Search Results (217)

Protein kinase inhibition can be achieved through various mechanisms, including blocking phosphorylation activity or disrupting regulatory interactions. While small molecule inhibitors have shown promise, their selectivity remains challenging due to the structural similarities among kinase catalytic sites. To design selective kinase inhibitors based on peptide terminal tail interactions with the activation segment, focusing on five kinases with different conformational states: GSK3, PAK4, TTN (OUT conformation) and PKB, FLT3 (IN conformation). Three-dimensional structures from RCSB PDB were optimized using MODELLER version 9.0. Peptide sequences were designed with PeptiDerive (Rosetta) and RosettaDesign version 3.5, followed by pharmacophore modeling based on key interaction residues. Virtual screening was then conducted with PyRx 0.8 and molecular docking with AutoDock Vina 1.1.2. Molecular dynamics simulations were performed using Desmond v6.6 (Schrödinger Suite 2016, Multisim v3.8.5.19) (100 ns, NPT ensemble, 300 K). Analysis of the five kinases revealed distinct interaction profiles with designed peptidomimetic compounds. Kinases displaying the IN conformation of the activation segment (PKB and FLT3) consistently showed superior stability and stronger interaction profiles compared to those in the OUT conformation. The designed compounds formed key hydrogen bonds and hydrophobic interactions with critical residues in the activation segment binding pocket. The most promising inhibitors demonstrated stability throughout the molecular dynamics simulations, with IN conformation kinases maintaining more consistent conformational profiles than their OUT conformation counterparts. Kinases with IN conformation of the activation segment demonstrated superior stability and interaction profiles compared to OUT conformations. These findings contribute to our understanding of selective kinase inhibition and provide a framework for developing novel inhibitors, particularly for PKB and FLT3. The implications of this study extend to rational drug design approaches that leverage natural regulatory mechanisms for therapeutic intervention, though further optimization is needed for GSK-3β, PAK4, and TTN to improve stability and binding affinity. Full article

This review develops a unified geometric framework for synthesizing global asymptotic laws, termed classical entanglement. The central tool is the entanglement operator, a Minkowski–$L_a$ metric blend that couples asymptotic regimes through an index $a>1$, producing a nonlinear global state whose intermediate region is metrically non-separable and cannot be written as a linear combination of its limits. The framework reveals a universal transition knee whose curvature scales linearly with a, independent of amplitudes or local scales. We show that this geometric mechanism encompasses Orlicz norms, weighted Hölder metrics, and iterated Hölder constructions, the latter being structurally isomorphic to self-similar root approximants. A conceptual “Rosetta Stone” links practitioner terminology, geometric meta-language, and functional-analytic structures, clarifying how classical entanglement unifies empirical blending, metric curvature, and Calderón-type interpolation. Applications to turbulence (Darcy friction factor), fractional dynamics, and scale-dependent diffusion illustrate how classical entanglement provides stable, asymptotically consistent global states across multi-scale systems. Full article

Background: Shiga toxin (Stx), produced by enterohemorrhagic Escherichia coli (EHEC), is a highly potent exotoxin responsible for severe complications such as hemolytic uremic syndrome (HUS). Among its isoforms, Stx2 exhibits stronger cytotoxicity and poses greater clinical risk, yet no effective therapy currently exists. Methods: In this study, two human monoclonal antibodies, YG12-1 and YG12-2, were identified from a phage display library and systematically characterized using an integrated modeling–validation workflow. Results: Structural modeling with ImmuneBuilder and Rosetta revealed that YG12-2 possessed a longer CDRH3 topology, more short-range hydrogen bonds, and stronger electrostatic complementarity, corresponding to lower binding energy and higher apparent affinity in ELISA and SPR. Although YG12-2 had a better affinity, YG12-1 shows better protective activity in a murine model of acute peritoneal infection. This paradox highlights a non-linear relationship between structural affinity and biological efficacy, emphasizing the importance of functional epitope accessibility and pharmacokinetic behavior in determining neutralization outcomes. Conclusions: Overall, these results indicated that targeting Stx2 with YG12-1 and YG12-2 could serve as a promising protective strategy against E. coli O157:H7 infection. Full article

The metal-binding periplasmic protein CusF has been proposed as a bifunctional tag that enhances the solubility of recombinant proteins and enables purification using Cu affinity chromatography. However, evidence for its performance remains limited to a few model proteins. Here, we evaluated CusF as a solubility tag for two heterologous proteins: a putative poly(A)-polymerase from Enterococcus faecalis (Efa PAP) and the red fluorescent protein mCherry. The proteins were fused to CusF, expressed in E. coli BL21 (DE3) pLysS and Rosetta 2 (DE3) strains, and assessed for solubility and IMAC binding. Native Efa PAP was completely insoluble under all tested conditions, and fusion to CusF did not improve its solubility. Similarly, CusF–mCherry accumulated predominantly in the insoluble fraction, with only trace amounts detectable in soluble lysates. Soluble CusF–mCherry did not bind Cu²⁺-charged IMAC resin, while moderate binding to Ni²⁺-charged resin was attributable to the vector-encoded His tag rather than CusF. These results indicate that CusF does not universally enhance protein solubility and may not consistently bind Cu-based IMAC resin. Our findings expand empirical knowledge of solubility tag performance and emphasize the necessity of testing multiple tags to identify optimal strategies for recombinant protein production. Full article

Carotid atherosclerosis remains one of the primary etiological factors underlying ischemic stroke, contributing to adult neurological disability and mortality. In recent years, non-coding RNAs (ncRNAs) have emerged as key regulators of gene expression, actively modulating molecular pathways involved in atherogenesis. This systematic review, the first to be exclusively focused on carotid atherosclerosis, aimed at synthesizing current findings on the differential expression of ncRNAs throughout the natural history of the disease, thus providing the first comprehensive attempt to delineate a stage-specific ncRNA expression profile in carotid disease. A comprehensive literature search was conducted in PubMed and Scopus databases in January 2025, following PRISMA guidelines. Original studies involving human subjects with carotid atherosclerosis, evaluating the expression of intracellular or circulating ncRNAs, were included and then categorized according to their association with cardiovascular risk factors, carotid intima-media thickness (cIMT), presence of atherosclerotic plaques, plaque vulnerability, clinical symptoms, and ischemic stroke. Out of 148 articles initially identified, 49 met the inclusion criteria and were analyzed in depth. Among the different classes of ncRNAs, microRNAs (miRNAs) were the most frequently reported as dysregulated, followed by circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs). Notably, the majority of identified ncRNAs were implicated in key pathogenic mechanisms such as inflammatory signaling, vascular smooth muscle cell (VSMC) phenotypic modulation, and ABCA1-mediated cholesterol efflux. Collectively, the evidence underscores the association and possible involvement of ncRNAs in the initiation and progression of carotid atherosclerosis and its cerebrovascular complications. Their relative stability in biological fluids and cell-specific expression profiles highlight their strong potential as minimally invasive biomarkers and—possibly—novel therapeutic targets. Full article

Melamine, a nitrogen-rich industrial chemical, has raised increasing concern as an emerging environmental contaminant with potential reproductive toxicity. While its nephrotoxic effects are well established, the direct impact of melamine on human sperm remains poorly defined. In this study, we investigated the in vitro effects of melamine on human sperm, under both capacitating and non-capacitating conditions. Functional analyses revealed that the exposure to 0.8 mM melamine, the highest non-cytotoxic concentration in vitro, significantly compromised sperm motility and disrupted key capacitation processes, including tyrosine phosphorylation patterns, cholesterol efflux, and the acrosome reaction. Molecular assessments demonstrated melamine-induced mitochondrial dysfunction, characterized by COX4I1 downregulation, reduced mitochondrial membrane potential, and altered reactive oxygen species production. In parallel, gene expression analyses revealed the activation of apoptotic pathways, with the upregulation of BAX and downregulation of BCL2, changes that were more pronounced during capacitation. Furthermore, melamine exposure significantly increased sperm DNA fragmentation and denaturation, indicating genotoxic stress. Collectively, these findings demonstrate that even low, non-cytotoxic concentrations of melamine compromise sperm function by disrupting capacitation, mitochondrial activity, and genomic integrity. This study identifies capacitation as a critical window of vulnerability and underscores the need to consider melamine as a potential environmental risk factor for male reproductive health. Full article

The Clausius–Mossotti (CM) factor underpins the theoretical description of dielectrophoresis (DEP) and is widely used in micro- and nano-scale systems for frequency-dependent particle and cell manipulation. It has further been proposed as an “electrophysiology Rosetta Stone” capable of linking DEP spectra to intrinsic cellular electrical properties. In this paper, the mathematical foundations and interpretive limits of this proposal are critically examined. By analyzing contrast factors derived from Laplace’s equation across multiple physical domains, it is shown that the CM functional form is a universal consequence of geometry, material contrast, and boundary conditions in linear Laplacian fields, rather than a feature unique to biological systems. Key modelling assumptions relevant to DEP are reassessed. Deviations from spherical symmetry lead naturally to tensorial contrast factors through geometry-dependent depolarisation coefficients. Complex, frequency-dependent CM factors and associated relaxation times are shown to inevitably arise from the coexistence of dissipative and storage mechanisms under time-varying forcing, independent of particle composition. Membrane surface charge influences DEP response through modified interfacial boundary conditions and effective transport parameters, rather than by introducing an independent driving mechanism. These results indicate that DEP spectra primarily reflect boundary-controlled field–particle coupling. From an inverse-problem perspective, this places fundamental constraints on parameter identifiability in DEP-based characterization. The CM factor remains a powerful and general modelling tool for micromachines and microfluidic systems, but its interpretive scope must be understood within the limits imposed by Laplacian field theory. Full article

Modeling the water cycle in the critical zone requires understanding interactions between the soil–vegetation–atmosphere compartments. Mechanistic modeling of soil water flow relies on the accurate determination of hydrodynamic parameters that control hydraulic conductivity and water retention curves. These parameters can be derived either using pedotransfer functions (PTFs), using soil properties obtained from field samples, or through inverse modeling, which allows the parameters to be adjusted to minimize differences between simulations and observations. While PTFs are widely used due to their simplicity, inverse modeling requires specific instrumentation and advanced numerical tools. This study, conducted at the Hydro-Geochemical Environmental Observatory (Strengbach forested catchment) in France, aims to determine the optimal hydrodynamic parameters for two contrasting forest plots, one dominated by spruce and the other by beech. The methodology integrates granulometric data across multiple soil layers to estimate soil parameters using PTFs (Rosetta). Water content and conductivity data were then corrected to account for soil stoniness, improving the KGE and NSE metrics. Finally, inverse parameter estimation based on water content measurements allowed for refinement of the evaluation of α, Ks, and n. This framework to estimate soil parameter was applied on different time periods to investigate the influence of the calibration chronicles on the estimated parameters. Results indicate that our methodology is efficient and that the optimal calibration period does not correspond to one with the most severe drought conditions; instead, a balanced time series including both wet and dry phases is preferable. Our findings also emphasize that KGE and NSE must be interpreted with caution, and that long simulation periods are essential for evaluating parameter robustness. Full article

Pine wilt disease (PWD), caused by the pine wood nematode (PWN) Bursaphelenchus xylophilus, is a devastating pine disease that is characterized by rapid transmission, high lethality, and limited control options. In our previous study, the fucosyltransferase gene (fut) which encoded fucosyltransferase (FUT) was found to be a putative virulence determinant in PWN, which regulates pathogenicity of nematodes. To investigate the functional role of the fut gene in PWN, a comprehensive analysis was conducted to understand its molecular structure and biological activity. The full-length open reading frame (ORF) of fut was amplified using reverse transcription PCR (RT-PCR) and successfully ligated into the pET-28a expression vector. Heterologous expression of the recombinant FUT was achieved in Escherichia coli Rosetta (DE3) through induction with 1.0 mM isopropyl-β-D-thiogalactoside (IPTG), followed by purification via nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. Biochemical characterization revealed that the recombinant FUT exhibited optimal enzymatic activity at 30 °C and pH 8.0, respectively. Furthermore, RNA interference (RNAi) validated by RT-qPCR was used to explore the biological functions of fut in PWN, and results indicated that downregulation of the fut gene could significantly reduce the vitality, reproduction, pathogenicity, development, and lifespan of PWN. Furthermore, gallic acid as an inhibitor of FUT displayed a strong inhibitory effect on recombinant FUT activity and nematicidal activity against PWNs in vitro and could alleviate the wilt symptom of pine seedlings inoculated with PWNs at a concentration of 100 μg/mL, indicating that it has the potential to be a novel nematicide. Collectively, these results establish fut as a critical virulence determinant in PWN and highlight its potential as a molecular target for controlling pine wilt disease. Full article

BPM1, a representative of the plant MATH-BTB protein family comprises three conserved domains—MATH, BTB, and BACK—that facilitate diverse protein–protein interactions central to developmental processes. However, recombinant production of BPM1 and its variants in Escherichia coli are frequently constrained by low solubility and poor stability. In this study, we systematically optimized E. coli-based expression strategies to enable soluble production and purification of domain-truncated BPM1 variants (BPM1ΔBTB, BPM1ΔMATH, and BPM1ΔBACK). A combinatorial approach was employed: varying induction temperature, medium composition, affinity tag selection, bacterial strain, and solubility-enhancing supplements. Expression outcomes were highly dependent on specific parameter combinations. Notably, BPM1ΔBTB—previously the most recalcitrant variant—showed a marked solubility improvement when expressed as a GST fusion in E. coli Rosetta (DE3) cultivated in TB medium supplemented with MgCl₂. By contrast, BPM1ΔMATH and BPM1ΔBACK displayed enhanced solubility when expressed in BL21 (DE3) cultivated in 4 × YT medium instead Rosetta (DE3) in 2 × YT medium. Constructs with N-terminal His-tags consistently resulted in poor solubility or failed expression. These results establish a framework for producing otherwise insoluble BPM1 variants and highlight a broadly applicable strategy for handling unstable proteins through tailored E. coli expression systems. Full article

Skin aging is influenced by both internal and external factors, resulting in wrinkles, decreased elasticity and irregular pigmentation. Hyaluronic acid (HA), a key component of the extracellular matrix, is essential for skin hydration and structural support. Peptides, short amino acid chains, have gained attention in cosmetics due to their multifunctional biological activities. This study explored the moisturizing and metal-chelating properties of Chrono Control Penta (S-Cannabis Sativa-pentapeptide-1), a novel plant-derived peptide whose sequence is WVSPL. In vitro, it chelated iron ions up to 17.86 ± 2.50% and copper ions up to 47.08 ± 1.49% at 10 mM and 3 mM, respectively. Western blot and Enzyme-Linked Immunosorbent Assay (ELISA) analysis showed that, under H₂O₂-induced stress, Chrono Control Penta increased hyaluronan synthase 2 (HAS2) production by 81.72% in BJ-5ta fibroblasts and enhanced HA secretion by 20.11% compared to simulated aging conditions alone, respectively. Furthermore, experiments carried out with the Franz diffusion cell and human full thickness skin demonstrated the peptide’s ability to penetrate the skin layers and even diffuse laterally with a quantified peptide skin biodistribution accounting for 0.095/0.06 nM/mg in 6 h. Advanced AI-based modeling (AlphaFold2, RosettaFold) and docking analysis revealed stable peptide-peptide transporter 2 (PEPT2) interactions, supporting carrier-mediated skin permeation and linking computational predictions with experimental diffusion data. Hence, this study extends previous evidence on the cosmetic efficacy of Chrono Control Penta by (i) adding mechanistic insights into metal chelation and HAS2/HA modulation, (ii) rigorously quantifying local skin penetration and lateral diffusion with HPLC-MS/MS, and (iii) providing a plausible mechanistic link between skin biodistribution and PEPT2-mediated transport based on deep learning structural models. Full article

Keratin is a fibrous structural protein found in various natural materials such as hair, feathers, and nails. Its high stability and cross-linked structure make it resistant to degradation by common proteases, leading to the accumulation of keratinous waste in various industries. In this study, we developed and validated an effective bioinformatics-driven strategy for mining novel keratinase genes from the Esmatlas (ESM Metagenomic Atlas) macrogenomic database. Two candidate genes, ker820 and ker907, were identified through sequence alignment, structural modeling, and phylogenetic analysis, and were subsequently heterologously expressed in Escherichia coli Rosetta (DE3) with the assistance of a solubility-enhancing chaperone system. Both enzymes belong to the Peptidase S8 family. Enzymatic characterization revealed that GST-tagged ker820 and ker907 exhibited strong keratinolytic activity, with optimal conditions at pH 9.0 and temperatures of 60 °C and 50 °C, respectively. Both enzymes showed significant degradation of feather and cat-hair keratin. Kinetic analysis showed favorable catalytic parameters, including $K_m$ values of 9.81 mg/mL (ker820) and 5.25 mg/mL (ker907), and $V_{max}$ values of 120.99 U/mg (ker820) and 89.52 U/mg (ker907). Stability tests indicated that GST-ker820 retained 70% activity at 60 °C for 120 min, while both enzymes remained stable at 4 °C for up to 10 days. These results demonstrate the high catalytic capacity, thermal stability, and substrate specificity of the enzymes, supporting their classification as active keratinases. This study introduces a promising strategy for efficiently discovering novel functional enzymes using an integrated computational and experimental approach. Beyond keratinases, this methodology could be extended to screen for enzymes with potential applications in environmental remediation. Full article

Dielectrophoresis (DEP) has been used for decades to estimate the passive electrical properties of cells. However, the body of work on cell electrophysiology derived from Clausius–Mossotti analysis of DEP-derived data pales to insignificance against the wider backdrop of cell electrophysiology based on the Goldman–Hodgkin–Katz equation measured by patch clamp, which focuses on membrane potential V_m—a parameter which does not appear in the Clausius–Mossotti model—and values of patch clamp-derived membrane conductance which, shorn of double-layer conductivity, are often orders of magnitude lower than those derived from DEP. Conversely, the body of work on DEP analysis is more substantial than that reporting the electrical properties of the extracellular (ζ) potential. To address this, several studies have recently been published into the interconnections between the electrical properties determined by the Clausius–Mossotti model, V_m, and ζ-potential, which analyzed the effect of varying the suspending medium conductivity over a wide range, from below 50 mSm⁻¹ to above 1.5 Sm⁻¹. The results of these studies identified relationships between the cytoplasm conductivity, V_m, membrane conductance and capacitance, surface conductance, whole-cell resistance, and ζ-potential. Significantly, many of these relationships only become apparent when analyzed as a function of the conductivity of the suspending medium. This paper assembles these interconnections, using several separate studies approaching different parameter connections, to draw together a set of equations which collectively form a “cellular electrome”. This demonstrates that analysis of the Clausius–Mossotti factor across multiple conductivities allows determination of not only passive electrical properties, but also the membrane and ζ-potential, and accurately predicts DEP behavior at higher conductivity for the first time. Full article

Cytochrome P450 enzymes (CYPs) are versatile biocatalysts capable of performing selective oxidation reactions valuable for industrial and pharmaceutical applications. However, their catalytic efficiency is often constrained by dependence on costly electron donors, the requirement for redox partners, and uncoupling reactions that divert reducing power toward reactive oxygen species. Improving electron transfer efficiency through optimized redox partner interactions is therefore critical for developing effective CYP-based biocatalysts. In this study, we investigated the interaction between CYP119, a thermophilic CYP from Sulfolobus acidocaldarius, and putidaredoxin (Pdx), the redox partner of P450cam. Using rational design and computational modeling with PyRosetta 3, 14 CYP119 variants were modeled and analyzed by docking simulations on the Rosie Docking Server. Structural analysis identified three key mutations (N34E, D77R, and N34E/D77R) for site-directed mutagenesis. These mutations (N34E, D77R, and N34E/D77R) enhanced Pdx binding affinity by 20-, 3-, and 12-fold, respectively, without affecting substrate binding. Catalytic assays using lauric acid and indirect assays to monitor electron transfer revealed that, despite improved complex formation, the N34E variant showed reduced electron transfer efficiency compared to D77R. These findings highlight the delicate balance between redox partner binding affinity and catalytic turnover, emphasizing that fine-tuning electron transfer interfaces are essential for engineering efficient CYP biocatalysts. Full article

Despite global vaccination efforts, poliomyelitis continues to cause paralytic cases, highlighting the need for alternative therapeutic approaches. Nanobodies offer significant advantages over conventional antibodies due to their small size, stability, and low immunogenicity, yet few have been developed specifically against poliovirus. This study presents a fully computational pipeline for de novo design of nanobodies targeting Virus Protein 3 (VP3) of the Poliovirus I Sabin strain. Our integrated approach employed Ig-VAE for scaffold generation, ProteinMPNN and RFantibody for sequence design, tFold-Ab/Ag for structure prediction, multi-platform molecular docking (Rfantibody, Rosetta3, ClusPro2, ReplicaDock 2.0), molecular dynamics simulations, and humanization tools. The pipeline identified three humanized nanobodies (scFv-0389-304-6H, scFv-0389-459-5H, and scFv-0743-166-7/H) that demonstrated strong binding to VP3 with binding free energies of −37.66 ± 10.35, −40.11 ± 20.01, and −48.62 ± 11.21 kcal/mol, respectively. All designs exhibited favorable physicochemical properties and high solubility. Notably, nanobodies humanized prior to CDR-loop design (scFv-0743-166-7/H) showed superior stability, binding affinity, and structural similarity to experimentally validated nanobodies. This work demonstrates the feasibility of a fully computational approach for designing promising nanobodies against viral targets, providing an alternative to traditional methods with potential applications in drug design. Full article