Hypoxia and cold temperatures are major limiting factors for animals reared at high altitudes. Previous adaptation studies have primarily focused on genetic and genomic aspects, while the mechanisms by which the gut microbiome contributes to this adaptation are still not fully understood. We used ruminants as both naturally adapted (yaks) and non-adapted (Holstein cows) models to investigate the role of gut microbiome in high-altitude adaptation by applying multi-omics approaches. First, 20 yaks and 20 Holstein cows that had been reared at approximately 4000 m altitude since birth were fed the same diet for 44 days prior to sampling to eliminate the short-term effects of nutrition and altitude adaptation. The yak rumen microbiome showed significant enrichment in carbon metabolism, particularly central carbon metabolism pathways, such as glycolysis/gluconeogenesis, pyruvate metabolism, and the pentose phosphate pathway, whereas that of Holstein cows was enriched in starch, sucrose, pentose, and glucuronate interconversions. Compared with those of Holstein cows kept at high altitudes for their entire life, the yak rumen epithelial cells, as determined by single-nucleus RNA sequencing, exhibited higher elevated scores for ketone body biosynthesis and fatty acid beta-oxidation. Second, mixed rumen fluid was transplanted from 10 yaks to 10 Holstein cows. Holstein cows then showed better milk production performance. A progressive decline in carbon metabolism activity from 6 h to 7 and 28 days post-transplantation was verified. In conclusion, the rumen microbiome and host epithelial function appear to support high-altitude adaptation by improving the energy supply of the host.

Data storage using large DNA fragments enables low-cost in vivo replication and offers a promising strategy for distributed data applications. However, data readout from massive, unordered sequencing reads requires alignment based on overlapping regions and is complicated by diverse sequencing errors, especially insertion and deletion (indel) errors. Here, we propose a fast and reliable readout framework in a bootstrap manner tailored for data storage using watermarked large DNA fragments. Our scheme transforms the de novo readout into a resequencing-like workflow through multiple-fold hidden references, substantially reducing readout complexity. The framework is compatible with sequencing platforms exhibiting diverse error profiles. For technologies with low indel rates, we employ correlation-based identification and bit-wise consensus to enable rapid decoding. For indel-prone platforms, we incorporate progressive read alignment using multiple-fold hidden references and the forward-backward algorithm to ensure robust recovery. In vivo experiments on large DNA fragments with different coding efficiencies validated the proposed framework. Error-free recovery was achieved using Illumina reads (raw error rate of ~0.2%) at a coverage of 0.6–2.5× and using nanopore reads (raw error rate ~5%) at a coverage of 1.6× or 4.3×. These results demonstrate the practicality and scalability of large-fragment DNA storage for real-world applications.

The early discovery of covalent drugs is frequently inspired by, or derived from, natural sources, with such compounds often showing favorable safety profiles and a comparatively lower risk of clinical failure. However, a straightforward, high-throughput technique for screening covalent-binding molecules directly from complex medicinal plant extracts remains unavailable. In this study, we introduce an integrated strategy that combines protein microarrays with bioorthogonal click chemistry (Ccc-Chip). This platform includes a differential scanning fluorimetry (DSF)-based pre-screening step to enhance efficiency, with the Ccc-Chip serving as the core confirmation tool. It provides simple and intuitive readouts, enabling synchronous, high-throughput screening of covalent ligands targeting multiple proteins through detection of their competitive binding with cysteine-reactive probes. To validate the approach, we constructed a mutant isocitrate dehydrogenase 1 (mIDH1) protein microarray and used the integrated workflow to screen 110 medicinal plants. Our results led to the identification of flavokawain C (Flc), a covalent inhibitor of mIDH1, from Piper methysticum Forst. Subsequent in vivo experiments showed that Flc significantly reduced 2-hydroxyglutarate (2-HG) levels in an mIDH1-driven orthotopic tumor model and enhanced CD8⁺ T cell activity. Notably, when combined with a programmed cell death protein 1 (PD-1) blocking antibody, Flc synergistically augmented antitumor immunity, resulting in suppressed tumor growth. This work not only supports the high-throughput utility of the Ccc-Chip strategy but also provides a practical framework for combining bioorthogonal labeling with protein microarray technology, facilitating the discovery of bioactive covalent molecules from plant sources for challenging therapeutic targets.

Curcuminoids, including curcumin (CUR) and demethoxycurcumin (DMC), are known for their antiviral properties, but their underlying antiviral targets remain unclear, and the relationship between curcuminoids and the type I interferon (IFN-I) signaling pathway has not been fully elucidated. Here, we explored the regulatory effects of DMC and CUR on the IFN-I pathway in an EV-D68-infected murine model and employed multiomics analysis to identify key drug targets and their interaction networks. FTIR analysis indicated that DMC has better physicochemical stability than CUR, exhibiting greater stability under changes in light, temperature, and pH. In both in vitro and neonatal mouse models, DMC and CUR effectively inhibited EV-D68 replication by suppressing viral 2A gene expression and the release of proinflammatory cytokines. Both compounds upregulated the molecular chaperone CRYAB (αB-crystallin), which translocates to the nucleus and acts as a central regulator of host metabolism and antiviral immunity during EV-D68 infection. Further multiomics analyses revealed that CRYAB overexpression inhibited purine metabolism and upregulated interferon-stimulated genes. Proteomic profiling identified RBM26 as a key CRYAB-interacting target. CRYAB stabilizes RBM26 by inhibiting virus-induced ubiquitination, which leads to enhanced IFN-I responses. DMC and CUR activated the mtDNA-cGAS-STING pathway via RBM26, stimulating downstream signaling and antiviral effects. RBM26 reconstitution altered the splicing of cytidine/uridine monophosphate kinase 2 (CMPK2), resulting in increased nucleotide turnover and reduced cytidine levels, impairing viral replication. DMC/CUR treatment or CRYAB overexpression similarly reduced intracellular cytidine and uridine levels, increasing antiviral activity. Additionally, DMC/CUR restored mtDNA levels suppressed by EV-D68 infection in an RBM26-dependent manner, stimulating cGAS-mediated cGAMP production and activating the STING-TBK1-IRF3 axis. These findings not only clarify the molecular mechanisms underlying the antiviral effects of curcuminoids but also highlight their therapeutic potential as host-directed antiviral agents.

Clostridium difficile infection (CDI) is a major complication of inflammatory bowel disease (IBD), but the complicated treatment strategy of antimicrobial drugs and immunosuppressive drugs cannot effectively control the syndrome of IBD and CDI. Glycolysis in intestinal epithelial cells (IECs) leads to excessive production of lactic acid, which is considered to be a key factor in the destruction of the intestinal barrier by toxin B of Clostridium difficile (TcdB). In this study, we revealed that uncoupling protein 2 (UCP2) was highly expressed in IECs, and disrupted mitochondrial function to reprogram metabolism toward glycolysis, thereby weakening the resistance of IECs to TcdB. We developed an oral proton-reprogrammed nanomedicine (OPR) to convert the metabolism of IECs from glycolysis to oxidative phosphorylation, which can effectively alleviate IBD and CDI syndromes. OPR blocks the rapid delivery of protons to the mitochondrial matrix induced by UCP2, which restores mitochondrial membrane potential (MMP) and drives protons to reenter the matrix through ATP synthase, mediating the recovery of mitochondrial function. In mouse IBD and IBD complicated CDI (IBD-CDI) models, OPR selectively targeted inflammatory colonic lesions, alleviated mitochondria-dependent pyroptosis of IECs, inhibited macrophage STING pathway activation, and reduced lactic acid levels. Especially in the treatment of IBD-CDI syndrome, the therapeutic effect of OPR is far better than the combination of antibiotics and immunosuppressive drugs. In summary, our study establishes UCP2-mediated mitochondrial disability as a key mechanism for the functioning of TcdB and suggests that IEC metabolic reprogramming could be a therapeutic target.

Ionizing radiation-induced intestinal injury (IRIII) reduces survival in nuclear accident victims and compromises the efficacy of abdominal radiotherapy, and current treatment options remain limited. Human defensin 5 (HD5)-derived fragments are endogenous regulators of the gut microbiota, which affects host responses to radiation. However, whether these fragments influence intestinal radiosensitivity or can serve as lead compounds for IRIII therapeutics remains unclear. In this study, we investigated the role of HD5-derived fragments in IRIII and developed AT9(C/G), a potent radioprotective oligopeptide based on the lead fragment AT9. Fecal metagenomic and metabolomic analyses revealed that the oral administration of AT9(C/G) enriches Bifidobacterium pseudolongum and increases lithocholic acid (LCA) levels in the intestine. Both murine and clinical studies demonstrated a negative correlation between IRIII severity and fecal LCA levels. The radioprotective effect of LCA was further validated in both mouse models and human small intestinal organoids. Mechanistically, LCA suppresses ferroptosis in irradiated cells by remodeling lipid metabolism. Specifically, LCA activates Takeda G protein-coupled receptor 5 (TGR5), leading to the upregulation of sterol regulatory element-binding protein 1 (SREBP1), which transcriptionally modulates stearoyl-CoA desaturase 1 (SCD1) to catalyze monounsaturated fatty acid production. Pharmacological inhibition of SCD1 or genetic ablation of G-protein coupled bile acid receptor 1 (Gpbar1, encodes TGR5) attenuates the protective effects of AT9(C/G) in mice. This study establishes that an oligopeptide can modulate gut microbiota-derived LCA to confer intestinal radioprotection, presenting a promising preventive strategy against IRIII.

With an increasing global cancer burden, the regulatory function of the human microbiome and its metabolites in tumor epigenetics has garnered significant interest. Microbial metabolites are not merely passive byproducts but serve as signaling molecules and epigenetic modulators, contributing to tumor progression through multiple overlapping pathways. Short-chain fatty acids (SCFAs) such as butyrate directly inhibit histone deacetylases to reactivate tumor suppressor genes, while secondary bile acids (BAs) induce gene silencing via DNA methylation remodeling by altering the FXR/TGR5 signaling pathway. Folate and vitamin B12 serve as substrates for DNA and histone methylation through one-carbon metabolism. A complex bidirectional feedback loop exists between microbial metabolism and tumor epigenetics: reprogramming driven by hypoxia or oncogenes alters metabolite flux, generating molecules such as lactate and succinate that not only remodel chromatin and the tumor microenvironment (TME) but also selectively promote the growth of metabolically adapted microbial species, thereby reinforcing epigenetic dysregulation. Despite growing mechanistic insights, establishing causality and correlating spatiotemporal dynamics and dose responses within the highly heterogeneous TME remain major challenges. Data integration across multi-omics remains limited by methodological and computational constraints. Resolving these issues will be critical for understanding the microbe–metabolite–epigenetic axis and advancing personalized precision oncology.