[The skin surface microcirculation of conception vessel, governor vessel and thoroughfare vessel in patients with primary dysmenorrhea].

OBJECTIVE: To observe the skin surface microcirculation of acupoints of conception vessel, governor vessel and thoroughfare vessel in patients with primary dysmenorrhea using laser speckle contrast imaging (LSCI), and provide acupoint selection basis of acupuncture-moxibustion for primary dysmenorrhea. METHODS: Ninety-nine healthy female college students with regular menstrual cycles (normal group) and 94 female college students with primary dysmenorrhea (dysmenorrhea group) were recruited. Before menstrual period, on the first day of menstruation, and on the third day after menstruation, LSCI was used to observe the surface microcirculation at the abdominal acupoints of conception vessel, i. e. Yinjiao (CV 7), Qihai (CV 6), Shimen (CV 5), Guanyuan (CV 4), Zhongji (CV 3) and Qugou (CV 2), acupoints of thoroughfare vessel, i. e. Huangshu (KI 16), Zhongzhu (KI 15), Siman (KI 14), Qixue (KI 13), Dahe (KI 12), Henggu (KI 11) and acupoints of lumbosacral region of governor vessel, i. e. Xuanshu (GV 5), Mingmen (GV 4), Yaoyangguan (GV 3), Yaoshu (GV 2) as well as two non-acupoints. RESULTS: Before menstrual period, there was no significant difference in the surface blood perfusion of the acupoints between the dysmenorrhea group and the normal group (P>0.05). On the first day of menstruation, the surface blood perfusion of Xuanshu (GV 5), Mingmen (GV 4), Yaoyangguan (GV 3) and right Huangshu (KI 16) in the dysmenorrhea group was higher than that in the normal group (P<0.05, P<0.01). On the third day after menstruation, the surface blood perfusion of the right Henggu (KI 11) in the dysmenorrhea group was lower than that in the normal group (P<0.05). CONCLUSION: In patients with primary dysmenorrhea, on the first day of menstruation, the surface blood perfusion of Xuanshu (GV 5), Mingmen (GV 4), Yaoyangguan (GV 3) of governor vessel, and the right Huangshu (KI 16) of thoroughfare vessel is increased, while on the third day after menstruation, the surface blood perfusion of the right Henggu (KI 11) of thoroughfare vessel is decreased. These findings might provide a basis for acupoint selection in the acupuncture-moxibustion treatment of primary dysmenorrhea.

Metabolomic and regular analysis reveal phytotoxic mechanisms of sterigmatocystin in Amaranthus retroflexus L.

Sterigmatocystin (STE) is a common hepatotoxic and nephrotoxic contaminant in cereals, however, its phytotoxicity and mechanisms are poorly understood. Here, the phytotoxic mechanisms of STE were investigated via the metabolomics of Amaranthus retroflexus L. A total of 140 and 113 differential metabolites were detected in the leaves and stems, respectively, among which amino acids, lipids, and phenolic compounds were significantly perturbed. Valine, leucine, isoleucine, and lysine biosynthesis were affected by STE. These metabolic responses revealed that STE might be toxic to plants by altering the plasma membrane and inducing oxidative damage, which was verified by measuring the relative electrical conductivity and quantification of reactive oxygen species. The elevated amino acids, as well as the decreased of D-sedoheptuiose-7-phosphate indicated increased proteolysis and carbohydrate metabolism restriction. Furthermore, the IAA level also decreased. This study provides a better understanding of the impacts of STE on the public health, environment and food security.

2E,4E-Decadienoic Acid, a Novel Anti-Oomycete Agent from Coculture of Bacillus subtilis and Trichoderma asperellum.

Phytophthora nicotianae is an oomycete pathogen of global significance threatening many important crops. It is mainly controlled by chemosynthetic fungicides, which endangers ecosystem and human health; thus, there is an urgent need to explore alternatives for these fungicides. In this study, a new anti-oomycete aliphatic compound, 2E,4E-decadienoic acid (DDA), was obtained through coculture of Bacillus subtilis Tpb55 and Trichoderma asperellum HG1. Both in vitro and in vivo tests showed that DDA had a strong inhibitory effect against P. nicotianae. In addition, rhizosphere microbiome analysis showed that DDA reduced the relative abundance of Oomycota in rhizosphere soil. Transcriptome sequencing (RNA-Seq) analysis revealed that treatment of P. nicotianae with DDA resulted in significant downregulation of antioxidant activity and energy metabolism, including antioxidant enzymes and ATP generation, and upregulation of membrane-destabilizing activity, such as phospholipid synthesis and degradation. The metabolomic analysis results implied that the pathways influenced by DDA were mainly related to carbohydrate metabolism, energy metabolism, and the cell membrane. The biophysical tests further indicated that DDA produced oxidative stress on P. nicotianae, inhibited antioxidant enzyme and ATPase activity, and increased cell membrane permeability. Overall, DDA exerts inhibitory activity by acting on multiple targets in P. nicotianae, especially on the cell membrane and mitochondria, and can therefore serve as a novel environment-friendly agent for controlling crop oomycete disease. IMPORTANCE P. nicotianae is an oomycete pathogen that is destructive to crops. Although some oomycete inhibitors have been used during crop production, most are harmful to the ecology and lead to pathogen resistance. Alternatively, medium-chain fatty acids have been reported to exhibit antimicrobial activity in the medical field in previous studies; however, their potential as biocontrol agents has rarely been evaluated. Our in vivo and in vitro analyses revealed that the medium-chain fatty acid 2E,4E-decadienoic acid (DDA) displayed specific inhibitory activity against oomycetes. Further analysis indicated that DDA may acted on multiple targets in P. nicotianae, especially on the cell membrane and mitochondria. Our findings highlight the potential of DDA in controlling oomycete diseases. In conclusion, these results provide insights regarding the future use of green and environment-friendly anti-oomycete natural products for the prevention and control of crop oomycete diseases.

Antifungal Nafuredin and Epithiodiketopiperazine Derivatives From the Mangrove-Derived Fungus Trichoderma harzianum D13.

A new polyketide derivative, nafuredin C (1), a novel heterocyclic dipeptide, trichodermamide G (3), together with four known biogenetically related compounds nafuredin A (2), trichodermamide A (4), aspergillazin A (5), and peniisocoumarin H (6), were isolated from the mangrove-derived fungus Trichoderma harzianum D13. Their structures, including their absolute configurations, were determined by spectroscopic analysis and time-dependent density functional theory-electronic circular dichroism (ECD) calculations. Trichodermamide G was found to be a novel epithiodiketopiperazine derivative with an unprecedented cyclic system containing a sulfur bridge, and nafuredin C represented the third nafuredin derivative of these homologous compounds. The new compound nafuredin C exhibited obvious antifungal activity against Magnaporthe oryzae with a minimum inhibitory concentration (MIC) of 8.63 µM, which is on the same order of magnitude as the positive control carbendazim (MIC = 3.27 µM).

Characterization of a novel cyfluthrin-degrading bacterial strain Brevibacterium aureum and its biochemical degradation pathway.

Brevibacterium aureum DG-12, a new bacterial strain isolated from active sludge, was able to degrade and utilize cyfluthrin as a growth substrate in the mineral medium. Response surface methodology using central composite rotatable design of cultural conditions was successfully employed for optimization resulting in 88.6% degradation of cyfluthrin (50mgL(-1)) within 5days. The bacterium degraded cyfluthrin by cleavage of both the carboxylester linkage and diaryl bond to form 2,2,3,3-tetramethyl-cyclopropanemethanol, 4-fluoro-3-phenexy-benzoic acid, 3,5-dimethoxy phenol, and phenol, and subsequently transformed these compounds with a maximum specific degradation rate, half-saturation constant and inhibition constant of 1.0384day(-1), 20.4967mgL(-1), and 141.9013mgL(-1), respectively. A novel degradation pathway for cyfluthrin was proposed based on analysis of these metabolites. In addition, this strain was found capable of degrading a wide range of synthetic pyrethroid insecticides. Our results suggest that B. aureum DG-12 may be an ideal microorganism for bioremediation of the pyrethroid-contaminated environments.

The global regulator Crc plays a multifaceted role in modulation of type III secretion system in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa utilizes type III secretion system (T3SS) to translocate effector proteins into eukaryotic host cells that subvert normal host cell functions to the benefit of the pathogen, and results in serious infections. T3SS in P. aeruginosa is controlled by a complex system of regulatory mechanisms and signaling pathways. In this study, we described that Crc, an RNA-binding protein, exerts a positive impact on T3SS in P. aeruginosa, as evidenced by promoter activity assays of several key T3SS genes, transcriptomics, RT-PCR, and immunoblotting in crc mutant. We further demonstrated that the regulatory function of Crc on the T3SS was mediated through the T3SS master regulator ExsA and linked to the Cbr/Crc signaling system. Expression profiling of the crc mutant revealed a downregulation of flagship T3SS genes as well as 16 other genes known to regulate T3SS gene expression in P. aeruginosa. On the basis of these data, we proposed that Crc may exert multifaceted control on the T3SS through various pathways, which may serve to fine-tune this virulence mechanism in response to environmental changes and nutrient sources.

A novel two-component system BqsS-BqsR modulates quorum sensing-dependent biofilm decay in Pseudomonas aeruginosa.

Pseudomonas aeruginosa can grow either as planktonic- or biofilm-form in response to environmental changes. Recent studies show that switching from biofilm to planktonic lifestyle requires rhamnolipids. Here we report the identification of a novel twocomponent system BqsS-BqsR that regulates biofilm decay in P. aeruginosa. BqsS is a multidomain sensor kinase and BqsR is an OmpR-like response regulator. Deletion of either bqsS or bqsR in P. aeruginosa mPAO1 resulted in a significant increase in biofilm formation. Time course analysis showed that the bqsS-bqsR mutants were defective in biofilm dispersal and in rhamnolipid production. Mutation of the BqsS-BqsR two-component system did not affect the biosynthesis of long chain quorum sensing (QS) signal N-3-oxo-dodecanoyl-homoserine lactone (3OC12HSL) but resulted in reduced production of the short chain QS signal N-butyryl-L-homoserine lactone (C4HSL) and the Pseudomonas quinolone signal (PQS). Exogenous addition of C4HSL, PQS or rhamnolipids to the bqsS mutant reduced the biofilm formation to the wild-type level. Evidence suggests that the BqsS-BqsR two-component system might promote conversion of anthranilate to PQS. Taken together, these results establish BqsS-BqsR as a novel two-component system that regulates biofilm decay in P. aeruginosa by modulating biosynthesis of QS signals and rhamnolipids.

VqsM, a novel AraC-type global regulator of quorum-sensing signalling and virulence in Pseudomonas aeruginosa.

Human pathogen Pseudomonas aeruginosa uses quorum-sensing (QS) signalling systems to synchronize the production of virulence factors. There are two interrelated QS systems, las and rhl, in P. aeruginosa. In addition to this complexity, a number of transcriptional regulators were shown to have complicated interplays with las and rhl central QS components. Here, we describe a novel virulence and QS modulator (VqsM) that positively regulates the QS systems in P. aeruginosa. Mutation in vqsM resulted in much reduced production of N-acylhomoserine lactones (AHLs) and extracellular enzymes. Sequence analysis revealed that vqsM encodes a transcriptional regulator with an AraC-type helix-turn-helix DNA binding domain at the C-terminal of the peptide. Global gene expression profile analysis showed at least a total of 302 genes to be influenced, directly or indirectly, by VqsM. Among the 203 VqsM-promoted genes, 52.2% were known to be QS upregulated. Several genes encoding the key regulators implicated in QS, such as rhlR, rsaL, vqsR, mvfR, pprB and rpoS, and two AHL synthesis genes, lasI and rhlI, were suppressed in the vqsM mutant. Similar to the 'AHL-blind' phenotype of vqsR and pprB mutants, vqsM mutant did not respond to external addition of N-3-oxo-dodecanoyl-homoserine lactone signals. Moreover, overexpression of vqsR in vqsM mutant more or less restored the production of both AHL and virulence factors. The results demonstrate that VqsM, largely through modulation of vqsR expression, plays a vital role in regulation of QS signalling in P. aeruginosa.

The two-component response regulator PprB modulates quorum-sensing signal production and global gene expression in Pseudomonas aeruginosa.

The response regulator PprB and its cognate sensor PprA were recently reported as a two-component regulatory system that controls membrane permeability and antibiotic sensitivity of Pseudomonas aeruginosa. We found that a Tn5 insertion mutation in pprB caused a drastic reduction in virulence factor production and cell motility. A transcriptome analysis revealed that 175 genes were regulated by PprB. Among the 113 PprB-activated genes, 85.5% are known to be activated by N-3-oxo-dodecanoyl-homoserine lactone (OdDHL) and N-butanoyl-homoserine lactone (BHL). In particular, the expression of lasI, rhlI and rhlR, which encode key components of the las and rhl quorum-sensing (QS) systems, were significantly decreased in the pprB mutant. These data suggest that PprB might regulate QS signal production. Measurement of OdDHL and BHL in cultures of the mutant sustained this hypothesis. By using various OdDHL- or BHL-responsive QS reporter systems, including lasB-lacZ, lasI-lacZ and rsaL-lacZ, we found that the mutation in pprB resulted in a large decrease in the sensitivity of P. aeruginosa to exogenous OdDHL. However, there was no difference in sensitivity to BHL. Further analysis showed that the OdDHL influx was significantly reduced in the pprB mutant. We conclude that PprB is a novel QS modulator that positively regulates N-acylhomoserine lactone production probably by affecting the OdDHL signal influx and thereby influences global expression of the QS-dependent genes.

Insecticidal Bacillus thuringiensis silences Erwinia carotovora virulence by a new form of microbial antagonism, signal interference.

It is commonly known that bacteria may produce antibiotics to interfere with the normal biological functions of their competitors in order to gain competitive advantages. Here we report that Bacillus thuringiensis suppressed the quorum-sensing-dependent virulence of plant pathogen Erwinia carotovora through a new form of microbial antagonism, signal interference. E. carotovora produces and responds to acyl-homoserine lactone (AHL) quorum-sensing signals to regulate antibiotic production and expression of virulence genes, whereas B. thuringiensis strains possess AHL-lactonase, which is a potent AHL-degrading enzyme. B. thuringiensis did not seem to interfere with the normal growth of E. carotovora; rather, it abolished the accumulation of AHL signal when they were cocultured. In planta, B. thuringiensis significantly decreased the incidence of E. carotovora infection and symptom development of potato soft rot caused by the pathogen. The biocontrol efficiency is correlated with the ability of bacterial strains to produce AHL-lactonase. While all the seven AHL-lactonase-producing B. thuringiensis strains provided significant protection against E. carotovora infection, Bacillus fusiformis and Escherichia coli strains that do not process AHL-degradation enzyme showed little effect in biocontrol. Mutation of aiiA, the gene encoding AHL-lactonase in B. thuringiensis, resulted in a substantial decrease in biocontrol efficacy. These results suggest that signal interference mechanisms existing in natural ecosystems could be explored as a new version of antagonism for prevention of bacterial infections.