Co-occurrence network and functional profiling of the bacterial community in the industrial pile fermentation of Qingzhuan tea: Understanding core functional bacteria.

The distinct quality of Qingzhuan tea is greatly influenced by the bacterial community but was poorly characterized. Therefore, this study investigated the Co-occurrence network and functional profiling of the bacterial community, with special attention paid to core functional bacteria in the industrial pile fermentation. Microbiomics analysis indicated that Klebsiella and Pantoea dominated raw tea leaves, and were rapidly replaced by Pseudomonas in pile fermentation, but substituted mainly by Burkholderia and Saccharopolyspora in final fermented tea. Bacterial taxa were grouped into 7 modules with the dominant in module I, III, and IV, which were involved in flavor formation and biocontrol production. Functional profiling revealed that "penicillin and cephalosporin biosynthesis" increased in pile fermentation. Twelve bacterial genera were identified as core functional bacteria, in which Klebsiella, Pantoea, and Pseudomonas also dominated the pile fermentation. This work would provide theoretical basis for its chemical biofortification and quality improvement by controlling bacterial communities.

Exploring core functional fungi driving the metabolic conversion in the industrial pile fermentation of Qingzhuan tea.

The distinct sensory quality of Qingzhuan tea is mainly formed in pile fermentation by a group of functional microorganisms but the core functional ones was poorly characterized. Therefore, this study investigated the dynamic changes in the fungal community and metabolic profile by integrating microbiomics and metabolomics, and explored the core functional fungi driving the metabolic conversion in the industrial pile fermentation of Qingzhuan tea. Indicated by microbiomics analysis, Aspergillus dominated the entire pile-fermentation process, while Thermoascus, Rasamsonia, and Cylindrium successively abounded in the different stages of the pile fermentation. A total of 50 differentially changed metabolites were identified, with the hydrolysis of galloyl/polymeric catechins, biosynthesis of theabrownins, oxidation of catechins, N-ethyl-2-pyrrolidinone substitution of catechins, and deglycosylation of flavonoid glucosides. Nine fungal genera were identified as core functional fungi, in which Aspergillus linked to the hydrolysis of polymeric catechins and insoluble polysaccharides as well as biosynthesis of theabrownins, while Thermoascus participated in the biosynthesis of theabrownins, deglycosylation of flavonoid glucosides, and N-ethyl-2-pyrrolidinone substitution of catechins. These findings would advance our understanding of the quality formation of Qingzhuan tea and provide a benchmark for precise inoculation for its quality improvement.

Construction, Characterization, and Application of an Ammonium Transporter (AmtB) Deletion Mutant of the Nitrogen-Fixing Bacterium Kosakonia radicincitans GXGL-4A in Cucumis sativus L. Seedlings.

Nitrogen is an important factor affecting crop yield, but excessive use of chemical nitrogen fertilizer has caused decline in nitrogen utilization and soil and water pollution. Reducing the utilization of chemical nitrogen fertilizers by biological nitrogen fixation (BNF) is feasible for green production of crops. However, there are few reports on how to have more ammonium produced by nitrogen-fixing bacteria (NFB) flow outside the cell. In the present study, the amtB gene encoding an ammonium transporter (AmtB) in the genome of NFB strain Kosakonia radicincitans GXGL-4A was deleted and the △amtB mutant was characterized. The results showed that deletion of the amtB gene had no influence on the growth of bacterial cells. The extracellular ammonium nitrogen (NH4+) content of the △amtB mutant under nitrogen-free culture conditions was significantly higher than that of the wild-type strain GXGL-4A (WT-GXGL-4A), suggesting disruption of NH4+ transport. Meanwhile, the plant growth-promoting effect in cucumber seedlings was visualized after fertilization using cells of the △amtB mutant. NFB fertilization continuously increased the cucumber rhizosphere soil pH. The nitrate nitrogen (NO3-) content in soil in the △amtB treatment group was significantly higher than that in the WT-GXGL-4A treatment group in the short term but there was no difference in soil NH4+ contents between groups. Soil enzymatic activities varied during a 45-day assessment period, indicating that △amtB fertilization influenced soil nitrogen cycling in the cucumber rhizosphere. The results will provide a solid foundation for developing the NFB GXGL-4A into an efficient biofertilizer agent.

Physiological and transcriptome profiling revealed defense networks during Cladosporium fulvum and tomato interaction at the early stage.

Tomato leaf mold caused by Cladosporium fulvum (C. fulvum) is a serious fungal disease which results in huge yield losses in tomato cultivation worldwide. In our study, we discovered that ROS (reactive oxygen species) burst was triggered by C. fulvum treatment in tomato leaves. RNA-sequencing was used to identify differentially expressed genes (DEGs) induced by C. fulvum inoculation at the early stage of invasion in susceptible tomato plants. Gene ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were used to annotate functions of DEGs in tomato plants. Based on our comparative analysis, DEGs related to plant-pathogen interaction pathway, plant hormone signal transduction pathway and the plant phenylpropanoid pathway were further analyzed. Our results discovered that a number of core defense genes against fungal invasion were induced and plant hormone signal transduction pathways were impacted by C. fulvum inoculation. Further, our results showed that SA (salicylic acid) and ABA (abscisic acid) contents were accumulated while JA (jasmonic acid) content decreased after C. fulvum inoculation in comparison with control, and quantitative real-time PCR to detect the relative expression of genes involved in SA, ABA and JA signaling pathway further confirmed our results. Together, results will contribute to understanding the mechanisms of C. fulvum and tomato interaction in future.

Are shared streets acceptable to pedestrians and drivers? Evidence from Virtual Reality experiments.

While the development of cities tends to focus on improving traffic mobility, it has gradually neglected people's demand for safety and comfort walking on the streets. To address this problem, shared streets that can integrate traditional street life and traffic mobility are getting more attention as pedestrian-friendly development. In order to measure the performance of shared streets, it is essential to identify how people feel when driving and walking around. However, investigating the various factors that influence the real world is not straightforward because of cost, time-consuming, and safety problems. Virtual reality and the Human-in-the-loop (HITL) have become valuable tools for conducting experiments without compromising them. The experiments are performed on both pedestrians' and drivers' sides. The three shared street layouts in a virtual environment are designed according to Europe's real shared street cases. To evaluate shared street effects, questions in five aspects: amenity, walking or driving experience, safety, economy or priority, and environmental perception are asked to participants, respectively. MPR, EWM, and Fuzzy Comprehension Evaluation methods are used to assess the performance. The result revealed that different groups of people have different sensitivity and preferences for each evaluation criteria. However, the results of the comprehensive evalutation showed that scenario C with the largest isolation measurement is preferable in both pedestrian and driver's groups based on shared street design elements. The city planners can get help from this shared street analysis, where the new design and layout could be tested in advance.

Gut Microbiota Differentially Mediated by Qingmao Tea and Qingzhuan Tea Alleviated High-Fat-Induced Obesity and Associated Metabolic Disorders: The Impact of Microbial Fermentation.

Although dark tea is a unique microbial-fermented tea with a high reputation for having an antiobesity effect, little is known about the effect of microbial fermentation on tea leaves' antiobesity properties. This study compared the antiobesity effects of microbial-fermented Qingzhuan tea (QZT) and unfermented Qingmao tea (QMT), providing insight into their underlying mechanisms associated with gut microbiota. Our results indicated that the supplementation of QMT extract (QMTe) and QZT extract (QZTe) displayed similar antiobesity effects in high-fat diet (HFD)-fed mice, but the hypolipidemic effect of QZTe was significantly stronger than that of QMTe. The microbiomic analysis indicated that QZTe was more effective than QMTe at regulating HFD-caused gut microbiota dysbiosis. Akkermansiaceae and Bifidobacteriaceae, which have negative correlations with obesity, were enhanced notably by QZTe, whereas Faecalibaculum and Erysipelotrichaceae, which are positively correlated with obesity, were decreased dramatically by QMTe and QZTe. A Tax4Fun analysis of QMTe/QZTe-mediated gut microbiota revealed that QMTe supplementation drastically reversed the HFD-induced upregulation of glycolysis and energy metabolism, whereas QZTe supplementation significantly restored the HFD-caused downregulation of pyruvate metabolism. Our findings suggested that microbial fermentation showed a limited effect on tea leaves' antiobesity, but enhanced their hypolipidemic activity, and QZT could attenuate obesity and associated metabolic disorders by favorably modulating gut microbiota.

Dynamic changes of metabolic profile and taste quality during the long-term aging of Qingzhuan Tea: The impact of storage age.

Qingzhuan tea (QZT) with longer aging year is usually believed to have higher quality and commercial value. In this study, a 20 years sequence of aged QZT were subjected to an electronic tongue and liquid chromatography-mass spectrometry to investigate the effect of storage age on its metabolic profile and taste quality. The changes in both taste quality and metabolic profile exhibited a parabolic trend in the 20 years of QZT aging and reached the maximum at the 10th year. A total of 47 compounds were identified as critical metabolites responsible for the age variation of QZT quality, with the methylation of catechins, glycosylation of flavonoids, degradation of flavoalkaloids, biosynthesis of triterpenoids, and formation of theabrownins. These results suggested that the taste of QZT was improved after 10 years of storage, with the reduction of bitterness and astringency and a general increase of key quality-related compounds.

Screening for Candidate Genes Associated with Biocontrol Mechanisms of Bacillus pumilus DX01 Using Tn5 Transposon Mutagenesis and a 2-DE-Based Comparative Proteomic Analysis.

A total of 1467 mutants of the biocontrol bacterium Bacillus pumilus DX01 were obtained by Tn5 insertional mutagenesis and subjected to the determination of antagonistic capabilities. Compared with the wild-type strain DX01, the mutant M25 was identified to have the most significant reduction in antagonistic capability against the phytopathogen Bipolaris maydis and extracellular proteinase activity. The integration site of the exogenous T-DNA in the genome of mutant M25 was revealed in the coding region of malony CoA-ACP transacylase (MCAT) gene (mcat), which belongs to a polyketide synthase (PKS) gene cluster, DX01pks of B. pumilus DX01. Furthermore, the whole DX01pks gene cluster was cloned using Illumina Solexa sequencing technology, and it has a modular framework different from the other two gene clusters involved in polyketide synthesis in B. amyloliquefaciens FZB42 (pks1) and B. subtilis 168 (pksX). Finally, in order to gain more insights into the molecular mechanisms of biocontrol of B. pumilus DX01, the changes in the relative level of expression of total proteins between the original strain DX01 and the mutant M25 were detected by 2-DE-based proteomic analysis. A total of twenty differentially expressed proteins were identified upon the mcat gene transposition mutagenesis. Of these proteins, seven proteins were up-regulated in expression level and the other proteins were down-regulated. Taken together, the results in this study showed that Tn5 transposon mutagenesis of B. pumilus DX01 can lead to a significant change of antiphytopathogen ability, and the DX01pks gene cluster possibly play a potential role in the biocontrol processes of this bacterium.

Colorimetric aptasensor for progesterone detection based on surfactant-induced aggregation of gold nanoparticles.

This paper proposes an aptasensor for progesterone (P4) detection in human serum and urine based on the aggregating behavior of gold nanoparticles (AuNPs) controlled by the interactions among P4-binding aptamer, target P4 and cationic surfactant hexadecyltrimethylammonium bromide (CTAB). The aptamer can form an aptamer-P4 complex with P4, leaving CTAB free to aggregate AuNPs in this aptasensor. Thus, the sensing solution will turn from red (520 nm) to blue (650 nm) in the presence of P4 because P4 aptamers are used up firstly owing to the formation of an aptamer-P4 complex, leaving CTAB free to aggregate AuNPs. However, in the absence of P4, CTAB combines with aptamers so that AuNPs still remain dispersed. Therefore, this assay makes it possible to detect P4 not only by absorbance measurement but also through naked eyes. By monitoring the variation of absorbance and color, a CTAB-induced colorimetric assay for P4 detection was established with a detection limit of 0.89 nM. Besides, the absorbance ratio A650/A520 has a linear correlation with the P4 concentration of 0.89-500 nM. Due to the excellent recoveries in serum and urine, this biosensor has great potential with respect to the visual and instrumental detection of P4 in biological fluids.

Colorimetric detection of bisphenol A based on unmodified aptamer and cationic polymer aggregated gold nanoparticles.

In this study, a colorimetric method was exploited to detect bisphenol A (BPA) based on BPA-specific aptamer and cationic polymer-induced aggregation of gold nanoparticles (AuNPs). The principle of this assay is very classical. The aggregation of AuNPs was induced by the concentration of cationic polymer, which is controlled by specific recognition of aptamer with BPA and the reaction of aptamer and cationic polymer forming "duplex" structure. This method enables colorimetric detection of BPA with selectivity and a detection limit of 1.50 nM. In addition, this colorimetric method was successfully used to determine spiked BPA in tap water and river water samples.

The cytotoxic effect of the NOS-mediated oxidative stress in MCF-7 cells after PbCl2 exposure.

The potential Pb-induced cytotoxicity in various tissues and biological systems has been reported. Some evidences also indicate that the Pb-caused cytotoxicity may be associated with the nitric oxide synthase (NOS). However, there remains uncertainty about the role of the NOS signaling pathway during the Pb-induced cytotoxicity. In this report, we provide data showing that PbCl2 treatment depresses the expressions of the three distinct NOS isoforms: neuronal nitric oxide synthase (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS) on both transcriptional and translational levels in MCF-7 cells. The down-regulation of NOSs expressions by PbCl2 exposure leads to reduced NOS activity and nitric oxide (NO) production. Meanwhile, the intracellular reactive oxygen species (ROS) level is elevated after PbCl2 exposure, which leads to the alpha subunit of eukaryotic initiation factor 2 (elF2α) phosphorylation. The reduction effects of the free radical scavenger N-acetyl-L-cysteine or the NOS substrate l-arginine on the Pb-induced ROS generation suggest that the NOS signaling pathway plays a key role in the Pb-induced oxidative stress, which further results in the elF2α phosphorylation and cytotoxicity.

Determining soil enzyme activities for the assessment of fungi and citric acid-assisted phytoextraction under cadmium and lead contamination.

Microorganism or chelate-assisted phytoextraction is an effective remediation tool for heavy metal polluted soil, but investigations into its impact on soil microbial activity are rarely reported. Consequently, cadmium (Cd)- and lead (Pb)-resistant fungi and citric acid (CA) were introduced to enhance phytoextraction by Solanum nigrum L. under varied Cd and Pb pollution levels in a greenhouse pot experiment. We then determined accumulation of Cd and Pb in S. nigrum and the soil enzyme activities of dehydrogenase, phosphatase, urease, catalase, sucrase, and amylase. Detrended canonical correspondence analysis (DCCA) was applied to assess the interactions between remediation strategies and soil enzyme activities. Results indicated that the addition of fungi, CA, or their combination enhanced the root biomass of S. nigrum, especially at the high-pollution level. The combined treatment of CA and fungi enhanced accumulation of Cd about 22-47 % and of Pb about 13-105 % in S. nigrum compared with the phytoextraction alone. However, S. nigrum was not shown to be a hyperaccumulator for Pb. Most enzyme activities were enhanced after remediation. The DCCA ordination graph showed increasing enzyme activity improvement by remediation in the order of phosphatase, amylase, catalase, dehydrogenase, and urease. Responses of soil enzyme activities were similar for both the addition of fungi and that of CA. In summary, results suggest that fungi and CA-assisted phytoextraction is a promising approach to restoring heavy metal polluted soil.

Expression and Characterization of a Recombinant Laccase with Alkalistable and Thermostable Properties from Streptomyces griseorubens JSD-1.

Streptomyces griseorubens JSD-1 is a novel actinomycete that could grow efficiently upon lignin, and the ligninolytic genes active in this biotransformation were expected to be crucial. To investigate the molecular mechanism of utilizing lignin, genome sequencing was carried out to obtain its draft genome, which was deposited at GenBank under the accession No. JJMG00000000. Multiple copper oxidase (MCO) was obtained, which proved to be an extracellular enzyme and have relative high expression with the stimulation of ligninolytic materials. Judging from its putative 3D structure, the N-terminal of MCO was bared, which was fit for the linkage of poly-HIS10 tag. As a result, heterogeneous expression conditions of recombinant laccase was achieved with TransB(DE3) grown in a modified terrific broth (TB) medium with an extra addition of 0.5% glucose at 30 °C until optical density at 600 nm (OD600) reached 0.8 when expression was induced by 25 µM isopropyl ß-D-1-thiogalactopyranoside (IPTG) and also 100 µM copper sulphate as supplement. Finally, it exhibited special characters of thermal robustness, alkaline activity profiles, high resistance to metallic ions and chemical inhibitors as well as dye decolourization. In summary, our findings illustrated the genetic basic of utilizing lignin in this isolate. Additionally, a novel laccase expected to be potential in agricultural and industrial application was expressed and characterized as well.

The role of NOS-mediated ROS accumulation in an early phase Cu-induced acute cytotoxicity in MCF-7 cells.

Copper (Cu) ion is essential for the biological systems, however, high level of CuCl2 exposure causes detrimental effects, which leads to cell apoptosis. Nitric oxide (NO) is an efficient cell signal messenger, which plays an important role in cell apoptosis. However, the potential mechanism of an early phase Cu-induced acute cytotoxicity through the nitric oxide synthase (NOS) signaling pathway and its interaction has not been studied. In this report, we provide data showing that high level of CuCl2 could rapidly decrease the NO production with the release of Ca(2+) and Zn(2+), and then modulate the transcriptional and translational expression of NOSs in MCF-7 cells. The reactive oxygen species (ROS) level in cells was increased after high level of CuCl2 exposure, which led to the alpha subunit of eukaryotic initiation factor 2 phosphorylation. By using the free radical scavenger N-acetyl-L-cysteine or the NOS substrate L-arginine, it demonstrated that NOS played a critical role on the Cu-induced ROS generation, which further led to the oxidative stress and cell apoptosis. These results suggested that Cu-induced apoptosis was associated with the oxidative stress, and through the NOS-mediated signaling pathway.

The role of nitric oxide synthase in an early phase Cd-induced acute cytotoxicity in MCF-7 cells.

Literature to date has confirmed that cadmium (Cd) can accomplish its toxic effects via the free radical-induced damage, but Cd itself cannot generate free radicals directly. Nitric oxide (NO) is a fundamental molecule that interplays with reactive oxygen species (ROS), which may be associated with the Cd-induced cytotoxicity. However, the role of nitric oxide synthase (NOS) in an early phase Cd-induced acute cytotoxicity and its interaction has not been studied. In this report, we provide data showing that CdCl2 (10 µM, 100 µM, 1 mM) could modulate NOS activity in terms of NO production which was first suppressed with the release of Ca(2+) and Zn(2+), then induced with the transcriptional and translational activation of the three NOS isoforms in a possible feedback manner. The ROS level in cells was increased after CdCl2 exposure. By using the free radical scavenger N-acetyl-L-cysteine (LNAC) or the NOS activity inhibitor N(G)-methyl-L-arginine (LNMMA), it was demonstrated that NOS played a critical role on the Cd-induced ROS generation. The Cd-induced cytotoxicity was associated with the NOS-mediated oxidative stress in MCF-7 cells.

The role of nitric oxide synthase signaling pathway in the Zn-induced cellular responses in MCF-7 cells.

Trace amount zinc plays key roles in biological systems, while in excessive amount it causes toxic effects. Evidence shows that there exists a crosstalk between NO and Zn apoptotic signal transduction pathway. However, the potential mechanism of Zn-induced cellular responses through the NOS signaling pathway has not been determined yet. In this research, trace amount ZnCl2 (1nM) could induce the NO production, however it appears that this influence does not extend to genetic level in MCF-7 cells. Whereas, excess ZnCl2 (100µM, 1mM) could lead to a decreased NO production first with the release of Ca(2+), and then induce the NO production with the transcriptional and translational activation of NOSs. The ROS generation was also induced by excess ZnCl2, causing the elF2α phosphorylation. The alleviation effect of N-acetyl-l-cysteine or l-arginine on the Zn-induced ROS generation and apoptosis suggested that Zn-induced apoptosis was associated with the NOS-mediated oxidative stress.

Nanoparticles assembled by aptamers and crystal violet for arsenic(III) detection in aqueous solution based on a resonance Rayleigh scattering spectral assay.

Aptamer-assembled nanomaterials have captured much attention from the field of analytical chemistry in recent years. Although they have been regarded as a promising tool for heavy metal monitoring, report involving aptamer-based biosensors for arsenic detection are rare. Herein we developed a highly sensitive and selective aptamer biosensor for As(iii) detection based on a Resonance Rayleigh Scattering (RRS) spectral assay. Prior to As(iii) detection, we firstly assembled a variety of nanoparticles with different sizes via controlling the concentration of arsenic-binding aptamers in crystal violet (CV) solutions. The results of photon correlation spectroscopy (PCS) and scanning probe microscope (SPM) testified that the introduction of As(iii) had indeed changed the size of nanoparticles, which caused a great variation in the RRS intensity at 310 nm. In the presence of 100 ppb As(iii), a maximum decline in the ratio of RRS intensity was achieved for large nanoparticles assembled from 200 nM of aptamers and CV molecules, where the average size of nanoparticles had decreased from 273 nm to 168 nm. In the case of small nanoparticles, the maximum increase ratio of the RRS intensity was obtained when the concentration of aptamer was over 600 nM. Combined with an RRS spectral assay, an effective biosensor has been developed for As(iii) detection, using the above large and small nanoparticles as the target recognition element. The present biosensor has a detection limit as low as 0.2 ppb, a dynamic range from 0.1 ppb to 200 ppb, and high selectivity over other metal ions. Such an efficient biosensor will play an important role in environmental detection.

A simple and label-free sensor for mercury(II) detection in aqueous solution by malachite green based on a resonance scattering spectral assay.

Mercury ions (Hg(2+)) can specifically interact with the thymine-rich Hg(2+) aptamer and malachite green (MG) to form the Hg(2+) aptamer-MG-Hg(2+) complex, inducing the increase of resonance scattering (RS) intensity at 611 nm, which enables the label-free detection of Hg(2+) in aqueous solution with high selectivity and a detection limit of 1.7 nM.

Three new flavonol triglycosides from Derris trifoliata.

Three new flavonol triglycosides, kaempferol-3-O-alpha-L-rhamnopyranosyl-(1 --> 6)-beta-D-glucopyranosyl-(1 --> 3)-beta-D-glucopyranoside (1), quercetin-3-O-alpha-L-rhamnopyranosyl-(1 --> 6)-beta-D-glucopyranosyl-(1 --> 3)-beta-D-glucopyranoside (2), quercetin-3-O-alpha-L-rhamnopyranosyl-(1 --> 6)-beta-D-glucopyranosyl-(1 --> 2)-beta-D-glucopyranoside (3), together with the two known flavonol glycosides, kaempferol-3-O-alpha-L-rhamnopyranosyl-(1 --> 6)-beta-D-glucopyranoside and kaempferol-3-O-alpha-L-rhamnopyranosyl-(1 --> 6)-beta-D-glucopyranosyl-(1 --> 2)-beta-D-glucopyranoside, were isolated from the aerial parts of Derris trifoliata. Their structures were determined on the basis of chemical and spectroscopic analyses.

A new dihydrochalcone from dragon's blood, red resin of Dracaena cochinchinensis.

A new dihydrochalcone, 4'-hydroxy-4,2'-dimethoxy-dihydrochalcone, was isolated from Chinese dragon's blood, the red resin of Dracaena cochinchinensis. Its structure was established by spectrum analysis.