Fusobacterium nucleatum-triggered neutrophil extracellular traps facilitate colorectal carcinoma progression.

BACKGROUND: Fusobacterium nucleatum (Fn) acts as a procarcinogenic bacterium in colorectal carcinoma (CRC) by regulating the inflammatory tumor microenvironment (TME). Neutrophil extracellular traps (NETs), which can be generated by persistent inflammation, have been recently considered to be significant contributors in promoting cancer progression. However, whether NETs are implicated in Fn-related carcinogenesis is still poorly characterized. Here, we explored the role of NETs in Fn-related CRC as well as their potential clinical significance. METHODS: Fn was measured in tissue specimens and feces samples from CRC patients. The expression of NET markers were also detected in tissue specimens, freshly isolated neutrophils and blood serum from CRC patients, and the correlation of circulating NETs levels with Fn was evaluated. Cell-based experiments were conducted to investigate the mechanism by which Fn modulates NETs formation. In addition, we clarified the functional mechanism of Fn-induced NETs on the growth and metastasis of CRC in vitro and in vivo experiments. RESULTS: Tissue and blood samples from CRC patients, particularly those from Fn-infected CRC patients, exhibited greater neutrophil infiltration and higher NETs levels. Fn infection induced abundant NETs production in in vitro studies. Subsequently, we demonstrated that Fn-induced NETs indirectly accelerated malignant tumor growth through angiopoiesis, and facilitated tumor metastasis, as manifested by epithelial-mesenchymal transition (EMT)-related cell migration, matrix metalloproteinase (MMP)-mediated basement membrane protein degradation, and trapping of CRC cells. Mechanistically, the Toll-like receptor (TLR4)-reactive oxygen species (ROS) signaling pathway and NOD-like receptor (NOD1/2)-dependent signaling were responsible for Fn-stimulated NETs formation. More importantly, circulating NETs combined with carcinoembryonic antigen (CEA) could predict CRC occurrence and metastasis, with areas under the ROC curves (AUCs) of 0.92 and 0.85, respectively. CONCLUSIONS: Our findings indicated that Fn-induced NETs abundance by activating TLR4-ROS and NOD1/2 signalings in neutrophils facilitated CRC progression. The combination of circulating NETs and CEA was identified as a novel screening strategy for predicting CRC occurrence and metastasis.

Transcriptome-based analysis of the effects of compound microbial agents on gene expression in wheat roots and leaves under salt stress.

Introduction: Salt stress inhibits the beneficial effects of most plant growth-promoting rhizobacteria. The synergistic relationship between beneficial rhizosphere microorganisms and plants helps achieve more stable growth-promoting effects. This study aimed 1) to elucidate changes in gene expression profiles in the roots and leaves of wheat after inoculation with compound microbial agents and 2) to determine the mechanisms by which plant growth-promoting rhizobacteria mediate plant responses to microorganisms. Methods: Following inoculation with compound bacteria, transcriptome characteristics of gene expression profiles of wheat, roots, and leaves at the flowering stage were investigated using Illumina high-throughput sequencing technology. Gene ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on the genes that were significantly differentially expressed. Results: The expression of 231 genes in the roots of bacterial preparations (BIO) -inoculated wheat changed significantly (including 35 upregulated and 196 downregulated genes) compared with that of non-inoculated wheat. The expression of 16,321 genes in leaves changed significantly, including 9651 upregulated genes and 6670 downregulated genes. The differentially expressed genes were involved in the metabolism of carbohydrates, amino acids, and secondary compounds as well as signal transduction pathways. The ethylene receptor 1 gene in wheat leaves was significantly downregulated, and genes related to ethylene-responsive transcription factor were significantly upregulated. GO enrichment analysis showed that metabolic and cellular processes were the main functions affected in the roots and leaves. The main molecular functions altered were binding and catalytic activities, among which the cellular oxidant detoxification enrichment rate was highly expressed in the roots. The expression of peroxisome size regulation was the highest in the leaves. KEGG enrichment analysis showed that linoleic acid metabolism expression was highest in the roots, and the expression of photosynthesis-antenna proteins was the highest in leaves. After inoculation with a complex biosynthesis agent, the phenylalanine ammonia lyase (PAL) gene of the phenylpropanoid biosynthesis pathway was upregulated in wheat leaf cells while 4CL, CCR, and CYP73A were downregulated. Additionally, CYP98A and REF1 genes involved in the flavonoid biosynthesis pathway were upregulated, while F5H, HCT, CCR, E2.1.1.104, and TOGT1-related genes were downregulated. Discussion: Differentially expressed genes may play key roles in improving salt tolerance in wheat. Compound microbial inoculants promoted the growth of wheat under salt stress and improved disease resistance by regulating the expression of metabolism-related genes in wheat roots and leaves and activating immune pathway-related genes.

Elevated neutrophil extracellular traps by HBV-mediated S100A9-TLR4/RAGE-ROS cascade facilitate the growth and metastasis of hepatocellular carcinoma.

BACKGROUND: Neutrophil extracellular traps (NETs) are considered significant contributors to cancer progression, especially metastasis. However, it is still unclear whether NETs are involved in hepatitis B virus (HBV)-related hepatocarcinogenesis and have potential clinical significance during evaluation and management for hepatocellular carcinoma (HCC). In this study, we aimed to investigate the functional mechanism of NETs in HBV-related hepatocarcinogenesis and their clinical significance. METHODS: A total of 175 HCC patients with and without HBV infection and 58 healthy controls were enrolled in this study. NETs were measured in tissue specimens, freshly isolated neutrophils and blood serum from these patients, and the correlation of circulating serum NETs levels with malignancy was evaluated. The mechanism by which HBV modulates NETs formation was explored using cell-based studies. In addition, in vitro and in vivo experiments were further performed to clarify the functional mechanism of NETs on the growth and metastasis of HCC. RESULTS: We observed an elevated level of NETs in blood serum and tissue specimens from HCC patients, especially those infected with HBV. NETs facilitated the growth and metastasis of HCC both in vitro and in vivo, which were mainly dominated by increased angiogenesis, epithelial-mesenchymal transition (EMT)-related cell migration, matrix metalloproteinases (MMPs)-induced extracellular matrix (ECM) degradation and NETs-mediated cell trapping. Inhibition of NETs generation by DNase 1 effectively abrogated the NETs-aroused HCC growth and metastasis. In addition, HBV-induced S100A9 accelerated the generation of NETs, which was mediated by activation of toll-like receptor (TLR4)/receptor for advanced glycation end products (RAGE)-reactive oxygen species (ROS) signaling. Further, circulatory NETs were found to correlate with viral load, TNM stage and metastasis status in HBV-related HCC, and the identified NETs could predict extrahepatic metastasis, with an area under the ROC curve (AUC) of 0.83 and 90.3% sensitivity and 62.8% specificity at a cutoff value of 0.32. CONCLUSIONS: Our findings indicated that activation of RAGE/TLR4-ROS signaling by HBV-induced S100A9 resulted in abundant NETs formation, which subsequently facilitated the growth and metastasis of HCC cells. More importantly, the identified circulatory NETs exhibited potential as an alternative biomarker for predicting extrahepatic metastasis in HBV-related HCC.

Biocontrol and plant growth promotion by combined Bacillus spp. inoculation affecting pathogen and AMF communities in the wheat rhizosphere at low salt stress conditions.

Applying plant growth-promoting rhizobacteria (PGPR) improves the efficiency of soil-borne disease control and is considered a sustainable practice. However, the effect of PGPR on the fungal community, especially pathogenic fungi and arbuscular mycorrhizal fungi (AMF), remains unclear. In this study, we examined the effects of a compound microbial agent (consisting of Bacillus subtilis HG-15 and Bacillus velezensis JC-K3) on the incidence and yield of wheat under low salt stress, as well as compared the diversity and community composition of the rhizosphere fungal and AMF communities of wheat in the CK (not inoculated bacterial agent) and BIO (inoculated with a bacterial agent) groups. Chlorophyll relative content (SPAD), net photosynthesis rate (Pn), transpiration rate (Tr), leaf water use efficiency (WUE L), grains per spike and wheat yield in the BIO group increased more than in the CK group. The number of diseased plants and disease incidence was observed to be reduced. The relative efficacy reached 79.80%. We classified 1007 fungal operational taxonomic units (OTU) based on Miseq sequencing data: 11 phyla, 173 families, 319 genera, and 521 species. Fifty-four OTUs were classified from the AMF effective sequences, including 1 phylum, 3 families, 3 genera, and 17 species. The inoculation of bacterial agents reduced the relative abundance of pathogen genera such as Gibberella, Fusarium, Cladosporium, and Alternaria in wheat rhizosphere. It increased the relative abundance of AMF species such as Glomus-group-B-Glomus-lamellosu-VTX00193, Glomus-viscosum-VTX00063, and Glomus-Glo2-VTX00280. In addition, pH, EC, exchangeable K, available N, total N, organic matter, and olsen P were the main driving forces for shaping wheat rhizosphere fungi. The pH value was positively correlated with the relative abundance of fungal communities in soil, especially Gibberella, Cladosporium, Fusarium, and Alternaria. In summary, inoculation with Bacillus subtilis HG-15 and Bacillus velezensis JC-K3 affected wheat yield, incidence, rhizosphere soil chemical properties, rhizosphere fungi, and AMF fungal diversity and community. The findings may provide a theoretical foundation and strain support for constructing efficient PGPR-community and clarifying its mechanism of pathogenic bacteria inhibition.

S100A8-Mediated NLRP3 Inflammasome-Dependent Pyroptosis in Macrophages Facilitates Liver Fibrosis Progression.

NLRP3 inflammasome-dependent pyroptosis has been implicated in liver fibrosis progression. However, the definite intrahepatic cell types that undergo pyroptosis and the underlying mechanism as well as the clinical importance remain unclear. Here, augmented levels of pyroptosis-related indicators GSDMD, IL-1ß, and IL-18 were verified in both liver fibrosis patients and CCl4-induced fibrotic mouse model. Confocal imaging of NLRP3 with albumin, F4/80 or α-SMA revealed that enhanced NLRP3 was mainly localized to kupffer cells (KCs), indicating that KCs are major cell types that undergo pyroptosis. Targeting pyroptosis by inhibitor MCC950 attenuated the severity and ameliorated liver function in fibrosis models. In addition, elevated S100A8 in liver fibrosis patients was correlated with pyroptosis-related indicators. S100A8 stimulated pyroptotic death of macrophages, which resulted in activation of human hepatic stellate cell line LX-2 cells and increased collagen deposition. Mechanistically, S100A8 activated TLR4/NF-κB signaling and upregulated its target genes NLRP3, pro-IL-1ß, and pro-IL-18 expression, and induced reactive oxygen (ROS) abundance to activate NLRP3 inflammasome, finally leading to pyroptotic cell death in macrophages. More importantly, circulating GSDMD had the optimal predicting value for liver fibrosis progression. In conclusion, S100A8-mediated NLRP3 inflammasome-dependent pyroptosis by TLR4/NF-κB activation and ROS production in macrophages facilitates liver fibrosis progression. The identified GSDMD has the potential to be a biomarker for liver fibrosis evaluation.

Genetic Diversity and in vitro Activity of Aztreonam/Avibactam and Ceftazidime/Avibactam Against Carbapenem-Resistant Enterobacterales: A Multi-Center Study in Southwest China.

Purpose: This study aimed to understand the distribution characteristics of carbapenemase genes and assess the antimicrobial activities of aztreonam/avibactam (ATM/AVI) and ceftazidime/avibactam (CAZ/AVI) against carbapenem-resistant Enterobacterales (CRE) isolates in Chongqing, Southwest China. Methods: CRE isolates and their clinical information were collected from 22 hospitals covering all the five regions across Chongqing between January 1, 2016 and December 31, 2017. PCR was used to screen for common carbapenemase genes. And minimum inhibitory concentrations (MICs) were determined by broth microdilution method. Results: A total of 312 unduplicated CRE isolates (eg, 206 Klebsiella pneumoniae, 43 Escherichia coli, and 42 Enterobacter cloacae) were collected during the two-year study period. Among these CRE isolates, 92.3% carried carbapenemase genes, with a majority of isolates carrying single bla KPC-2 (47.1%) or single bla NDM/IPM (36.2%) and 8.9% of isolates carrying two or three carbapenemase genes. Notably, 95.6% (197/206) K. pneumoniae, 86.0% (37/43) E. coli and 88.1% (37/42) E. cloacae harbored carbapenemase genes. In addition, bla KPC-2 was prevalent in K. pneumoniae (70.4%), while bla NDM was predominant in E. coli (83.7%) and E. cloacae (78.6%). Besides, only metallo-ß-lactamase (MBL) genes were detected in the CRE isolates from children. Overall, 0.0%, 48.1%, 59.0%, 61.5% and 63.1% of the CRE isolates were resistant to ATM/AVI, CAZ/AVI, nitrofurantoin, amikacin and trimethoprim/sulfamethoxazole, respectively. 99.7% of the total 312 isolates could be killed by ATM/AVI with the MIC 1 µg/mL, whereas CAZ/AVI showed good antibacterial activity (98.0% susceptible) against the bla KPC-2-carriers with the MIC50/90 values of 1/4 µg/mL. Conclusion: The distribution features of carbapenemase genes in Chongqing were comprehensively illustrated in terms of species and sources of CRE for the first time in this multi-center study that covered all the geographical locations across Chongqing. ATM/AVI showed superior activity against all CRE isolates regardless of their genotype, whereas CAZ/AVI was active against almost all KPC-producers.

Long transcripts minus touchdown qPCR (LTMT-qPCR): a simplified and convenient method for the screening and quantification of microRNA profiles.

Due to the short length and differences in abundance of microRNAs, microRNA profile screening and quantification is challenging. In this study, we found that size selection magnetic beads could be employed to easily and efficiently remove long RNA transcripts. After removing the long transcripts, the remaining small RNAs could be concentrated and then reverse-transcribed using universal stem-loop primers (USLP), with six randomized nucleotides at the 3' end region. The efficiency of reverse transcription decreased when the number of randomized nucleotides was reduced. In addition, we found that touchdown qPCR improved microRNA profile detection, with lower CT values and better detection efficiency than the regular qPCR protocol, especially for those low-abundance microRNAs. Finally, we incorporated these observations to create a new protocol we named long transcripts minus touchdown qPCR (LTMT-qPCR). We performed a side-by-side comparison of LTMT with USLP and traditional stem-loop primer (TSLP) protocols. We found that LTMT has higher detection efficiency than USLP, especially for the detection of low-abundance microRNAs. Although LTMT was equivalent to TSLP in terms of microRNA profile detection, LTMT is more convenient, user-friendly, and cost-effective. Taken together, the present data indicate that LTMT is a simple, rapid, and user-friendly approach that has higher precision, accuracy, and sensitivity than the previously described methods, making it more suitable for microRNA profile screening and quantification.

The Novel Methylation Biomarker SCARA5 Sensitizes Cancer Cells to DNA Damage Chemotherapy Drugs in NSCLC.

BACKGROUND: Scavenger Receptor Class A Member 5 (SCARA5), also known as TESR, is expressed in various tissues and organs and participates in host defense. Recent studies have found SCARA5 to produce an anti-tumor effect for multiple tumors, although the mechanistic basis for the effect is unknown. METHODS: Bioinformatics, methylation-specific polymerase chain reaction (MSP), quantitative real-time PCR, and immunohistochemistry were used to assess promoter methylation and expression of SCARA5 in lung cancer tissues and cell lines. The biological effect of SCARA5 on lung cancer cells was confirmed by the CCK8 assay, colony formation assay, and flow cytometry. GSEA, Western blot, RNA sequencing, and luciferase-based gene reporter assay were used to explore the mechanistic basis for the anti-tumor effect of SCARA5. Chemosensitivity assays were used to evaluate the anti-tumor effect of SCARA5 in conjunction with chemotherapeutic drugs. RESULTS: We found SCARA5 to be downregulated in lung cancer cell lines and tissues with SCARA5 levels negatively related to promoter methylation. Ectopic expression of SCARA5 suppressed proliferation of lung cancer both in vitro and in vivo through upregulation of HSPA5 expression, which inhibited FOXM1 expression resulting in G2/M arrest of the A549 cell line. SCARA5 also improved susceptibility of A549 cells to chemotherapeutic drugs that damage DNA. CONCLUSION: SCARA5 was silenced in NSCLC due to promoter methylation and could be a potential tumor marker in NSCLC.

Fusobacterium nucleatum Facilitates M2 Macrophage Polarization and Colorectal Carcinoma Progression by Activating TLR4/NF-κB/S100A9 Cascade.

Fusobacterium nucleatum (Fn) has been considered as a significant contributor in promoting colorectal carcinoma (CRC) development by suppressing host anti-tumor immunity. Recent studies demonstrated that the aggregation of M2 macrophage (Mφ) was involved in CRC progress driven by Fn infection. However, the underlying molecular mechanisms are poorly characterized. Here, we investigated the role of Fn in Mφ polarization as well as its effect on CRC malignancy. Fn infection facilitated differentiation of Mφ into the M2-like Mφ phenotype by in vitro study. Histological observation from Fn-positive CRC tissues confirmed the abundance of tumor-infiltrating M2-like Mφ. Fn-induced M2-like Mφ polarization was weakened once inhibiting a highly expressed damage-associated molecular pattern (DAMP) molecule S100A9 mainly derived from Fn-challenged Mφ and CRC cells. In addition, Fn-challenged M2-like Mφ conferred CRC cells a more malignant phenotype, showing stronger proliferation and migration characteristics in vitro and significantly enhanced tumor growth in vivo, all of which were partially inhibited when S100A9 was lost. Mechanistic studies further demonstrated that activation of TLR4/NF-κB signaling pathway mediated Fn-induced S100A9 expression and subsequent M2-like Mφ activation. Collectively, these findings indicate that elevated S100A9 in Fn-infected CRC microenvironment participates in M2-like Mφ polarization, thereby facilitating CRC malignancy. Furthermore, targeting TLR4/NF-κB/S100A9 cascade may serve as promising immunotherapeutic strategy for Fn-associated CRC.

Identification and Bioactivity of Compounds from the Mangrove Endophytic Fungus Alternaria sp.

Racemic new cyclohexenone and cyclopentenone derivatives, (±)-(4R*,5S*,6S*)-3-amino-4,5,6-trihydroxy-2-methoxy-5-methyl-2-cyclohexen-1-one (1) and (±)-(4S*,5S*)-2,4,5-trihydroxy-3-methoxy-4-methoxycarbonyl-5-methyl-2-cyclopenten-1-one (2), and two new xanthone derivatives 4-chloro-1,5-dihydroxy-3-hydroxymethyl-6-methoxycarbonyl-xanthen-9-one (3) and 2,8-dimethoxy-1,6-dimethoxycarbonyl-xanthen-9-one (4), along with one known compound, fischexanthone (5), were isolated from the culture of the mangrove endophytic fungus Alternaria sp. R6. The structures of these compounds were elucidated by analysis of their MS (Mass), one and two dimensional NMR (nuclear magnetic resonance) spectroscopic data. Compounds 1 and 2 exhibited potent ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)] scavenging activities with EC50 values of 8.19 ± 0.15 and 16.09 ± 0.01 µM, respectively. In comparison to Triadimefon, compounds 2 and 3 exhibited inhibitory activities against Fusarium graminearum with minimal inhibitory concentration (MIC) values of 215.52 and 107.14 µM, respectively, and compound 3 exhibited antifungal activity against Calletotrichum musae with MIC value of 214.29 µM.