Qiyao Wang

The efficient utilization of industrial one-carbon (C1) gases, such as carbon dioxide (CO₂) and carbon monoxide (CO), has attracted widespread interest in recent years. Clostridium ljungdahlii, a key chemoautotrophic bacterium, can ferment syngas (a mixture of CO₂, CO and H₂) into value-added chemicals, making it a subject of extensive research. However, despite advances in genetic tool development for C. ljungdahlii, the rapid identification of genes underlying crucial cellular functions remains a bottleneck, hindering both a deeper understanding and successful genetic engineering of this bacterium. In this study, we constructed a genome-wide random mutagenesis library of C. ljungdahlii using a mariner transposon system, enabling the identification of numerous functional genes associated with cellular fitness to syngas. We overcame the library's relatively low plasmid curing efficiency by employing an IPTG-regulated conditional replicon. Subsequent high-throughput transposon insertion sequencing (Tn-seq) of this library allowed for the rapid exploration of a broad set of genes potentially involved in growth on syngas. Experimental validation of these candidate genes revealed several that affect the production of ethanol and acetate (two major metabolites) of C. ljungdahlii in gas fermentation. The data generated in this study provide a valuable resource for future research and optimization of C. ljungdahlii. Furthermore, the strategy presented here can be applied to identify genes associated with other important phenotypes in this bacterium.

Stimuli-responsive DNA nanostructures can be programmatically switched from an initial sensing-inert to a sensing-ready conformation, enabling precise bioimaging with high spatiotemporal control. The use of endogenous stimuli, rather than external triggers, offers considerable advantages for smart theranostic applications. Herein, we propose a novel Mg2+-activated strategy to program a DNA tetrahedron (MTD) for sensitive and accurate in situ imaging of mitochondrial adenosine triphosphate (ATP). This design features ATP aptamer sequence and Mg2+-specific DNAzyme integrated into a tetrahedral DNA framework, further bioconjugation with a mitochondria-targeting peptide MTP-131. Notably, the 3′ end of the ATP aptamer is designed within the cleavage domain of the DNAzyme. In the absence of Mg2+, the Cy5-labeled ATP aptamer strand and BHQ2 modified strands remain in close proximity resulting in quenched red fluorescence. Upon Mg2+ activation, the DNAzyme cleaves its substrate strands, triggering the partial release of the ATP aptamer strand. Subsequent binding of ATP induces a significant recovery of red fluorescence. The DNA nanodevice demonstrates low cytotoxicity, high specificity, and outstanding stability. Importantly, intracellular studies confirm the effective lysosomal escape, precise mitochondrial location and dynamic ATP monitoring capability upon chemical modulation including Ca2+ stimulation and oligomycin inhibition. This smart DNA nanodevice integrates precise targeting ability with dual-responsive sensing units, establishing a powerful tool to decipher mitochondrial energy metabolism in live cells, with potential applications in diagnostics and drug screening related to metabolic disorders.

To address the challenges such as expensive equipment, limited space, and high risks in the teaching of internships in biomedicine under the emerging engineering education paradigm, this study established a AI-empowered and virtually-physically integrated teaching model. This model follows the teaching logic of progressing from virtual to physical operations step-by-step. It starts with virtual simulation projects to help students build a comprehensive understanding of process workflows. Subsequently, AI-empowered process design and decision-making training are introduced, allowing students to simulate the entire production process from strain construction to final product output by adjusting parameters and optimizing strategies, thereby cultivating their data-driven process development capabilities. Following this, students proceed to hands-on training with real materials in on-campus practical training centers, achieving a safe and smooth transition from virtual to physical operations and enhancing their practical skills. Finally, through diversified field internships in enterprises, students integrate knowledge, skills, and professional literacy in real industrial settings. This model successfully creates a high-fidelity, low-risk, repeatable, and comprehensive teaching environment for internships, effectively bridges the gap between theoretical education and industrial requirements, and significantly enhances students' abilities of engineering innovation and practical application.

Visceral white nodules disease (VWND), caused by Pseudomonas plecoglossicida, poses a significant threat to the large yellow croaker (Larimichthys crocea) aquaculture industry. Developing live attenuated vaccines (LAVs) requires a fine balance between safety and immunogenicity, which remains a primary challenge. In this study, we developed a stable and effective LAV by constructing a markerless triple-gene deletion mutant ΔmlaEΔompRΔdeoR of P. plecoglossicida. The mutant exhibited high genetic stability and maintained growth kinetics compared to the wild-type (WT) strain under host-relevant conditions. Vaccination trials demonstrated that the triple mutant was significantly attenuated and safe for L. crocea. Following WT challenge, the triple mutant provided a relative percent survival (RPS) of 80.43% via intraperitoneal (i.p.) injection and 76.92% via immersion. For i.p.-vaccinated group, a significant reduction in splenic bacterial colonization and the absence of macroscopic white nodules were observed post-challenge. Furthermore, i.p.-vaccination triggered a coordinated immune response, characterized by the early upregulation of pro-inflammatory cytokines followed by the sustained activation of adaptive immune-related genes and elevated serum-specific IgM levels. These findings demonstrate that the ΔmlaEΔompRΔdeoR mutant is a promising LAV candidate, providing a viable strategy for the integrated control of P. plecoglossicida infection.

Visceral white nodules disease (VWND), caused by Pseudomonas plecoglossicida, poses a significant threat to the large yellow croaker (Larimichthys crocea) aquaculture industry. Developing live attenuated vaccines (LAVs) requires a fine balance between safety and immunogenicity, which remains a primary challenge. In this study, we developed a stable and effective LAV by constructing a markerless triple-gene deletion mutant ΔmlaEΔompRΔdeoR of P. plecoglossicida. The mutant exhibited high genetic stability and maintained growth kinetics compared to the wild-type (WT) strain under host-relevant conditions. Vaccination trials demonstrated that the triple mutant was significantly attenuated and safe for L. crocea. Following WT challenge, the triple mutant provided a relative percent survival (RPS) of 80.43% via intraperitoneal (i.p.) injection and 76.92% via immersion. For i.p.-vaccinated group, a significant reduction in splenic bacterial colonization and the absence of macroscopic white nodules were observed post-challenge. Furthermore, i.p.-vaccination triggered a coordinated immune response, characterized by the early upregulation of pro-inflammatory cytokines followed by the sustained activation of adaptive immune-related genes and elevated serum-specific IgM levels. These findings demonstrate that the ΔmlaEΔompRΔdeoR mutant is a promising LAV candidate, providing a viable strategy for the integrated control of P. plecoglossicida infection.

Edwardsiella piscicida is a major pathogen in marine aquaculture, and co-infection with Vibrio anguillarum dramatically exacerbates disease severity and mortality. However, the molecular mechanisms by which pathogens cooperatively exploit host factors during co-infection remain elusive. Cyclophilin A (CypA), a key regulator of intercellular communication, is often subverted by pathogens to promote infection. Here, we report that co-infection with E. piscicida and V. anguillarum markedly upregulates and promotes the extracellular secretion of host CypA. Transcriptomic analysis of infected Scophthalmus maximus spleen (SMSP) cells revealed that this CypA upregulation is linked to the modulation of the Ras-PI3K-Akt/MAPK signaling axes. Activation of these pathways leads to actin cytoskeleton remodeling, which in turn facilitates the invasion of E. piscicida strain EIB202. Collectively, these findings demonstrate that V. anguillarum promotes E. piscicida co-infection by hijacking the host's CypA-dependent signaling pathways, leading to actin remodeling. This work identifies CypA as a critical node in polymicrobial synergy and a promising therapeutic target for combating bacterial co-infections in aquaculture.

Outer membrane vesicles (OMV) are bioactive extracellular vesicles(EVs,but their low yield and purity limit their widespread application. This study found that adding the sterile supernatant of Escherichia coli to a mixed solution of Polyethylene Glycol (PEG) and Ammonium Sulfate ((NH₄)₂SO₄) rapidly generated a precipitate. Upon standing, the PEG / (NH₄)₂SO₄ system formed an aqueous two-phase system(ATPS）, with the precipitate enriched at the phase interface. Its efficiency for OMV separation was significantly higher than that of ultracentrifugation (UC). Based on this discovery, we designed an OMV purification process: ultrafiltration concentration - separation (UC or aqueous two-phase precipitation (ATPP)) - size-exclusion chromatography (SEC). This process maintained high recovery while removing impurities and residual precipitants generated by ATPP. OMV obtained through this process using ATPP method showed a 5-fold higher yield compared to those enriched by UC, while also exhibiting intact morphology and uniform distribution. Furthermore, we applied this method to extract OMV from Neisseria meningitidis serogroup B, a strain with application in OMV vaccines, and successfully obtained the corresponding OMV, indicating the promising application potential of this method.

Pseudomonas plecoglossicida is the etiological agent of visceral white spot disease, which induces significant mortality in economically important fish such as the large yellow croaker. In this study, we integrated recombinase polymerase amplification (RPA) with CRISPR/Cas12a-mediated detection to establish a fluorescence-based assay for rapid identification of P. plecoglossicida. The complete single-tube, two-stage RPA–CRISPR/Cas12a workflow can be performed within ~45 min. Using purified genomic DNA, the assay achieved an analytical detection limit of 1.65 copies μl−1 and showed no cross-reactivity with several other common fish pathogens. Its applicability was further evaluated using crude DNA extracts from spleen, liver, and kidney tissues of experimentally infected large yellow croakers. Overall, with its rapid turnaround, minimal equipment requirement, and high sensitivity, the RPA–CRISPR/Cas12a assay represents a promising diagnostic tool for rapid detection of P. plecoglossicida, thereby helping to control the spread of infection.

We reported the complete genome sequence of turbot circovirus (TurCV) strain TurCV10LN-18/2021, detected in a diseased turbot ( Scophthalmus maximus ) in China. TurCV is responsible for the emerging acute hemorrhagic syndrome (EAHS) in farmed turbot. The availability of the complete genome sequence facilitates the investigation of circovirus epidemiology in turbot.