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.
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.
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.
Understanding macrophage phenotype regulation by mechanical stimuli is a promising way to elucidate the body's inflammatory response and design new therapies. However, creating dynamic interfaces that allow precise, real-time, and reversible control over mechanical cues remains a challenge. In this study, we report the immunomodulatory effects of dynamic liquid crystal (LC) polymer films on in vitro macrophage responses. By utilizing reversible light-induced LC surface topographies, we generate dynamic mechanical stimuli on cells during topography formation and removal, enabling on-demand and reversible reprogramming of cell behavior. Our findings reveal a strong topographical shape-dependent cell response by examining the effects of flat, pillared, and grooved LC films on THP-1-derived macrophages. A strong increase in both pro- and anti-inflammatory markers is observed on grooves, while pillars maintain the anti-inflammatory profile without broad activation. Macrophages on LC film-generated topographies furthermore present distinct cytokine expression profiles. Notably, light-induced grooves triggered a stronger pro-remodeling cellular response, while pillars appeared to exert an inhibitory effect on macrophage activation. The dynamic topographies remarkably induced distinct changes in the macrophage membrane morphology, triggering migration-associated blebbing of the cell membrane in all cases except for grooves that promoted an increased degree of lamellipodia and filopodia formation. Overall, these results demonstrate that light-responsive LC surfaces provide a controllable platform for topography-dependent and adaptive immune modulation, opening opportunities for rational design of immunoregulatory scaffolds that exploit macrophage plasticity for regenerative medicine.
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.
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.
Extracellular vesicles (EVs) have emerged as promising therapeutic agents for tissue repair due to their intrinsic abilities in intercellular communication, immunomodulation, and regeneration promotion. However, the clinical translation of EV-based therapies for wound healing remains limited by challenges such as rapid clearance, poor retention at the wound site, and uncontrolled bioactivity. Recently, DNA hydrogels have attracted increasing attention as programmable biomaterials with excellent biocompatibility, structural tunability, and stimuli-responsive properties. Integrating EVs with DNA hydrogels offers a novel strategy to construct intelligent therapeutic platforms that enhance EV stability, enable sustained release, and provide spatiotemporal control of therapeutic signaling. In this review, we summarize recent advances in extracellular vesicle-functionalized DNA hydrogels as next-generation platforms for wound healing and management. We discuss the design principles of DNA hydrogels, strategies for EV incorporation and functionalization, and the synergistic mechanisms by which these hybrid systems regulate inflammation, promote angiogenesis, and accelerate tissue regeneration. In addition, emerging intelligent features such as stimuli-responsive release, biosensing capability, and programmable therapeutic delivery are highlighted. Finally, we address current challenges, including large-scale manufacturing, standardization, and translational barriers, and outline future perspectives for the clinical application of EV-functionalized DNA hydrogel systems in regenerative medicine.
The engineering cartilage scaffolds with biomimetic structural features hold critical importance for effective repair and regeneration of damaged cartilage tissue. Recent advancements in 3D printing technology have facilitated the fabrication of multi-layered, gradient scaffolds with precisely controlled macrostructural geometries. However, current 3D-printed scaffolds still fall short of replicating the full spectrum of biomechanical and functional properties inherent to native cartilage. In this study, a 5Gt-7Alg-3HA composite scaffold featuring vertical-oriented microstructures was fabricated by integrating template-freezing orientation and 3D printing technologies. The influence of vertical-oriented microstructures on the scaffold's multifaceted properties was evaluated through comparative analysis with non-oriented scaffolds in the control group. The results revealed that, compared to non-oriented scaffolds, the vertical-oriented scaffolds exhibited a more uniform and interconnected 3D network structure, higher porosity, more suitable swelling and degradation rates, as well as superior biocompatibility. Notably, significant improvements were observed in the mechanical properties of the vertical-oriented scaffolds. In conclusion, these findings may offer a strategic approach for developing next-generation cartilage scaffolds with biomimetic properties.
This study addresses the challenge of antimicrobial resistance in Gram-negative bacteria by developing a novel class of biodegradable polymeric antibiotic adjuvants. We have engineered cationic polyesters functionalized with guanidinium and phenylboronic acid (PBA) groups through the one-pot thiol-Michael addition post-modification to create a dual-targeting mechanism against the bacterial outer membrane (OM). This design combines the membrane-disruptive action of guanidinium with the specific binding capability of PBA to lipopolysaccharides. The relationships between structure of the polymers and their antibacterial ability or cytocompatibility were explored. The optimized polymer, G95-BA5, exhibited potent intrinsic bactericidal activity and good biocompatibility. It also demonstrated remarkable in vitro synergy with rifampicin against multidrug-resistant pathogens, achieving fractional inhibitory concentration indices as low as 0.06 by significantly enhancing the outer membrane permeability. In a lethal murine peritonitis model, the combination of G95-BA5 and rifampicin provided 100% survival, drastically reduced bacterial burden in vital organs, and controlled systemic inflammation. This work establishes an effective polymer platform for overcoming the OM permeability-based resistance and revitalizing existing antibiotics.
Silk fibroin nanoparticles (SFNPs) have shown great promise as oral drug delivery carriers due to their favorable biocompatibility and tunable release properties. However, their effects on the gut microbiota under physiological conditions remain poorly understood. Herein, we systematically investigated the impact of orally administered SFNPs on the composition and temporal dynamics of the gut microbiome in healthy mice. SFNPs were thoroughly characterized, showing stability in intestinal fluid and structural transition to β-sheet conformation. Our results revealed that SFNP administration induced significant, dose-dependent shifts in microbial communities, notably increasing the Firmicutes/Bacteroidota ratio and enriching potentially beneficial genera such as Faecalibaculum and Dubosiella, while reducing taxa associated with inflammation and metabolic disorders. Medium doses (2.4 and 12 mg/kg) promoted sustained potentially beneficial effects, whereas a high dose (60 mg/kg) led to transient dysbiosis and enrichment of the inflammation-related genus Enterorhabdus. These findings underscore the dose-responsive modulatory effects of SFNPs on gut microbiota and highlight the importance of microecological safety in the design of nanocarrier systems for oral administration. This study provides critical insights into the gut-nanoparticle interface and supports the potential of SFNPs in microbiome-based therapeutics.
To address the global threat of increasing antimicrobial resistance, the investigation and development of rapid identification and detection methods for bacteria and infection sensing approaches have become major areas of research. Expanding on earlier work on enzyme-sensitive biopolymer-based autonomous sensing materials, the impact of different morphologies and concomitantly surface-to-volume ratio on the sensitivity of the detection of the enzyme β-glucuronidase (β-Gus) from pathogenic and non-pathogenic E. coli was unraveled. The enzymatic hydrolysis of microbeads and freeze-dried scaffolds was compared to that of neat films equipped by post-fabrication modification with 4-methylumbelliferyl-β-D-glucuronide hydrate (MUG) as a β-Gus reporting functionality. Fluorescence spectroscopy and in vitro tests with planktonic MACH-1 and NCTC strains revealed that the scaffolds outperformed the films with a close to ten-fold increased sensitivity, while the beads showed an enhanced rate of only a factor of 2.4. For an observation time of 60 min, the scaffold, beads, and film exhibited a limit of detection (LOD) of 3.3, 13.4, and 17.5 nm, respectively. These results support the hypothesis that increased surface-to-volume ratios enhance the sensitivity, but also point to the role of surface porosity and accessibility of enzyme-labile sites in swollen materials, which may be compromised due to the fabrication process.
Intestinal fistula refers to an abnormal anatomical channel between the intestines or between the intestine and another organ, commonly resulting from factors such as intestinal incision leakage, anastomotic leak, inflammatory bowel disease, and radiation enteropathy. Following its occurrence, various complications may arise, including abdominal infection, sepsis, intra-abdominal hypertension, massive abdominal hemorrhage, necrotizing fasciitis of the abdominal wall, chronic critical illness, and multiple organ dysfunction syndrome. In most cases, surgical resection of the intestinal fistula combined with thorough irrigation and drainage is required, though healing remains challenging. Hydrogels, as cross-linked polymer materials, are widely used in pharmaceuticals, biomedical implants, tissue engineering, regenerative medicine, and bioadhesive barriers. Based on these characteristics and functions, hydrogel materials also play a significant role in the treatment of intestinal fistulas. This review will systematically outline the design strategies, application mechanisms, and research progress of hydrogel materials for treating intestinal fistulas, and discuss current challenges as well as future development trends.
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.
Carbon dots (CDs), as a burgeoning class of zero-dimensional carbon-based nanomaterials, have garnered significant attention in biomedicine due to their various advantages. While extensive studies have focused on their fluorescence and photothermal characteristics, their potential as advanced photosensitizers for photodynamic therapy (PDT) is increasingly recognized. This review provides a systematic and focused summary of recent advances in CD-based tumor PDT. Design strategies for CDs with photodynamic properties are comprehensively categorized, with the donor-acceptor strategy highlighted as the first-ever summary of this rational approach. By improving tumor targeting, alleviating hypoxia, and depleting overexpressed glutathione, the unfavorable tumor microenvironment for CD-based PDT can be effectively circumvented. To overcome the limitations of monotherapies, CD-based PDT is often integrated with photothermal therapy, chemotherapy, and immunotherapy, particularly in combination with immunogenic cell death and immune checkpoint blockade therapy. Such synergistic strategies enable effective eradication of primary tumors while simultaneously establishing long-term immune surveillance against circulating tumor cells. Furthermore, by integrating imaging guidance with therapeutic function, imaging-guided CD-based PDT offers a theranostic platform that paves the way for precise tumor therapy. Collectively, this review offers a comprehensive roadmap for the rational design and translational development of CD-based PDT.
Levan is a microbial fructan type exopolysaccharide that has attracted increasing attention due to its biocompatibility, biodegradability, low toxicity, and versatile functional properties. However, the direct application of native levan is often limited due to poor mechanical strength, moisture sensitivity, and instability under processing or storage conditions. To overcome these limitations, levan has been integrated with natural polymers, synthetic polymers, nanoclays, and metal or metal oxide nanoparticles to develop advanced hybrid composites with enhanced physicochemical and biological performance. These levan based composites exhibit improved tensile strength, thermal stability, barrier properties, controlled swelling, and tunable surface characteristics, making them promising materials for biomedical and healthcare applications. Recent studies have highlighted their potential in drug delivery, wound healing, antimicrobial and antibiofilm coatings, tissue engineering scaffolds, biosensors, and anticancer systems. This review critically discusses levan structure, biosynthesis, production strategies, and the key factors governing successful composite formation, including interfacial compatibility, filler dispersion, and molecular architecture. In addition, major challenges such as high production cost, batch-to-batch variability, scalability, long-term stability, and regulatory concerns are addressed. Overall, levan inspired hybrid composites represent a promising platform for next-generation sustainable biomaterials, although further process optimization and translational studies are required for commercial realization.
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.
The activity of many antimicrobial peptides (AMPs), a promising alternative to classical antibiotics, depends strongly on their aggregation-propensity and weak peptide association favor antimicrobial function, whereas stable assembly can reduce activity. Thus, modulation of intermolecular interactions offers a feasible way to enhance the potency of existing AMPs. However, a general strategy to achieve this objective has not been established. Here, we demonstrate that the spectrum of activity of Gram-positive-specific self-assembling AMP Fmoc-phenylalanine can be broadened by co-assembly with non-antibiotic Fmoc-glutamic acid. Biophysical assays confirmed that the co-assembled system disrupted bacterial membrane integrity, leading to cell death. The enhanced potency correlated with the reduced mechanical rigidity of the co-assembled hydrogel, as determined by rheological measurements, and molecular dynamics simulations further revealed that heterogeneous non-covalent interactions were detrimental to the fibril stability. These findings suggest that rationally designed co-assembling partners that weaken stabilizing non-covalent interactions can serve as a common strategy to enhance the antibacterial efficacy of existing AMPs.