Yuchao Gu

Enteroviruses causing hand, foot, and mouth disease, including currently circulating Coxsackievirus A10 (CVA10) and Coxsackievirus A6 (CVA6), rely on KREMEN1 (KRM1) for cell entry, making the virus–KRM1 interface a potential target for antiviral intervention. We screened GLMarNP, an in-house library of about 3000 natural products and derivatives. Candidates targeting the KRM1 ectodomain were selected by blind docking, drug-likeness assessment, and scaffold diversity analysis. To enable high-throughput antiviral screening, we inserted eGFP into a full-length infectious CVA10 cDNA clone to generate a reporter virus designated eGFP-A10. The virus was rescued in HEK293T cells and produced robust fluorescence in RD cells over serial passages. Among 29 candidate compounds, LM-10 showed the strongest antiviral activity and exhibited low cytotoxicity. Mechanistically, LM-10 bound KRM1 and acted early in the viral life cycle. Molecular docking suggested that LM-10 binds the KR domain at a site overlapping the VP2 EF-loop contact region. KRM1 also acts as a DKK1 receptor to regulate Wnt/β-catenin signaling. LM-10 did not significantly affect this pathway in the TOP-GFP reporter assay or in transgenic zebrafish embryos. These findings establish a practical CVA10 reporter-virus platform and support steric blockade of the receptor interface as a potential antiviral strategy.

ERα36, a membrane-associated splice variant of estrogen receptor α, mediates rapid non-genomic estrogen signaling and is frequently overexpressed in aggressive and therapy-resistant breast cancers. Although ERα36 represents a promising therapeutic target, its clinical exploitation remains limited due to the lack of high-affinity and highly specific targeting ligands capable of recognizing its extracellular domain. In this paper, we constructed a shark-derived variable new antigen receptor (VNAR) phage display immune library and isolated two high-affinity VNAR single-domain antibodies (FR6 and TH4) targeting distinct epitopes on ERα36. The TH4 VNAR was further engineered by fusion with a truncated Pseudomonas aeruginosa exotoxin (PE24) to generate a recombinant immunotoxin (TH4-PE24), characterized by (1) high specificity and affinity for ERα36 (K_D = 0.751 nmol/L), (2) potent and selective cytotoxicity against ERα36-positive breast cancer cells (IC₅₀ = 1.54 nmol/L), (3) a dual mechanism involving ERα36 degradation and suppression of PI3K/AKT and MAPK/ERK signaling pathways, and (4) significant in vivo antitumor efficacy in xenograft models without observable systemic toxicity. With this immunotoxin, we further investigated how ERα36 targeting could remodel tumor metabolism. ERα36-mediated metabolic regulation in breast cancer is a new research direction with many aspects unknown. We found that TH4-PE24 treatment led to extensive metabolic reprogramming, including disruption of nucleotide metabolism, ABC transporters, and central carbon metabolism. Overall, we report a novel shark VNAR-based immunotoxin platform that can achieve precise and potent antitumor effects by targeting the previously intractable membrane receptor ERα36. The modular design features make it translational for various other studies, such as targeting additional immune checkpoint molecules and developing next-generation precision therapeutics against membrane receptors that are inaccessible to conventional antibody modalities. The schematic diagram illustrates the screening of shark-derived variable new antigen receptors (VNARs) targeting estrogen receptor α, the identification and functional characterization of antigen-binding epitopes, and the construction of a VNAR-based immunotoxin, together with its in vitro and in vivo antitumor efficacy and safety evaluation. Estrogen receptor α36 (ERα36) is a membrane-associated splice variant of estrogen receptor α that primarily mediates non-genomic estrogen signaling. Owing to the α-helical structure of its C-terminal 27 amino acids, ERα36 has long been considered difficult to target with functionally active antibodies. Shark VNARs possess a unique and extended CDR3 region, enabling the recognition of complex and conformational epitopes. Taking advantage of this structural feature, we achieved functional targeting of ERα36 and identified two VNARs that recognize distinct linear and conformational epitopes, respectively. Among them, the conformational epitope-recognizing VNAR, TH4, was further employed to construct a novel immunotoxin. This immunotoxin not only suppresses ERα36 expression in breast cancer cells but also inhibits AKT and ERK signaling pathways, leading to reduced breast cancer cell proliferation and attenuation of tumorigenicity in ERα36-positive breast cancer.

Chitosan–astaxanthin nanoparticles (CS–ASTNPs) exhibited an anti-colitis activity, but their poor mobility restricts anti-inflammatory efficacy. Chlamydomonas reinhardtii (C. reinhardtii) was employed to load CS–ASTNPs to construct astaxanthin-loaded microalgal motors (CS–AST@CR). The particle size of CS–AST@CR was 4.2 ± 0.21 μm and its swimming speed was 89.3 ± 4.4 µm/s, which was slower than native C. reinhardtii (117.2 ± 2.7 µm/s), though their movement trajectories remained comparable. Compared to CS–ASTNPs, CS–AST@CR notably alleviated chronic colitis symptoms. It enhanced colon barrier function by promoting ZO-1, occludin, and MUC2 expression. Furthermore, CS–AST@CR markedly reduced TNF-α, IL-1β, and NLRP3 inflammasome production while increasing IL-10 levels. The oxidative response was effectively suppressed via activating the Nrf2/HO-1 pathway. Additionally, CS–AST@CR significantly increased Lactobacillus and Enterorhabdus abundance; whereas, CS–ASTNPs elevated Akkermansia and Bifidobacteriaceae richness. Collectively, CS–AST@CR integrated the advantages of microalgal motility and nanoparticle functionality, showing great potential for development as functional foods or therapeutic agents for colitis treatment.

Background: The rising global incidence of inflammatory bowel disease (IBD) creates an urgent need for safer, gut-targeted therapies. Current treatments, from small-molecule drugs to systemic anti-tumor necrosis factor-alpha (TNF-α) biologics, are frequently limited by off-target immunosuppression, heightened infection risk, and poor mucosal bioavailability. Engineered probiotic-based live biotherapeutics offer a compelling alternative by enabling localized drug production within the inflamed intestine. Methods: We engineered Escherichia coli Nissle 1917 (EcN) to secrete the anti-TNF-α nanobody MT1, creating the streamlined, single-strain platform EcN-MT1. Five signal peptides were screened, and plasmid-based and CRISPR-Cas9-mediated chromosomal integration strategies were compared. Structural modeling and molecular dynamics simulated MT1−murine TNF-α (mTNF-α) binding. Binding affinity and anti-inflammatory activity were assessed by ELISA and in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. Therapeutic efficacy was further evaluated in a dextran sulfate sodium (DSS)-induced murine colitis model by assessing body weight, disease activity index (DAI), colon length, histopathology, colonic pro-inflammatory cytokines, and 16S rRNA gut microbiota profiling. Results: Among the tested signal peptides, α-hemolysin (HlyA) achieved highest secretion (4.6 mg/L), and the plasmid-based strain markedly outperformed genomic integrants without impairing growth. Simulations confirmed stable complementarity-determining regions (CDR)-mediated binding, consistent with the high affinity (EC ₅₀ 27.9 nM) and potent suppression of LPS-induced mRNA expression of Tnf and interleukin-1β ( Il1b ) in macrophages. In the DSS-induced murine colitis model, oral administration of EcN-MT1 significantly attenuated weight loss, improved DAI scores, and preserved colon length. Histopathological analysis revealed reduced mucosal ulceration, crypt loss, and immune cell infiltration, accompanied by downregulated colonic Tnf and Il1b mRNA. Notably, EcN-MT1 treatment restored gut microbial diversity, corrected dysbiosis, and enriched beneficial taxa linked to butyrate production, barrier enhancement, and anti-inflammatory effects. Conclusion: This study establishes EcN-MT1 as a potent, orally deliverable live biotherapeutic that achieves localized TNF-α neutralization while concurrently promoting microbial and mucosal homeostasis, offering a novel and translatable strategy for IBD treatment.

The escalating global crisis of antimicrobial resistance urgently demands innovative antimicrobial agents beyond conventional antibiotics. Metal‐based nanomaterials, including metal/metal oxide nanoparticles (NPs) and metal‐organic frameworks (MOFs), represent a promising class of broad‐spectrum antibacterial agents. However, their practical application is often hindered by intrinsic limitations such as aggregation, instability, and cytotoxicity. Integrating them with structurally and chemically tunable silica nanoparticles has been demonstrated as a promising strategy to mitigate the limitations and engineer advanced nanohybrids with synergistic functionalities. This review highlights how tailored interfacial chemistry achieves precise architectural control over silica&metal‐based nanohybrids. The controlled nanoarchitecture is essential to fully exploit the structural and functional contributions of silica components to overcome the physiochemical limitations of meta‐based material. We then examine the biological performance of these heterostructures mainly in antibacterial fields, including membrane disruption, stimuli‐responsive activation, biofilm penetration/eradication, and receptor‐mediated active targeting to pathogenic bacteria. Finally, challenges and future research directions are outlined based on our own perspectives, providing a design framework for next‐generation antimicrobial nanotherapeutics.