Formate is an important solar fuel, with large application potential in bioconversion. Especially, the win-win collaboration is achieved when formate is applied to the cultivation of microalgae, which combines the advantages from both artificial and natural photosynthesis. However, the inhibition of formate on the photosynthetic electron transport hinders the application of formate at high concentrations. The engineering or directed evolution of the regulation pathway is a case-by-case and time-consuming strategy. Here, we developed a new strategy by introducing a Stenotrophomonas sp. strain which was isolated and identified from the long-term self-evolution process of Chlamydomonas reinhardtii for adapting to high concentrations of formate. The co-culture with the strain or the fermentation broth relieved the inhibition of formate (50 mmol/L) on C. reinhardtii and promoted the growth of the microalga. Especially, the protein content increased significantly to nearly 50% of the dried weight. In addition, the co-culture also benefited the growth of both Chlorella pyrenoidesa and Synechocystis sp. PCC 6803 exposed to formate, which indicated broader applicability of this strategy. This strategy provides the opportunity to overcome the bottleneck in the formate-mediated artificial-natural hybrid photosynthesis and to aid the development of technologies for solar energy-driven production of bulk biomass, including proteins, by carbon dioxide reduction.
Photosynthesis/physiology* ; Formates/pharmacology* ; Stenotrophomonas/growth & development* ; Microalgae/metabolism* ; Chlamydomonas reinhardtii/growth & development*

Flavonoids are key bioactive components for evaluating the pharmacological activities of Chimonanthus praecox. Exploring the potential flavonoids and pharmacological mechanisms of C. praecox lays a foundation for the rational development and efficient utilization of this plant. This study employed ultra-performance liquid chromatography-tandem mass spectrometry-based widely targeted metabolomics to comprehensively identify the flavonoids in C. praecox. Network pharmacology was employed to explore the bioactive flavonoids and their mechanisms of action. Molecular docking was adopted to validate the predicted results. Finally, the content of bioactive flavonoids in different varieties of C. praecox was measured. The widely targeted metabolomics analysis identified 387 flavonoids in C. praecox, and the flavonoids varied among different varieties. Network pharmacology predicted 96 chemical components including 19 bioactive compounds, 181 corresponding targets and 2 504 disease targets, among which 99 targets were shared by the active components and the disease. Thirty-three core targets were predicted, involving 229 gene ontology terms and 99 pathways (P≤0.05), which indicated that the flavonoids components of C. praecox exhibited pharmacological activities including antioxidant, anti-inflammatory, antimicrobial, and antiviral activities. Topological analysis screened out five core components (salvigenin, laricitrin, isorhamnetin, quercetin, and 6-hydroxyluteolin) and five core targets (SRC, PIK3R1, AKT1, ESR1, and AKR1C3). The predicted bioactive flavonoids from C. praecox stably bound to key targets, which indicated that these flavonoids possessed potential bioactivities in their interactions with the targets. The flavonoids in C. praecox exerted pharmacological activities in a multi-component, multi-target, and multi-pathway manner. The combined application of metabolomics and network pharmacology provides a theoretical basis for in-depth studies on the pharmacological effects and mechanisms of C. praecox.
Flavonoids/metabolism* ; Network Pharmacology ; Metabolomics/methods* ; Molecular Docking Simulation ; Calycanthaceae/chemistry* ; Tandem Mass Spectrometry ; Drugs, Chinese Herbal/chemistry*

Biosynthesized selenium nanoparticles (SeNPs) have attracted much attention because of their unique physical, chemical, and biological properties. The microbial reduction of selenium salts to SeNPs has great potential, while there is a lack of elite strains. In this study, we explored the reduction of Na₂SeO₃ by Streptomyces avermitilis into SeNPs. The colonies and hyphae of the strain and the synthesized SeNPs were characterized by optical microscopy, scanning electron microscopy (SEM), transmission electron microscope (TEM), energy dispersive spectrometry (EDS), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). At the same time, the inhibitory activity of SeNPs on Fusarium oxysporum, the main pathogen causing root rot of Lycium barbarum, was studied. The results showed that S. avermitilis converted Na₂SeO₃ into SeNPs and tolerated 300 mmol/L Na₂SeO₃, demonstrating strong tolerance. S. avermitilis synthesized spherical SeNPs in the cytoplasm, and most of SeNPs had a diameter of about 100 nm and were released by hyphal fracture. The SeNPs synthesized by S. avermitilis were amorphous, and their surfaces were dominated by C and Se, with the existence of O, N and other elements. SeNPs had functional groups such as -OH, C=O, C-N, and C-H, which were closely related to the stability and biological activity of SeNPs. The SeNPs synthesized by S. avermitilis showcased significant inhibitory activity on F. oxysporum, and 25.0 μmol/mL SeNPs showcased the inhibition rate of 77.61% and EC₅₀ of 0.556 μmol/mL. In conclusion, S. avermitilis can tolerate high Na₂SeO₃ stress and mediate the synthesis of SeNPs. The synthesized SeNPs have good stability and strong inhibitory activity, demonstrating the potential application value in the preparation of SeNPs and the control of L. barbarum root rot.
Streptomyces/metabolism* ; Fusarium/drug effects* ; Lycium/microbiology* ; Selenium/metabolism* ; Nanoparticles/chemistry* ; Plant Diseases/microbiology* ; Metal Nanoparticles/chemistry* ; Antifungal Agents/pharmacology*

Due to the wide application of silver-containing dressings and silver-coated medical devices in clinical treatment; the extensive use of antibacterial agents and heavy metal agents in feed factories, Escherichia coli has formed the tolerance to silver ions. To systematically understand the known silver ion resistance mechanisms of E. coli, this article reviews the complex regulatory network and various physiological mechanisms of silver ion tolerance in E. coli, including the regulation of outer membrane porins, energy metabolism modulation, the role of efflux systems, motility regulation, and silver ion reduction. E. coli reduces the influx of silver ions by missing or mutating outer membrane porins such as OmpR, OmpC, and OmpF. It adapts to high concentrations of silver ions by altering the expression of ArcA/B and enhances the efflux capacity of silver ions under high-concentration silver stress via the endogenous Cus system and exogenous Sil system. Furthermore, the motility of bacteria is related to silver tolerance. E. coli has the ability to reduce silver ions, thereby alleviating the oxidative stress induced by silver ions. These findings provide a new perspective for understanding the formation and spread of bacterial tolerance and provide directions for the development of next-generation silver-based antimicrobials and therapies.
Escherichia coli/genetics* ; Silver/pharmacology* ; Drug Resistance, Bacterial ; Anti-Bacterial Agents/pharmacology* ; Porins/metabolism*

Reactive oxygen species (ROS) are major contributors to radiation therapy-induced side effects in cancer patients. A fusion antioxidant enzyme comprising glutathione S-transferase (GST), superoxide dismutase 1 (SOD1), and a transmembrane peptide has been shown to effectively mitigate ROS-induced damage. To enhance its targeting capability, the fusion protein was further modified by incorporating a matrix metalloproteinase-2/9 substrate peptide (X) and the transmembrane peptide R9, yielding the antioxidant enzyme GST-SOD1-X-R9 (GS1XR). This modification reduced its transmembrane ability in tumor cells, thereby selectively protecting normal cells from oxidative stress. However, the use of non-human GST poses potential immunogenicity risks. In this study, we employed seamless cloning technology to construct an expression vector containing the human GST gene to replace the non-human GST gene, and then expressed and purified novel fusion antioxidant enzymes GS1R and GS1XR. The protective effects of newly constructed GS1R and GS1XR against hydrogen peroxide (H₂O₂)-induced oxidative damage in L-02 cells were then evaluated using GS1 as a control. Enzymatic activity assays revealed that the specific activity of GST in GS1XR remained unchanged compared to the unmodified protein, while SOD activity was enhanced. Exposure to 200 μmol/L H₂O₂ transiently activated the nuclear factor-erythroid 2-related factor 2 (Nrf2) pathway; however, this activation diminished after 24 h, reducing cell viability to 48.4%. Both GS1R and GS1XR effectively scavenged intracellular ROS, directly counteracting oxidative stress and promoting Nrf2 nuclear translocation, thereby activating antioxidant pathways and restoring cell viability to normal levels. The two enzymes showed comparable efficacy. In contrast, GS1, lacking transmembrane capability, was restricted to scavenging extracellular ROS and provided only limited protection. In conclusion, both novel fusion antioxidant enzymes demonstrated significant potential in safeguarding normal cells from ROS-mediated oxidative damage. The findings provide a foundation for further investigation in related field.
Humans ; Oxidative Stress/drug effects* ; Hydrogen Peroxide ; Antioxidants/metabolism* ; Glutathione Transferase/metabolism* ; Recombinant Fusion Proteins/pharmacology* ; Superoxide Dismutase-1 ; Reactive Oxygen Species/metabolism* ; Superoxide Dismutase/biosynthesis*

SoxR, one of bacterial transcriptional regulators, plays a crucial role in bacterial responses to oxidative stress induced by unfavorable environmental conditions. So far, the understanding of bacterial responses to oxidative stress mainly stems from a handful model bacteria such as Escherichia coli and the studies on non-model bacterial responses to oxidative stress are limited. In this study, Citrobacter braakii JPG1, a commonly occurring strain of enterobacteria, was used as a model for the first time to explore the role of SoxR in the responses to aerobic/anaerobic-menadione stress. First, we analyzed the phylogenetic relationship of SoxR based on the whole genome and constructed the soxR-deleted strain (ΔsoxR). Then, the cell counts of the wild type (WT) and ΔsoxR were compared under aerobic/anaerobic-menadione stress. The results showed that the cell count of WT exposed to the aerobic-low concentration menadione (0.1 mmol/L) stress for 24 h increased by 4.2 times compared with that at the time point of 0 h, while that of ΔsoxR only increased by 1.3 times. The vast majority of WT and ΔsoxR cells died after exposure to the aerobic-high concentration menadione (0.3 mmol/L) stress for 24 h, with the cell counts only 29% and 0.2% of those at the time point of 0 h, respectively. Interestingly, the cell counts of WT showed no significant difference between the anaerobic-menadione stress and the control (P > 0.05), and the same was true for ΔsoxR. All these results indicated that SoxR of C. braakii JPG1 only has a regulatory effect on the redox cycling compound menadione under aerobic conditions and enhance the antioxidant capacity. Under anaerobic conditions, menadione failed to activate SoxR. The findings from this study provide new insights into understanding both the physiological responses to menadione stress and the regulatory role of SoxR under different oxygen conditions.
Bacterial Proteins/physiology* ; Anaerobiosis ; Aerobiosis ; Vitamin K 3/pharmacology* ; Citrobacter/metabolism* ; Transcription Factors/physiology* ; Oxidative Stress ; Gene Expression Regulation, Bacterial

Hormesis effect has been observed in the secondary metabolite synthesis of microorganisms induced by rare earth elements. However, the underlying molecular mechanism remains unclear. To analyze the molecular mechanism of the regulatory effect of Rhodotorula mucilaginosa in the presence of lanthanum chloride, different concentrations of lanthanum chloride were added to the fermentation medium of Rhodotorula mucilaginosa, and the carotenoid content was subsequently measured. It was found that the concentrations of La³⁺ exerting the promotional and inhibitory effects were 0-100 mg/L and 100-400 mg/L, respectively. Furthermore, the expression of 33 genes and the synthesis of 55 metabolites were observed to be up-regulated, while the expression of 85 genes and the synthesis of 123 metabolites were found to be down-regulated at the concentration range of the promotional effect. Notably, the expression of carotenoid synthesis-related genes except AL1 was up-regulated. Additionally, the content of β-carotene, lycopene, and astaxanthin demonstrated increases of 10.74%, 5.02%, and 3.22%, respectively. The expression of 5 genes and the synthesis of 91 metabolites were up-regulated, while the expression of 35 genes and the synthesis of 138 metabolites were down-regulated at the concentration range of the inhibitory effect. Meanwhile, the content of β-carotene, lycopene, and astaxanthin decreased by 21.73%, 34.81%, and 35.51%, respectively. In summary, appropriate concentrations of rare earth ions can regulate the synthesis of secondary metabolites by modulating the activities of various enzymes involved in metabolic pathways, thereby exerting the hormesis effect. The findings of this study not only contribute to our comprehension for the mechanism of rare earth elements in organisms but also offer a promising avenue for the utilization of rare earth elements in diverse fields, including agriculture, pharmaceuticals, and healthcare.
Lanthanum/pharmacology* ; Rhodotorula/genetics* ; Carotenoids/metabolism* ; Hormesis/drug effects* ; Fermentation ; Multiomics

This study established the mouse podocyte clone-5 (MPC5) with Smad3 knockout and studied the effect of transforming growth factor-beta 1 (TGF-β1) on the dedifferentiation of the MPC5 cells with Smad3 knockout, aiming to provide a cell tool for studying the role of Smad3 in mouse podocytes. The single-guide RNA (sgRNA) sequence targeting Smad3 was designed according to the principles of CRISPR/Cas9 design. The pX458-Smad3 vector was constructed and introduced into competent cells, and then the vector was extracted and used to transfect MPC5 cells. The successfully transfected cells were sorted by a flow cytometer. After single-cell clone expansion, PCR amplification of sequences adjacent to the edition site of Smad3 and sequencing were performed to identify potential cells with gene knockout. Western blotting was employed to verify the knockout efficiency of Smad3. Finally, the effect of Smad3 knockout on TGF-β1-induced dedifferentiation of MPC5 cells was analyzed by reverse transcription-polymerase chain reacting (RT-PCR), Western blotting, and the immunofluorescence method. The sgRNA was designed to target the fifth exon of Smad3. EGFP expression was observed 24 h after transfection of the pX458-Smad3 plasmid into MPC5 cells, with the transfection efficiency of 0.1% as determined by flow cytometry. From the transfected cells, 21 cell clones were obtained through flow cytometric sorting and single-cell clone expansion. PCR amplification and sequencing of the region around the sgRNA target site in Smad3 identified two cell clones with biallelic frameshift mutations. Western blotting results confirmed the absence of Smad3 expression in these clones, indicating successful establishment of the MPC5 cell line with Smad3 knockout. In normal MPC5 cells, TGF-β1 stimulation promoted the expression of fibrosis-related genes fibronectin and Col1a1 (collagen I) and inhibited the expression of the podocyte marker proteins synaptopodin and podocin, which suggested epithelial-mesenchymal transition and podocyte injury. However, in the two MPC5 cell lines with Smad3 knockout, TGF-β1-induced expression of epithelial-mesenchymal transition markers was significantly suppressed. The MPC5 cell lines with Smad3 knockout that were constructed by CRISPR/Cas9 provide a valuable cell model for functional studies of Smad3 protein and highlight the critical role of Smad3 in cell dedifferentiation.
Animals ; Smad3 Protein/genetics* ; CRISPR-Cas Systems/genetics* ; Mice ; Podocytes/metabolism* ; Transforming Growth Factor beta1/pharmacology* ; Cell Line ; Gene Knockout Techniques ; RNA, Guide, CRISPR-Cas Systems/genetics*

The culture of suspended Vero cells is facing difficulties such as low cell viability and long doubling time. To investigate the main reasons for the slow growth and low viability of suspended Vero cells, this study conducted transcriptomic analysis of suspended Vero cells (Vero-XF) and adherent Vero cells (Vero-AD) to screen the differentially expressed genes (DEGs) affecting the growth of suspended cells. In addition, epidermal growth factor (EGF) was supplemented to the culture system to improve the growth of Vero-XF. The results showed that compared with the Vero-AD group, the Vero-XF group had 7 376 significant DEGs. Kyoto encyclopedia of genes and genomes enrichment analysis revealed that the DEGs were mainly enriched in the autophagy and mitophagy pathways. Eleven DEGs were selected and verified by quantitative real-time PCR, which showed up-regulated expression of ATG9B, WIPI2, LAMP2, OPTN, Rab7a, and DEPTOR and down-regulated expression of ATG4D, being consistent with the results of transcriptomic analysis. In addition, the Vero-XF group showed significantly up-regulated expression of ATG101, ATG2A, and STX17 and insignificant change in the expression of NBR1, compared with the Vero-AD group. The protein levels of LC3 and P62 in Vero-XF and Vero-AD were determined by Western blotting, which showed up-regulated expression of LC3Ⅱ/Ⅰ and down-regulated expression of P62 in Vero-XF, indicating a higher level of autophagy. Finally, the exogenous supplementation of EGF at 10, 20, and 30 μg/L in the culture system reduced the autophagy level of Vero-XF by 22.35%, 48.15%, and 71.29%, increased the specific growth rate by 15.48%, 33.33%, and 57.14%, and decreased the apoptosis rate by 2.84%, 15.46%, and 16.23%, respectively. The results of this study preliminarily reveal that the activation of autophagy is one of the reasons for the slow growth of Vero-XF, which provides reference for the subsequent culture of suspended Vero cells.
Animals ; Vero Cells ; Autophagy/genetics* ; Chlorocebus aethiops ; Epidermal Growth Factor/pharmacology* ; Gene Expression Profiling ; Transcriptome ; Cell Survival

Traditional Chinese medicine (TCM) plays an important role in preventing and treating diseases and improving human health. However, the complex bioactive components and regulation of signaling pathway and network restrict the elucidation of the mechanisms of action of TCM. A human being is regarded as a super "symbiont" composed of body cells and commensal microorganisms. Gut microbiota is the core commensal microorganism system of a human body, being considered as "the second genome" and the new "organ". Alterations in gut microbiota reflect the state of body health and progression of diseases. Recent investigations have revealed that the TCM rich in polysaccharides and polyphenols can modulate gut microbiota metabolites to rehabilitate gut homeostasis, thus ameliorating diseases via regulating gut-liver axis or gut-brain axis. This review summarizes the causal relationship and mechanisms of action of TCM in the treatment of diseases from the perspective of gut microbiota metabolites. Our findings are expected to provide new insights into the mechanisms of TCM in preventing and treating diseases and guidance for TCM-based drug discovery in the future.
Humans ; Gastrointestinal Microbiome/physiology* ; Medicine, Chinese Traditional/methods* ; Drugs, Chinese Herbal/therapeutic use* ; Polyphenols/pharmacology* ; Polysaccharides/pharmacology*