Response of methanogenic community and their activity to temperature rise in alpine swamp meadow at different water level of the permafrost wetland on Qinghai-Tibet Plateau.

Wetlands are an important source of atmospheric methane (CH4) and are sensitive to global climate change. Alpine swamp meadows, accounting for ~50% of the natural wetlands on the Qinghai-Tibet Plateau, were considered one of the most important ecosystems. Methanogens are important functional microbes that perform the methane producing process. However, the response of methanogenic community and the main pathways of CH4 production to temperature rise remains unknown in alpine swamp meadow at different water level in permafrost wetlands. In this study, we investigated the response of soil CH4 production and the shift of methanogenic community to temperature rise in the alpine swamp meadow soil samples with different water levels collected from the Qinghai-Tibet Plateau through anaerobic incubation at 5°C, 15°C and 25°C. The results showed that the CH4 contents increased with increasing incubation temperature, and were 5-10 times higher at the high water level sites (GHM1 and GHM2) than that at the low water level site (GHM3). For the high water level sites (GHM1 and GHM2), the change of incubation temperatures had little effect on the methanogenic community structure. Methanotrichaceae (32.44-65.46%), Methanobacteriaceae (19.30-58.86%) and Methanosarcinaceae (3.22-21.24%) were the dominant methanogen groups, with the abundance of Methanotrichaceae and Methanosarcinaceae having a significant positive correlation with CH4 production (p < 0.01). For the low water level site (GHM3), the methanogenic community structure changed greatly at 25°C. The Methanobacteriaceae (59.65-77.33%) was the dominant methanogen group at 5°C and 15°C; In contrast, the Methanosarcinaceae (69.29%) dominated at 25°C, and its abundance showed a significant positive correlation with CH4 production (p < 0.05). Collectively, these findings enhance the understanding of methanogenic community structures and CH4 production in permafrost wetlands with different water levels during the warming process.

Wnt5a/Ca2+ signaling regulates silica-induced ferroptosis in mouse macrophages by altering ER stress-mediated redox balance.

Silicosis is a chronic pulmonary disease characterized by diffuse fibrosis of lung caused by the deposition of silica dust (SiO2). The inhaled silica-induced oxidative stress, ROS production and macrophage ferroptosis are key drivers of the pathological process of silicosis. However, mechanisms that involved in the silica-induced macrophage ferroptosis and its contributions to pathogenesis of silicosis remain elusive. In the present study, we showed that silica induced murine macrophage ferroptosis, accompanied by elevation of inflammatory responses, Wnt5a/Ca2+ signaling activation, and concurrent increase of endoplasmic reticulum (ER) stress and mitochondrial redox imbalance in vitro and vivo. Mechanistic study further demonstrated that Wnt5a/Ca2+ signaling played a key role in silica-induced macrophage ferroptosis by modulating ER stress and mitochondrial redox balance. The presence of Wnt5a/Ca2+ signaling ligand Wnt5a protein increased the silica-induced macrophage ferroptosis by activating ER-mediated immunoglobulin heavy chain binding protein (Bip)-C/EBP homology protein (Chop) signaling cascade, reducing the expression of negative regulators of ferroptosis, glutathione peroxidase 4 (Gpx4) and solute carrier family 7 member 11 (Slc7a11), subsequentially increasing lipid peroxidation. The pharmacologic inhibition of Wnt5a signaling or block of calcium flow exhibited an opposite effect to Wnt5a, resulted in the reduction of ferroptosis and the expression of Bip-Chop signaling molecules. These findings were further corroborated by the addition of ferroptosis activator Erastin or inhibitor ferrostatin-1. These results provide a mechanism by which silica activates Wnt5a/Ca2+ signaling and ER stress, sequentially leads to redox imbalance and ferroptosis in mouse macrophage cells.

Comparative genomics analysis of the multidrug-resistant Aeromonas hydrophila MX16A providing insights into antibiotic resistance genes.

In this paper, the whole genome of the multidrug-resistant Aeromonas hydrophila MX16A was comprehensively analyzed and compared after sequencing by PacBio RS II. To shed light on the drug resistance mechanism of A. hydrophila MX16A, a Kirby-Bauer disk diffusion method was used to assess the phenotypic drug susceptibility. Importantly, resistance against ß-lactam, sulfonamides, rifamycins, macrolides, tetracyclines and chloramphenicols was largely consistent with the prediction analysis results of drug resistance genes in the CARD database. The varied types of resistance genes identified from A. hydrophila MX16A revealed multiple resistance mechanisms, including enzyme inactivation, gene mutation and active effusion. The publicly available complete genomes of 35 Aeromonas hydrophila strains on NCBI, including MX16A, were downloaded for genomic comparison and analysis. The analysis of 33 genomes with ANI greater than 95% showed that the pan-genome consisted of 9556 genes, and the core genes converged to 3485 genes. In summary, the obtained results showed that A. hydrophila exhibited a great genomic diversity as well as diverse metabolic function and it is believed that frequent exchanges between strains lead to the horizontal transfer of drug resistance genes.

Heme oxygenase-1 modulates ferroptosis by fine-tuning levels of intracellular iron and reactive oxygen species of macrophages in response to Bacillus Calmette-Guerin infection.

Macrophages are the host cells and the frontline defense against Mycobacterium tuberculosis (Mtb) infection, and the form of death of infected macrophages plays a pivotal role in the outcome of Mtb infections. Ferroptosis, a programmed necrotic cell death induced by overwhelming lipid peroxidation, was confirmed as one of the mechanisms of Mtb spread following infection and the pathogenesis of tuberculosis (TB). However, the mechanism underlying the macrophage ferroptosis induced by Mtb infection has not yet been fully understood. In the present study, transcriptome analysis revealed the upregulation of heme oxygenase-1 (HMOX1) and pro-ferroptosis cytokines, but downregulation of glutathione peroxidase 4 (GPX4) and other key anti-lipid peroxidation factors in the peripheral blood of both patients with extra-pulmonary tuberculosis (EPTB) and pulmonary tuberculosis (PTB). This finding was further corroborated in mice and RAW264.7 murine macrophage-like cells infected with Bacillus Calmette-Guerin (BCG). A mechanistic study further demonstrated that heme oxygenase-1 protein (HO-1) regulated the production of reactive oxygen species (ROS) and iron metabolism, and ferroptosis in BCG-infected murine macrophages. The knockdown of Hmox1 by siRNA resulted in a significant increase of intracellular ROS, Fe2+, and iron autophagy-mediated factor Ncoa4, along with the reduction of antioxidant factors Gpx4 and Fsp1 in macrophages infected with BCG. The siRNA-mediated knockdown of Hmox1 also reduced cell survival rate and increased the release of intracellular bacteria in BCG-infected macrophages. By contrast, scavenging ROS by N-acetyl cysteine led to the reduction of intracellular ROS, Fe2+, and Hmox1 concentrations, and subsequently inhibited ferroptosis and the release of intracellular BCG in RAW264.7 cells infected with BCG. These findings suggest that HO-1 is an essential regulator of Mtb-induced ferroptosis, which regulates ROS production and iron accretion to alter macrophage death against Mtb infections.

Microarray Profiling and Co-Expression Network Analysis of LncRNAs and mRNAs in Acute Respiratory Distress Syndrome Mouse Model.

BACKGROUND: Long noncoding RNAs (LncRNAs) play critical roles in many respiratory diseases. Acute respiratory distress syndrome (ARDS) is a destructive clinical syndrome of respiratory diseases. However, the potential mechanism of LncRNAs on ARDS remains largely unknown. METHODS: To identify the profiles of LncRNAs and mRNAs in the LPS-induced ARDS mouse model, the microarray analyses were hired to detect the expression of LncRNAs and mRNAs in present study. Subsequently, microarray data were verified by quantitative qRT-PCR. Functional annotation on DE mRNAs and LncRNAs were carried out by bioinformatics analysis. Furthermore, the role of selected DE LncRNAs on correlated genes was confirmed by si-RNA and Western blot. RESULTS: The expression of 2110 LncRNAs and 2690 mRNAs were significantly changed, which were further confirmed by qRT-PCR. GO and KEGG analysis indicated that the up-regulated mRNAs were mainly related to a defense response and tumor necrosis factor (TNF) signaling pathway, respectively. LncRNA-mRNA co-expression analyses showed that LncRNAs NR_003508, ENSMUST00000131638, ENSMUST00000119467, and ENSMUST00000124853 may correlate to MLKL, RIPK3, RIPK1, Caspase1, and NLRP3, respectively, or cooperatively, which were highly involved in the cell necroptosis process. Furthermore, siRNA for NR_003508 confirmed the co-expression analyses results. CONCLUSION: To summarize, this study implied that the DE LncRNAs could be potent regulators and target genes of ARDS and will provide a novel insight into the regulation of the pathogenesis of ARDS.

Corrigendum: Heme oxygenase-1 modulates ferroptosis by fine-tuning levels of intracellular iron and reactive oxygen species of macrophages in response to Bacillus Calmette-Guerin infection.

[This corrects the article DOI: 10.3389/fcimb.2022.1004148.].

Serum Levels of Seven General Cytokines in Acute Brucellosis Before and After Treatment.

PURPOSE: Human brucellosis is the most common bacterial zoonosis globally that poses a severe health threat. Despite the availability of antibiotic therapy for human brucellosis, its tendencies of chronicity and persistence may lead to severe debilitating and disabling conditions in patients. Comprehensive understanding of the immune response in brucellosis will be helpful in improving the treatment strategies. In this study, we measured serum levels of T helper cell type 1 (Th1), Th2, and Th17 cytokines in patients with acute brucellosis before and after treatment. PATIENTS AND METHODS: Overall, 30 patients with acute brucellosis from the Beijing Di Tan Hospital and 26 healthy controls were enrolled in this study. All the patients received a 6-week therapy regimen comprising ceftriaxone, doxycycline, and rifampicin. Serum samples were collected from patients with acute Brucella infection and healthy controls before and after treatment. Serum seven cytokine levels of Th1 (IL-2, IFN-γ, and TNF-α), Th2 (IL-4, IL-6, IL-10), and Th17 (IL-17A) were measured using cytometric bead array. RESULTS: In patients with acute infection, the IL-2, IFN-γ, and IL-10 levels were significantly increased compared with those in healthy controls (P < 0.001). After treatment, IL-2, IFN-γ, and IL-10 levels significantly decreased (P < 0.05) and the TNF-α level significantly increased compared with the corresponding baseline levels and those in healthy controls (P < 0.05). Furthermore, the IFN-γ, IL-4, and IL-10 levels were higher in patients after treatment than in healthy controls (P < 0.05). IL-2 and IL-6 levels exhibited a positive correlation with the C-reactive protein (CRP) level in acute brucellosis (P < 0.05). CONCLUSION: Levels of most serum Th1 and Th2 cytokines were simultaneously increased in acute infection, followed by reduction in the corresponding cytokine levels and residual cytokine response during treatment. This residual immune response could represent a therapeutic opportunity that may improve the long-term clinical outcomes in patients with acute brucellosis after treatment.

Impact of knockdown LincRNA-Cox2 on apoptosis of macrophage infected with Bacillus Calmette-Guérin.

Mycobacterium tuberculosis (Mtb)-induced apoptosis of alveolar macrophages plays an important role in the pathogenesis of tuberculosis. Previous studies indicated that massive LncRNAs could deteriorate MTB invasion or latent infection by regulating macrophage's apoptosis. However, whether LincRNA-Cox2 is involved in apoptosis of macrophage infected with Mtb is unclear. In this study, we found Bacillus Calmette-Guerin(BCG)infection induced cell apoptosis with a increasing LincRNA-Cox2 expression in RAW264.7 cells. Furthermore, the activation of TLR signal pathway elevated the expression of lincRNA-Cox2. In this regard, we used small interfering RNA to explore the role of LincRNA-Cox2 on regulating apoptosis of RAW264.7 cells infected with BCG. The results showed that si-LincRNA-Cox2 was capable of increased the expression of apoptosis-associated proteins and accumulation of ROS in BCG-infected RAW264.7 cells. Mechanically, si-LincRNA-Cox2 facilitated BCG-induced macrophage apoptosis by activating the intrinsic apoptotic pathway as well as increased the genes expression of PERK/eIF2α/CHOP. These results provide novel insights into host-pathogen interactions and highlight the potential role of LincRNA-Cox2 in regulating apoptosis induced by BCG-infection.

Autophagy plays a protective role during Pseudomonas aeruginosa-induced apoptosis via ROS-MAPK pathway.

Pseudomonas aeruginosa infection can induce alveolar macrophage apoptosis and autophagy, which play a vital role in eliminating pathogens. These two processes are usually not independent. Recently, autophagy has been found to interact with apoptosis during pathogen infections. Nevertheless, the role of autophagy in P. aeruginosa-infected cell apoptosis is unclear. In this study, we explored the impact of P. aeruginosa infection on autophagy and apoptosis in RAW264.7 cells. The autophagy activator rapamycin was used to stimulate autophagy and explore the role of autophagy on apoptosis in P. aeruginosa-infected RAW264.7 cells. The results indicated that P. aeruginosa infection induced autophagy and apoptosis in RAW264.7 cells, and that rapamycin could suppress P. aeruginosa-induced apoptosis by regulating the expression of apoptosis-related proteins. In addition, rapamycin scavenged the cellular reactive oxygen species (ROS) and diminished p-JNK, p-ERK1/2 and p-p38 expression of MAPK pathways in RAW264.7 cells infected with P. aeruginosa. In conclusion, the promotion of autophagy decreased P. aeruginosa-induced ROS accumulation and further attenuated the apoptosis of RAW264.7 cells through MAPK pathway. These results provide novel insights into host-pathogen interactions and highlight a potential role of autophagy in eliminating P. aeruginosa.

Knockdown of fatty acid binding protein 4 exacerbates Bacillus Calmette-Guerin infection-induced RAW264.7 cell apoptosis via the endoplasmic reticulum stress pathway.

Mycobacterial infection can induce alveolar macrophage apoptosis, which plays a vital role in the pathogenesis of tuberculosis. Accumulating evidence has demonstrated that fatty acid oxidation is involved in apoptosis during various pathological processes, including bacterial infection. However, whether fatty acid oxidation regulates mycobacterial infection-induced macrophage apoptosis remains unclear. Hence, the present study aimed to investigate the role of fatty acid binding protein 4 (FABP4) which is a carrier protein for fatty acids, in regulating apoptosis in RAW264.7 cells infected with Bacillus Calmette-Guerin (BCG). In our study, the impact of BCG infection on apoptosis and fatty acid oxidation in RAW264.7 cells was examined. Notably, we found that FABP4 was overexpressed during this process. Furthermore, small interfering RNAs targeting FABP4 were used to investigate the role of FABP4 in regulating apoptosis and fatty acid oxidation in BCG-infected RAW264.7 cells. The results indicated that mycobacterial infection promoted apoptosis and enhanced fatty acid oxidation in RAW264.7 cells. Moreover, FABP4 knockdown exacerbated BCG-induced apoptosis and upregulated the expression of p-PERK, p-eIF2α and chop, which are endoplasmic reticulum (ER) stress markers. In addition, FABP4 knockdown promoted fatty acid oxidation and ROS production, which result in the activation of ER stress. Our data suggested that FABP4 knockdown exacerbated BCG-induced apoptosis in RAW264.7 cells via the ER stress pathway.

Shifts in microbial community, pathogenicity-related genes and antibiotic resistance genes during dairy manure piled up.

The uncomposted faeces of dairy cow are usually stacked on cow breeding farms, dried under natural conditions and then used as cow bedding material or they may be continuously piled up. However, no information is available to evaluate variations in the human and animal pathogen genes and antibiotic resistance during the accumulation of fresh faeces of dairy cow to manure. Here, we present the metagenomic analysis of fresh faeces and manure from a dairy farm in Ning Xia, showing a unique enrichment of human and animal pathogen genes and antibiotic resistance genes (ARGs) in manure. We found that manure accumulation could significantly increase the diversity and abundance of the pathogenic constituents. Furthermore, pathogens from manure could spread to the plant environment and enphytotic pathogens could affect the yield and quality of crops during the use of manure as a fertilizer. Levels of virulence genes and ARGs increased with the enrichment of microbes and pathogens when faeces accumulated to manure. Accumulated manure was also the transfer station of ARGs to enrich the ARGs in the environment, indicating the ubiquitous presence of environmental antibiotic resistance genes. Our results demonstrate that manure accumulation and usage without effective manure management is an unreasonable approach that could enrich pathogenic microorganisms and ARGs in the environment. The manure metagenome structure allows us to appreciate the overall influence and interaction of animal waste on water, soil and other areas impacted by faecal accumulation and the factors that influence pathogen occurrence in products from dairy cows.

Magnesium sulfate attenuates lipopolysaccharides-induced acute lung injury in mice.

Acute lung injury (ALI) is a common and severe respiratory disease with high morbidity and mortality. Although some progress has been made in the past years, the pathogenesis of ALI is still poorly understood and the therapeutic outcome has still not been significantly improved. It is well-recognized that magnesium sulfate (MgSO4) possesses potent anti-inflammation capacity. The present study was designed to investigate the protective effects of MgSO4 in lipopolysaccharides (LPSs)-induced ALI taken into account that excessive inflammatory response plays critical role in the development of ALI. In this study, Kunming mice were intravenously injected with LPS through tail vein to establish the ALI model and in parallel, A549 cells were used to establish cell model. The lung wet-to-dry weight ratio, malondialdehyde (MDA) levels in lung tissue, lung permeability index, hematoxylin and eosin staining, cytokines in the serum and bronchoalveolar lavage fluid (BALF), neutrophil counts in BALF, LPS-induced A549 cell apoptosis as well as apoptosis-inducing factor (AIF), and Poly(ADP-ribose) polymerase-1 (PARP-1) expression in both mice and A549 cells were detected. Our results demonstrated that MgSO4 significantly attenuated the LPS-induced ALI, oxidative stress (decreased MDA levels), and lung inflammatory response. Moreover, MgSO4 exerted protective effects by mitigating LPS-induced A549 cell apoptosis. Furthermore, MgSO4 decreased the AIF and PARP-1 expression both in vivo and in vitro. Our results, taken together, demonstrated that MgSO4 is a potential therapeutic agent for ALI taken into consideration that MgSO4 is commonly used in clinical settings.

Characterization of Microbial Communities in a Dairy Farm Matrix in Ningxia, China, by 16S rDNA Analysis.

A large amount of dairy manure is produced annually in the Ningxia Hui Autonomous Region of China due to the increase in food-producing animal agriculture in this region. The presence of bovine-originated zoonotic, especially human, pathogenic bacteria in untreated manure poses a significant threat to the environment and to public health. However, little is known about the composition, diversity, and abundance of bacterial communities in untreated dairy manure in the Ningxia region. In this study, the microbial community structure of the dairy farm matrix was characterized through 16S rDNA sequencing. The impact of manure treatment methods on bacterial communities was also analyzed. The results showed that the microbial community in dairy manure contained both beneficial bacteria and pathogens, with Firmicutes, Bacteroidetes, Proteobacteria, Spirochaetes, and Actinobacteria as dominant phyla. The results also showed the diversity and variety of abundance of zoonotic pathogens among different matrices. The number of pathogens was found to increase significantly in the accumulated but untreated manure, which appeared to be the main matrix of dairy farms that accumulated pathogens including zoonotic pathogens. Findings from this study suggested that farm management, particularly proper treatment of manure, is essential to achieve a shift in the bacterial community composition and a reduction in the environmental load of pathogens including zoonotic pathogens.

A role for Wnt/ß-catenin signalling in suppressing Bacillus Calmette-Guerin-induced macrophage autophagy.

Mycobacterium tuberculosis (Mtb)-induced autophagy of alveolar macrophages has been confirmed to play a central role in the pathogenesis of tuberculosis. Growing evidence indicates that excessive or uncontrolled autophagic activity, which results in type II programmed cell death, can be regulated by many factors, including Wnt/ß-catenin signalling. Wnt/ß-catenin signalling has been demonstrated to be involved in multiple diseases through the regulation of autophagy; however, its exact role in regulating autophagy induced by Mtb remains unclear. Accordingly, this study examined the function of the Wnt/ß-catenin signalling pathway in regulating Mycobacterium bovis Bacillus Calmette-Guerin (BCG)-induced autophagy in RAW264.7 macrophage cell line. In the present study, we found that BCG induced the autophagy of RAW264.7 cells in a time- and dose-dependent manner along with an accumulation of LC3 (Microtubule-associated protein 1 light chain 3) protein. Intriguingly, Wnt3a, a Wnt/ß-catenin signalling ligand, significantly inhibited autophagy, with decreased autophagy rates and autophagic flux. An immunoblot analysis further revealed that Wnt/ß-catenin signalling was capable of inhibiting the expression of the LC3 and autophagy-associated gene (Atg) cascade proteins in BCG-infected cells. Mechanistically, Wnt/ß-catenin signalling may inhibit autophagy in BCG-infected macrophages by activating mTOR-dependent pathways. Our findings reveal the mechanisms of Wnt/ß-catenin signalling regulates cellular autophagy induced by Mtb and provide novel insights into physiological and immune control of tuberculosis by modulating autophagy processes.

The Generation and Characterization of Recombinant Protein and Antibodies of Clostridium perfringens Beta2 Toxin.

Introduction. Clostridium perfringens (C. perfringens) beta2 toxin (CPB2) is an important virulent factor of necrotic enteritis in both animals and humans. However, studies of its pathogenic roles and functional mechanisms have been hampered due to the difficulty of purification and lack of specific antibodies against this toxin. Methods. A recombinant His-tagged C. perfringens beta2 (rCPB2) toxin and monoclonal antibodies (McAbs) against CPB2 were generated and characterized by assays of cytotoxicity, immunoblotting, ELISA, neutralization, and immunofluorescence. Results. A His-tagged rCPB2 with integrity and cytotoxicity of native CPB2 was purified from E. coli expressing system, which exhibited a moderate cytotoxicity on NCM460 human intestinal epithelial cells. The rCPB2 could induce apoptotic cell death rather than necrotic death in part through a pathway involved in caspase-3 signaling. Mechanistically, rCPB2 was able to first bind to cell membrane and dynamically translocate into cytoplasm for its cytotoxic activity. Three McAbs 1E23, 2G7 and 2H7 were characterized to be able to immunologically react with CPB2 and neutralize rCPB2 cytotoxicity on NCM460 cells. Conclusion. These results indicated the rCPB2 and antibodies generated in this study are useful tools for studies of biological functions and pathogenic mechanisms of CPB2 in future, which warrants for further investigations.