Development of the Static and Dynamic Gene Expression Regulation Toolkit in Pseudomonas chlororaphis.

The advancement of metabolic engineering and synthetic biology has promoted in-depth research on the nonmodel microbial metabolism, and the potential of nonmodel organisms in industrial biotechnology is becoming increasingly evident. The nonmodel organism Pseudomonas chlororaphis is a safe plant growth promoting bacterium for the production of phenazine compounds; however, its application is seriously hindered due to the lack of an effective gene expression precise regulation toolkit. In this study, we constructed a library of 108 promoter-5'-UTR (PUTR) and characterized them through fluorescent protein detection. Then, 6 PUTRs with stable low, intermediate, and high intensities were further characterized by report genes lacZ encoding ß-galactosidase from Escherichia coli K12 and phzO encoding PCA monooxygenase from P. chlororaphis GP72 and thus developed as a static gene expression regulation system. Furthermore, the stable and high-intensity expressed PMOK_RS0128085UTR was fused with the LacO operator to construct an IPTG-induced plasmid, and a self-induced plasmid was constructed employing the high-intensity PMOK_RS0116635UTR regulated by cell density, resulting in a dynamic gene expression regulation system. In summary, this study established two sets of static and dynamic regulatory systems for P. chlororaphis, providing an effective toolkit for fine-tuning gene expression and reprograming the metabolism flux.

Glufosinate-ammonium causes liver injury in zebrafish by blocking the Nrf2 pathway.

Glufosinate-ammonium (GLA) is a widely used herbicide, but less research has been done on its harmful effects on non-target organisms, especially aquatic organisms. In this study, 600 adult zebrafish were exposed to different concentration of GLA (0, 1.25, 2.5, 5, 10, and 20 mg/L) for 7 days, and the livers were dissected on the eighth day to examine the changes in liver structure, function, oxidative stress, inflammation, apoptosis, and Nrf2 pathway, and finally to clarify the mechanism of GLA induced liver injury in zebrafish. The levels of alanine aminotransferase, aspartate aminotransferase, reactive oxygen species, malondialdehyde, inflammatory factors (IL-6 and TNF-α), and caspase-3 gradually increased, while the levels of superoxide dismutase, catalase, glutathione, and glutathione peroxidase gradually decreased with the increase of GLA concentration. The Nrf2 pathway was activated at low concentrations (1.25-5 mg/L) and significantly inhibited at high concentrations (10 and 20 mg/L). These results suggested that GLA could cause oxidative stress, inflammation, and apoptosis in zebrafish liver. Therefore, GLA can cause liver injury in zebrafish, and at high concentrations, the inhibition of Nrf2 pathway is one of the important causes of liver injury.

The relationship between negative workplace gossip and thriving at work among Chinese kindergarten teachers: the roles of psychological contract breach and bianzhi.

Introduction: Exploring the influencing factors and functioning mechanisms of thriving at work is of practical significance both for teachers and kindergartens. Based on the socially embedded model of thriving at work, this study aimed to examine the association between negative workplace gossip and thriving at work. The mediating role of psychological contract breach and the moderating role of bianzhi were also examined. Methods: A total of 1105 Chinese kindergarten teachers were chosen to complete a questionnaire on negative workplace gossip, psychological contract breach, and thriving at work. Results: The results demonstrated that negative workplace gossip was positively associated with psychological contract breach and negatively associated with thriving at work. In addition, psychological contract breach was negatively associated with thriving at work. According to the mediation model test, psychological contract breach was a mediating factor between negative workplace gossip and thriving at work. The impact of psychological contract breach on thriving at work could be further moderated by bianzhi. Conclusion: This study complements knowledge systems about the influential factors and functional mechanisms of thriving at work. In practical terms, this study offers a fresh and innovative perspective for kindergartens seeking to enhance teachers' thriving at work.

Comparative metabolomics and transcriptomics analyses provide insights into the high-yield mechanism of phenazines biosynthesis in Pseudomonas chlororaphis GP72.

AIMS: Phenazines, such as phenazine-1-carboxylic acid (PCA), phenazine-1-carboxamide (PCN), 2-hydroxyphenazine-1-carboxylic acid (2-OH-PCA), 2-hydroxyphenazine (2-OH-PHZ), are a class of secondary metabolites secreted by plant-beneficial Pseudomonas. Ps. chlororaphis GP72 utilizes glycerol to synthesize PCA, 2-OH-PCA and 2-OH-PHZ, exhibiting broad-spectrum antifungal activity. Previous studies showed that the addition of dithiothreitol (DTT) could increase the phenazines production in Ps. chlororaphis GP72AN. However, the mechanism of high yield of phenazine by adding DTT is still unclear. METHODS AND RESULTS: In this study, untargeted and targeted metabolomic analysis were adopted to determine the content of metabolites. The results showed that the addition of DTT to GP72AN affected the content of metabolites of central carbon metabolism, shikimate pathway and phenazine competitive pathway. Transcriptome analysis was conducted to investigate the changed cellular process, and the result indicated that the addition of DTT affected the expression of genes involved in phenazine biosynthetic cluster and genes involved in phenazine competitive pathway, driving more carbon flux into phenazine biosynthetic pathway. Furthermore, genes involved in antioxidative stress, phosphate transport system and mexGHI-opmD efflux pump were also affected by adding DTT. CONCLUSION: This study demonstrated that the addition of DTT altered the expression of genes related to phenazine biosynthesis, resulting in the change of metabolites involved in central carbon metabolism, shikimate pathway and phenazine competitive pathway. SIGNIFICANCE AND IMPACT OF THE STUDY: This work expands the understanding of high yield of phenazine by the addition of DTT and provides several targets for increasing phenazine production.

Preparation and characterisation of ecdysterone/hydroxypropyl-Β-cyclodextrin inclusion complex with enhanced oral and transdermal bioavailability.

To prepare ecdysterone (ES)/hydroxypropyl-ß-cyclodextrin (HP-ß-CD) inclusion complex, thus improving the water solubility and bioavailability of ES. Phase-solubility study was performed to study the mass ratio of ES and HP-ß-CD. Then, the ES/HP-ß-CD inclusion complex was prepared by the solvent evaporation method, and its physicochemical properties were characterised using the SEM, DSC, XRD, 1HNMR and FT-IR. In addition, in vitro dissolution and bioavailability (oral and transdermal) experiments were also conducted. The inclusion complex was formed with ES and HP-ß-CD at the mass ratio of 1:1. ES existed in an amorphous form in the inclusion complex. The equilibrium solubility of ES/HP-ß-CD inclusion complex in SGF (simulated gastric fluid) and SIF (simulated intestinal fluid) was 50.6 ± 0.11 mg/mL and 75.9 ± 0.38 mg/mL in SGF and SIF, which was 5.93 and 9.96 times higher than that of free ES, respectively. The ES/HP-ß-CD inclusion complex had better dissolution ability and transdermal permeability than the free ES. The oral bioavailability and the transdermal bioavailability were respectively increased by 2.97 times and 1.92 times compared with the free ES. These data suggest that the ES/HP-ß-CD inclusion complex can be developed as potential pharmaceutical product for future clinical applications.

Acmoxanthones A-E, New Lavandulated Xanthones from Hypericum acmosepalum N. Robson.

Acmoxanthones A-E (1-5), five new lavandulylated xanthones, were isolated from the aerial parts of Hypericum acmosepalum, together with four known xanthones. Their structures with absolute configurations were elucidated on the basis of analysis of MS, NMR and chiroptical properties. A bioassay against high glucose-induced damage on human umbilical vein endothelial cells (HUVECs) showed ananixanthone (6) and osajaxanthone (7) had potential antioxidative damage activity with EC50 values of 10.5 µg/mL and 7.6 µg/mL, respectively, while 3-hydroxy-2,4-dimethoxyxanthone (8) exhibited cytotoxic effect on the damaged cells with IC50 values of 7.1 µg/mL.

Structurally diverse polyprenylated acylphloroglucinols from Hypericum uralum Buch.-Ham. ex D. Don.

Uralins A - D, four undescribed polycyclic polyprenylated acylphloroglucinols (PPAPs) featuring an unprecedented fused hexacyclic architecture, a unique monocyclic tetra-seco-tetranor-b-PPAP, an oxidative b-PPAP and a rare norspiroindane-type m-PPAP, respectively, were isolated from the aerial parts of Hypericum uralum, along with ten known PPAPs. Their structures and absolute configurations were elucidated by extensive spectroscopic techniques (MS, NMR, [α]D, CD), conceivable biogenetic pathways and time-dependent density functional theory-based electronic circular dichroism (TDDFT-ECD) calculations. Biological assays showed three b-PPAPs had moderate antioxidative damage activities, while spiroindanes exhibited moderate cytotoxic effects.

Characterization and Engineering of Pseudomonas chlororaphis LX24 with High Production of 2-Hydroxyphenazine.

The take-all disease of wheat is one of the most serious diseases in the field of food security in the world. There is no effective biological pesticide to prevent the take-all disease of wheat. 2-Hydroxyphenazine (2-OH-PHZ) was reported to possess a better inhibitory effect on the take-all disease of wheat than phenazine-1-carboxylic acid, which was registered as "Shenqinmycin" in China in 2011. The aim of this study was to construct a 2-OH-PHZ high-producing strain by strain screening, genome sequencing, genetic engineering, and fermentation optimization. First, the metabolites of the previously screened new phenazine-producing Pseudomonas sp. strain were identified, and the taxonomic status of the new Pseudomonas sp. strain was confirmed through 16S rRNA and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Then, the new Pseudomonas sp. strain was named Pseudomonas chlororaphis subsp. aurantiaca LX24, which is a new subspecies of P. chlororaphis that can synthesize 2-OH-PHZ. Next, the draft genome of strain LX24 was determined, and clusters of orthologous group (COG) analysis, KEGG analysis, and gene ontology (GO) analysis of strain LX24 were performed. Furthermore, the production of 2-OH-PHZ increased to 351.7 from 158.6 mg/L by deletion of the phenazine synthesis negative regulatory genes rpeA and rsmE in strain LX24. Finally, the 2-OH-PHZ production of strain LX24 reached 677.1 mg/L after fermentation optimization, which is the highest production through microbial fermentation reported to date. This work provides a reference for the efficient production of other pesticides and antibiotics.

A Robust Mixed-Lanthanide PolyMOF Membrane for Ratiometric Temperature Sensing.

Temperature sensors play a significant role in biology, chemistry, and engineering, especially those that can work accurately in a noninvasive manner. We adopted a photoinduced post-synthetic copolymerization strategy to realize a membranous ratiometric luminescent thermometer based on the emissions of two lanthanide ions. This novel mixed-lanthanide polyMOF membrane exhibits not only the integrity and temperature sensing behaviour of the Ln-MOF powder but also excellent mechanical properties, such as flexibility, elasticity, and processability. Moreover, the polyMOF membrane shows remarkable stability under harsh conditions, including high humidity, strong acid and alkali (pH 0-14), which allowed the mapping of temperature distributions in extreme circumstances. This work highlights a simple strategy for polyMOF membrane formation and pushes forward the further practical application of Ln-MOF-based luminescent thermometers in various fields and conditions.

Engineering of glycerol utilization in Pseudomonas chlororaphis GP72 for enhancing phenazine-1-carboxylic acid production.

Glycerol is a by-product of biodiesel, and it has a great application prospect to be transformed to synthesize high value-added compounds. Pseudomonas chlororaphis GP72 isolated from the green pepper rhizosphere is a plant growth promoting rhizobacteria that can utilize amount of glycerol to synthesize phenazine-1-carboxylic acid (PCA). PCA has been commercially registered as "Shenqinmycin" in China due to its characteristics of preventing pepper blight and rice sheath blight. The aim of this study was to engineer glycerol utilization pathway in P. chlororaphis GP72. First, the two genes glpF and glpK from the glycerol metabolism pathway were overexpressed in GP72ANO separately. Then, the two genes were co-expressed in GP72ANO, improving PCA production from 729.4 mg/L to 993.4 mg/L at 36 h. Moreover, the shunt pathway was blocked to enhance glycerol utilization, resulting in 1493.3 mg/L PCA production. Additionally, we confirmed the inhibition of glpR on glycerol metabolism pathway in P. chlororaphis GP72. This study provides a good example for improving the utilization of glycerol to synthesize high value-added compounds in Pseudomonas.

Effect of sulfamethazine on surface characteristics of biochar colloids and its implications for transport in porous media.

Antibiotics are contaminants of emerging concern due to their potential effect on antibiotic resistance and human health. Antibiotics tend to sorb strongly to organic materials, and biochar, a high efficient agent for adsorbing and immobilizing pollutants, can thus be used for remediation of antibiotic-contaminated soil and water. The effect of ionizable antibiotics on surface characteristics and transport of biochar colloids (BC) in the environment is poorly studied. Column experiments of BC were conducted in 1 mM NaCl solution under three pH (5, 7, and 10) conditions in the presence of sulfamethazine (SMT). Additionally, the adsorption of SMT by BC and the zeta potential of BC were also studied. The experimental results showed that SMT sorption to BC was enhanced at pH 5 and 7, but reduced at pH 10. SMT sorption reduced the surface charge of BC at pH 5 and 7 due to charge shielding, but increased surface charge at pH 10 due to adsorption of the negatively charged SMT species. The mobility of BC was inhibited by SMT under acidic or neutral conditions, while enhanced by SMT under alkaline conditions, which can be well explained by the change of electrostatic repulsion between BC and sand grains. These findings imply that pH conditions played a crucial role in deciding whether the transport of BC would be promoted by SMT or not. Biochar for antibiotics remediation will be more effective under acidic and neutral soil conditions, and the mobility of BC will be less than in alkaline soils.

Toxicity and haemolytic activity of a newly described dinoflagellate, Heterocapsa bohainensis to the rotifer Brachionus plicatilis.

The algae Heterocapsa bohaiensis is a newly described species of dinoflagellate associated with Penaeus japonicus and larvae of Eriocheir sinensis in a coastal pond of Liaodong Bay China. The rotifer Brachionus plicatilis is used as live feed for aquaculture organisms including prawns and crabs larvae. To evaluate the potential toxicity of H. bohaiensis, the effects on Brachionus plicatilis and haemolytic activity were investigated in this study. The results showed that H. bohaiensis had significant toxic effect on B. plicatilis, and this effect was dependent on the cell concentration. Significant rotifer growth suppression was observed in the ruptured cells of H. bohaiensis with ultrasonic. Relatively similar rotifer mortalities were induced both in the light and in the dark. Interestingly, haemolysis to erythrocytes was also caused in a cell density-dependent and time-dependent manner, which meant the results of haemolytic activity were consistent with the toxicity. Therefore, haemolytic toxins were considered to be involved in the toxic mechanism of H. bohaiensis against rotifers. Then, the concentrations of calcium were measured in the mastax, stomach and ovary of B. plicatilis. Obviously increased fluorescence intensity was found in the stomach, which indicated the alteration of calcium homeostasis and membrane permeability after ingesting H. bohaiensis. These results implicated haemolytic activity as a causative factor linked to the toxicity of H. bohaiensis against B. plicatilis. The results contributed to research the production and control of H. bohaiensis toxins.

Two new noroleanane-type triterpenoid saponins from the stems of Stauntonia chinensis.

Two new noroleanane-type triterpenoid saponins, 3ß,20α,24-trihydroxy-29-norolean-12-en-28-oic acid 24-O-ß-L-fucopyranosyl-(1→2)-[ß-D-xylopyranosyl-(1→3)]-ß-D-glucopyranoside (1) and 3ß,20α,24-trihydroxy-29-norolean-12-en-28-oic acid 24-O-ß-D-glucopyranosyl-(1→2)-[α-L-arabinopyranosyl-(1→3)]-ß-D-glucopyranoside (2) were isolated from the stems of Stauntonia chinensis DC., together with three known compounds, brachyantheraoside B2 (3), eupteleasaponin Ⅷ (4) and fargoside B (5). Their structures were elucidated by spectroscopic and chemical methods. The cytotoxic activities of compounds 1 and 2 were evaluated against five human tumor cell lines (HCT-116, HepG2, BGC-823, NCI-H1650, and A2780). Compounds 1 and 2 showed moderate cytotoxic activities toward the tested cell lines with IC50 values ranging from 12.71 to 32.04 µM.

Cytotoxic triterpenoid saponins from the defatted seeds of Camellia oleifera Abel.

Five new oleanane-type triterpenoid saponins, oleiferasaponins D1-D5 (1-5), were isolated from the defatted seeds of Camellia oleifera Abel. Their structures were elucidated by spectroscopic and chemical methods. The cytotoxic activities of compounds 1-5 were evaluated against five human tumor cell lines (HCT-116, HepG2, BGC-823, NCI-H1650, and A2780). Compounds 1-2 exhibited cytotoxic activity against five human cancer cell lines, with IC50 values ranging from 3.31 to 10.23 µM. Compounds 3-5 showed moderate cytotoxic activities toward the tested cell lines.

Prognostic significance of Tspan9 in gastric cancer.

Tetraspanins are a large superfamily of glycoproteins, which are engaged in a wide range of specific molecular interactions by forming tetraspanin-enriched microdomains. Tetraspanin 9 (Tspan9) is a previously poorly studied tetraspanin gene, which was predominantly identified as an amplified gene in serous Fallopian tube carcinoma. However, the expression and role of Tspan9 in gastric cancer have yet to be fully elucidated. The aim of the present study was to evaluate the expression and clinical significance of Tspan9 in gastric cancer. In the present study, 105 gastric cancer tissue samples and corresponding adjacent normal samples were detected for Tspan9 expression using immunohistochemistry; furthermore, the association between clinical characteristics and Tspan9 expression was also analyzed. Tspan9 expression was determined to be significantly lower in cancer samples compared with those in corresponding adjacent normal samples (P<0.001). However, its increased levels of expression in cancer samples appeared to demonstrate a poorer prognostic tendency, which is associated with deeper tumor depth (P=0.025), more nodal involvement (P=0.01), more advanced tumor/lymph node/metastasis (TNM) stages (P=0.017) and a larger tumor size (P=0.026). Additionally, multivariate analysis demonstrated that high expression of Tspan9 was an independent prognostic factor for poor overall survival (P<0.01). These results suggested that Tspan9 may be used as a potential prognostic factor in gastric cancer.

DNA hypermethylation of the vimentin gene inversely correlates with vimentin expression in intestinal- and diffuse-type gastric cancer.

The vimentin gene is a hallmark of epithelial-to-mesenchymal transition and has been observed to be overexpressed in various types of tumor cell line and tissue. Previous studies have reported correlations between vimentin DNA methylation levels and subsequent vimentin expression levels in solid tumors, including breast and colorectal cancer; however, to the best of our knowledge, such a correlation has not been reported for gastric cancer (GC) using Lauren classification. Therefore, the present study aimed to quantify DNA methylation levels of the vimentin gene using quantitative (q) methylation-specific polymerase chain reaction (PCR) in intestinal-type GC cell lines (MKN-28, AGS and MKN-1), diffuse-type GC cell lines (SGC-7901, SNU-5 and KATO III), the GES-1 immortalized human non-neoplastic gastric epithelial cell line, as well as in tumor and paratumor normal tissue samples. Furthermore, the present study analyzed the messenger RNA expression of the vimentin gene in these cell lines and tissues by reverse transcription-qPCR. A comparison of the clinicopathological features was conducted between patients, grouped according to the Lauren classification. The present study identified that the vimentin promoter region was hypermethylated in all GC cell lines and tumor tissue samples when compared with immortalized normal gastric epithelial cells and paratumor normal tissues. In addition, vimentin promoter methylation levels were observed to be higher in intestinal-type cell lines when compared with those of diffuse-type lines and tissues. Correspondingly, vimentin expression levels were lower in intestinal-type gastric cell lines compared with those of diffuse-type cell lines and tissues, and were lowest in the non-neoplastic gastric cell line and paratumor normal tissues. Patients with diffuse-type GC were on average younger (P=0.023), and exhibited higher tumor (P=0.020), node (P=0.032) and TNM classification of malignant tumor stage (P=0.039) than those with intestinal-type GC. Following treatment of AGS cells (which demonstrated the highest methylation level of the vimentin gene) with 5-aza-2'-deoxycytidine, vimentin expression was restored significantly. Thus, the present study revealed that vimentin promoter methylation levels are inversely correlated with vimentin expression levels in GC (according to Lauren classification). High levels of methylation in the vimentin gene promoter region may be involved in carcinogenesis and the development of GC, and may provide a novel molecular classification for GC.

Prognostic significance of the co-expression of nucleophosmin and trefoil factor 3 in postoperative gastric cancer patients.

Although a number of studies have indicated that the positive expression of nucleophosmin (NPM) and trefoil factor 3 (TFF3) is associated with oncogenesis and poor prognosis in several tumor types, the prognostic value of the co-expression of NPM and TFF3 in gastric cancer (GC) has not been fully elucidated. Therefore, in this study, we aimed to investigate the role of NPM and TFF3 in GC and determine their prognostic value. We retrospectively reviewed 108 patients who had undergone radical gastric tumor resection. The expression of NPM and TFF3 was detected by immunohistochemistry and the association of NPM and TFF3 with clinicopathological characteristics was investigated using the Chi-square test. Furthermore, univariate and multivariate analyses were conducted to determine the prognostic value of these markers. Of the 108 samples, NPM was positive in 57 (53%) and TFF3 was positive in 54 samples (50%). The positive expression of NPM was correlated with advanced tumor stage and recurrence (P=0.0333 and P<0.0001, respectively), whereas the expression of TFF3 was associated with larger tumor size (P=0.0005), poor differentiation (P=0.0435), lymph node metastasis (P=0.0116), advanced tumor stage (P=0.0244) and recurrence (P=0.0116). The univariate analysis revealed that the expression of NPM, the expression of TFF3 and the co-expression of the two were associated with poor survival (P=0.0004, 0.0028 and 0.0020, respectively). By multivariate analysis, all three factors were identified as independent prognostic factors in postoperative GC patients (hazard ratio = 1.970, 2.021 and 2.339, respectively). In conclusion, the expression of NPM and TFF3 and, particularly, the co-expression of the two, may serve as independent prognostic factors in postoperative GC patients.