Objective To investigate the effects of Chlamydia trachomatis plasmid protein pORF5 on cellular mitophagy and mitochondrial fission and to elucidate whether its mechanism is related to Drp1 activation. Methods HeLa cells stably expressing pORF5 and control cells were constructed by lentiviral transfection. After serum starvation treatment, the expression levels of autophagy-related proteins—microtubule-associated protein 1 light chain 3 (LC3), Beclin-1, and p62—were determined by Western blotting. Co-localization of LC3 and translocase of outer mitochondrial membrane 20 (TOMM20) was assessed by indirect immunofluorescence. Mitochondria were stained with MitoTracker Red CMXRos, and mitochondrial morphology was observed and analyzed through confocal laser scanning microscopy. Dynamin-related protein 1 (Drp1) phosphorylation and its translocation to mitochondria were examined by Western blotting and indirect immunofluorescence. To explore the role of Drp1 in autophagy and mitochondrial fission, we pretreated cells with the Drp1-specific mitochondrial division inhibitor 1 (Mdivi-1). Changes in mitochondrial morphology and Drp1 translocation were evaluated by confocal microscopy and indirect immunofluorescence. Then, Western blotting was employed to determine the expression levels of autophagy-related proteins, and indirect immunofluorescence assay to analyze LC3 fluorescence intensity and its co-localization with TOMM20. Results Compared with the control group, pORF5 significantly upregulated the expression of LC3-Ⅱ and Beclin-1, downregulated the expression of p62, and enhanced the co-localization of LC3 and TOMM20. pORF5 expression led to the fragmentation of the mitochondrial network structure. It promoted Drp1 phosphorylation at Ser616 and enhanced Drp1 translocation to mitochondria. Inhibition of Drp1 with Mdivi-1 attenuated Drp1 phosphorylation and translocation, resulting in elongated mitochondrial morphology. In addition, the Mdivi-1 inhibitor group showed downregulated expression of LC3-Ⅱ and Beclin-1, upregulated the expression of p62, and attenuated co-localization of LC3 and TOMM20. Conclusion The C. trachomatis plasmid protein pORF5 may induce mitophagy and mitochondrial fission by promoting Drp1 phosphorylation and its mitochondrial translocation.

Phytoplankton are key primary producers in marine ecosystems, and their community composition is influenced not only by environmental factors but also by biological factors such as parasitic organisms. However, the relationships between parasitic eukaryotes and phytoplankton in coastal bays remain poorly understood. Objective To investigate the community composition, seasonal succession, and interactions of dominant phytoplankton, including diatoms, dinoflagellates, and their parasitic eukaryotes, Syndiniales, in Sanmen Bay, Zhejiang Province during summer and autumn. Methods Surface water samples were collected from Sanmen Bay in September (summer) and November (autumn) 2019. High-throughput sequencing was performed for the V4 region of the 18S rRNA gene of eukaryotic microbes. Principal coordinate analysis, co-occurrence network analysis, and bipartite network analysis were employed to explore the succession and interactions of diatom, dinoflagellate, and Syndiniales communities. Results Diatom communities exhibited pronounced seasonal variations, with the relative abundance of 36.9% in summer and 5.5% in autumn, being dominated by Cyclotella, Skeletonema, and Thalassiosira. Dinoflagellates showed similar relative abundance in summer (28.0%) and autumn (28.5%), being dominated by Gyrodinium, Heterocapsa, and Gymnodinium. Four Syndiniales groups (group I-IV) were detected in Sanmen Bay, with higher relative abundance in autumn (10.5%) than in summer (7.6%). Among Syndiniales, group I dominated in summer (49.1%), while group Ⅱ dominated in autumn (75.2%). Salinity, nitrate, nitrite, N:P ratio, and Si:N ratio were identified as the main drivers of the dominant phytoplankton and Syndiniales communities. Network analysis indicated that Syndiniales mainly interacted with diatoms and dinoflagellates, with higher interaction frequency in autumn than in summer. Syndiniales were identified as keystone taxa in the interaction network. Conclusion Seasonal succession of diatoms in Sanmen Bay was more pronounced than that of dinoflagellates. Syndiniales groups I and Ⅱ exhibited significant seasonal succession and maintained close interactions with diatoms and dinoflagellates, serving as key taxa in the interaction network. High-throughput sequencing overcame the limitations of microscopy in detecting Syndiniales, providing systematic insights into the community features and potential interactions of dominant phytoplankton and Syndiniales in Sanmen Bay. This study offers important insights into phytoplankton adaptation and interspecies interactions in eutrophic coastal bays.

Objective To examine the anti-lung cancer activity of Cordyceps militaris and predict its potential quality markers. Methods We systematically reviewed the current studies on the anti-lung cancer effects of C. militaris and summarized its chemical components. Homo sapiens lung cancer microarray data were integrated with network pharmacology to build a “component-target-pathway” network, followed by molecular docking analysis. On this basis, the potential quality markers of C. militaris for lung cancer treatment were predicted. Results We predicted 11 potential quality markers, which were grouped into six categories: (1) cordycepin and its analog O^5′-acetylcordycepin; (2) adenosine and its analogs, including N⁶-[β-(acetylcarbamoyloxy)ethyl]-adenosine, N⁶-(2-hydroxyethyl)-adenosine, N⁶-(4-methylbutyrate)-adenosine, and 5′-(3″-deoxy-β-D-ribofuranosyl)-3′-deoxyadenosine; (3) ergosta-7,22-dien-3β,5α-dihydroxy-6-one; (4) cordycepisosalt A; (5) pentostatin; and (6) cordyrrole B. Conclusion This study integrates literature review and bioinformatics analysis to predict potential anti-lung cancer quality markers of C. militaris. The suggested mechanisms and candidate components are theoretical and need further experimental validation to confirm their effectiveness. This work offers a reference for developing a quality standard system of C. militaris for anti-lung cancer applications.

Meloidogyne incognita is one of the most destructive plant-parasitic nematodes worldwide, causing severe economic losses in agricultural production. Biocontrol bacteria can effectively control M. incognita, with significant differences in control efficacy among different strains. However, the mechanisms underlying differences in control efficacy remain unclear. Objective To explore the mechanisms responsible for the different efficacy of various biocontrol bacteria against nematodes. Methods The differences in nematicidal activity between two biocontrol bacterial strains, B133 and B104, were analyzed. Comparative genomics and metabolomics techniques were employed to investigate the genetic composition and metabolic mechanisms influencing the nematicidal activity of the two strains. Result From 24 h to 120 h of fermentation, the nematicidal activity of strain B133 was significantly higher than that of strain B104, reaching peaks of 77% and 54%, respectively, at the time point of 60 h. Whole-genome comparative analysis revealed that strain B133 possessed a larger genome size and a greater number of coding genes than strain B104. The phylogenetic trees conducted based on 16S rRNA gene or the housekeeping gene gyrB indicated that strains B133 and B104 were two different subspecies of Priestia megaterium. Predictions based on the virulence factors database (VFDB) and Kyoto encyclopedia of genes and genomes (KEGG) database showed that strain B133 harbored 22 unique virulence genes and 75 unique metabolism genes compared with strain B104. Meanwhile, the metabolites in the fermentation filtrate (60 h) were determined. Principal component analysis demonstrated significant differences in metabolite profiles between the two strains. Compared with that of strain B104, the fermentation filtrate of strain B133 had 40 increased metabolites (P<0.05), such as galactinol, 4-aminobenzoic acid, lumichrome, anthranilic acid, trehalose, and 3-methylthiopropionic acid. Moreover, through integrated genomics-metabolomics analyses, cysteine and methionine metabolism was identified as a key pathway influencing nematicidal activity. This pathway involves an L-lactic dehydrogenase (LDH) gene unique to strain B133 and 3-methylthiopropanoic acid with an elevated level and a positive correlation with the nematicidal effect of the strain. Conclusion By coupling genomics and metabolomics, this study reveals the different functional gene clusters and potential related metabolites of different subspecies of P. megaterium, laying a theoretical foundation and a practical basis for the targeted screening, modification, and industrial development of efficient biocontrol agents for nematodes.

A reverse genetics platform for foot-and-mouth disease virus (FMDV) is an indispensable tool for studying the pathogenic mechanism, protein function, and vaccine development. However, the conventional method of constructing infectious clones of FMDV is usually laborious, time-consuming, and costly. Objective To establish a new reverse genetics platform for rapid rescue of FMDV based on infectious subgenomic amplicons (ISA), which can avoid in vitro ligation and bacterial cloning. Methods The whole gene of FMDV O/GDLeiZh/2020 strain was divided into five overlapping fragments and then individually amplified by high-fidelity PCR. The T7 promoter sequence was added to the 5′-end gene and the poly(A) tail was introduced at the 3′-end. At the same time, the poly(C) sequence and molecular markers were introduced by fusion PCR. Two large fragments covering the whole gene of FMDV were obtained by multiple rounds of fusion PCR amplification and co-transfected into BSR/T7 cells expressing T7 RNA polymerase. The cell supernatant was collected 72 h post-transfection. The rescued virus was identified and characterized by RT-PCR, indirect immunofluorescence, electron microscopy, plaque assay, and one-step growth curve assay. Results The typical cytopathic effect of FMDV was observed 60 h post-transfection. Sequencing, immunofluorescence, and electron microscopy collectively confirmed that infectious FMDV was successfully rescued. Furthermore, one-step growth curve and plaque assays demonstrated that the rescued virus retained replication kinetics and biological characteristics comparable to those of the wild-type virus. Conclusion This study successfully establishes a new method for rapid and efficient rescue of FMDV based on ISA, which will lay a solid foundation for further improving FMDV rescue technology and rapidly expanding its application in the future.

Sugarcane smut is a severe fungal disease caused by Sporisorium scitamineum, resulting in yield reduction and economic losses. Reversible protein phosphorylation plays a crucial role in the sexual mating and pathogenicity of S. scitamineum. Protein phosphatases, as key regulators of reversible protein phosphorylation, remain poorly characterized in S. scitamineum. Objective To elucidate the biological functions of the protein phosphatase SsPpe1 in S. scitamineum, providing a potential target for effective control of sugarcane smut. Methods We constructed overexpression mutants OE-Ssppe1 by Agrobacterium-mediated genetic transformation technology and analyzed the sporidium morphology, sexual mating ability, stress tolerance, and pathogenicity. Results The OE-Ssppe1 sporidia exhibited pseudohyphal morphology with multiple nuclei and abnormal chitin accumulation. The OE-Ssppe1 mutants showed reduced tolerance to NaCl and SDS, sexual mating, and pathogenicity. RT-qPCR and RNA-seq analyses revealed that Ssppe1 overexpression affected the expression of genes related to pheromone response, MAPK, and cAMP-PKA signaling pathways. In addition, Ssppe1 overexpression affected protein synthesis and folding process. Conclusion The protein phosphatase SsPpe1 is involved in regulating the sporidium morphology, stress responses, sexual mating, and pathogenicity of S. scitamineum. These findings provide a theoretical basis for thoroughly elucidating the pathogenic mechanisms of S. scitamineum and developing targeted disease control strategies.

Objective Southern blight, caused by Sclerotium rolfsii Sacc., is a major soil-borne disease that limits peanut production. At present, its management relies mainly on chemical fungicides. To improve the field control efficacy, reduce fungicide application rates, and lower pesticide residues in peanuts, this study screened antagonistic microorganisms against S. rolfsii and evaluated their synergistic effects with chemical fungicides in the control of peanut southern blight. The results are expected to provide technical support for the green and sustainable management of this disease. Methods This study first used a plate-based screening assay to obtain biocontrol strains showing strong antagonistic activity against S. rolfsii and high compatibility with commonly used fungicides. Next, a fungicide screening assay was conducted to identify control agents that effectively inhibited S. rolfsii without adversely affecting the growth of the biocontrol strains. Subsequently, an in vitro combined toxicity assay was performed. The pathogen was inoculated onto PDA plates containing different concentrations of thifluzamide (0.15 and 0.30 μg/mL), sterile 10% fermentation broth of H02, or their mixtures. Fungal growth was observed, and the inhibition rate and inhibition ratio (IR) were calculated to evaluate the interaction type of the combined treatment. Finally, a pot experiment was carried out to compare the control efficacy against peanut southern blight among the fermentation broth, the full-dose fungicide (recommended field rate), and the combination of fermentation broth with a half-dose fungicide. Results A Burkholderia gladioli strain H02 with high antagonistic activity and good fungicide compatibility was screened out, exhibiting an inhibition rate of 76.03%. Concurrently, thifluzamide was identified as a fungicide with strong inhibitory activity against S. rolfsii and no negative impact on the growth of strain H02, exhibiting an EC₅₀ of 0.151 3 μg/mL. Results from the in vitro combined toxicity assay indicated that the combination of the H02 fermentation broth and thifluzamide showed an IR greater than 1, exhibiting a synergistic effect. Moreover, the combination of half the concentration of thifluzamide with the fermentation broth significantly demonstrated higher inhibition rates against pathogen mycelia and sclerotia (74.91% and 95.58%, respectively) than the full concentration of thifluzamide alone (68.04% and 83.67%, respectively). Pot experiment results showed that the combination of H02 fermentation broth and half dosage of thifluzamide had the control efficacy comparable to that of the full dosage of thifluzamide (68.94% vs. 66.63%), whereas the single application of H02 fermentation broth provided the control efficacy of 61.17%. Conclusion The combined application of B. gladioli H02 and thifluzamide can achieve synergistic control of peanut southern blight and reduce chemical fungicide usage while maintaining effective disease control, thus showing promising potential for practical application.

Objective To investigate the antagonistic activity of Bacillus amyloliquefaciens X60 against tobacco phyllosphere microorganisms and its effects on the phyllosphere microbial community of tobacco. Methods Bioactivity assays were conducted to evaluate the antagonistic effects of B. amyloliquefaciens X60 against 20 species of pathogenic fungi, 15 species of non-pathogenic fungi, 2 specialized forms of pathogenic bacteria, and 15 species of non-pathogenic bacteria. Amplicon sequencing was employed to assess the influence of this strain on the phyllosphere microbial community structure. Results B. amyloliquefaciens X60 exhibited strong antagonistic activity (inhibition rates of 60.00%-80.00%) against 13 species of pathogenic fungi (e.g., Rhizopus oryzae) and 12 species of non-pathogenic fungi (e.g., Trichoderma harzianum). Moderate antagonism (inhibition rates of 10.00%-59.00%) was observed against 7 species of pathogenic fungi (e.g., Alternaria tenuissima) and 3 species of non-pathogenic fungi (e.g., Thielavia microspora). Significant antibacterial activity (inhibition zone diameter >20 mm) was detected against 2 specialized forms of pathogenic bacteria (Pseudomonas syringae pv. tabaci and pv. angulata) and 7 non-pathogenic bacteria (e.g., Exiguobacterium). After application, X60 showed the control efficacy of 52.35% against tobacco leaf spot. Following treatment, the relative abundance of Pantoea—a genus of opportunistic bacteria dominating the infected tissue—increased, whereas bacterial diversity and richness initially declined and then recovered. Fungal richness decreased throughout the observation period, while fungal diversity exhibited a transient decrease followed by a rebound. The relative abundance of phytopathogenic fungi declined from 44.87% to 6.71%. Conclusion B. amyloliquefaciens X60 possesses a broad antimicrobial spectrum and exerts strong antagonistic activity against 25 fungal and 9 bacterial species colonizing the tobacco phyllosphere. Under field conditions, the strain provided 52.35% control of tobacco leaf spot and significantly reduced the abundance of foliar phytopathogens, demonstrating the potential as a biocontrol agent for the management of this disease.

Objective Cyanocobalamin (CN-CbI) requires to be converted into adenosylcobalamin (Ado-CbI) to exert neuroprotective effects, yet its conversion efficiency is impaired under conditions such as chronic sleep deprivation (CSD). This study aimed to obtain a bacterial strain with high efficiency in converting CN-CbI to Ado-CbI that could enhance neuroprotective effects. Methods Lactobacillus paragasseri CCFM1526, a strain capable of converting CN-CbI to Ado-CbI, was isolated via UPLC. A mouse model of CSD was established, and the cognitive functions of mice were evaluated by the novel object recognition and Morris water maze tests. The neuronal structure in the hippocampal dentate gyrus (DG) region was observed via histological staining. Tissue vitamin B₁₂ levels were measured, and the ERK/mTOR signaling pathway along with related neural protein expression was analyzed to assess the neuroprotective mechanism of the fermentation broth. Results Compared with CN-CbI supplementation alone, the fermentation broth of L. paragasseri CCFM1526 significantly improved the cognitive function and alleviated the structural damage in the hippocampal DG region of CSD mice. Intervention with the fermentation broth increased the total vitamin B₁₂ content in the liver, serum, and brain by 10.2%, 16.3%, and 29.0%, respectively (P<0.05). Meanwhile, it activated the ERK/mTOR signaling pathway, leading to increases of 21.5%, 52.4%, 17.3%, and 19.7% in the content of myelin basic protein, postsynaptic density protein 95, brain-derived neurotrophic factor, and nerve growth factor, respectively (P<0.05). Conclusion L. paragasseri CCFM1526 converts CN-CbI into Ado-CbI through fermentation, subsequently activating the ERK/mTOR signaling pathway and upregulating the expression of neurotrophic and myelin repair-related proteins, thereby alleviating CSD-induced nerve injury.

Objective To achieve the targeted isolation of the cyclooctapeptides, surugamides, from the deep-sea-derived Streptomyces sp. NA13 and explore their biological activities. Methods An approach integrating genome mining and LC-MS/MS molecular networking was employed to discover cyclopeptides from Streptomyces sp. NA13. Through systematic natural product isolation and characterization, these compounds were identified as surugamides. Their growth-promoting effects on Oryza sativa and Zea mays were assessed. Results Four cyclooctapeptides (surugamides A, B, D, and E) were isolated and identified. They had significant effects of promoting root growth in Z. mays and O. sativa seedlings. Notably, surugamide A at a concentration of 0.1 µmol/L demonstrated particularly outstanding growth-promoting effects on Z. mays roots. Conclusion This study uncovers the novel plant growth-promoting activity of surugamides, offering lead compounds for the development of innovative marine microbial-derived plant growth regulators.