Search Results (353)

Extracellular traps (ETs) are a novel host defense mechanism used to immobilize and eliminate invading microorganisms, and their formation depends on the recognition of foreign pathogens by membrane receptors. Previous studies have demonstrated that Vibrio splendidus flagellin can induce the release of ETs in coelomocytes of the sea cucumber Apostichopus japonicus, yet the underlying regulatory mechanism remains unclear. Here, we identify another Toll-like receptor (TLR) homolog, AjTLR2, in Apostichopus japonicus, which is composed of an extracellular LRR domain, a transmembrane domain, and an intracellular TIR domain. As a membrane receptor, AjTLR2 is upregulated upon infection with Vibrio splendidus AJ01, which is isolated from diseased Apostichopus japonicus. The extracellular LRR domain exhibits binding activity toward LPS, PGN, and MAN. In addition to these ligands, AjTLR2 recognizes flagellin C of AJ01 (AJ01-FliC), whereas other AjTLRs, such as AjToll and AjTLR3, do not. Further functional analysis reveals that knockdown of AjTLR2 results in a reduction in the typical weblike DNA structures of ETs, accompanied by a significant decrease in the expression of the ET-associated antimicrobial proteins H2A, H2B, and lysozyme. Furthermore, AjTLR2 knockdown similarly inhibits ET formation induced by recombinant AJ01-FliC protein. Mechanistically, the Apostichopus japonicus proto-oncogene tyrosine-protein kinase Src homolog (AjSRC), previously identified in our laboratory, is a downstream signaling molecule of AjTLR2 and is recruited via the TIR domain of AjTLR2. Knockdown of AjSRC also suppresses AJ01-FliC-induced ET formation. Collectively, our results indicate that the recruitment of AjSRC by AjTLR2 represents a potential regulatory pathway for AJ01-FliC induced ET generation. Full article

The SARS-CoV-2 JN.1 subvariant has attracted attention due to the accumulation of mutations in the Spike (S) glycoprotein, particularly within the receptor-binding domain (RBD), contributing to the structural heterogeneity of the Spike protein. However, the extent to which conformational dynamics influence ligand-recognition patterns across functional Spike states remains insufficiently characterized. In this study, we applied a hierarchical computational framework combining homology modeling, molecular dynamics simulations, and molecular docking to determine whether conformational sampling modifies the location, accessibility, and recurrence of ligand-interaction regions in the JN.1 Spike protein. Closed, semi-closed, and open conformations were modeled and subjected to triplicate simulations, including an initial 100 ns phase followed by extended 200 ns simulations. Representative conformations were obtained by trajectory clustering and used for docking analyses. Two flavonoids, hesperitin-7-O-rutinoside (H7R) and flavanone-7-O-glucoside (F7G), were employed as structural probes to assess how ligand interaction patterns vary across conformational states. Comparative analyses revealed that conformational sampling modifies the location, accessibility and spatial distribution of ligand-interaction regions while generating distinct docking poses and recurrent interaction patterns not fully captured by static structural models. These findings highlight the importance of dynamics-informed structural ensembles for docking analyses in flexible viral proteins. Rather than predicting absolute binding affinities, this study provides an exploratory computational framework for evaluating ligand-recognition behavior in structurally dynamic viral systems. Full article

In silico structural characterization of carbohydrate-active enzymes (CAZymes) in Bacillus subtilis T7 reveals mechanistic insight into the strain’s capacity for consolidated bioprocessing of untreated lignocellulosic biomass. Homology models for 13 CAZymes were constructed using SWISS-MODEL, with Cu²⁺ and FAD cofactors incorporated into the AA10 lytic polysaccharide monooxygenase and AA3 oxidoreductase models, respectively. Blind molecular docking across the full protein surface identified energetically favorable binding pockets on GH9 endoglucanase and Abhydrolase_1. Among 12 enzyme–ligand pairs screened, Abhydrolase_1 exhibited the highest affinity for xylotetraose (−7.7 kcal/mol) and GH9 showed the strongest preference for cellotetraose (−7.0 kcal/mol). Site-specific docking confirmed six hydrogen bonds with Gly32, Phe33, Thr34, Ser36, Arg179, and His256, supplemented by two carbon–hydrogen bonds with Ile180 and Ser39, anchoring xylotetraose within the Abhydrolase_1 binding cavity, and seven hydrogen bonds stabilizing cellotetraose in the GH9 catalytic groove, with key contacts at Tyr141, Trp145, Asp194, Trp193, Arg254, Tyr255, and Tyr354. One-hundred nanosecond all-atom molecular dynamics simulations (GROMACS 2023.2, CHARMM36 force field, triplicate runs) confirmed overall structural integrity for both proteins: Abhydrolase_1 maintained a compact conformation (Rg = 18.11 ± 0.09 Å; backbone RMSD 2–3 Å), while GH9 was similarly stable (Rg = 30.36 ± 0.33 Å; RMSD 2–5 Å). Ligand dynamics were more variable—xylotetraose remained bound within the Abhydrolase_1 active site for approximately 75 ns before partial displacement, whereas cellotetraose exhibited dynamic association along the GH9 catalytic channel, consistent with processive substrate translocation in endoglucanases. These computational findings line up with the strain’s experimentally observed hydrolytic clearance zones (cellulase 24.5 mm; xylanase 11.6 mm), 63.4% alkali lignin decolorization, transient accumulation of ferulic acid and vanillin, and a hydrogen yield of 1.41 mol H₂/mol substrate from untreated food waste. Together they give a molecular-level picture of substrate-specific CAZyme recognition in B. subtilis T7 and support its potential as a pretreatment-free platform for lignocellulosic biohydrogen production. Full article

Oligonucleotide therapeutics represent an expanding class of medicines that can regulate gene expression, RNA processing, protein translation, immune signaling, and biomolecular recognition through sequence-specific or structure-dependent mechanisms. Despite clinical progress, their application remains constrained by nuclease degradation, rapid clearance, inefficient tissue and cellular delivery, endosomal sequestration, off-target activity, immune recognition, and mechanism-specific requirements for target engagement. Chemical modification is central to oligonucleotide therapeutic development because it can mitigate some of these limitations while influencing target affinity, protein binding, pharmacokinetics, and intracellular activity. This review examines chemical modification strategies to improve the biological stability and functional performance of therapeutic oligonucleotides and is organized around major classes of chemical modification, including phosphate and backbone-linkage modifications, sugar and conformational modifications, backbone-replacement analogs, and conjugation-based approaches. Rather than presenting these chemistries as uniformly beneficial, this review emphasizes that the same modification can be enabling in one therapeutic mechanism and disruptive in another, so its value cannot be judged apart from the modality and molecular architecture in which it is placed. Clinically successful oligonucleotide designs are likely to rely on combinations of chemical features, including modified backbones, modified sugars, stereochemical control, terminal stabilization, and ligand- or formulation-based delivery strategies. Understanding how these features interact is essential to develop more predictable and mechanism-appropriate oligonucleotide therapeutics. Full article

Graphene oxide (GO)-based nanomaterials functionalized with targeting ligands are promising platforms for selective cancer drug delivery. Among relevant targets, integrin α_Vβ₃ is a highly overexpressed receptor in several solid tumors and is commonly targeted using cyclic Arg-Gly-Asp (cRGD) peptides. However, the molecular details governing the interaction between cRGD-functionalized GO dots and integrins remain poorly understood. In this work, all-atom molecular dynamics simulations are employed to investigate the interaction between integrin α_Vβ₃ and a nanocarrier composed of a GO dot coated with polyethylene glycol (PEG) and functionalized with cRGD ligands. Multiple 1 μs simulation replicas are used to characterize both specific ligand recognition and non-specific nanocarrier/receptor interactions. The simulations show that cRGD binding within the integrin-binding pocket is stable, indicating that the nanocarrier does not impair receptor recognition. Beyond cRGD-mediated binding, both PEG-cRGD chains and GO itself establish additional contacts with the protein, whose nature and distribution are modulated by the relative orientation of the GO plane. Overall, the structural dynamics of integrin α_Vβ₃ remains preserved upon nanocarrier binding. These findings provide atomistic insights into the interplay between ligand-mediated and multivalent surface-mediated interactions of GO-based nanocarriers with integrins for the rational design of selective nanocarriers for cancer therapy. Full article

Neurogenic bladder is a condition associated with impaired voiding, leading to urinary stasis and increased susceptibility to urinary tract infections, predominantly caused by Escherichia coli. In this context, bacterial iron acquisition systems represent attractive targets for alternative antimicrobial strategies. This study aimed to investigate the interactions of deferoxamine with E. coli iron acquisition proteins, combining an experimental model of neurogenic bladder with molecular analyses. The experimental model of neurogenic bladder was induced by complete spinal cord transection in rats, followed by urine collection by cystocentesis and microbiological characterization of uropathogens. Subsequently, molecular docking and energetic analyses were performed to evaluate the binding of deferoxamine and its Fe-DFO complex to the FhuE receptor of the ferric hydroxamate uptake pathway, with FhuA and FhuD prepared as correlated targets of the same pathway for structural context. The animals presented urinary retention and bacterial colonization, with E. coli identified as the pathogen. The results of the molecular docking revealed geometrically plausible accommodation of Fe-DFO within siderophore recognition pockets, involving residues associated with siderophore recognition and transport, as well as binding affinity scores consistent with weak-to-moderate structural complementarity compared to reference ligands. It is concluded that the neurogenic bladder model provides a biologically relevant framework for the study of urinary tract infections and that deferoxamine exhibits molecular interactions consistent with the ferric hydroxamate uptake system of E. coli. Because the present analysis was restricted to the Fhu pathway, these findings cannot be extrapolated to overall bacterial iron homeostasis, which involves multiple parallel acquisition systems. The current work is explicitly positioned as a proof-of-concept investigation; in vivo administration of DFO in the neurogenic bladder model, functional assays of iron uptake, transporter specificity experiments, and molecular dynamics analyses are identified as priority directions for future work. Full article

The zinc finger protein 36 (ZFP36) family, including ZFP36/tristetraprolin (TTP), ZFP36 CCCH-type-like 1 (ZFP36L1), and ZFP36 CCCH-type-like 2 (ZFP36L2), consists of conserved CCCH-type tandem zinc-finger RNA-binding proteins. These proteins recognize AU-rich elements (AREs) in target mRNAs and promote deadenylation, decay, and translational repression. In this review, we use the term post-transcriptional immune checkpoint in a restricted conceptual sense: ZFP36 family proteins are intracellular, RNA-level negative regulators that tune the magnitude, duration, and resolution of immune effector programs, rather than classical receptor-ligand immune checkpoints such as programmed cell death protein 1 (PD-1)/ programmed death-ligand 1 (PD-L1) or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). We summarize structural features, ARE-recognition mechanisms, mRNA decay pathways, translational repression mechanisms, and post-translational regulation of the ZFP36 family, while explicitly distinguishing mechanisms established for ZFP36 from those inferred for ZFP36L1 and ZFP36L2. We then review cell-type-specific roles in innate and adaptive immunity, including myeloid inflammatory responses, barrier tissue inflammation, innate lymphoid cell function, T cell activation and effector differentiation, regulatory T cell stability, B cell development, and antiviral immunity. In cancer, ZFP36 family members show context-dependent functions that should be separated into tumor-cell-intrinsic effects and immune-microenvironment-dependent effects. They suppress tumor progression by destabilizing pro-inflammatory, angiogenic, metabolic, and epithelial–mesenchymal transition (EMT)-associated transcripts, yet may also restrict antitumor immune responses or promote immune evasion in selected tumor contexts. Finally, we discuss autoimmune and inflammatory diseases, allergic disorders, transplant immunity, neuroimmune relevance, and therapeutic strategies, emphasizing the current evidentiary limits, preclinical status, and safety concerns of ZFP36 family modulation. Full article

Carbohydrate-active enzymes (CAZymes) encoded by the human gut microbiome are central mediators of dietary glycan metabolism and host–microbe biochemical homeostasis. Among these, β-glucuronidases represent functionally pivotal hydrolases implicated in metabolism, intestinal physiology, and therapeutic modulation. The present study performs an integrative in silico structural and functional interrogation of β-glucuronidases derived from Acidobacterium capsulatum (3VNY), Bacteroides ovatus (6D8K), and Faecalibacterium prausnitzii (6ED2). An integrated computational framework encompassing physicochemical parameters profiling, hierarchical structural prediction, tertiary-structure validation, salt-bridge energetics, functional domain and motif annotation, protein–protein interaction reconstruction, ligand-binding thermodynamics via molecular docking, and residue-resolved non-covalent interaction network mapping using the Protein Contacts Atlas (PCA) was employed. Physicochemical analyses indicated that all enzymes are thermostable, intracellular, and hydrophilic, while secondary-structure organization revealed a functional balance between helix-mediated rigidity and coil-driven flexibility. Structural validation metrics identified 6ED2 as the most conformationally stable architecture, whereas 6D8K displayed enhanced functional complexity, including enriched motif composition, membrane-associated features, and superior ligand-binding affinity. Docking simulations highlighted castanospermine and calcium saccharate as the most favorable interacting ligands across enzyme variants. Importantly, PCA-based interaction analysis revealed distinct ligand-centered atomic contact networks, with immediate contact counts of 57 (3VNY), 32 (6D8K), and 41 (6ED2), providing residue-level insight into stabilization mechanisms and interaction topology beyond conventional docking metrics. Collectively, these findings establish a multidimensional computational framework linking structural stability, functional diversification, ligand recognition, and atomic interaction networks in gut microbial β-glucuronidases, thereby supporting future biochemical validation, microbiome-targeted therapeutics, and biotechnological or cosmeceutical applications. Full article

The Zona Pellucida (ZP) and its structural analogs are evolutionarily ancient extracellular matrix components. These are essential for oocyte protection, species-specific gamete recognition, and prevention of polyspermy across Metazoa. Defined by the conserved ZP-domain—comprising ZP-N and ZP-C subdomains—these glycoproteins self-assemble into fibrillar matrices through tightly regulated polymerization. Mechanisms of the regulated polymerization involve furin cleavage, disulfide bonding, and hydrophobic interactions. Once considered a vertebrate innovation, the canonical ZP-domain—defined by its bipartite ZP-N/ZP-C architecture, eight conserved cysteine residues, and capacity for matrix polymerization—is now recognized as an ancient metazoan extracellular module, with homologs identified in basal lineages including Porifera, Cnidaria, and Placozoa. While ZP-like sequences have been reported in choanoflagellates such as Salpingoeca rosetta, these lack the complete canonical features and are considered distant structural relatives rather than true ZP-modules. There they function in cell adhesion and tissue integrity, suggesting an origin predating the evolution of specialized reproductive coats. Previous phylogenetic analyses across 97 metazoan species have revealed that vertebrate ZP genes arose from ancestral duplications of the canonical ZP-module. Accordingly, they give rise to eight subfamilies (ZP1–ZP4, ZPD, ZPAX, ZPX, ZPY), with lineage-specific expansions, losses, and pseudogenization reflecting adaptations to diverse reproductive strategies. Positive selection in sperm-binding regions of ZP2 and ZP3 drives a rapid adaptive evolution. It underscores coevolutionary arms races with sperm ligands, contributing to reproductive isolation and speciation. In invertebrates such as abalone and insects, ZP-domain proteins mediate analogous functions through lineage-specific elaborations, including tandem repeats and domain shuffling. Post-translational modifications, particularly glycosylation, fine-tune sperm receptor specificity and matrix stability. The functional transition from a general protective barrier in early metazoans to a sophisticated gamete recognition interface in vertebrates exemplifies modular evolution. This synthesis highlights the domain-level deep homology of ZP-domain proteins as a foundational element of metazoan extracellular matrices, repurposed through gene duplication, neofunctionalization, and selection to meet the demands of evolving reproductive modes. These insights bridge evolutionary biology, reproductive medicine, and developmental genetics. However, major gaps remain, including unresolved orthology between vertebrate and invertebrate ZP genes, the relative contribution of glycans versus protein backbone in sperm recognition, and the lack of functional evidence for canonical ZP-domain proteins in insects. Future studies integrating glycoproteomics, single-cell transcriptomics, and CRISPR-based models are needed to resolve these questions. Full article

Abiotic stresses, including drought, salinity, alkalinity, temperature extremes, flooding, heavy metals, and emerging pollutants, challenge plant growth and productivity by disturbing water relations, ion balance, redox homeostasis, membrane stability, energy metabolism, and developmental progression. Although substantial progress has been made in the identification of stress-responsive hormones, second messengers, kinases, transcription factors, transporters, and metabolic regulators, plant stress adaptation cannot be fully explained by linear signaling cascades or single tolerance genes. A major unresolved question is how early molecular events are reorganized into coordinated physiological and developmental outputs that support survival, recovery, and productivity. In this review, we propose an intermolecular interaction-driven adaptive remodeling framework for plant abiotic stress responses. This framework emphasizes that stress tolerance emerges from dynamic changes in receptor–ligand recognition, protein–protein interactions, calcium decoding, redox-sensitive modification, phosphorylation networks, transcriptional regulation, chromatin-associated control, and metabolite-mediated feedback. We further emphasize ROS as integrative redox switches that connect stress sensing, defense activation, senescence-related transitions, and recovery, and chromatin-associated mechanisms as regulators that may stabilize primed or memory-like adaptive states. We discuss how these interaction networks converge on core signaling hubs, including abscisic acid, reactive oxygen species, Ca²⁺, and kinase/phosphatase systems, and how they remodel stomatal behavior, root architecture, ion and pH homeostasis, redox buffering, metabolism, development, and reproductive resilience. We further highlight how natural variation, multi-omics, genome editing, high-throughput phenotyping, and field validation can translate interaction-centered stress biology into crop resilience. This perspective provides a conceptual bridge between molecular stress perception, network behavior, physiological adaptation, and climate-resilient agriculture. Full article

Background/Objectives: Mosquitoes, including Culex quinquefasciatus and Aedes aegypti, are important vectors of dengue fever, Zika virus, West Nile virus, Japanese encephalitis virus, Eastern equine encephalitis virus, etc. Biological control has always been urgent in mosquito prevention due to resistance developing to synthetic insecticides and environmental toxicity by insecticides. Methods: The leaf essential oil of Mosla. chinensis was isolated, and major components were identified via GC-MS, followed by olfactory behavior assays to evaluate its repellent activity against C. quinquefasciatus. Additionally, the odorant-binding protein 1 and odorant-binding protein 2 (CquiOBP1-2) genes were prokaryotically expressed, and their fluorescence competitive binding activities with the active components of essential oils were examined. Results: The bioassays indicated this essential oil greatly repels C. quinquefasciatus, which will significantly protect people against vector-borne diseases. In the fluorescence competitive binding experiments, the CquiOBP1-2 proteins exhibit great binding capacities to volatile components, including Citronellal, Citronellol, Geraniol, Limonene and Isopulegol. Furthermore, the behavioral experimental results also indicate that the mixture of these five ligand compounds has an obvious repellent effect on mosquitoes, highlighting that they may be applied as potential mosquito repellent agents. Moreover, molecular docking and site-directed mutation analysis further confirm Phe123 and Gln77 are both key amino acid residues of CquiOBP1-2 proteins involved in the olfactory recognition of repellent ligand compounds from M. chinensis essential oil. Conclusions: The behavioral experimental verification and the exploration of olfactory molecular mechanisms are helpful to promote the biological control of plant essential oils in mosquito pests. Full article

The dopamine D₃ receptor (D₃R) has long been considered a secondary target in the treatment of Parkinson’s disease (PD), with therapeutic strategies primarily focused on D₂ receptor–mediated motor control. However, accumulating evidence now supports D₃R as a functionally distinct dopaminergic receptor subtype with specific relevance to non-motor symptom domains and dopaminergic signaling under hypodopaminergic conditions. Recent advances in high-resolution structural biology have elucidated the molecular basis of D₃R/D₂R discrimination, revealing how subtle residue-level and microstructural differences within a conserved G protein–coupled receptor framework shape ligand recognition and receptor activation. In parallel, the emergence of ligand-dependent biased signaling has refined current understanding of D₃R pharmacology. Selected ligands can preferentially engage Gα_i/o-mediated pathways while limiting β-arrestin recruitment and associated regulatory processes, providing a mechanistic rationale for more stable modulation of mesolimbic dopaminergic circuits involved in affective and motivational regulation. Beyond symptomatic modulation, preclinical studies suggest that D₃R signaling may influence neuronal resilience, synaptic plasticity, and adaptive responses to dopaminergic injury; however, such effects remain experimental and have not been demonstrated in clinical PD. This review integrates recent structural, signaling, and functional insights into D₃R biology, with particular emphasis on biased agonism and emerging therapeutic concepts. Although D₃R-targeted strategies do not currently represent disease-modifying interventions, they offer a rational framework for the development of next-generation dopaminergic therapies aimed at improving precision, tolerability, and long-term signaling stability in Parkinson’s disease. Full article

The innate immune response is a critical defense mechanism by which vertebrates recognize and eliminate invading pathogens. Pattern recognition receptors (PRRs) detect pathogen-associated molecular patterns and activate downstream signaling pathways. NOD1, a classic PRR of the NLR family, recruits the adaptor protein RIPK2 to initiate antibacterial signaling. In this study, we cloned and characterized the NOD1 gene from snakehead (Channa argus). Briefly, the full-length NOD1 cDNA is 2829 bp encoding 943 amino acids, showing high homology with Perciformes. The qPCR analysis revealed widespread NOD1 gene expression in various tissues, with significant upregulation in the gill (p < 0.05) and spleen (p < 0.05) following bacterial infection. Overexpression of the NOD1 gene activated the NF-κB signaling pathway in a dose- and time-dependent manner, and specifically responded to the bacterial ligand iE-DAP but not to other tested ligands. Furthermore, NOD1 synergized with the downstream adaptor RIPK2 to enhance NF-κB activity, and direct protein interaction between NOD1 and RIPK2 was confirmed by co-immunoprecipitation. Taken together, these findings demonstrate that snakehead NOD1 plays a critical role in the host antimicrobial immune response. Full article

The selective detection of small, highly hydrophilic metabolites differing only in stereochemistry represents a major challenge in biosensor development. Here, we present a computational investigation to elucidate the molecular basis of the experimentally observed selectivity of a protein-based electrochemical biosensor toward myo-inositol over D-chiro-inositol. Although the two stereoisomers differ only in the orientation of a single hydroxyl group, they induce distinct dynamic effects on the protein recognition element. Molecular docking revealed comparable binding regions and similar affinity scores, indicating that selectivity does not arise from differences in binding site or docking energy. To investigate dynamic contributions, all-atom molecular dynamics simulations were performed in triplicate (3 × 100 ns) using the AMBER99SB force field and explicit TIP3P water. Trajectory analyses showed that myo-inositol forms a more persistent hydrogen bond network, resulting in reduced residue-level flexibility, more stable ligand–protein interactions, and enhanced local structural stabilization. Overall, these findings support a dynamic model of stereoselective recognition in which ligand-induced modulation of protein conformational ensembles, rather than static affinity, governs biosensor performance. This work highlights the value of molecular dynamics simulations in the rational design of biosensors targeting structurally similar analytes. Full article

DNA viruses rely extensively on host cellular machinery, including replication factors and transcriptional systems, to persist after infection. These mechanisms make studying and targeting DNA viral proteins challenging, as they also play key roles in mammalian processes. Traditional strategies include CRISPR-mediated gene disruption and small interfering RNA (siRNA) to target host proteins. However, Proteolysis Targeting Chimeras (PROTACs) offer a novel strategy by enabling the selective and rapid degradation of specific viral or host proteins involved in the DNA viral lifecycle. PROTACs are heterobifunctional molecules composed of three key components: a ligand that binds the target protein, a chemical linker, and a ligand that recruits an E3 ubiquitin ligase. By simultaneously binding both the target protein and the E3 ligase, PROTACs form a ternary complex. This proximity enables the E3 ligase to ubiquitinate the target protein, marking it for recognition and subsequent degradation by the intracellular proteasome. This approach represents a promising avenue for targeting previously undruggable proteins and improving therapeutic outcomes in virus-associated malignancies. In this perspective, we describe studies that use PROTACs as tools to modulate host proteins to investigate DNA viral processes with temporal control of host protein expression, as well as the use of PROTACs as antivirals to directly target DNA viral proteins. We also provide a detailed chart summarizing known host-targeting PROTACs and their potential applications across different stages of DNA viral lifecycles, highlighting opportunities for future DNA virus research. Full article