Tumor-derived exosomes are promising circulating biomarkers for early colorectal cancer (CRC) diagnosis, yet their low abundance and the complexity of biological fluids demand sensing strategies with both stringent specificity and amplified signal output. Here, we report an ultrasensitive electrochemical aptasensor for SW620 cell-derived exosomes (SW620-Exo) that integrates aptamer proximity ligation, a three-dimensional DNAzyme walker, an integrated sequential activation DNA (ISD) molecular circuit, and highly catalytic BiFeO3/g-C3N4 heterojunction nanosheets. Dual aptamer recognition of distinct epitopes on SW620-Exo brings split DNAzyme segments into proximity, displacing an inhibitory blocker and assembling an active Mg2+-dependent DNAzyme walker. The walker cyclically cleaves hairpin substrates immobilized on magnetic nanobeads to release abundant single-stranded primers to initiate the ISD circuit, in which the second hybridization chain reaction (HCR) module is preconfined within the first to prevent premature activation and reduce background leakage. The resulting methylene blue (MB)-tagged DNA assemblies are captured on the electrode through sequence hybridization, where BiFeO3/g-C3N4 nanosheets efficiently catalyze MB oxidation to generate amplified electrochemical currents. This multilevel design combines cooperative exosome recognition, low-leakage DNA circuitry, and catalytic signal enhancement, affording a linear response for SW620-Exo from 1 × 102 to 1 × 108 particles/mL and a detection limit of 119 particles/mL. The sensor also enables reliable SW620-Exo detection in diluted human sera, indicating its potential as a programmable bioanalytical platform for trace exosome analysis and proof-of-concept for liquid biopsy applications.

Post-myocardial infarction (MI) remodeling is accompanied by metabolic stress, but the myocardial genes associated with copper handling are poorly defined. We sought to prioritize a tissue-derived candidate rather than establish a copper-dependent mechanism. Regional MI transcriptomes and an in-house left anterior descending coronary artery ligation mouse RNA-sequencing cohort were integrated with protein quantitative trait locus-based Mendelian randomization (MR). Follow-up comprised local and external tissue validation, cardiac single-cell RNA sequencing, Genotype-Tissue Expression co-expression, computational perturbation, and CRIP2 knockdown or overexpression in H9c2 cells exposed to hypoxia/reoxygenation (H/R). CRIP2 showed a nominal protective-direction MR association (odds ratio 0.831, 95% confidence interval 0.735-0.939; p = 0.0031), but did not pass the Bonferroni threshold. Crip2 was lower in the local MI model (p = 0.0168; n = 5 per group) and in an independent dataset. A prespecified lipoylated-tricarboxylic-acid module was negatively enriched after MI (normalized enrichment score -1.63; false discovery rate 0.012), whereas the broader copper-homeostasis set was not significant. Single-cell data localized Crip2 mainly to cardiomyocytes, but were not adequately replicated for condition-level inference. Under H/R, Atp7a was the only copper-handling transcript whose knockdown-by-oxygen interaction remained significant after adjustment (q = 0.0405). CRIP2 overexpression was associated with higher Cell Counting Kit-8 metabolic activity during H/R (interaction p = 0.00551), whereas the knockdown interaction was not significant. Copper abundance, mitochondrial function, and cuproptosis markers were not measured. The data prioritize CRIP2 for mechanistic study, but do not show that it regulates copper flux or post-MI remodeling.

Wash-free immunoassays with enzyme-linked immunosorbent assay (ELISA)-level performance remain challenging because peroxidase-based signal generation typically requires multiple washing steps and the handling of unstable H2O2. Here, we report a bead-based and glucose-fueled ELISA-like platform that co-localizes target capture and catalytic amplification on biogenic magnetic nanoparticles (MNPs) with peroxidase-like activity from magnetotactic bacteria. To generate biogenic antibody-displaying magnetic nanoparticle (bioMagbody), we used the SpyCatcher/SpyTag (SC/ST) system as a covalent protein coupling module. SC was genetically displayed via fusion to the abundant membrane protein Mms13 on the biogenic MNPs, enabling covalent ligation of ST-fused single-chain variable fragments (scFvs) to generate bioMagbody. SC expression and scFv display preserved the intrinsic peroxidase-like activity and target binding. Using glucose oxidase (GOx)-scFv complex as the H2O2 donor and bioMagbody as the acceptor, C-reactive protein (CRP) was quantified in buffer with a limit of detection of 4.5 nM, and specific detection was demonstrated in human serum by simple mixing without washing steps. The wash-free operation arises from target-localized H2O2 generation by GOx-scFv and proximity-enabled transfer to bioMagbody, which drives luminol chemiluminescence upon glucose addition as the reaction initiator. The platform was readily reconfigured to detect inactivated SARS-CoV-2 by exchanging scFvs. This work establishes a genetically programmable biogenic nanozyme interface for wash-free immunoassays.

Syndecan-2 (SDC-2) is a key regulator of colon cancer progression, but its cooperation with integrins in cell motility is not fully understood. Here, we show that SDC-2 overexpression in HT29 colon cancer cells (HT29-S2W) enhances adhesion, spreading, and migration on laminin-332, an extracellular matrix component linked to poor prognosis. These effects correlate with increased basal membrane localization of integrin α6, as well as elevated expression and deposition of laminin-332 subunits (α3, β3, and γ2), thereby promoting laminin-332 assembly within the extracellular matrix. Proximity ligation assays revealed enhanced interactions between SDC-2 and integrin α6β1 following SDC-2 overexpression, indicating coordinated signaling mediated by laminin-332. Notably, HT29-S2W cells adhered more efficiently to full-length laminin-332 containing the SDC-2-binding LG4/5 modules of the laminin α3 chain than to truncated laminin-332 lacking these modules. Furthermore, both an anti-integrin α6 antibody and a laminin-α3-derived peptide (A3G75aR) significantly inhibited adhesion to full-length laminin-332. In metastatic DLD-1 cells, which express high levels of SDC-2 and laminin-332, combined treatment with the antibody and peptide markedly suppressed cell migration and invasion. Collectively, these findings identify an SDC-2-laminin-332-integrin α6β1 axis that drives invasive motility and may represent a promising therapeutic target in metastatic colorectal cancer.

Against the backdrop of green biomanufacturing, engineering methanol-utilizing Komagataella phaffii (K. phaffii) represents an effective strategy to expand the one carbon (C1) product profile and speed up the industrialization of C1-based bioeconomy. To address the technical challenges of low efficiency and cumbersome experimental procedures for multiplex gene editing and precise large-fragment integration during the reconstruction of complex metabolic pathways in K. phaffii, this study established a CRISPR toolkit - Efficient Multi-Gene Editing System 3.0 (EMGES 3.0) - which enabled one-step large-fragment integration coupled with multiplex gene knockout. EMGES 3.0 was constructed through the synergistic optimization of a repair-engineered chassis and an episomal CRISPR vector. For chassis engineering, five DNA repair modules: Δlig4 (DNA Ligase IV, non-homologous end joining end ligation), ppMRE11(The endogenous MRE11 gene from Pichia pastoris) overexpression (The Meiotic Recombination 11, DNA double-strand break end resection), Δrad9 (Radiation-Sensitive 9, DNA damage checkpoint regulation), Δmph1 (Mutator Phenotype Helicase 1, improvement of homologous recombinant strand extension), and PapRecT-PaSSB co-expression (stabilization of recombination intermediates) were integrated to generate the highly recombinogenic strain Y09. For vector engineering, cenARS was replaced by panARS and the endogenous promoter PGAP was employed to drive the double hammerhead ribozyme-single guide RNA-hepatitis delta virus ribozyme (double HH-sgRNA-HDV: dHgH)-mediated sgRNA expression, yielding the optimized vector Nov_pGAP_panARS_pLAT1_Cas9. These two features on K. phaffii together enhanced the EMGES 3.0 to a higher standard of transformation rate and editing efficiency. According to our results, EMGES 3.0 achieved dual-functional gene knockout efficiencies between 76.6% and 100%. For insertion of medium-long fragments (>4.5 kb), the efficiency achieved 93.3%. In addition, the one-step integration of ultra-long fragments (>16 kb) achieved 14.8%, which was reported for the first time. Furthermore, the efficiency of simultaneous long-fragment integration at three neutral loci reached 38.4% (>15 kb). We applied the system for one-step production of free fatty acids (FFAs, yield: 5.82 ∼ 7.30 mg/L/OD600) and resveratrol (yield: 1.14 ∼ 1.28 mg/L) using methanol as the sole carbon source. EMGES 3.0 provides a robust technical foundation for complex compounds biosynthesis and high-yield industrial strains, while also advancing K. phaffii as an industrial synthetic biology chassis for efficient C1 utilization.

The gut microbiota is increasingly recognized as a key regulator of cardiovascular health; however, its functional dynamics following myocardial infarction (MI) remain poorly defined. While traditional sequencing approaches focus on microbial composition, they cannot capture real-time functional activity. In this study, we established a rat model of MI via permanent ligation of the left anterior descending artery and collected fecal samples from MI and sham-operated cohorts at baseline and Days 2, 7, and 14 post-surgery. High-resolution metaproteomics was applied to quantify microbial and host proteins, integrating functional annotation, differential expression, and weighted gene co-expression network analysis (WGCNA). We observed an acute, generalized decline in microbial diversity at Day 2 across both groups, indicative of a physiological response to surgical stress. Crucially, MI-specific divergence emerged during the subacute phase (Day 7) and persisted into recovery (Day 14). At Day 7, functional perturbations peaked in the MI group, significantly involving carbohydrate metabolism, nucleotide biosynthesis, and oxidative stress pathways, accompanied by taxonomic shifts including the depletion of Akkermansia and enrichment of Odoribacter and Muribaculum. Concurrently, host-derived proteins displayed time-dependent alterations in lipid catabolism and redox regulation. WGCNA revealed co-regulated protein modules linked to MI status and the recovery phase, reflecting highly synchronized host-microbiome responses. This time-resolved metaproteomic study demonstrates that following initial surgical stress, MI triggers dynamic, stage-specific alterations in gut microbial function and coordinated host responses, providing mechanistic insights into the gut-heart axis and suggesting potential microbiota-targeted strategies to promote post-MI recovery.

This study aims to explore the protective role of Tongfu Pingchuan decoction (TFP) on sepsis. Thirty rats were randomly assigned to Sham, sepsis (cecal ligation and puncture, CLP), and CLP + Tongfu Pingchuan decoction (TFP) groups (n = 10 per group). TFP (1 mL/kg) or PBS was administered by gavage twice daily for 7 days. Cecum tissues were subjected to transcriptome sequencing (n = 6 per group), followed by network pharmacology and WGCNA analyses. Key findings were validated in rat cecum tissues and LPS-stimulated RAW264.7 macrophages. Compared with the Sham group, the Model group showed activation of TNF signaling, complement and coagulation cascades, fatty acid metabolism, and PPAR signaling pathways, along with suppression of cholinergic synapse, ECM-receptor interaction, and vascular smooth muscle contraction pathways. These alterations were largely reversed by TFP treatment. Integrated bioinformatics analyses identified Chrm4 and Pygm as overlapping differentially expressed genes between predicted TFP targets and a key WGCNA module. Chrm4 expression was significantly downregulated in septic rats and restored by TFP administration. Sepsis induced M1 macrophage polarization and suppressed M2 polarization in cecum tissues, whereas TFP reduced M1 markers and enhanced M2 markers. In LPS-stimulated RAW264.7 cells, TFP similarly promoted M2 polarization, reduced IL-6 and TNF-α levels, increased IL-10 expression, and suppressed IL1RN, p-ERK2/ERK2, and p-AKT/AKT signaling. These effects were abolished by Chrm4 silencing or treatment with the PCTR1 inhibitor Boc-2. These findings demonstrate that TFP alleviates sepsis by restoring intestinal Chrm4 expression, promoting macrophage polarization toward a pro-resolving phenotype, and modulating ERK/AKT-associated inflammatory signaling pathways.

Antimicrobial peptide (AMP)-based positive selection is a powerful strategy for rapid gene cloning. A notable example is the use of the Escherichia coli (E. coli) CcdB toxin to eliminate non-recombinant plasmids. Building on this, dual prokaryotic/mammalian expression vectors like pgMAX and pgMAX-II minimize subcloning by allowing direct expression after a single ligation. However, combining selection modules (e.g., CcdB and Microcin B17) may affect recovery, particularly when their mechanisms both target DNA gyrase. In this study, we evaluated the feasibility of cloning and expressing the porcine AMP protegrin-1 (PG-1) using an AMP-assisted selection framework. PG-1 is an 18-residue, cysteine-rich cathelicidin that adopts a disulfide bond-stabilized β-hairpin structure. It exhibits potent antibacterial activity by permeabilizing anionic lipid bilayers through pore formation. We demonstrate that protegrin-1 functions as an effective selectable marker for plasmid recombination within the pgMAX-II system, achieving high cloning efficiency. This novel framework enables simple, high-efficiency subcloning with dual-expression capability in both E. coli and mammalian cells.

Colicin E3 (E3-rRNAse) abolishes protein biosynthesis in bacteria by cleaving 16S rRNA in the decoding centre. The RtcB2-PrfH 16S rRNA repair module prevents cellular death upon exposure to E3-rRNAse in E. coli. Upon overexpression, RtcB2 RNA ligase alone was capable of relieving E. coli growth, which was inhibited by E3-rRNAse. Using in vitro ribosome repair system based on recombinant components, we demonstrated that RtcB2 alone could repair E3-rRNAse cleaved 30S subunits and 70S ribosomes. The peptide chain release factor homolog (PrfH), which is able to hydrolyse peptidyl-tRNA bound by colicin-cleaved ribosomes, boosts RtcB2 ligation efficiency for damaged ribosomes engaged in translation.

Skeletal muscle repair requires coordinated regulation of inflammation and protein synthesis, but the roles of ribosome biogenesis and protein composition remain poorly defined. To address this, mice underwent femoral artery ligation (FAL) to induce muscle regeneration over 28 days. In humans, tibialis anterior biopsies from patients with traumatic tibial fracture were subjected to RNA sequencing. Following FAL, c-Myc mRNA increased transiently, followed by increased ribosomal DNA transcription, leading to elevated total RNA levels. Skeletal muscle-specific ribosomal protein paralog RPL3L was replaced by the ubiquitously expressed RPL3 during the initial phases of recovery, but this shift was reversed by day 28. A substantial transcriptomic response was observed in human muscle injury, with heavy emphasis on MYC-induced anabolism and inflammation. This supports a model in which MYC-driven changes in ribosomal content and composition form a core anabolic module in skeletal muscle repair, potentially representing a targetable axis to enhance recovery after muscle injury.NEW & NOTEWORTHY Our findings establish ribosome biogenesis and ribosome remodeling as core components of the skeletal muscle regenerative program conserved across species. The rapid, MYC-driven induction of translational capacity, coupled with a transient switch in ribosomal protein composition, increases ribosome heterogeneity during skeletal muscle regeneration.

Cell-surface glycoRNA plays a crucial role in cellular behavior, yet its RNA substrate signatures and membrane transport mechanism remain unclear. Here, we developed GlycoRNA-Lighted In situ Nano-Tracking (GLINT), an approach for the visualization of distinct RNA-specific glycosignatures. GLINT employs a modular, localized concatenated DNA circuit based on a proximity ligation-mediated dual hierarchical hybridization chain reaction (HCR). Metabolically labeled sialic acid probe and RNA-specific probes are combined into a dual-recognition module through proximity ligation. The module subsequently initiates a dual hierarchical HCR cascade, enabling ultrasensitive in situ tracking of U1, U3, U35a, Y5, and U8 glycoRNAs at the single-cell level. Leveraging GLINT technology, glycoRNAs were confirmed to be transported intracellularly via a SNARE protein-mediated secretory extracellular mechanism. Furthermore, the identification of ten subtypes of breast cancer cells was achieved based on the level of distinct RNA-specific glycosignatures on the cell surface. GLINT demonstrates great potential for tracking RNA-specific glycosignatures, offering a powerful tool for in situ cell subtyping and exploration of RNA-related glycosylation processes.

Rolling Circle Replication (RCR) holds significant potential for detecting low-abundance nucleic acids. However, its practical application is often hindered by nonspecific ligation, which leads to high background signals and false-positive results. Herein, we designed a novel dual-locked circular template, termed no-bilateral-overhang dual-locked probe (ndRC). This probe employed steric hindrance from its dual hairpins lacking overhangs to create a "dual-locked" mechanism that specifically prevents off-target circularization. By integrating this template with exonuclease purification and a CRISPR/Cas12a module, we constructed the dual-locked RCT-CRISPR/Cas12a (DL-RCT-Cas12a) system, in which RCT generates long RNA transcripts containing crRNA repeats. These transcripts then activate Cas12a-mediated trans-cleavage, thereby amplifying the detection signal. The established assay achieved an ultralow detection limit of 40.31 aM, with a detection range from 100 aM to 1 μM. It demonstrated robust performance in complex matrices for the detection of partial HPV16 L1 gene sequences and exhibited a superior ability to discriminate high-concentration targets compared to qPCR. This work presents a versatile strategy to enhance the specificity of highly sensitive detection, improving performance in low-abundance nucleic acid analysis.