized conditions, the proposed method exhibited a linear dynamic range of 1 × 10⁻⁹–6 × 10⁻⁷ mol L⁻¹, with a correlation coefficient (R²) of 0.9917 and a limit of detection (LOD) of 3.2 × 10⁻¹⁰ mol L⁻¹; the LOD of this method is one order of magnitude lower than the LODs reported in contemporary Hg²⁺ detection methods. This method exhibits high selectivity, sensitivity, cost-effectiveness, and is label-free, thereby demonstrating significant potential for environmental applications. Full article

Background: Testicular irradiation presents technical challenges due to the temperature-dependent cremasteric reflex causing positional variability, yet detailed immobilization protocols addressing this issue and cone-beam computed tomography (CBCT)-based setup data remain lacking. This formative and preliminary single-patient descriptive technical report describes a temperature-controlled immobilization technique and reports preliminary setup observations from its clinical application. Methods: A 74-year-old male with primary testicular diffuse large B-cell lymphoma (DLBCL) received prophylactic contralateral testicular irradiation. The immobilization protocol combined a custom thermoplastic device with infrared warming to maintain the scrotal surface temperature at 36–36.5 °C, intended to facilitate a relaxed scrotal position prior to and during each fraction under temperature-controlled conditions. Treatment was delivered using a three-field three-dimensional conformal radiotherapy (3D-CRT) technique (30.6 Gy in 17 fractions), and seven CBCT scans were used to document interfraction setup measurements. Results: The treatment was completed as planned with adequate target coverage (clinical target volume [CTV] D97% = 100%) and minimal organ-at-risk (OAR) doses. Setup measurements showed a CTV root-mean-square displacement (RMS) of 3.8 mm and a mean Dice similarity coefficient (DSC) of 0.85, while the testis alone showed an RMS of 5.2 mm and a mean DSC of 0.73. Conclusions: The temperature-controlled immobilization technique was feasibly implemented, and the setup measurements observed during its application showed a CTV RMS of 3.8 mm and a mean DSC of 0.85. These findings may provide a practical reference for institutions encountering this rare clinical scenario. Full article

Antimicrobial resistance (AMR) is emerging as one of the most serious global health problems, necessitating the urgent development of alternative approaches to pathogen control. The present study describes the synthesis and characterization of a novel iodinated BODIPY derivative (BODIPY5), designed as a highly efficient photosensitizer for antimicrobial photodynamic inactivation (aPDI). The molecular design of the compound involves the introduction of two iodine atoms into the BODIPY5 core, which induces a “heavy atom effect”, accelerates the intersystem transition from the singlet to the triplet state, and leads to increased generation of singlet oxygen upon irradiation with visible light. Photophysical measurements show a significant fluorescence quenching of BODIPY5 compared to its unsubstituted counterpart, which is a direct indicator of increased photodynamic activity. The compound’s antimicrobial efficacy was tested in a homogeneous medium and after immobilization on cotton textiles via physical adsorption. In solution, BODIPY5 nearly eliminated the model bacterial strains B. cereus and P. aeruginosa at a low concentration of 10 µg/mL under light, with cell viability below 1%. The functionalized cotton fabric exhibits pronounced self-disinfection properties, retaining high photodynamic activity against the Gram-negative pathogen P. aeruginosa. Scanning electron microscopy results confirm extensive morphological damage and loss of structural integrity in bacterial cells on the treated textile following irradiation. The non-specific mechanism of action, which generates reactive oxygen species (¹O₂) in situ, prevents the development of bacterial resistance and makes the developed material a promising candidate for use in hospital environments, including antibacterial clothing and protective equipment. Full article

The toxicity of copper (Cu) severely affects the growth and physiological metabolism of plants. 2-(3,4-Dichlorophenoxy) triethylamine (DCPTA) is a plant growth regulator known to enhance plant tolerance to various abiotic stresses; however, its specific role in mitigating Cu toxicity via cell wall modulation and nitrogen metabolism remains unclear. “Zhongnong 26” (Cucumis sativus L.) seedlings were subjected to a randomized block design with four treatments: control (CK), 0.25 mg/L DCPTA, 50 μM Cu, and 50 μM Cu + 0.25 mg/L DCPTA, with three biological replicates per treatment. The results indicated that DCPTA application significantly alleviated Cu-induced growth inhibition. Specifically, DCPTA improved root system architecture by markedly increasing total root length (68.8%), surface area (68.7%), and the number and length of secondary lateral roots (69.6%, 173.2%). Furthermore, DCPTA enhanced the biosynthesis of cell wall polysaccharides—including pectin (24.3%), hemicellulose 1 (22.4%), hemicellulose 2 (23.7%) and cellulose (33.1%) in roots. Fourier Transform Infrared (FTIR) spectroscopy analysis revealed that DCPTA modified functional groups (e.g., –OH, –COOH) within the cell wall, enhancing their metal-chelating capacity. Consequently, DCPTA promoted the immobilization of Cu in the root cell wall fractions (particularly pectin and HC2) and shifted Cu into less toxic, pectate- and protein-bound forms, thereby reducing its translocation to leaves. Additionally, DCPTA restored the activities of key nitrogen metabolism enzymes in leaves and roots. Compared with Cu treatment alone, nitrate reductase (NR) activity increased by 77.7% and 90.6%, while glutamine synthetase (GS) activity remained stable, and glutamate synthase (GOGAT) activity increased by 10.3% and 71.3% in leaves and roots, respectively. In conclusion, DCPTA enhances copper sequestration in roots by coordinating the regulation of root structure and cell wall strengthening (with an increase in pectin and hemicellulose content). This is crucial for protecting the nitrogen metabolism within the cells (including the enzymes that drive the nitrate–ammonium reduction pathway) to maintain metabolic balance under Cu stress. Full article

Chlorothalonil is a widely used fungicide in agriculture, but its excessive application can lead to environmental contamination. This study investigated the biodegradation potential of Proteus terrae ZQ02 in free and immobilized forms. Under optimal conditions (37 °C, pH 7), free cells degraded 97.2–98.7% of chlorothalonil (50 mg/L) within seven days. Bacterial microcapsules were prepared using 3% sodium alginate, 2% calcium chloride, and 60 g/L wet biomass, with encapsulation times ranging from 6 to 12 h. The microcapsules displayed uniform size, high mechanical strength, porous structure, and excellent mass transfer, ensuring stable degradation activity. Encapsulated cells demonstrate enhanced tolerance to variations in pH, temperature, and salinity compared to free cells. In soil, microcapsules reduced chlorothalonil half-lives to 1.33–5.45 days for concentrations of 10–30 mg/L, achieving 92–96% degradation over 14–35 days. In tomato-planted soils, encapsulated and free cells degraded 96.3% and 81.6% of residues, respectively, after 28 days, significantly exceeding the control. These findings highlight that immobilization improves the stability, reusability, and efficiency of P. terrae ZQ02, making it a promising strategy for sustainable chlorothalonil biodegradation. The study demonstrates the potential of combining microbial strains with carrier materials for effective pesticide remediation and environmental protection, providing a foundation for large-scale applications in contaminated agroecosystems. Full article

Endocrine therapy is the mainstay for estrogen receptor (ER) α-positive breast cancer (BC), yet many patients display acquired resistance. We then screened natural compounds using human ERα-positive BC cells and identified perillyl alcohol (POH), a monoterpene from perilla, that reduces ERα protein levels. Chemoproteome analysis using POH-immobilized nanomagnetic beads revealed adenine nucleotide translocase 2 (ANT2), a mitochondrial inner membrane protein, as a direct target of POH. Molecular dynamics (MD) simulations predicted POH binding to the central pore of ANT2, which functions in ATP transport. ANT2 depletion reduced ERα levels, and public datasets indicate that high ANT2 expression correlates with poor prognosis in ERα-positive BC. POH also inhibited the growth of Tamoxifen- and Fulvestrant-resistant BC cells. RNA sequencing showed that fatty acid elongation-related genes were upregulated in Fulvestrant-resistant cells but downregulated by ANT2 depletion. Both ANT2 depletion and POH treatment led to the accumulation of intracellular lipid droplets in Fulvestrant-resistant cells, consistent with impaired fatty acid elongation. Finally, in silico screening using MD simulations identified venetoclax and nystatin as potential ANT2 pore binders. Both compounds reduced ERα levels in ERα-positive BC cells and increased lipid droplet formation in Fulvestrant-resistant cells. These findings highlight ANT2 as a druggable target against endocrine-resistant BC. Full article