We explore mass-resolved imaging of fragments generated from single macromolecular assembly (MMA) ions on a custom-built Orbitrap/time-of-flight (TOF) mass spectrometer with integrated UV photodissociation (UVPD) and a position- and time-sensitive Timepix3 imaging detector assembly. We postulated that the 2D detector images provide information about the 3D geometry of the MMAs in the gas phase as the TOF analyzer has the ability to retain the relative positions of the product ions following the fragmentation process and until they reach the imaging detector, when the fragmentation occurs at the level of single-precursor MMA ion. We demonstrate that the Orbitrap/TOF mass spectrometer enables fragmentation at the single-precursor MMA ion level using dimeric and tetrameric noncovalently bound assemblies. Timepix3-derived relative position data from single-precursor fragmentation events of two distinct tetrameric MMAs reveal different higher-order structural signatures that enable their differentiation. Furthermore, mapping these single-precursor fragmentation events to possible dissociation pathways provides insight into the underlying dissociation mechanisms. Overall, this study demonstrates the potential of single-ion mass-resolved imaging to understand UVPD dissociation mechanisms, fragmentation pathways of MMA ions, and their higher-order structure.
UV-induced polymerization and photocrosslinking have become versatile tools for engineering advanced biomaterials, providing rapid reaction kinetics, spatial-temporal control, and compatibility with sensitive biological environments. This review integrates the fundamental mechanisms of photopolymerization, cationic photopolymerization, and thiol-mediated photopolymerization with the design principles of key UV-responsive material classes, including hydrogels, smart stimuli-responsive systems, elastomers and thermosets, polymeric networks, and composite or hybrid matrices. Their expanding roles in biomedical technologies are highlighted through applications in drug delivery, bioactive coatings, scaffolds, and photoactivated bioadhesives, as well as in photopolymerization-based additive manufacturing strategies such as digital light processing (DLP) and stereolithography (SLA). The unique advantages of UV-activated systems, such as mild processing conditions, on-demand curing, and compatibility with in situ and minimally invasive procedures, are discussed alongside current constraints, including limited light penetration, oxygen inhibition, cytotoxicity, and application-specific barriers. By linking photochemical fundamentals with application-driven design, this review underscores the growing potential of UV-engineered polymer networks to enable next-generation solutions in tissue engineering, regenerative medicine, and targeted therapeutic delivery.
Colorimetric nucleic acid sensing platforms offer attractive advantages for low-instrumentation detection; however, their performance is strongly governed by the nature of macromolecular interactions involved in signal transduction. In this work, we present a comparative evaluation of two distinct colorimetric sensing strategies, gold nanoparticle (Au NP) agglomeration and toluidine blue O (TBO)-based metachromasia for oligonucleotide detection using synthetic, amplification-relevant targets. Specific oligonucleotide sequences derived from hepatitis C virus (HCV) genotypes 1 and 3 have been employed as representative model targets to enable controlled assessment of sequence discrimination and sensing behavior. Target oligonucleotides are selectively captured on probe-functionalized magnetite nanoparticles, followed by strand separation and release of single-stranded complementary DNA (cDNA) for downstream colorimetric detection. In the Au NP-based system, target-induced electrostatic screening and bridging effects have led to nanoparticle agglomeration, resulting in characteristic localized surface plasmon resonance shifts observable by UV-visible spectroscopy. In contrast, the TBO-based system has exhibited concentration-dependent metachromatic responses arising from electrostatically driven association and agglomeration of the cationic dye along the negatively charged phosphate backbone of the released cDNA. Direct comparison under identical experimental conditions revealed distinct differences in sensitivity, signal evolution, and sequence discrimination between the two platforms, highlighting how fundamentally different macromolecular interaction pathways govern colorimetric response generation. Rather than constituting a disease-specific diagnostic assay, this study provides mechanistic and design-level insights into post-amplification-compatible colorimetric oligonucleotide sensing, offering a generalizable framework for the rational development of colorimetric nucleic acid sensors.
Ribosomes are essential macromolecular machines that facilitate protein synthesis and have long been recognized as effective targets for antimicrobial agents. While structural differences between prokaryotic and eukaryotic ribosomes form the basis for selective antibiotics against bacteria, similar approaches for developing antifungal agents targeting ribosomes have remained limited due to the high sequence and structural conservation with human ribosomes. However, emerging insights into ribosome homeostasis, including ribosome biogenesis, turnover, and hibernation, have uncovered a set of ribosome-associated proteins whose function is critical yet display greater sequence divergence from their human counterparts. These observations suggest that these regulatory components may represent viable antifungal targets by disrupting fungal proteostasis. The present review aims to explore this developing concept by examining ribosome-associated factors and considering whether short ribosomal protein-derived peptides may eventually serve as druggable molecules for selectively modulating these pathways in fungal pathogens.
Protein Data Bank Japan (https://pdbj.org/) is the Asian hub of three-dimensional (3D) macromolecular structure data and a founding member of the global Protein Data Bank (PDB) network. Over two decades, we have curated and distributed experimentally determined structures, complementing international collaborations with Research Collaboratory for Structural Bioinformatics (RCSB) PDB, Biological Magnetic Resonance Data Bank, Protein Data Bank in Europe (PDBe), and Electron Microscopy Data Bank. In response to user demand for integrated structural and chemical data, we developed a new PubChem Portal that enables interactive exploration of compound-protein interactions. Users can view ligand binding poses in 3D via our Web Graphics Library (WebGL)-based Molmil viewer, with key interactions highlighted and key residues displayed in semi-transparent stick models, enhanced through integration with secondary databases (e.g., Dynamics DB, eF-site) for advanced insights into molecular dynamics and electrostatics. The system supports filtering by UniProt ID, Enzyme Commission (EC) number, Pfam ID, or PROSITE ID to identify structurally related compounds and visualizes protein-ligand interactions. A dynamic two-dimensional (2D) Japan Agency for Medical Research and Development representation enables real-time atom-level navigation, with clickable atoms linking to 3D structures. This tool allows users to explore compound-protein interaction landscapes, identify potential binding modes, and guide experimental design, such as mutagenesis or crystallization. The portal offers a comprehensive, user-centered ecosystem that bridges chemical and structural data, enhancing access to biological insights through integrated visualization and analysis.
Diabetes remains a major global health burden, necessitating advanced therapies beyond insulin injections, such as closed-loop systems mimicking pancreatic beta-cell function. To address the limitations of current glucose-responsive insulin delivery platforms, particularly limited injectability and suboptimal biocompatibility, we developed injectable, glucose-responsive G-quartet/protein hydrogels (GPHGs) via supramolecular self-assembly and iminoboronate chemistry. GPHGs exhibit tunable viscoelasticity, with storage moduli ranging from 17 to 50 kPa across three formyl-phenylboronic acid (FPBA)-based crosslinkers (2FPBA, 3FPBA, and 4FPBA), thereby imparting structural integrity and stimulus responsiveness to the network. Under high glucose conditions (100 mM glucose), GPHG derived using 2FPBA exhibited a 0.28-fold decrease in storage modulus (G') and a 0.21-fold reduction in loss modulus (G″), indicating glucose-mediated softening of the network. This mechanical response corresponds with accelerated insulin release, yielding ∼85% insulin release in 24 h under 100 mM glucose compared to ∼45% insulin release under 0 mM glucose. GPHG1 exhibits remarkable self-healing and negligible cytotoxicity at a concentration of 10 mg/mL in HaCaT, MCF7, and HCT116 cells. In type 1 diabetic rats, insulin-loaded GPHG1 sustained normoglycemia (120-150 mg/dL or 6.6-8.3 mM) for up to seven days, outperforming subcutaneous insulin. These findings highlight GPHG as a minimally invasive strategy for innovative insulin delivery systems, harnessing macromolecular and supramolecular design to advance blood glucose control.
The assembly of cellular microtubules relies heavily on γ-tubulin ring complex (γTuRC), a macromolecular assembly of γ-tubulin and associated proteins that serves as a nucleation template. Here, we identify that within γTuRC, γ-tubulin undergoes mitosis-specific phosphorylation at the conserved residue Ser364. This phosphorylation is mediated by Cdk1/cyclin B and occurs exclusively in cytoplasmic γTuRC, but not in γTuRC associated with mitotic spindles. Functionally, Ser364 phosphorylation strongly suppresses the microtubule-nucleating activity of γTuRC. Although γTuRC activity is essential for spindle microtubule assembly, disrupting Ser364 phosphorylation by expressing a non-phosphorylatable γ-tubulin mutant leads to defective spindle formation and chromosome segregation. Ser364 phosphorylation establishes spatial control over microtubule nucleation by inactivating cytoplasmic γTuRC, while spindle-associated γTuRC remains unphosphorylated and functionally active, consistent with the recently identified inhibitory control of spindle-localized Cdk1/cyclin B. This γTuRC regulation acts together with other Cdk1/cyclin B actions to eliminate non-spindle microtubules and support spindle assembly. Our findings reveal that Ser364 phosphorylation provides precise microtubule control for mitotic progression.
The extended hypoxic microenvironment and ongoing infection in chronic wounds are two interrelated factors that significantly impede the healing process. The presented study develops a multifunctional bilayer hydrogel wound dressing that addresses these issues by combining oxygen production, oxygen transport, and antibacterial properties within a 3D printed bilayer hydrogel system. The bilayer hydrogel system is constructed with an upper layer of alginate loaded with oxygen-releasing CaO2 and a lower layer of antibacterial polymer integrated with perfluorocarbon-based oxygen-adsorbing and transporting nanoparticles (PMOF). In addition, the resultant scaffold demonstrates 3D printability and enhanced mechanical and degradation properties. Under hypoxic conditions, continuous oxygen release (5.5 mg/L O2 over 5 days) is achieved. Correspondingly, the bilayer system shows strong antibacterial activity against Gram Positive S. aureus and modest antibacterial activity against Gram negative E. coli bacteria. Furthermore, in vitro cell experiments show enhanced healthy cell viability after 7 days of incubation under hypoxic conditions. Lipid peroxidation results suggested that the bilayer system may provide antibacterial efficacy while maintaining oxidative cellular compatibility. Consistent with these findings, results from the in vitro scratch assay indicate that the Ge-PMOF/Alg-CaO2 scaffold promotes wound closure, achieving around 90% and 95% closure under normoxic and hypoxic conditions, respectively.
With the continuous growth of the Protein Data Bank archive, the Worldwide Protein Data Bank (wwPDB) partnership anticipates that the entire complement of possible four-character PDB accession codes (for example 1ABC) will be exhausted by 2028. wwPDB is, therefore, revising the PDB accession code (PDB ID) to 12 characters by extending its length and prepending `pdb_' (for example pdb_1000axyz) in lower case. This change will enable the robust detection of references to PDB entries in published literature. On or about July 21st 2027, the PDB will convert to releasing entries with extended PDB IDs only, which will not be compatible with the legacy PDB format. A beta version of the PDB Archive (PDB Beta Archive) is now available to help communities adapt to and embrace the extended PDB IDs and PDBx/mmCIF format during a transition phase. All files in the current PDB archive are reorganized in the Beta Archive with extended PDB IDs (including file naming and directories) on an entry-level basis, mirroring the data organization of the PDB Versioned Archive. wwPDB encourages scientific journals, PDB community members and users to transition to the PDBx/mmCIF format and adopt the new PDB ID format as early as possible. The PDB Beta Archive will replace the current public archive, and 12-character PDB IDs will be solely assigned to all newly deposited PDB entries.
Nanoparticle (NP) carriers are critical for enhancing the stability and cellular uptake of mRNA, enabling its therapeutic potential. However, conventional mRNA NP carriers enter cells via endocytosis, followed by endosome-lysosome trafficking, which leads to inefficient mRNA release into the cytoplasm. This insufficient endosomal escape has become a challenge in improving mRNA delivery efficiency for polymer and lipid NP carriers. In this study, we design a novel class of lipoic acid derivative (LA-Der) composite NPs with surface disulfide bonds that anchor onto cell membranes through disulfide exchange with cell surface thiols, bypassing endosomal degradation and directly releasing mRNA into the cytoplasm. We synthesized 25 LA-Ders with diverse chemical structures via amidation and incorporated 10 kDa polyethyleneimine (PEI) to form composite NPs for optimal mRNA loading. In vitro optimization identified three LA-Der/PEI formulations, LA-4A1, LA-5A1, and LA-6A2, as the most effective for mRNA delivery. Mechanistic studies revealed that thiol-mediated uptake is the predominant internalization pathway, facilitating the bypass of endosomal barriers. In vivo experiments confirmed high luciferase expression in the spleen and liver following intravenous injection of NPs carrying luciferase mRNA. These LA-Der/PEI composite NPs provide a promising strategy for overcoming intracellular endosomal escape barriers, advancing the clinical translation of mRNA therapeutics.
Bacterial wound infections are often complicated by biofilm formation, which leads to persistent inflammation and multidrug resistance, severely impeding the healing process. Conventional antibiotic therapies are challenged by both overuse and rising antimicrobial resistance. To address this, we designed and fabricated a dual-mode responsive delivery system (CSOAC@MN) based on chitosan (CS) and oleanolic acid (OA) composite microneedles for synergistic sono-photodynamic therapy (SPDT). This system employs chlorin e6 (Ce6), which exhibits both photodynamic and sonodynamic activities, as the core antibacterial component. It utilizes the self-assembly properties of OA to form nanoparticles for Ce6 encapsulation, and reinforces the microneedle mechanical strength through composite formation with CS, enabling efficient transdermal delivery. In vitro experiments demonstrated that under combined laser and ultrasound irradiation, the system exhibited strong antibacterial activity against both standard strains (Staphylococcus aureus and Escherichia coli) and a clinically relevant drug-resistant strain and effectively disrupted pre-formed biofilms. The antibacterial effect is attributed to the reactive oxygen species generated during synergistic SPDT. Cytocompatibility assays confirmed good biocompatibility with normal fibroblast cells at effective antibacterial concentrations. This study presents an integrated strategy combining transdermal microneedles with SPDT to combat biofilm-associated infections, showing promising potential for clinical translation.
Urethral stricture is a common urological condition in men that significantly affects quality of life. Autografts remain the gold standard for substitution urethroplasty; however, they present unavoidable limitations that tissue engineering seeks to overcome. One of the main challenges in long-segment urethral strictures is achieving an effective graft capable of providing adequate oxygen and nutrient delivery, thereby preventing graft necrosis and failure. Therefore, both angiogenesis induction and mechanical integrity of the grafts are critical for successful urethroplasty. This review provides an integrated framework encompassing three complementary strategies for promoting angiogenesis in urethral tissue engineering: (i) angiogenesis guided substrates, (ii) growth factor delivery systems, and (iii) cell-based therapies. Unlike previous reviews that focus on isolated aspects, our work provides a comprehensive and comparative discussion of these approaches within the specific context of urethroplasty. In addition, we highlight the translational challenges and pitfalls associated with each strategy, underscoring the need for standardized scaffold design, optimized cell culture protocols, and rigorous clinical validation. Despite ongoing progress, no optimal graft has yet been developed to ensure adequate vascularization for long segment urethral defects. Nevertheless, current findings suggest that angiogenesis induction holds promising potential for advancing urethral tissue engineering and improving clinical outcomes.
Biocompatibility of biomaterials is essential for their application in the biomedical field, particularly in tissue engineering and drug delivery systems. This study focuses on VECOLLAN, a recombinant, non-animal-derived collagen-like protein (CLP), which exhibits excellent biocompatibility while promoting in vitro cell proliferation and wound healing. We previously optimized electrospinning parameters and employed a coaxial crosslinking approach to produce VECOLLAN-based fibers with tunable dissolution, swelling behavior, and elasticity suitable for biomedical applications. The current study aims to assess the compatibility of these fibers with cells (NIH/3T3), investigate chemical leachables from three different formulations of DMTMM cross-linked VECOLLAN-fibers (according to ISO 10993-18), and conduct spectroscopic analysis to confirm crosslinking efficacy. Results indicate that most CLP-based nonwoven mats maintained cell viability above the 70% safety threshold as per ISO-10993-5. The sample with a CLP:DMTMM ratio of 1:0.1 demonstrated the most favorable cell compatibility and effective crosslinking. While the coaxial crosslinking method showed efficiency, residual crosslinker molecules and unexpected derivatives are identified. Spectroscopic investigations gave hints of successful crosslinking, although a direct correlation between crosslinker concentration and spectral band intensity is not established. Future research shall explore additional crosslinkers and cell types to further investigate the biocompatibility and potential applications of VECOLLAN-based nonwoven mats.
Photopolymerizable and degradable poly(ethylene glycol) (PEG) hydrogels are a promising platform to deliver chondrocytes for cartilage tissue engineering. Previous studies reported that cells are exposed to and react with free-radicals produced during encapsulation, creating a pericellular region of reduced crosslink density measured by the distance Rd. This study tests the hypothesis that increasing Rd improves spatial elaboration of deposited extracellular matrix (ECM) (i.e., micro-tissue) in a degrading hydrogel. Chondrocytes pre-treated or not with the antioxidant ascorbic acid atlow (1 nM) and high (1000 nM) concentrations prior to encapsulation resulted in increasingly larger Rd's: 3.3 (0 nM), 6.7 (1 nM), and 10 (1000 nM) µm. Encapsulated chondrocytes when cultured up to ten weeks, produced micro-tissues within the hydrogels. The median micro-tissue area increased by 29% (1 nM) and 570% (1000 nM) compared to 0 nM condition. This work shows that bolstering antioxidant defense mechanisms enhances chondrocyte inhibition of the polymerization immediately around the cell, resulting in larger regions of reduced crosslinking. This local variation in network structure, which degrades more quickly, led to improved neotissue assembly and larger micro-tissues, comprised primarily of sulfated glycosaminoglycans and collagen type II. This approach offers a novel way to improve ECM elaboration and interconnectivity in free-radical polymerized hydrogels for tissue engineering.
Human natural killer (NK) cells represent promising therapeutic agents for cancer and immune diseases. However, cryopreservation-induced stress leads to high post-thaw mortality and impaired cytotoxic function. While cryoprotectant (CPA) loading is one of the most critical processes for subsequent cell preservation, the high osmotic pressure and chemical toxicity significantly compromise cell viability. Here, we investigate osmotic stress responses in primary PBNK cells during CPA loading under varying osmotic conditions. We develop a mathematical model to simulate the osmotic processes and water transport dynamics across the cell membrane and enable quantitative assessment of non-osmotic protective components, i.e., Human Serum Albumin (HSA) and Dextran-40 (Dex-40). The experimental results demonstrate that our optimized protocol reduces ​osmotic and toxic stress damage by more than 30%. Simulation further indicates that HSA and Dex-40 act synergistically to modulate membrane transport dynamics, reducing free water loss and stabilizing cell volume against osmotic damage. This work establishes the predictive model of CPA loading damage in NK cells, which is potential to provide a new perspective and approach for improving immune cell cryopreservation.
Advanced biomaterials-based in vitro platforms are increasingly required to overcome the limited predictive power of conventional 2D cell cultures in colorectal cancer (CRC) drug screening. Herein, we report the development of a biomimetic, multicellular 3D CRC model based on gelatin methacrylate (GelMA) hydrogels, designed to recapitulate key structural and biological features of the tumor microenvironment. The platform integrates a vascularized hydrogel compartment with human endothelial cells, combined with cancer-associated fibroblasts and macrophages, enabling controlled tumor-stroma-vessel interactions within a physiologically relevant architecture. The GelMA hydrogels were comprehensively characterized, and their role in regulating endothelial viability, migration, and angiogenic marker expression was systematically evaluated. The drug screening capability of the platform was assessed using 5-fluorouracil (5-FU). Comparative analyses revealed that 3D cultures exhibited attenuated cytotoxicity, oxidative stress, and apoptotic responses relative to 2D monolayers, particularly at lower drug concentrations and prolonged exposure times. These findings demonstrate that GelMA-based microenvironment actively modulates cellular drug responses through multicellular interactions and diffusion-mediated effects, rather than acting as a passive scaffold. Overall, this study establishes a functional 3D in vitro platform that provides improved physiological relevance and predictive capability for preclinical CRC drug screening, while offering a human-relevant alternative aligned with the principles of the 3Rs.
Wound healing is a complex physiological process with different demands at different stages. In this work, a pH-responsive GA/Gelatin/ZnS (Glycyrrhizic Acid/Gelatin/Zinc Sulfide) hydrogel that can automatically release drugs was constructed in a facile way. It was fabricated by amide bonds, hydrogen bonds, and metal ion coordination among ZnS nanoparticles, glycyrrhizic acid (GA), and gelatin, which was confirmed by the molecular dynamics (MD) simulations and characterization results. Taking advantage of the pathological microenvironments at the wound site, it can synergize with multi-substance therapies at different stages, thus promoting wound healing. The as-prepared hydrogel can not only exhibit excellent coagulation ability in the hemostatic phase, but also release zinc ion (Zn2+) and hydrogen sulfide (H2S) due to the weak acid microenvironment at the wound site, thus boosting inflammatory stage to proliferative phase. Meanwhile, benefit from its good re-epithelialization and angiogenesis properties in proliferative phase, the as-prepared hydrogel can increase collagen deposition during the remodeling phase, thereby accelerating wound healing. It would open a new perspective for effective wound management.
Poly(2-oxazoline)s (POx) are an emerging class of synthetic polymers with potential in biomedical applications as most of them are characterized by biocompatibility, stealth properties, and structural tunability. Similarly, microgels gain attention for cell encapsulation, drug delivery, and as building blocks for physical hydrogels and tissue constructs. However, the predominant cross-linking methods for both POx and microgels rely on UV light and radicals, which can harm cells. This study aims to integrate the trends of POx and microgels and to overcome limitations of UV-based methods. It introduces a radiation-free cross-linking mechanism via thiol-Michael-addition for POx-based microgels, tailored for cell-friendly cell encapsulation. Therefore, a hybrid polymer system of thiolated POx, gelatin, and acrylated hyaluronic acid is chosen and its cross-linking kinetics is optimized for microfluidic procedures. Subsequently, hydrogels and microgels of different molar ratios of the functional groups are prepared. These differ in stiffness and degradation. Cell encapsulation tests with fibroblasts show cell viabilities >90% and that the gel systems support cell spreading and proliferation, irrespective of molar ratio. This confirms that the proposed cross-linking strategy is effective for creating POx-based microgels suitable for cell-friendly cell encapsulation.
As the primary barrier between the human body and the external environment, skin is susceptible to damage from multiple factors, making wound healing management a significant clinical challenge. Traditional wound care approaches mainly rely on passive dressings or mechanical closure devices, which lack the ability to dynamically monitor the wound microenvironment or regulate therapeutic interventions. In recent years, smart microneedle (MN) systems have emerged as a novel platform for advanced wound management because they provide minimally invasive access to the wound microenvironment while enabling localized drug delivery and biochemical sensing. However, the effective integration of sensing, therapy, and intelligent decision-making remains a critical challenge. In this review, we summarize recent advances in AI-assisted smart MN systems for advanced wound management from four interconnected perspectives: material innovation, MN structural engineering, fabrication technologies, and multifunctional integration. We further highlight emerging applications including therapeutic drug delivery, antibacterial intervention, wearable sensing platforms, and AI-driven closed-loop regulation. Finally, current challenges and future opportunities toward intelligent, personalized, and data-driven advanced wound management are highlighted.
Conventional therapies still provide only limited true tissue regeneration for periodontal diseases, particularly periodontitis, which are highly prevalent chronic inflammatory conditions associated with irreversible loss of tooth-supporting tissues and relevant systemic consequences. In this context, hydrogels based on natural polymers have been widely explored in periodontal tissue engineering as biomimetic matrices capable of modulating the inflammatory response, enabling localized delivery of bioactive agents, and offering temporary structural support. This review summarizes recent advances in hydrogels composed of alginate, collagen, chitosan, and other natural or semi-synthetic polymers applied to periodontal regeneration. The influence of polymer origin, crosslinking strategies, physicochemical and rheological properties, and processing approaches, including injectable formulations, self-healing systems, and bioinks for three-dimensional bioprinting, is discussed in relation to cell adhesion, angiogenesis, osteogenesis, and functional restoration of periodontal tissues. Hybrid platforms such as interpenetrating polymer networks, ceramic-reinforced composites, and systems designed for controlled delivery of drugs, growth factors, exosomes, and stem cells are also examined, with emphasis on immunomodulatory and stimuli-responsive designs tailored to the periodontal microenvironment. Despite robust preclinical evidence demonstrating coordinated regeneration of cementum, periodontal ligament, and alveolar bone, major challenges remain. These include the scarcity of well-controlled clinical trials, limitations in standardization, and regulatory barriers to translation.