Characterization, Pathogenicity, Phylogeny, and Comparative Genomic Analysis of Pseudomonas tolaasii Strains Isolated from Various Mushrooms in China.

Since 2016, devastating bacterial blotch affecting the fruiting bodies of Agaricus bisporus, Cordyceps militaris, Flammulina filiformis, and Pleurotus ostreatus in China has caused severe economic losses. We isolated 102 bacterial strains and characterized them polyphasically. We identified the causal agent as Pseudomonas tolaasii and confirmed the pathogenicity of the strains. A host range test further confirmed the pathogen's ability to infect multiple hosts. This is the first report in China of bacterial blotch in C. militaris caused by P. tolaasii. Whole-genome sequences were generated for three strains: Pt11 (6.48 Mb), Pt51 (6.63 Mb), and Pt53 (6.80 Mb), and pangenome analysis was performed with 13 other publicly accessible P. tolaasii genomes to determine their genetic diversity, virulence, antibiotic resistance, and mobile genetic elements. The pangenome of P. tolaasii is open, and many more gene families are likely to emerge with further genome sequencing. Multilocus sequence analysis using the sequences of four common housekeeping genes (glns, gyrB, rpoB, and rpoD) showed high genetic variability among the P. tolaasii strains, with 115 strains clustered into a monophyletic group. The P. tolaasii strains possess various genes for secretion systems, virulence factors, carbohydrate-active enzymes, toxins, secondary metabolites, and antimicrobial resistance genes that are associated with pathogenesis and adapted to different environments. The myriad of insertion sequences, integrons, prophages, and genome islands encoded in the strains may contribute to genome plasticity, virulence, and antibiotic resistance. These findings advance understanding of the determinants of virulence, which can be targeted for the effective control of bacterial blotch disease.

Characterization and relative quantification of phospholipids based on methylation and stable isotopic labeling.

Phospholipids (PLs), one of the lipid categories, are not only the primary building blocks of cellular membranes, but also can be split to produce products that function as second messengers in signal transduction and play a pivotal role in numerous cellular processes, including cell growth, survival, and motility. Here, we present an integrated novel method that combines a fast and robust TMS-diazomethane-based phosphate derivatization and isotopic labeling strategy, which enables simultaneous profiling and relative quantification of PLs from biological samples. Our results showed that phosphate methylation allows fast and sensitive identification of the six major PL classes, including their lysophospholipid counterparts, under positive ionization mode. The isotopic labeling of endogenous PLs was achieved by deuterated diazomethane, which was generated through acid-catalyzed hydrogen/deuterium (H/D) exchange and methanolysis of TMS-diazomethane during the process of phosphate derivatization. The measured H/D ratios of unlabeled and labeled PLs, which were mixed in known proportions, indicated that the isotopic labeling strategy is capable of providing relative quantitation with adequate accuracy, reproducibility, and a coefficient of variation of 9.1%, on average. This novel method offers unique advantages over existing approaches and presents a powerful tool for research of PL metabolism and signaling.

Profiling and relative quantitation of phosphoinositides by multiple precursor ion scanning based on phosphate methylation and isotopic labeling.

Phosphoinositides, the phosphorylated derivatives of phosphatidylinositol (PtdIns), are key regulators of many fundamental biological processes, including cell growth, proliferation, and motility. Here, we present a novel method for rapid, sensitive, and simultaneous profiling of phosphatidylinositol trisphosphate (PtdInsP3), phosphatidylinositol bisphosphate (PtdInsP2), and phosphatidylinositol phosphate (PtdInsP) of different fatty acid compositions. This method is based on a technique called "charged diacylglycerol fragment ion-specific multiple precursor ion scanning" (DAG(+)-specific MPIS), coupled with prior phosphate methylation. Using DAG(+)-specific MPIS, we were able to identify 32 PtdIns, 28 PtdInsP, 30 PtdInsP2, and 3 PtdInsP3 molecular species from bovine brain extracts or prostatic cancer cell lines in an efficient and time-saving manner. Our analysis revealed a large range of fatty acyl compositions in phosphoinositides not obtained previously from mammalian samples. We also developed a method that involves isotopic labeling of endogenous phosphoinositides with deuterated diazomethane (CD2N2) for quantitation of phosphoinositides. CD2N2 was generated in situ through acid-catalyzed H/D exchange and methanolysis of trimethylsilyl diazomethane (TMS-diazomethane). Phosphoinositides, extracted from a PC3 prostatic cancer cell line, were labeled either with CH2N2 or CD2N2 and mixed in known proportions for DAG(+)-specific MPIS-based mass spectrometry (MS) analysis. The results indicate that isotopic labeling is capable of providing accurate quantitation of PtdInsP3, PtdInsP2, and PtdInsP with adequate linearity as well as high reproducibility with an average coefficient variation of 18.9%. More importantly, this new methods excluded the need for multiple phosphoinositide internal standards. DAG(+)-specific MPIS and isotopic labeling based MS analysis of phosphoinositides offers unique advantages over existing approaches and presents a powerful tool for research of phosphoinositide metabolism.

Genomic Features of Cladobotryum dendroides, Which Causes Cobweb Disease in Edible Mushrooms, and Identification of Genes Related to Pathogenicity and Mycoparasitism.

Cladobotryum dendroides, which causes cobweb disease in edible mushrooms, is one of the major fungal pathogens. Our previous studies focused on the genetic and morphological characterization of this fungus, as well as its pathogenicity and the identification of appropriate fungicides. However, little is known about the genome characters, pathogenic genes, and molecular pathogenic mechanisms of C. dendroides. Herein, we reported a high-quality de novo genomic sequence of C. dendroides and compared it with closely-related fungi. The assembled C. dendroides genome was 36.69 Mb, consisting of eight contigs, with an N50 of 4.76 Mb. This genome was similar in size to that of C. protrusum, and shared highly conserved syntenic blocks and a few inversions with C. protrusum. Phylogenetic analysis revealed that, within the Hypocreaceae, Cladobotryum was closer to Mycogone than to Trichoderma, which is consistent with phenotypic evidence. A significant number of the predicted expanded gene families were strongly associated with pathogenicity, virulence, and adaptation. Our findings will be instrumental for the understanding of fungi-fungi interactions, and for exploring efficient management strategies to control cobweb disease.

High rate of burnout among residents under standardized residency training in a tertiary teaching hospital of middle China: Results from a cross-sectional survey.

Burnout has been commonly observed in health care workers. Though research has been conducted involving burnout among doctors in China, few studies have focused on residents during standardized residency training (SRT). The professional status of the residents during SRT remains largely unclear. The present study was conducted in order to clarify the prevalence and potential risk factors of burnout in residents during SRT.An electronic questionnaire comprised 2 parts. The first part was designed to collect some basic characteristic information. Maslach Burnout Inventory-Human Services Survey was included in the second part.As many as 71.05% residents had at least 1 scale of burnout. Emotional exhaustion (EE) was found in 55.6% residents, depersonalization (DP) in 29.7% and reduced personal accomplishment (RPA) in 41.6%. Being older than 27, senior SRT year, working time more than 60 hours per week and poor sleeping quality was independently associated with at least on scale of burnout. Being unable to receive timely supervisor support significantly increased the probability of DP and EE. Not having friends at work or feeling cared for by the hospital were independently related to all 3 symptoms of burnout as well as overall burnout.Burnout rate is high in residents under SRT from middle part of China. Organizational, professional, and social support was demonstrated critical by the potential roles in protecting against burnout. Residents with burnout were prone to considering turnover. Strategies for managing burnout related factors among residents should be focused in future studies.

Dexmedetomidine alleviated neuropathic pain in dorsal root ganglion neurons by inhibition of anaerobic glycolysis activity and enhancement of ROS tolerance.

Neuropathic pain is a kind of chronic pain that is triggered or caused primarily by damage to the nervous system and neurological dysfunction. It's known that dexmedetomidine is a new type of highly selective alpha2-adrenoceptor agonist with sedation, anti-anxiety, analgesic and other effects. However, the function and mechanism of dexmedetomidine on neuropathic pain are not clear. Rat DRG neurons were isolated and identified using immunofluorescence assay. Following treatment with H2O2, dexmedetomidine or ROS inhibitor (NAC), the apoptosis and ROS levels were examined by flow cytometery; apoptosis- and anaerobic glycolysis-related proteins were determined by Western blot assay; glucose consumption, pyruvic acid, lactic acid and ATP/ADP ratios were also measured. The results revealed that dexmedetomidine inhibited H2O2-induced apoptosis and reactive oxygen species (ROS) in rat DRG neurons and in addition, dexmedetomidine down-regulated the expression levels of anaerobic glycolysis-related proteins, significantly reduced glucose, pyruvic acid and lactic acid levels. It also increased the ATP/ADP ratio in H2O2-treated rat dorsal root ganglion (DRG) neurons. Moreover, we also demonstrated that ROS inhibitor (NAC) also inhibited H2O2-induced apoptosis and anaerobic glycolysis in rat DRG neurons. In conclusion, dexmedetomidine suppressed H2O2-induced apoptosis and anaerobic glycolysis activity by inhibiting ROS, in rat DRG neurons. Therefore, dexmedetomidine might play a pivotal role in neuropathic pain by the inhibition of ROS.

Dexmedetomidine Attenuates Orthotopic Liver Transplantation-Induced Acute Gut Injury via α 2-Adrenergic Receptor-Dependent Suppression of Oxidative Stress.

Patients with orthotopic liver transplantation (OLT) frequently develop acute gut injury (AGI), and dexmedetomidine (Dex) has been reported to exert a protective effect against AGI. We investigated whether Dex protects against AGI through antioxidative stress effects by the Nrf2/HO-1 antioxidative signaling pathway. Rats were randomly allocated into a sham group and six orthotopic autologous liver transplantation (OALT) groups receiving different doses of Dex together with/without α 2-adrenergic receptor (AR) blockers. Intestinal tissues were collected to visualize the barrier damage and to measure the indexes of oxidative stress. For in vitro studies, rat intestinal recess epithelial cells (IEC-6) underwent hypoxia/reoxygenation (H/R), and the protective role of Dex was evaluated after α 2A-AR siRNA silencing. OALT resulted in increased oxidative stress, significant intestinal injury, and barrier dysfunction. Dex attenuated OALT-induced oxidative stress and intestinal injury, which was abolished by the pretreatment with the nonspecific α 2A-AR siRNA blocker atipamezole and the specific α 2A-AR siRNA blocker BRL-44408, but not by the specific 2B/C-AR siRNA blocker ARC239. Silencing of α 2A-AR siRNA also attenuated the protective role of Dex on alleviating oxidative stress in IEC-6 cells subjected to H/R. Dex exerted its protective effects by activating Nrf2/HO-1 antioxidative signaling. Collectively, Dex attenuates OALT-induced AGI via α 2A-AR-dependent suppression of oxidative stress, which might be a novel potential therapeutic target for OALT-induced AGI.

Macrophage Foam Cell-Derived Extracellular Vesicles Promote Vascular Smooth Muscle Cell Migration and Adhesion.

BACKGROUND: A new mechanism for intercellular communication has recently emerged that involves intercellular transfer of extracellular vesicles (EVs). Several studies have indicated that EVs may play a potential role in cell-to-cell communication between macrophage foam cells and vascular smooth muscle cells (VSMCs) in atherosclerotic lesion. METHODS AND RESULTS: This study involved the comparison of circulating EVs from atherosclerotic patients and control participants. The results showed that the circulation of the patients contained more leukocyte-derived EVs and that these EVs promoted more VSMC adhesion and migration than those of healthy participants. We then established a macrophage foam cell model and characterized the EVs from the macrophages. We used flow cytometric analyses and cell migration and adhesion assays and determined that the foam cells generated more EVs than the normal macrophages and that the foam cell-derived EVs were capable of promoting increased levels of VSMC migration and adhesion. Furthermore, we performed a proteomic analysis of the EVs. The data showed that the foam cell-derived EVs may promote VSMC adhesion and migration by regulating the actin cytoskeleton and focal adhesion pathways. In addition, Western blotting revealed that foam cell-derived EVs could promote the phosphorylation of ERK and Akt in VSMCs in a time-dependent manner. We also found that foam cell-derived EVs could enter the VSMCs and transfer integrins to the surface of these cells. CONCLUSIONS: The data in our present study provide the first evidence that EVs from foam cells could promote VSMC migration and adhesion, which may be mediated by the integration of EVs into VSMCs and the subsequent downstream activation of ERK and Akt.

[Groundwater organic pollution source identification technology system research and application].

Groundwater organic pollutions are found in large amount of locations, and the pollutions are widely spread once onset; which is hard to identify and control. The key process to control and govern groundwater pollution is how to control the sources of pollution and reduce the danger to groundwater. This paper introduced typical contaminated sites as an example; then carried out the source identification studies and established groundwater organic pollution source identification system, finally applied the system to the identification of typical contaminated sites. First, grasp the basis of the contaminated sites of geological and hydrogeological conditions; determine the contaminated sites characteristics of pollutants as carbon tetrachloride, from the large numbers of groundwater analysis and test data; then find the solute transport model of contaminated sites and compound-specific isotope techniques. At last, through groundwater solute transport model and compound-specific isotope technology, determine the distribution of the typical site of organic sources of pollution and pollution status; invest identified potential sources of pollution and sample the soil to analysis. It turns out that the results of two identified historical pollution sources and pollutant concentration distribution are reliable. The results provided the basis for treatment of groundwater pollution.

[In situ experimental research on natural attenuation of oil pollutants in a gas station].

The natural attenuation rules of oil pollutants are investigated by carrying out a field experiment in a gas station in Beijing, using a set of soil gas sampling and monitoring device designed independently. The samples of soil gas were collected in the field and tested to examine the content of O2, CO2 and VOCs in the unsaturated zone. The results show that after 381 days' natural attenuation, the concentration of the contaminants at the polluted point deceased by 99.2%, while the ratio of BTEX to TVOC decreased from 17.0% to 12.1%. Moreover, the content of O2 decreased while that of CO2 grew as the soil depth increases at G3 point. Further theoretical analysis indicated that: (1) The test results of first-step experiment show that there was a certain level of contamination in G3 area. And after 381 days' natural attenuation, the content of the BTEX at G3 point fell below the environmental standard level, which means the control measures could be removed. (2) The origin of the contamination was proved as short-term sources, and no long-term sources of leakage existed. (3) An effective removal of contaminants in the soil was found under the influence of the natural attenuation. This method could be applied in long-term monitoring for similar contaminated sites. (4) The test of O2and CO2 contents was proved to be an effective approach to determine if the aerobic degradation happens.

[Research of the natural attenuation capacity of oil pollutants based on in-situ experiment].

Based on the results of the in-situ experiment carried out in a Beijing gas station, the ways of natural attenuation were evaluated as both aerobic and anaerobic biodegradation via oxygen and carbon equilibrium calculation. And the research got the rates of the natural attenuation and half-life of the oil pollutants byusing the first-order decay model. Based on the analysis, the capability of monitored natural attenuation and the environmental quality were evaluated. The results show that the actual oxygen consumption of contaminated site is less than that needed theoretically in aerobic degradation of all pollutants, while the amount of CO2 detected is far less than that in the aerobic degradation by theoretical formation. And the half-life of petroleum contaminants in contaminated site is about 50 days. The analysis indicated that: (1) The ways of natural attenuation were proved as both aerobic and anaerobic biodegradation; (2) The contamination of the BTEX tested by the fist-step experiment fell below the environmental standard level after about 250 days' natural attenuation, which means the environmental risk in this gas station had been eliminated and the control measures could be removed.

[Physicochemical properties of Guanting Reservoir sediment and its land application].

Surface sediment of Guanting Reservoir was dredged up and dewatered in field, and pollutant and physicochemical characterizations were mensurated. The stabilization and agricultural land use of the sediment was also studied in the field. Results showed that the sediments have a higher clay content, bulk density (1.89 g x cm(-3)) and lower porosity (23.8%), higher deoxidize material and available nitrogen, phosphorus concentration. Heavy metal and organochlorinated pesticides concentration was lower than the class II of national standard for soil. Stabilized the sediment with sand soil and straw could improve the physical property and decrease the concentration of deoxidize material and available nitrogen, phosphorus. Stabilized sediment could be a suitable medium for alfalfa, tree and corn growth and used for agricultural land.

[Performance of treatment wetland systems for surface water quality improvement].

Intercropped with Phragmites communis and Typha angustifolia, subsurface flow constructed wetland systems (CWs) with the surface area of 3 x 20m x 2m were established beside Guanting Reservoir, an important source water base of Beijing. The treatment performance of the systems with different season were studied, the impacts of influent concentration, hydraulic loading rate and water temperature on contaminations removal were analyzed. The result showed that the subsurface flow CWs had the better decontamination effect to micro-pollution surface water. The relationship between the concentrations of CODMn and NH4+ -N in inflow and outflow followed the linear equation. The removal rates of total nitrogen (TN) and total phosphorus (TP) in the systems were 20%-60% and 30%-45%, respectively. The removal rates of contaminations were reduced with the decrease of water temperature and the increase of hydraulic loading rate, the removal rates of CODMn, N4+ -N and TN showed the positive correlation with their inflow concentration, but the removal rate of TP showed the negative correlation with its inflow concentration. Operation and management considerations of the subsurface flow CWs in winter were investigated in this study.