Sesquiterpenoids with PTP1B Inhibitory Activity and Cytotoxicity from the Edible Mushroom Pleurotus citrinopileatus.

One new perhydrobenzannulated 5,5-spiroketal sesquiterpene, pleurospiroketal F (1), as well as six new modified bisabolene sesquiterpenes pleurotins A-F (2-7) were isolated from solid-state fermentation of Pleurotus citrinopileatus. The structures of compounds 1-7 were determined by NMR and MS spectroscopic analysis. The absolute configuration of 1 was determined by X-ray diffraction analysis, while the absolute configurations of 3-7 were assigned using the in situ dimolybdenum circular dichroism method and circular dichroism data comparison. Protein tyrosine phosphatase 1B plays a crucial role as a negative regulator of the insulin-dependent signal cascades. Therefore, the protein tyrosine phosphatase 1B inhibitor can be used for treating type 2 diabetes mellitus and obesity. Compounds 2 and 6 showed moderate inhibitory effects on protein tyrosine phosphatase 1B with IC50 s of 32.1 µM and 30.5 µM, respectively. The kinetic study confirmed compound 2 to be a noncompetitive inhibitor. Compounds 1-7 did not show cytotoxic activity against cancer cell lines (IC50 > 50 µM).

Anaerostipes hadrus, a butyrate-producing bacterium capable of metabolizing 5-fluorouracil.

Anaerostipes hadrus (A. hadrus) is a dominant species in the human gut microbiota and considered a beneficial bacterium for producing probiotic butyrate. However, recent studies have suggested that A. hadrus may negatively affect the host through synthesizing fatty acid and metabolizing the anticancer drug 5-fluorouracil, indicating that the impact of A. hadrus is complex and unclear. Therefore, comprehensive genomic studies on A. hadrus need to be performed. We integrated 527 high-quality public A. hadrus genomes and five distinct metagenomic cohorts. We analyzed these data using the approaches of comparative genomics, metagenomics, and protein structure prediction. We also performed validations with culture-based in vitro assays. We constructed the first large-scale pan-genome of A. hadrus (n = 527) and identified 5-fluorouracil metabolism genes as ubiquitous in A. hadrus genomes as butyrate-producing genes. Metagenomic analysis revealed the wide and stable distribution of A. hadrus in healthy individuals, patients with inflammatory bowel disease, and patients with colorectal cancer, with healthy individuals carrying more A. hadrus. The predicted high-quality protein structure indicated that A. hadrus might metabolize 5-fluorouracil by producing bacterial dihydropyrimidine dehydrogenase (encoded by the preTA operon). Through in vitro assays, we validated the short-chain fatty acid production and 5-fluorouracil metabolism abilities of A. hadrus. We observed for the first time that A. hadrus can convert 5-fluorouracil to α-fluoro-ß-ureidopropionic acid, which may result from the combined action of the preTA operon and adjacent hydA (encoding bacterial dihydropyrimidinase). Our results offer novel understandings of A. hadrus, exceptionally functional features, and potential applications. IMPORTANCE: This work provides new insights into the evolutionary relationships, functional characteristics, prevalence, and potential applications of Anaerostipes hadrus.

Gut commensal Christensenella minuta modulates host metabolism via acylated secondary bile acids.

A strong correlation between gut microbes and host health has been observed in numerous gut metagenomic cohort studies. However, the underlying mechanisms governing host-microbe interactions in the gut remain largely unknown. Here we report that the gut commensal Christensenella minuta modulates host metabolism by generating a previously undescribed class of secondary bile acids with 3-O-acylation substitution that inhibit the intestinal farnesoid X receptor. Administration of C. minuta alleviated features of metabolic disease in high fat diet-induced obese mice associated with a significant increase in these acylated bile acids, which we refer to as 3-O-acyl-cholic acids. Specific knockout of intestinal farnesoid X receptor in mice counteracted the beneficial effects observed in their wild-type counterparts. Finally, we showed that 3-O-acyl-CAs were prevalent in healthy humans but significantly depleted in patients with type 2 diabetes. Our findings indicate a role for C. minuta and acylated bile acids in metabolic diseases.

AidC, a novel N-acylhomoserine lactonase from the potato root-associated cytophaga-flavobacteria-bacteroides (CFB) group bacterium Chryseobacterium sp. strain StRB126.

N-acylhomoserine lactones (AHLs) are used as quorum-sensing (QS) signal molecules by many gram-negative bacteria. We have reported that Chryseobacterium sp. strain StRB126, which was isolated from the root surface of potato, has AHL-degrading activity. In this study, we cloned and characterized the aidC gene from the genomic library of StRB126. AidC has AHL-degrading activity and shows homology to several metallo-ß-lactamase proteins from Bacteroidetes, although not to any known AHL-degrading enzymes. Purified AidC, as a maltose-binding fusion protein, showed high degrading activity against all tested AHLs, whether short- or long-chain forms, with or without substitution at carbon 3. High-performance liquid chromatography (HPLC) analysis revealed that AidC functions as an AHL lactonase catalyzing AHL ring opening by hydrolyzing lactones. An assay to determine the effects of covalent and ionic bonding showed that Zn(2+) is important to AidC activity both in vitro and in vivo. In addition, the aidC gene could also be PCR amplified from several other Chryseobacterium strains. In conclusion, this study indicated that the aidC gene, encoding a novel AHL lactonase, may be widespread throughout the genus Chryseobacterium. Our results extend the diversity and known bacterial hosts of AHL-degrading enzymes.

Metabolite profiling of human-originated Lachnospiraceae at the strain level.

The human gastrointestinal (GI) tract harbors diverse microbes, and the family Lachnospiraceae is one of the most abundant and widely occurring bacterial groups in the human GI tract. Beneficial and adverse effects of the Lachnospiraceae on host health were reported, but the diversities at species/strain levels as well as their metabolites of Lachnospiraceae have been, so far, not well documented. In the present study, we report on the collection of 77 human-originated Lachnospiraceae species (please refer hLchsp, https://hgmb.nmdc.cn/subject/lachnospiraceae) and the in vitro metabolite profiles of 110 Lachnospiraceae strains (https://hgmb.nmdc.cn/subject/lachnospiraceae/metabolites). The Lachnospiraceae strains in hLchsp produced 242 metabolites of 17 categories. The larger categories were alcohols (89), ketones (35), pyrazines (29), short (C2-C5), and long (C > 5) chain acids (31), phenols (14), aldehydes (14), and other 30 compounds. Among them, 22 metabolites were aromatic compounds. The well-known beneficial gut microbial metabolite, butyric acid, was generally produced by many Lachnospiraceae strains, and Agathobacter rectalis strain Lach-101 and Coprococcus comes strain NSJ-173 were the top 2 butyric acid producers, as 331.5 and 310.9 mg/L of butyric acids were produced in vitro, respectively. Further analysis of the publicly available cohort-based volatile-metabolomic data sets of human feces revealed that over 30% of the prevailing volatile metabolites were covered by Lachnospiraceae metabolites identified in this study. This study provides Lachnospiraceae strain resources together with their metabolic profiles for future studies on host-microbe interactions and developments of novel probiotics or biotherapies.

Genome sequence of Microbacterium testaceum StLB037, an N-acylhomoserine lactone-degrading bacterium isolated from potato leaves.

Microbacterium testaceum is an endophytic Gram-positive bacterium that resides within plant hosts. M. testaceum StLB037 was isolated from potato leaves and shows N-acylhomoserine lactone-degrading activity. Here, we present the 3.98-Mb complete genome sequence of StLB037, with an average GC content of 70.28%.

Role of circular RNA expression in the pathological progression after spinal cord injury.

Differential expression of non-coding RNA after traumatic spinal cord injury (TSCI) is closely related to the pathophysiological process. The purposes of this study were to systematically profile and characterize expression of circular RNA (circRNA) in the lesion epicenter of spinal tissues after TSCI, and predict the structure and potential function of the regulatory circRNA/miRNA network. Forty-eight C57BL/6 mice were randomly and equally assigned to two groups: one subjected to TSCI at T8-10 with an Allen's drop impactor, and a second subjected to laminectomy without TSCI. Spinal cord samples were stained with hematoxylin and eosin, sequenced, and validated. RNA-Seq, Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis, and network analyses (Targetscan and miRanda) were used to predict and annotate the circRNA/miRNA/mRNA network. Luciferase reporter, quantitative reverse transcription polymerase chain reaction, and western blot assays were used to profile expression and regulation patterns of the network in mouse models of TSCI. Hematoxylin-eosin staining revealed severe damage to the blood-spinal cord barrier after TSCI. Differentially expressed circRNA and miRNA profiles were obtained after TSCI; differentially expressed circRNAs, which were abundant in the cytoplasm, were involved in positive regulation of transcription and protein phosphorylation. miR-135b-5p was the most significantly downregulated miRNA after TSCI; circRNAAbca1 and KLF4 were predicted to be its target circRNA and mRNA, respectively. Subsequently, the circAbca1/miR-135b-5P/KLF4 regulatory axis was predicted and constructed, and its targeted binding was verified. After inhibiting circAbca1, GAP43 expression was upregulated. Differential expression of circRNAs might play an important role after TSCI. circAbca1 plays a neuroinhibitory role by targeted binding of the miR-135b-5P/KLF4 axis. The identified circRNA/miRNA/mRNA network could provide the basis for understanding pathophysiological mechanisms underlying TSCI, as well as guide the formulation of related therapeutic strategies. All animal protocols were approved by the Research Ethics Committee of West China Hospital of China (approval No. 2017128) on May 16, 2017.

AiiM, a novel class of N-acylhomoserine lactonase from the leaf-associated bacterium Microbacterium testaceum.

N-Acylhomoserine lactones (AHLs) are used as quorum-sensing signal molecules by many Gram-negative bacteria. We have reported that Microbacterium testaceum StLB037, which was isolated from the leaf surface of potato, has AHL-degrading activity. In this study, we cloned the aiiM gene from the genomic library of StLB037, which has AHL-degrading activity and shows high homology with the alpha/beta hydrolase fold family from Actinobacteria. Purified AiiM as a maltose binding fusion protein showed high degrading activity of AHLs with both short- and long-chain AHLs with or without substitution at carbon 3. High-performance liquid chromatography analysis revealed that AiiM works as an AHL lactonase that catalyzes AHL ring opening by hydrolyzing lactones. In addition, expression of AiiM in the plant pathogen Pectobacterium carotovorum subsp. carotovorum reduced pectinase activity markedly and attenuated soft rot symptoms on potato slices. In conclusion, this study indicated that AiiM might be effective in quenching quorum sensing of P. carotovorum subsp. carotovorum.

Neuroprotective mechanisms of rutin for spinal cord injury through anti-oxidation and anti-inflammation and inhibition of p38 mitogen activated protein kinase pathway.

Rutin has anti-inflammatory, antioxidant, anti-viral, anti-tumor and immune regulatory effects. However, the neuroprotective effects of rutin in spinal cord injury are unknown. The p38 mitogen activated protein kinase (p38 MAPK) pathway is the most important member of the MAPK family that controls inflammation. We assumed that the mechanism of rutin in the repair of spinal cord injury is associated with the inhibition of p38 MAPK pathway. Allen's method was used to establish a rat model of spinal cord injury. The rat model was intraperitoneally injected with rutin (30 mg/kg) for 3 days. After treatment with rutin, Basso, Beattie and Bresnahan locomotor function scores increased. Water content, tumor necrosis factor alpha, interleukin 1 beta, and interleukin 6 levels, p38 MAPK protein expression and caspase-3 and -9 activities in T8-9 spinal cord decreased. Oxidative stress related markers superoxide dismutase and glutathione peroxidase levels increased in peripheral blood. Rutin exerts neuroprotective effect through anti-oxidation, anti-inflammation, anti-apoptosis and inhibition of p38 MAPK pathway.

Chemical diversity from the Tibetan Plateau fungi Penicillium kongii and P. brasilianum.

Two new secondary metabolites, kongiilines A and B (1, 7), and two asperphenamate derivatives, asperphenamates B and C (5-6), together with 16 known compounds (2-4, 8-20), were isolated from Tibetan Plateau fungi Penicillium kongii and Penicillium brasilianum. This is the first report on asperphenamates B and C as naturally occurring compounds, and that aspterric acid is isolated from P. brasilianum for the first time. Their structures were elucidated by different spectroscopic techniques including high-resolution electrospray ionisation mass spectrum, 1D nuclear magnetic resonance (NMR), and 2D NMR as well as electronic circular dichroism. Compounds 4, 5, and 10 exhibited cytotoxicity activities against human colon carcinoma HCT116 cell line with IC50 values of 88.16, 77.68, and 36.92 µM, respectively. Fungi from Tibetan Plateau represent important and rich resources for the investigation of new chemicals.

[Lung cancer diagnosis based on urinary modified nucleoside metabolic profiling].

OBJECTIVE: To study the feasibility of modified urinary nucleosides metabolic profiling on lung cancer diagnoses. METHODS: The modified urinary nucleosides metabolic profiling from 42 normal adults and 80 patients with lung cancer were determined by a coupled-column high performance liquid chromatography system. Partial least squares-discriminant analysis (PLS-DA) models were used to class differentiation between the lung cancer patients and controls and to discover potential biomarkers. RESULT: The PLS-DA model results showed that there was a clear differentiation between normal adults and lung cancers patients, with the value of prediction (Q2) equals to 0.744. CONCLUSION: Modified urinary nucleosides metabolic profiling method is useful for lung cancer diagnoses.

Complete Genome Sequence of Pseudomonas chlororaphis subsp. aurantiaca Reveals a Triplicate Quorum-Sensing Mechanism for Regulation of Phenazine Production.

Pseudomonas chlororaphis subsp. aurantiaca StFRB508 regulates phenazine production through N-acyl-l-homoserine lactone (AHL)-mediated quorum sensing. Two sets of AHL-synthase and AHL-receptor genes, phzI/phzR and aurI/aurR, have been identified from the incomplete draft genome of StFRB508. In the present study, the complete genome of StFRB508, comprising a single chromosome of 6,997,933 bp, was sequenced. The complete genome sequence revealed the presence of a third quorum-sensing gene set, designated as csaI/csaR. An LC-MS/MS analysis revealed that StFRB508 produced six types of AHLs, with the most important AHL being N-(3-hydroxyhexanoyl)-l-homoserine lactone (3-OH-C6-HSL). PhzI mainly catalyzed the biosynthesis of 3-OH-C6-HSL, while AurI and CsaI catalyzed that of N-hexanoyl-l-homoserine lactone and N-(3-oxohexanoyl)-l-homoserine lactone, respectively. A mutation in phzI decreased phenazine production, whereas that in aurI or csaI did not. A phzI aurI csaI triple mutant (508ΔPACI) did not produce phenazine. Phenazine production by 508ΔPACI was stimulated by exogenous AHLs and 3-OH-C6-HSL exerted the strongest effects on phenazine production at the lowest concentration tested (0.1 µM). The plant protection efficacy of 508ΔPACI against an oomycete pathogen was lower than that of wild-type StFRB508. These results demonstrate that the triplicate quorum-sensing system plays an important role in phenazine production by and the biocontrol activity of StFRB508.

[Isolation of Quorum Quenching Bacteria and Their Function for Controlling Membrane Biofouling].

Interspecies quorum quenching by bacterial cells has been reported as a novel approach for mitigating the biofouling via restraining quorum sensing(QS). Five indigenous quorum quenching bacterial strains were isolated from activated sludge taken from wastewater treatment plant. Strain HG10 showed high degrading activity against C6-HSL(N-hexanoyl-L-Homoserine lactone). The result of 16S rDNA sequencing showed that the isolated strain HG10 belonged to the genus Bacillus cereus. Strain HG10 was immobilized in sodium alginate(SA), and the biofouling inhibition of SA-HG10 in membrane filtration treatment system was investigated. The results showed that the membrane flux in experimental group B(provided with SA-HG10)was 181.29 L·(m2·h)-1, and the membrane flux in control group A (without the beads) was 110.64 L·(m2·h)-1, The membrane flux in group B was 63.86% higher than group A. The content of extracellular polymeric substances (EPS) in the biofilm on the membrane filter was also measured. The results showed that the contents of polysaccharides and proteins in the experimental group B decreased by 29% and 48% respectively than those of group A. A large decrease in the content of hydrophobic proteins was the main reason for the decrease of membrane fouling. The content of EPS in the membrane surface decreased by 43%, indicating that SA-HG10 could inhibit biofilm formation on the membrane filter and effectively improve the filtering performance.

Complete Genome Sequence of Chryseobacterium sp. Strain StRB126, an N-Acylhomoserine Lactone-Degrading Bacterium Isolated from Potato Root.

Chryseobacterium sp. strain StRB126 was isolated from a potato root and showed N-acylhomoserine lactone-degrading activity. Here, we present the complete 5,503,743-bp genome sequence of StRB126, which has a G+C content of 35.6% and carries 4,828 protein-coding genes, six rRNA operons, and 80 tRNA genes.

Evaluation of intraspecies interactions in biofilm formation by Methylobacterium species isolated from pink-pigmented household biofilms.

Concern regarding household biofilms has grown due to their widespread existence and potential to threaten human health by serving as pathogen reservoirs. Previous studies identified Methylobacterium as one of the dominant genera found in household biofilms. In the present study, we examined the mechanisms underlying biofilm formation by using the bacterial consortium found in household pink slime. A clone library analysis revealed that Methylobacterium was the predominant genus in household pink slime. In addition, 16 out of 21 pink-pigmented bacterial isolates were assigned to the genus Methylobacterium. Although all of the Methylobacterium isolates formed low-level biofilms, the amount of the biofilms formed by Methylobacterium sp. P-1M and P-18S was significantly increased by co-culturing with other Methylobacterium strains that belonged to a specific phylogenetic group. The single-species biofilm was easily washed from the glass surface, whereas the dual-species biofilm strongly adhered after washing. A confocal laser scanning microscopy analysis showed that the dual-species biofilms were significantly thicker and tighter than the single-species biofilms.

Phenazine antibiotic production and antifungal activity are regulated by multiple quorum-sensing systems in Pseudomonas chlororaphis subsp. aurantiaca StFRB508.

A number of gram-negative bacteria have a quorum-sensing system and produce the N-acylhomoserine lactone (AHL) as a signal molecule. Pseudomonas chlororaphis subsp. aurantiaca StFRB508 produces one of the phenazine derivatives, phenazine-1-carboxylic acid (PCA). Whole-genome sequencing of StFRB508 revealed the presence of two sets of AHL-synthase and AHL-receptor gene, phzIR and aurIR. The mutation of phzI drastically decreased PCA production, but the mutation of aurI did not affect PCA production. The phzI and aurI double mutant did not show any PCA production. StFRB508 produces three major AHLs, N-butyryl-L-homoserine lactone (C4-HSL), N-hexanoyl-L-homoserine lactone (C6-HSL), and N-(3-hydroxyhexanoyl)-L-homoserine lactone (3-hydroxy-C6-HSL). As the results of TLC analysis, PhzI mainly catalyzes the biosynthesis of 3-hydroxy-C6-HSL, and AurI catalyzes the biosynthesis of C4-HSL and C6-HSL. PCA production in the phzI and aurI double mutant was restored by exogenous AHLs and the most active AHL was 3-hydroxy-C6-HSL. StFRB508 showed high inhibitory activity of the development of mycelia of plant pathogenic fungi, Fusarium oxysporum f. sp. conglutinans. However, the phzI and aurI double mutant could not inhibit the development of mycelia. These results demonstrated that the multiple quorum-sensing system play an important role in PCA production and antifungal activity in StFRB508.

Tricycloalternarenes F-H: three new mixed terpenoids produced by an endolichenic fungus Ulocladium sp. using OSMAC method.

Three new mixed terpenoids, tricycloalternarenes (TCAs) F-H (1-3), together with ten known tricycloalternarenes (4-13), were isolated from the Czapek's culture of an endophytic fungus Ulocladium sp. Their structures were identified by extensive spectroscopic experiments (NMR and MS) and comparison with literature data. TCA 1b (5) showed weak activity against the Bacille Calmette-Guerin strain with the MIC of 125µg/mL. TCA 9b (10) exhibited strong cytotoxic activity against Hela cell line with IC50 of 8.58µM.

Diversity and distribution of N-acylhomoserine lactone (AHL)-degrading activity and AHL-lactonase (AiiM) in genus microbacterium.

N-Acylhomoserine lactone (AHL)-degrading enzyme, AiiM, was identified from the potato leaf-associated Microbacterium testaceum StLB037. In this study, we cloned eight aiiM gene homologues from other AHL-degrading Microbacterium strains. The similarity of the chromosomal locus of the aiiM gene is associated with the phylogenetic classification based on 16S rRNA. Degenerate PCR revealed that the aiiM gene was only conserved in AHL-degrading Microbacterium strains, but not in fifteen Microbacterium type strains or two Microbacterium isolates from other plants. These results suggested that the high level of AHL-degrading activity in Microbacterium strains was caused by the aiiM gene encoded on their chromosome.

Tumor necrosis factor-α increases angiopoietin-like protein 2 gene expression by activating Foxo1 in 3T3-L1 adipocytes.

Angiopoietin-like protein 2 (Angptl2) is a key adipocyte-derived inflammatory mediator linking obesity to systemic insulin resistance, which is overexpressed in obesity and related metabolic diseases. However, its regulatory mechanism remains unclear. In this study, we showed that tumor necrosis factor (TNF)-α treatment increased the expression of Angptl2 gene in 3T3-L1 adipocytes. The cloning and sequence analysis of the Angptl2 gene promoter revealed the presence of several putative-binding sites for transcriptional factors, including two IREs. Insulin suppressed Angptl2 mRNA expression in dose-dependent manners, which could be attenuated by a phosphoinositide 3-kinase (PI3K) inhibitor LY294002. The interactions between IRE sites within Angptl2 promoter and forkhead transcription factor Foxo1 were identified by EMSA and ChIP assay. Furthermore, lentivirus-mediated knockdown of Foxo1 expression inhibited the transcriptional activity of Angptl2 promoter and decreased Angptl2 mRNA expression. Finally, TNF-α inhibited Foxo1 phosphorylation and enhanced its transcriptional activity, through which TNF-α increased the expression of Angptl2 in adipocytes. These results suggest that TNF-α up-regulates Angptl2 mRNA expression via PI3K/Foxo1 pathway in 3T3-L1 adipocytes, which may be involved in obesity-induced inflammation and insulin resistance.

[Clinical features and therapy of subjective benign paroxysmal positional vertigo].

OBJECTIVE: To evaluate the clinical features and therapy of subjective benign paroxysmal positional vertigo (S-BPPV). METHODS: By retrospectively analyzing the results of clinical features and therapy in 12 patients with S-BPPV from January 2003 to September 2006, the results were compared with 24 patients with objective benign paroxysmal positional vertigo (O-BPPV) of posterior semicircular canal. RESULTS: S-BPPV patients suffered from attack of transient vertigo with sudden onset triggered by head motion but no concomitant nystagmus in Dix-Hallpike test. The latency and duration of vertigo attack were (4.42 +/- 2.02) s and (8.67 +/- 4.31) s in S-BPPV, (3.2 +/- 1.18) s and (14.75 +/- 4.97) s in O-BPPV of posterior semicircular canal. The differences between the two groups were all significant (t = 2.30, P < 0.05 and t = 3.61, P < 0.01). The symptoms disappeared in 11 patients after a single therapy of particular repositioning maneuver and 1 patient after 2 times therapy in S-BPPV. The one-stage success rate was 91.7% in S-BPPV and 79.2% in O-BPPV of posterior semicircular canal, but the difference between these two groups was not significant. The number of circulation therapy in first management was (1.75 +/- 1.08) times in S-BPPV and (3.38 +/- 1.06) times in O-BPPV of posterior semicircular canal, while the difference was significant (t = 4.32, P < 0.01). There were 2 patients recurred during follow-up in S-BPPV and 7 patients in O-BPPV of posterior semicircular canal, but the difference wasn't significant. CONCLUSIONS: Longer latency, shorter duration and need less circulation therapy are achieved in S-BPPV compared with O-BPPV of posterior semicircular canal, which indicate that the effectiveness of S-BPPV seems to be more favorable than that of O-BPPV of posterior semicircular canal.