Search Results (1,338)

Terminal deoxynucleotidyl transferase (TdT) is a unique DNA polymerase that catalyzes template-independent nucleotide addition at the 3′-end of DNA, playing a critical role in generating immune receptor diversity. While the structural importance of Loop1 in blocking template strand binding and enabling this activity is established, the precise molecular contribution of hydrophobic interactions within Loop1 to the catalytic mechanism of human TdT remains unclear. In the present study, we aim to elucidate the roles of hydrophobic Loop1 residues (L397, F400, F404) in the structural organization and catalytic function of TdT. We engineered alanine and tryptophan substitutions at these positions and systematically analyzed the resulting mutant forms using molecular dynamics simulations and pre-steady-state kinetic measurements. Our results show that substitutions L397A and F400A increase Loop1 flexibility and significantly reduce catalytic activity, particularly for purine nucleotide incorporation, while F404A completely abolishes enzymatic function. The F404W mutant largely preserves activity. All mutant forms retain the ability to bind single-stranded DNA and dNTP, but in some cases, their affinity and thermal stability were reduced. These findings demonstrate that hydrophobic interactions in Loop1 are essential for maintaining the catalytically competent conformation of TdT, ensuring precise substrate positioning and active site stability. Full article

The purpose of this study is to enhance the performance of the mesohabitat model MesoHABSIM by lowering the necessary hydraulic modelling effort. This proof-of-concept study tests an application of the MesoHydraulics model to simulate the hydraulic characteristics of hydromorphological units (HMUs) occurring in a regulated river at different low discharges. In this quantitative approach, hydraulic patterns are transferred from a source site, where depth and velocity distributions were derived from field measurements and a 2D hydrodynamic model, to a target site, where only a single field hydrometric survey was conducted. Instead of modelling changes in individual hydraulic measurement values to estimate hydraulic responses to discharge, the model relies on statistical distributions of these values within HMUs. We were testing whether changes in the distribution of HMU’s and their hydraulics can be transferred between morphologically comparable river sections to serve as a sufficient hydraulic input for mesoscale habitat modelling. The hydrodynamic component of the River2D software (V.0.95a), routinely used in MesoHABSIM, served as a baseline for testing the MesoHydraulic model’s performance and for producing source data for deriving distribution functions. The test was conducted using data from two one-kilometre sites on the upper Oder River (Poland). The model transfers the HMU area distributions, along with corresponding depth and velocity frequency distributions, for a number of flows from one site (the source) to another (the target). The hydraulics at both sites were surveyed under single-discharge conditions. For the source site, the hydrodynamic model was applied to classify the HMU mosaic at three additional discharge stages. At the target reach, the HMU mapping was conducted based on survey data, and statistical frequency functions were used to model distributions of hydraulic patterns at discharges modelled for the source. The hydraulic model’s performance was evaluated at the target reach by comparing simulated hydraulics and HMU patterns with those modelled using River2D. Finally, both models were used to calculate habitat availability for the fish communities, and dissimilarities were observed. The resulting hydraulic distributions were similar, with an average affinity index of 90%. Higher affinity indices were reached at flows close to the measured value, with increasing model disagreement toward flow extremes, most notably for Run and Backwater units. Regardless, habitat models for the fish community were also highly correlated with R² = 0.98 for amounts of suitable habitat and almost identical habitat distribution among the species. Yet, the MesoHydraulics-based model slightly, but consistently, overestimated habitat availability. While the model was tested in a large and regulated river system, its accuracy may vary depending on the natural river morphology. Further research should evaluate modelling uncertainties and their applicability in less-modified water bodies. Full article

Background/Objectives: High fructose consumption is a significant risk factor for glomerular podocyte injury. This study aimed to identify the underlying mechanism of fructose-induced podocyte injury and explore the protective effect of the natural polyphenol morin. Methods: In vivo, high-fructose-diet-fed rats were used to evaluate podocyte injury through ultrastructural structure analysis, urinary albumin-to-creatinine ratio (UACR), and synaptopodin expression. In vitro, adenosine 5′-monophosphate deaminase (AMPD) expression and activity, mitochondrial function, and glycolytic flux were measured in mouse podocyte clone-5 (MPC5) exposed to 5 mM fructose, with molecular docking and siRNA interference assays validating morin’s regulatory role. Results: High fructose significantly increased AMPD activity in the purine nucleotide cycle (PNC), leading to mitochondrial dysfunction and a compensatory activation of glycolysis in podocytes. Morin effectively mitigated podocyte injury and suppressed the upregulation of AMPD activity, potentially through targeting AMPD2, as evidenced by molecular docking, which demonstrated a strong binding affinity between morin and AMPD2. Similarly, AMPD2 knockdown markedly alleviated mitochondrial impairment and glycolysis activation, confirming the pivotal role of AMPD2 in fructose-induced podocyte injury. In high-fructose-diet-fed rats, morin substantially improved ultrastructural damage, as shown by reduced podocyte foot process effacement, decreased UACR, restored glomerular synaptopodin expression, and suppressed AMPD activity in the renal cortex. Conclusions: Morin alleviated high-fructose-induced podocyte injury by inhibiting AMPD activity in the PNC, highlighting AMPD2 as a potential therapeutic target for podocyte injury caused by high fructose intake. This study provides novel mechanistic insights into how morin counteracts mitochondrial energy disturbance in podocyte injury. Full article

Background: Short peptide epitopes are valuable for mechanistic studies, yet their intrinsic low immunogenicity and lack of commercial antibodies hinder rapid antibody generation. Methods: We developed a reproducible, sequence-level workflow combining cross-species/structural triage, independent MHC-I/II prioritization, and conservative heteroclitic-style substitutions to enhance predicted MHC affinity while preserving native epitope features. Using visfatin as a model, two optimized fragments were conjugated to KLH and tested in mice for antibody titers, isotype profiles, and binding kinetics. Results: Mutant peptides improved MHC-binding prediction, elicited stronger antibody titers, and promoted isotype maturation (increased IgG1). Importantly, antibodies maintained measurable binding to wild-type sequences, indicating preserved cross-recognition. Similar effects were reproduced with additional antigens. Conclusions: This proof-of-concept study, based on small exploratory mouse cohorts (n = 3 per group), demonstrates that strategic, minimal sequence edits can significantly enhance peptide immunogenicity while preserving native epitope recognition. This streamlined workflow provides a low-barrier route to generate epitope-directed antibodies when commercial reagents are unavailable. Full article

Background: The Na⁺/K⁺-ATPase (NKA) maintains electrochemical gradients by exporting Na+ and importing K⁺ at the expense of ATP hydrolysis. Although NKA inhibition is a well-established strategy for increasing cardiac contractility, existing inhibitors such as cardiotonic steroids (CTS) are limited by serious adverse effects. N106 is a small molecule previously shown to enhance cardiac lusitropy by promoting SERCA2a SUMOylation and, intriguingly, also exerts positive inotropic effects, suggesting additional mechanisms of action. Methods: To test whether N106 directly modulates NKA, we combined ATPase activity assays with molecular docking and microsecond-scale molecular dynamics simulations. Results: Biochemical measurements showed that N106 partially inhibits NKA, achieving ~80% maximal inhibition with an IC50 of 7 ± 1 µM, while leaving the pump’s apparent affinity for Na⁺, K⁺, and ATP unchanged. Computational analyses suggest that N106 binds within the canonical CTS-binding pocket but undergoes intermittent unbinding events, consistent with the partial inhibition observed experimentally. Conclusions: These findings identify N106 as a first-in-class dual modulator of cardiac ion pumps, partially inhibiting NKA while previously shown to activate SERCA2a through enhanced SUMOylation. This combined mechanism likely underlies its positive inotropic and lusitropic effects and positions the N106 scaffold as a promising lead for developing next-generation dual-target therapeutics for heart failure. Full article

Fluorescence Anisotropy (FA) is a sensitive and efficient technique for quantifying biomolecular interactions, offering advantages such as minimal sample requirements and elimination of separation of bound from unbound species. Thus, it is well suited for aptamer–protein binding affinity studies. However, accurately determining equilibrium dissociation constants (K_D) in FA requires low concentrations of fluorescently labeled aptamers to prevent ligand depletion. A significant challenge arises at low aptamer concentrations due to an unexpected and physically nonmeaningful increase in apparent anisotropy, which impairs accurate data fitting. This anomalous increase in apparent anisotropy may arise from non-specific adsorption of aptamers to surfaces. In this study, we investigated the use of non-ionic surfactants to mitigate these effects and stabilize the anisotropy signal at low aptamer concentrations using the thrombin aptamer as a model system. We evaluated the impact of varying concentrations of two surfactants (Tween 20 and Triton X-100) on plots of anisotropy as a function of aptamer concentration and determined aptamer–protein binding affinities. Addition of 0.1% Tween 20 corrects the anomalous increase in anisotropy at low aptamer concentrations, enabling the use of aptamer concentrations as low as 5 nM in binding assays. Triton X-100 was less effective. By incorporating optimized concentrations of Tween 20, we demonstrated improved assay reproducibility and accuracy in K_D determination, expanding the dynamic range of usable aptamer concentrations in FA-based binding affinity studies. Similar benefits were observed with the clinically relevant aptamer s10yh2 and human serum albumin. These findings provide a practical strategy for enhancing the robustness of FA measurements and may be applicable to other aptamer–target systems and high-throughput assay formats. Full article

Acne vulgaris is a common chronic inflammatory skin condition. Conventional acne treatments are often limited by adverse effects, driving interest in alternative therapies. This study explored the multifunctional bioactivities of a lyophilized cell-free supernatant (LCFS) derived from Lacticaseibacillus paracasei T0901, isolated from fermented palm sap, with a focus on its antimicrobial, antibiofilm, and antimelanogenic potential for dermatological applications. Antimicrobial activity was evaluated using agar well diffusion and broth microdilution assays against acne-associated pathogens, while antibiofilm effects were quantified via crystal violet staining. Antimelanogenic activity was assessed in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16F10 melanoma cells by measuring melanin content and tyrosinase activity. Whole-genome sequencing was performed to identify genes linked to observed bioactivities, and molecular docking was used to predict metabolite–protein interactions. The LCFS exhibited strong inhibitory activity against acne-associated bacteria, with inhibition zones of C. acnes (10.67 ± 0.58 mm), S. epidermidis (21.00 ± 0.00 mm), and S. aureus (20.00 ± 0.00 mm), and a minimum inhibitory concentration of 25 mg/mL. Biofilm formation was significantly reduced by 62.98 ± 3.54%. In α-MSH-stimulated B16F10 cells, LCFS treatment (10 mg/mL) significantly decreased melanin content (73.23 ± 2.36%) and intracellular tyrosinase activity (68.19 ± 6.29%) relative to control. Genomic analysis revealed antioxidant-related genes (sodA, trxB, nox), pigmentation regulators (mco, fcbD), and buk (butyrate kinase), supporting the observed bioactivities. Molecular docking further demonstrated strong binding affinities of LCFS-derived metabolites to tyrosinase and MITF, suggesting modulation of melanogenic pathways. Collectively, these results indicate that L. paracasei T0901 produces safe postbiotic compounds with potent antimicrobial, antibiofilm, and antimelanogenic activities, highlighting its promise as a multifunctional ingredient in probiotic-based skincare formulations. Full article

Proton binding (i.e., charging) isotherms of halloysite nanotubes (HNT) were determined from cycled acid-base potentiometric titrations in KCl solution at constant ionic strengths (0.01, 0.10, 1.00 mol dm⁻³). The isotherms measured in the pH cycle from 3 to 11 and back exhibit a pronounced hysteresis with respect to the direction of pH change, which is accurately reproducible when the cycle is repeated. The hysteresis is absent if the cycled titration is performed within a narrow pH range between 5 and 9. These results align with the dissolution rates of alumina and silica, which form the two surfaces of the rolled kaolinite sheet in HNT, and clearly point to reversible partial dissolution-deposition processes in the HNT interior during a titration cycle, outside the above pH range (alumina dissolution below pH ≈ 5 and silica dissolution above pH ≈ 8.5). In the studied titration experiments, these processes produce partially dissolved surface-bound, rather than completely dissolved species (reversible surface etching). Under the applied conditions, reversible surface etching is less pronounced in the acidic part of the titration cycle. Charging isotherms recorded in the decreasing pH titrations at varied ionic strength exhibit a common intersection point very close to zero charge (point of zero charge) around pH ≈ 8.1, characteristic for an amphoteric solid surface. These isotherms were reasonably well fitted by applying the surface protonation model in the HNT interior, which invokes the Stern model of the electric double layer (EDL), by summing the surface charges calculated for alumina and silica as separate components (surfaces). The model surface charge isotherms for alumina surface in the HNT interior exhibit a point of zero charge at pH = 9.0, while the silica surface has a negative charge above pH > 8.5, which is in very good agreement with the values reported in the literature: as for these two surfaces, thus for kaolinite nanoparticles. The best-fit protonation site density for both surfaces is equal to 8.0 nm⁻², while the best-fit intrinsic pK_a for alumina and silica surfaces of HNT are equal to 9.0 and 8.5, respectively. The pH-dependence of electrophoretic mobility, measured by means of electrophoretic light scattering, reveals a more acidic behavior of the outermost silica surface than within the inner HNT phase, which is consistent with the literature result reported for kaolinite. The results reported herein confirm that the inner and outer surfaces of the HNT are oppositely charged below pH < 8.0 and negatively charged above that value, and importantly, they reveal new details about the protonation affinities and EDL parameters at active surfaces of HNT, important for the colloidal stability of HNT suspensions and the functionalization of HNT through the electrostatic binding of active molecules. Full article

Background and Clinical Significance: Calcinosis cutis is a rare condition that can develop through several mechanisms. These include dystrophic, calciphylaxis (classical, metastatic, and iatrogenic), and idiopathic mechanisms. Idiopathic calcinosis cutis is rare and always a diagnosis of exclusion. A particularly rare site for the development of idiopathic calcinosis cutis is the penis. Case Presentation: A previously healthy 18-year-old male presented to our institution with a three-month history of a painless, firm swelling on the outer layer of the prepucium in the area of the commissure. Histopathology of the excised specimen showed a varying caliber of calcium deposits within the dermis, ranging from small psammoma-like bodies to larger calcium deposits measuring up to 2.5 mm. The deposits were freely dispersed within the dermal collagen and did not exhibit vascular affinity, nor surrounding foci of inflammation. The epidermis was not involved, with only mild reactive hyperkeratosis. The results of detailed physical, imaging, and laboratory tests were normal, and hence the diagnosis of idiopathic calcinosis cutis of the penis was established. Conclusions: Penile calcinosis cutis is a rare condition that falls within the broader group of genital calcinosis cutis. The condition is typically present in young males and has an excellent prognosis after excision. Full article

Background: Rabies virus (RABV) causes approximately 59,000 human deaths annually. Current pre- and post-exposure vaccination relies on inactivated vaccines (INVs) with limited yield and immunogenicity. We engineered a dual-cationic LNP-based nucleocapsid-like nanostructure (NLS) that co-encapsulates RABV G-mRNA and recombinant RABV-N to engage MHC-I/II pathways and enhance protection. Methods: A pVAX-RABV-G plasmid containing 5′/3′UTRs, Kozak, and poly(A) was transcribed in vitro. RABV-N with an N-terminal 6× His tag was expressed in E. coli BL21(DE3) and purified by Ni-Sepharose affinity chromatography. Dual-cationic LNPs (DHA, DOTAP Cl, mPEG-DTA2K, DOPC) were formulated by microfluidics at a 4:1 (G-mRNA:RABV-N) mass ratio. Vaccine quality was assessed by encapsulation efficiency, DLS, PDI, zeta potential, and TEM. Mice received empty LNPs, INV, G-mRNA, or NLS under varied schedules and doses. ELISA measured RABV-G/N-IgG; RFFIT determined neutralizing antibody (nAb) titers; ELISPOT quantified CTL response; qPCR assessed T-cell activation genes. On day 35 after the first immunization of vaccines, mice were challenged intramuscularly with 25 LD₅₀ of CVS-24. Results: G-mRNA purity was >95% and drove strong RABV-G expression in 293T cells. Purified RABV-N was approximately 52 kDa, >90% pure, and reactive to anti-His and anti-N antibodies. NLS achieved >95% encapsulation, a diameter of 136.9 nm, PDI 0.09, and a +18.7 mV zeta potential. A single dose yielded approximately 10 IU mL⁻¹ nAb by day 7; two doses peaked at approximately 1000 IU mL⁻¹. Mice showed 100% survival and no viral rebound in brain, spinal cord, and sciatic nerve. NLS induced stronger MHC-I/II-linked cellular immunity and higher RABV G/N-specific IFN-γ spot frequencies than G-mRNA or INV. Conclusions: The dual-antigen NLS vaccine co-delivering G-mRNA and RABV-N via dual-cationic LNPs robustly activates MHC-I/II, rapidly generates high-titer nAb (≥10 IU mL⁻¹ within 1 week), and sustains potent CD8⁺ CTL and CD4⁺ Th responses. A two-dose regimen (days 0 and 21) conferred complete protection, supporting the NLS platform as a next-generation rabies vaccine candidate. Full article

Biomethane is one of the controllable Renewable Energy Sources. It may be derived from biogas, a multicomponent gas mixture, using, among others, membrane processes. The proper optimization of such a process requires the knowledge of the phenomena accompanying each specific biogas–membrane separation system. Therefore, the solubility, permeance and diffusion of CO₂, CH₄, O₂ and N₂ in a polyimide-based sample were described and analyzed using the Dual Mode Sorption and partial immobilization models. The parameters of the models were determined based on pure gas sorption isotherms measured gravimetrically and experimental permeances of the four gases. The membrane swelling caused by CO₂ was observed at temperatures of 293 and 303 K and for pressures higher than 3 bar. The adsorption of CH₄, O₂ and N₂ in the fractional free volume (FFV) has a dominant (>50%) share in their total solubility in the entire pressure range. This makes them sensitive to the presence of CO₂, whose affinity is the strongest towards the tested polyimide-based sample. The diffusion of O₂ is the fastest which makes it competitive with CO₂ in permeation through the membrane, despite its low solubility. The ideal CO₂/O₂ selectivity is thus relatively low (2.3–5.1). Methane, which is competitive in solubility compared to CO₂, was found to diffuse the slowest and as a result, it is also the slowest permeating gas. This translates into the very high CO₂/CH₄ ideal selectivity (33–95.7), which is, however, strongly dependent on temperature and pressure. Full article

The study of the conformational stability of protein layers at the interface between gold surfaces and lipid membranes is crucial for determining the biological activity of these systems and understanding their interactions. The surfaces differ significantly in hardness: gold is a rigid substrate, while the POPC/POPS liposome layer is highly flexible. A quartz crystal microbalance with dissipation (QCM-D) monitoring method and multi-parametric surface plasmon resonance (MP-SPR) were used to determine the adsorption efficiency of lysozymes, the level of layer hydration, and changes occurring within the secondary structure and the thickness of the formed protein layer. In both methods, lysozyme adsorption on the gold surface was more effective at pH 4.0 than at pH 7.4. The lysozyme adsorption efficiency on the surface of the lipid layer was the same for both measurement conditions. In contrast, the affinity of lysozyme molecules to the lipid surface was higher than that of the gold surface. The composition of the secondary structure of lysozymes was monitored using the FT-IR method. Deconvolution of the Amide I band confirms the existence of different mechanisms underlying lysozyme molecule immobilization depending on the type of adsorption surface. Along with the change in the surface, there is a transition from the dominance of electrostatic to hydrophobic interactions, which significantly affects the structure of the interphase layer. High content of random structures on the lipid surface is evident, while, in the case of the gold surface, there is a decrease in random structures and the presence of antiparallel β-sheets. Interaction with the surface induces the transition of amyloidogenic domains of the protein to conformations, which are particularly susceptible to aggregation, consequently leading to oligomerization. Full article

Aptamers are promising synthetic molecular receptors that bind to specific targets by adopting a unique tertiary structure. However, their selection using standard SELEX protocols often does not allow the achievement of high affinity to the targets. Due to the lack and difficulty of obtaining data on the 3D structure of aptamers and their complexes, the design of known aptamers based on simple rules and software is in demand. The presented work considers the comparative characterization and design of DNA aptamers specific to the antibiotic kanamycin based on complementary interactions and structural motifs (bulges, mismatches, loops) predicted by NUPACK, RNAfold, and UNAFold software. The design included the elimination of non-functional parts of the aptamers and the stabilization of the kanamycin-binding loop. Seven novel aptamers, chosen based on these predictions, were synthesized, and their affinities were measured using an isothermal titration calorimetry technique. The prediction of end stem and hairpin loop structures was confirmed by comparison with circular dichroism data. As a result of sequential design with truncation of unnecessary nucleotides, a novel optimal 42-base-long aptamer was designed and demonstrated a dissociation constant of 109 ± 15 nM, which is 4.7-fold lower than the initial preparation (470 ± 40 nM) and overcomes all known aptamers to kanamycin. Full article

This work examined the effects of four plasticizers, glycerol (GLY), potassium phosphate (PHOS), polyethylene glycol (PEG), and soy lecithin (SL), on the structural, surface, thermal, optical, and mechanical properties of polyhydroxybutyrate (PHB) films. FTIR spectra demonstrated that these plasticizers maintained the PHB molecular structure, while X-ray diffraction data proved that PHB crystallinity decreased upon adding SL, GLY, and PHOS. Under SEM, we discovered several defects in the plasticized samples, most of which were holes of distinct sizes and forms. The thermal analyses evaluated the impact of plasticization on PHB thermal processability, demonstrating that the material’s thermal stability improved, easing thermal processing due to the reduced melting peak temperatures (T_m) caused by all the additives assessed. While PEG, GLY, and PHOS reduced the hydrophilicity of the film, SL enhanced its affinity to water, as shown by the contact angle measurements. Reduced transparency resulted from adding 20% plasticizers with an increase of 345% in elongation at break and a decrease of 67% in elastic modulus compared to pristine PHB. Thus, SL proved to be the most promising of the four plasticizers used in terms of mechanical properties, crucial for PHB-based films for food packaging. Full article

Beta-antithrombin (β-AT), the isoform of antithrombin (AT) with a higher affinity for heparin, constitutes 5–10% of total AT in plasma. There are limited data regarding β-AT activity levels in thrombotic disorders. In our study, we analyzed samples from 200 non-AT-deficient patients who had experienced venous thromboembolism (VTE) compared to 200 healthy controls. Total AT activity was measured using a chromogenic anti-factor Xa assay. To measure β-AT, we used elevated NaCl (1.1 M) in the reagent to inhibit the heparin binding of α-AT. There were no significant differences in total AT activity (median (IQR)) levels between the control and VTE groups (100 (93–109)% and 99 (94–109)%, respectively; p = 0.955). However, the β-AT activity levels (median (IQR)) and the ratio of β-AT to total AT (mean ± SD) were significantly higher in the VTE group compared to the control group (93.3 (90.3–97.3)% vs. 89.3 (84.0–95.0)% and 9.34 ± 0.68% vs. 8.86 ± 0.88%; p < 0.001). β-AT activity levels and the ratios in the upper third were strongly associated with a higher risk of VTE (OR (95% CI): 5.78 (3.08–10.87) and 6.15 (3.36–11.24), respectively). Our study demonstrated an elevation of plasma levels of β-AT in patients with VTE. Further research is necessary to clarify the pathophysiological significance of this finding. Full article