Objective To compare the stress tolerance of recombinant Mycobacterium smegmatis strains Ms-PPE61 and Ms-Vec under different external stress conditions, investigate the activation/inhibition levels of the mitogen-activated protein kinase (MAPK)/nuclear factor (NF)-κB signaling pathway following their infection of macrophages, and explore differences in inflammatory cytokine expression after infection of RAW264.7 cells. Methods Ms-Vec and Ms-PPE61 were constructed and cultured to the logarithmic growth phase before being subjected to acidic, SDS, and H₂O₂ conditions. Colony-forming units (CFUs) were measured at different time points. Proteins were extracted from cells collected 1-48 h post-infection (hpi), and the expression levels of signaling pathway marker molecules were determined by Western blotting. The interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1β concentrations in the supernatants of RAW264.7 cells infected with Ms-Vec and Ms-PPE61 were measured by ELISA at 24 hpi and 48 hpi. GraphPad Prism 7.0 was used for analysis of variance of the data, and P<0.05 was considered significant. Results PCR revealed the presence of a target band in Ms-PPE61 but not in Ms-Vec. Coomassie brilliant blue staining confirmed consistent protein loading. Western blotting showed that Ms-PPE61 expressed a ~42 kDa Flag fusion protein, while Ms-Vec did not. Ultra-high-speed centrifugation was performed to separate the components of M. smegmatis. Western blotting revealed that the cytoplasmic marker protein GroES was expressed in the cytoplasmic fractions of both Ms-Vec and Ms-PPE61, while the Flag-tagged target protein was exclusively present in the cell wall of Ms-PPE61. After treatment under acidic conditions (pH 3.0) for 3 h, the survival rate of Ms-PPE61 was higher than that of Ms-Vec (P<0.000 1), while the survival rate showed no significant difference after treatment for 6 h and 9 h (P>0.05). After treatment with 0.2% SDS for 3, 6, and 9 h, the survival rate of Ms-PPE61 was higher than that of Ms-Vec (P<0.000 1). Similarly, after H₂O₂ treatment for 3 h and 6 h, the survival rate of Ms-PPE61 was higher than that of Ms-Vec (P<0.000 1). Western blotting showed that the Ms-PPE61 group had significantly lower p-p38 and p-ERK levels at 48 hpi and higher IκB-α levels at all time points than the Ms-Vec group. ELISA results indicated no differences in TNF-α secretion between the Ms-PPE61 and Ms-Vec groups at 24 hpi and 48 hpi (P>0.05), while the Ms-PPE61 group had lower IL-6 levels at 24 hpi and 48 hpi (P<0.000 1) and lower IL-1β level at 48 hpi (P<0.01) than the Ms-Vec group. Conclusion PPE61 can enhance the tolerance of recombinant Mycobacterium smegmatis to acidic, SDS and H₂O₂ stress, inhibit the MAPK and NF-κB signaling pathways by down-regulating the expression of p-p38 and p-ERK and up-regulating the expression of IκB-α, and reduce the secretion of IL-6 (significantly at both 24 h and 48 h) and IL-1β (significantly at 48 h) in macrophages, but has no significant effect on the secretion of TNF-α.

Objective To investigate the mechanism by which the endophytic fungus Trichoderma harzianum Rifai help the seedlings of the rare medicinal plant Camphora migao (H. W. Li) Y. Yang, Bing Liu & Zhi Yang to defend against drought stress. Methods We simulated different drought stress gradients using the potted weighing method after inoculation of C. migao seedlings with T. harzianumvia rhizosphere injection and investigated the plant growth, physiological, and biochemical indexes. Results Under different drought conditions, inoculation with T. harzianum significantly increased the growth indexes such as biomass, plant height, and root growth of C. migao seedlings, compared with the uninoculated control group. Furthermore, T. harzianum significantly increased the activities of antioxidant enzymes and the content of osmotically regulation substances, reduced the malondialdehyde content, and elevated the content of photosynthetic pigments in the seedling leaves, which effectively mitigated the drought stress effects on the growth and development of C. migao seedlings. Conclusion T. harzianum improved the physiological responses of C. migao seedlings to drought stress by regulating osmotic balance and maintaining the stability of antioxidant system.

Heterotrophic nitrifying-aerobic denitrifying (HN-AD) bacteria can simultaneously complete nitrification and denitrification processes under aerobic conditions, significantly simplifying wastewater treatment procedures. These strains exhibit rapid growth, tolerance to extreme environments, and are widely applied in various wastewater treatments. This review summarizes the nitrogen removal pathways of HN-AD bacteria and highlights the potential of novel immobilization carriers (composite materials, magnetic nanocarriers, and biochar) for nitrogen removal across diverse wastewater sectors. It focuses on elucidating the fundamental principles, application cases, current research status, and future prospects of emerging immobilization technologies, including biomimetic mineralization immobilization, electrospun fiber immobilization, and 3D printing carrier immobilization. The enhanced effects of novel immobilization strategies on improving nitrogen removal efficiency and system stability are discussed. Finally, challenges and future prospects for HN-AD bacterial immobilization technology during fabrication and application are outlined.

Objective To explore the application of hutC sequence analysis in the identification of Burkholderia cepacia complex (Bcc) at the species level. Methods We compared the sequences of hutC and the amino acid sequences of the encoded proteins and conducted phylogenetic analysis to theoretically assess the feasibility of using hutC for species-level identification of Bcc. Primers targeting the hutC of Bcc were designed, and the amplification conditions were optimized. With those of standard strains as templates, the hutC sequences of representative Bcc species were amplified. The sequencing results were compared with NCBI sequences for phylogenetic analysis to validate the theoretical hypothesis. Additionally, single nucleotide polymorphism analysis of hutC sequences was performed to identify species-level characteristic barcodes of Bcc. Results The hutC gene was relatively conserved between Bcc and non-Bcc. With the designed primers and amplification conditions, a 692 bp fragment of hutC was successfully amplified from 12 Bcc standard strains. Except for one strain with taxonomic errors, the remaining 11 strains had the comparison results consistent with those from the culture collection centers. The phylogenetic analysis based on hutC showed that different Bcc species could be clustered with high bootstrap values. A 12 nucleotide characteristic barcode of hutC was identified, which can rapidly distinguish different species of Bcc. Conclusion The gene hutC can serve as a new housekeeping gene target for accurate identification of Bcc at the complex and species levels.

Objective We compared the rhizosphere microbial interaction network structure and keystone taxon identification arising from distinct network construction algorithms, aiming to clarify the characteristics and advantages of each algorithm in inferring microbial interactions and identifying keystone taxa, thereby providing a theoretical basis for methodological selection. Methods Taking the rhizosphere microbial community of Camphora migao (a rare plant) as the model system, we constructed molecular ecological networks with three mainstream algorithms: sparse correlations for compositional data (SparCC), random matrix theory (RMT), and co-occurrence network (CoNet). We comprehensively compared network structural features and keystone taxon identification across algorithms by integrating PICRUSt2 functional prediction with keystone taxa-environmental factor correlation analysis. Results Network construction algorithms significantly influenced the topological properties of networks. SparCC generated highly modular networks (relative modularity index, RM=1.31) with distinct interaction segregation (edge connectivity=0). RMT produced a single-module structure (RM=0.78) and homogeneous connectivity (closeness centralization index=0.22). Integration of 26.0% negative correlations in CoNet reduced modularity (RM=0.95), increased network diameter (33.22 steps), and decreased robustness. Keystone taxon identification was method-dependent. Specifically, CoNet, SparCC, and RMT identified 224.00, 44.00, and 19.00 keystone taxa, respectively, with<9.2% cross-method overlap. Rhizobiales and Acidobacteriales were consistently identified as core keystone taxa by all methods, demonstrating cross-algorithm stability. The correlation analysis with environmental factors confirmed that these shared taxa significantly correlated with β-glucosidase activity, validating their role in cellulose degradation and highlighting methodological consistency in identifying key ecological processes. Conclusion The three algorithms exhibited complementary strengths: CoNet resolved complex competitive interactions; SparCC reliably assessed functional stability; RMT uncovered core functional modules. The correlation analysis with environmental factors validated the cellulose degradation function of keystone taxa, with high cross-method consistency in core ecological process identification. Our work provides a theoretical foundation for elucidating plant-microbe interactions and optimizing microbial network construction.

Agrobacteriumtumefaciens, a classic model organism for plant-microbe interaction research, is a valuable transgenic tool for plants. Phenolic acids secreted by plants after injury can affect the infection of the host by A. tumefaciens. Objective This study investigated the transcription factor PcaR of A. tumefaciens regarding its effects on the metabolism of simple phenolic acids, regulation of the target gene, and effect on the bacterial tumorigenicity in host plants. Methods The A. tumefaciens strain with atu4546 knockout (Δatu4546) and the complement strain C-Δatu4546 were constructed via the suicide plasmid pEX18Km and the plasmid pUCA19 with a strong promoter, respectively. Both Δatu4546 and C-Δatu4546 were tested for growth with p-hydroxybenzoic acid or protocatechuic acid as the sole carbon source and tumorigenicity on carrot stems and Kalanchoe pinnata leaves. In the wild-type strain C58 and Δatu4546, the reporter gene was in situ inserted into the downstream region of the metabolic target gene atu4549. The regulatory link between atu4546 and the target gene was examined based on the β-galactosidase activity. To investigate the self-regulation of PcaR, we constructed the atu4546 self-promoter reporter plasmid. To identify the binding sites of PcaR, we constructed the upstream promoter region reporter plasmid of the target gene to remove or replace the predicted binding sites and then determined the β-galactosidase activity. Results The knockout of atu4546 did not affect the growth of A. tumefaciens on sucrose, but led to the inability to use p-hydroxybenzoic acid or protocatechuic acid as the sole carbon source. The growth was restored after atu4546 was complemented. The tumor weights of carrot stems and K. pinnata leaves infected by Δatu4546 decreased by 34.90% and 52.58%, respectively, and the number of colonies per 0.1 g tumor decreased by 72.19% and 80.54%, respectively. The knockout of atu4546 led to a 102.04% increase in its own promoter activity, which suggested that atu4546 negatively regulated its own expression. Atu4546 boosted the expression of the atu4547-atu4549 gene cluster, as evidenced by a 74.86% decrease in β-galactosidase activity downstream of the target gene in Δatu4546 compared with that in the wild type. The promoter region sequence alteration experiment identified GTGCGATATATACGAAC as the binding site of PcaR. Conclusion This study shows that the transcription factor PcaR is involved in phenolic acid catabolism, negatively regulates itself and stimulates the transcription of the downstream gene pcaIJF. The binding site of PcaR to the target gene is GTGCGATATACGAAC. The knockout of PcaR attenuates the pathogenicity of A. tumefaciens. This study reveals the dual regulation mechanism in the phenolic acid metabolism-pathogenic signaling pathway and expands the theoretical cognition of plant-microbe interactions.

In recent years, the widespread application of new technologies such as viral metagenomics has expanded our understanding of viral diseases in giant pandas. In addition to the previously reported infections with canine distemper virus, rotavirus, and parvovirus, the viruses carried by giant pandas exhibit increasing diversity and unique genetic variation characteristics. The growing population size and density of giant pandas post serious challenges to the population biosecurity, especially the prevention and control of viral diseases. To better understand the epidemiological characteristics of viral diseases in giant pandas, as well as the current status of the prevention and control for the development of effective strategies, we review the studies about viral diseases in giant pandas. This paper systematically elucidates the infection characteristics and hazards of major viral pathogens, as well as diagnostic methods, treatment measures, and preventive strategies. Additionally, the paper explores the bottlenecks and challenges encountered in developing and applying vaccines for giant pandas and the difficulties faced in viral disease research and proposes future research directions and recommendations, aiming to provide a scientific basis for preventing and controlling viral diseases in giant pandas.

Objective The formation of neutrophil extracellular traps (NETs) induced by influenza A virus (IAV) subtype H1N1 was investigated both qualitatively and quantitatively. Methods Mouse bone marrow neutrophils were isolated, purified, and characterized. NETs were induced in vitro using lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA). Additionally, IAV groups with three different titers: one hundred 50% tissue culture infective doses (100 TCID₅₀), 50 TCID₅₀, and 25 TCID₅₀ as well as the normal control group were established, and the intracellular nucleoprotein (NP) mRNA expression levels of the IAV groups were detected using reverse transcription quantitative polymerase chain reaction (RT-qPCR). The effect of each factor on neutrophils was assessed by measuring the concentration of circulating cell-free DNA (cfDNA) in the supernatant of each group using the quantitative SYTOX Green staining method. The NETs structure in each group of cells was observed under a fluorescence microscope after Hoechst 33342 staining. An immunofluorescence assay was performed to detect the expression levels of NET characteristic markers citrullinated histone H3 (CitH3), peptidylarginine deiminase 4 (PAD4), myeloperoxidase (MPO), and neutrophil elastase (NE) proteins, as well as the nuclear co-localization and fluorescence intensity of PAD4 with CitH3, and MPO with NE in each group. The levels of reactive oxygen species (ROS) were determined by using a fluorescent probe assay, and the levels of intracellular CitH3 protein formation were determined by using Western blotting. Results The activity of neutrophils isolated from mouse bone marrow reached 98%, with purities of ≥87%. The expression levels of NP mRNA in the IAV groups were significantly higher than those in the control group. Compared with the control group, the cfDNA levels of the PMA, LPS, and IAV groups were significantly increased, with significant increases in the web-like structures of NETs. The immunofluorescence assay showed that the relative expression levels of MPO, NE, PAD4, and CitH3 proteins were elevated to varying degrees, with the co-localization of PAD4/CitH3 or MPO/NE increased after IAV infection. Moreover, the peak of MPO protein expression was observed before that of NE protein, whereas CitH3 expression paralleled that of PAD4 protein. Additionally, the ROS level was elevated, and the level of CitH3 protein formation was also significantly increased. Conclusion Stimulation of neutrophils by IAV (H1N1) induces NET formation, which may be related to the increased intracellular ROS and PAD4 levels.

Viruses are known as the most abundant and diverse biological entities on Earth and regarded as key ecological drivers in ecosystems. The discovery of giant viruses has challenged the conventional understanding of virology and the definition of life with their microscale-virions, megabase-genome sizes, and remarkably numerous eukaryote-specific genes, which were once considered to be hallmark genes of cellular life but barely seen in viruses. Therefore, these biological characteristics of giant viruses blur the boundary between viruses and cellular life. Metagenomics studies have revealed that giant viruses are globally distributed in marine, freshwater, and soil ecosystems, and their geographical distribution is influenced by environmental factors such as temperature, latitude, and host range. Giant virus genomes include core metabolic genes, which enhance environmental adaptability by regulating host metabolism. In addition, giant viruses may even be involved in the horizontal transfer of antibiotic resistance genes. We review the research progress in giant viruses in terms of their diversity, biogeographic distribution, ecological relationships with hosts and intracellular parasites, reprogramming of host cell metabolic systems, driving forces in biogeochemical cycles, and potential impacts on human health to explore the ecological roles of giant viruses from multiple dimensions. This review aims to revolutionize our knowledge of viruses by revealing the ecological significance of giant viruses and their roles in global biogeochemical cycles.

Helicobacter pylori infection is a major causative factor for chronic gastritis and gastric cancer, while current antibiotic therapies are facing increasingly severe resistance. Probiotics have emerged as a promising approach for anti-H. pylori research due to their high safety. Notably, certain Lactobacillus strains have been demonstrated to effectively alleviate H. pylori-induced inflammatory responses, yet their underlying molecular regulatory mechanisms remain unclear. Objective To investigate the molecular mechanism by which Lactiplantibacillus plantarum ZJ316 inhibits the H. pylori-induced inflammatory response by modulating the p38 mitogen-activated protein kinase (MAPK) signaling pathway in the host cells and assess the regulatory effect of this strain on gastric microecological homeostasis, thus providing a theoretical basis for the development of probiotic therapeutics targeting H. pylori. Methods We integrated cell experiments (human gastric adenocarcinoma cell line AGS) and animal experiments (C57BL/6 mice) and employed Western blotting (to determine the phosphorylation level of p38 MAPK), transcriptome sequencing and RT-qPCR (to analyze differential gene expression), ELISA [to determine the levels of inflammatory cytokines interleukin (IL)-8 and IL-10], 16S rRNA gene sequencing (to unveil the gastric flora structure), and hematoxylin-eosin staining (to observe gastric mucosal damage) to systematically study the intervention effect of L. plantarum ZJ316 on H. pylori infection. Results At the cellular level, L. plantarum ZJ316 inhibited H. pylori-induced p38 MAPK phosphorylation, with the inhibition rates of 21.95% and 33.72% at the time points of 1 h and 2 h, respectively (P<0.01). It down-regulated the expression of pathway genes such as MAP3K8 and FOS, and lowered the mRNA levels of the pro-inflammatory cytokines interferon-γ, tumor necrosis factor-α, and IL-6 by 43.26%, 35.95%, and 51.91%, respectively (P<0.01). The combination of this strain with adezmapimod, a p38 MAPK-specific inhibitor, further enhanced the inhibitory effect. In animal experiments, L. plantarum ZJ316 significantly attenuated gastric mucosal pathological injury and inflammatory response, and 16S rRNA gene sequencing revealed that ZJ316 reduced the relative abundance of pathogenic Pseudomonadota and significantly increased the relative abundance of Bacillota [(54.8±9.9)% vs. (27.8±5.9)%, P<0.01] in the stomach. When ZJ316 was combined with adezmapimod, the relative abundance of Bacteroidota was elevated [(58.5±5.2)% vs. (47.8±6.9)%, P<0.05], and specific beneficial genera such as Alistipes were synergistically enriched (an increase of 69.52% compared with the H. pylori group). Conclusion L. plantarum ZJ316 alleviated the inflammatory response triggered by H. pylori infection by inhibiting the p38 MAPK pathway and remodeled the gastric microecological structure. The findings provide a theoretical basis for the inhibition of H. pylori-induced inflammation by lactobacilli and the development of probiotic-based functional foods.