'Candidatus Liberibacter asiaticus' secretory protein SDE3 inhibits host autophagy to promote Huanglongbing disease in citrus.

Antimicrobial acroautophagy/autophagy plays a vital role in degrading intracellular pathogens or microbial molecules in host-microbe interactions. However, microbes evolved various mechanisms to hijack or modulate autophagy to escape elimination. Vector-transmitted phloem-limited bacteria, Candidatus Liberibacter (Ca. Liberibacter) species, cause Huanglongbing (HLB), one of the most catastrophic citrus diseases worldwide, yet contributions of autophagy to HLB disease proliferation remain poorly defined. Here, we report the identification of a virulence effector in "Ca. Liberibacter asiaticus" (Las), SDE3, which is highly conserved among the "Ca. Liberibacter". SDE3 expression not only promotes the disease development of HLB and canker in sweet orange (Citrus sinensis) plants but also facilitates Phytophthora and viral infections in Arabidopsis, and Nicotiana benthamiana (N. benthamiana). SDE3 directly associates with citrus cytosolic glyceraldehyde-3-phosphate dehydrogenases (CsGAPCs), which negatively regulates plant immunity. Overexpression of CsGAPCs and SDE3 significantly inhibits autophagy in citrus, Arabidopsis, and N. benthamiana. Intriguingly, SDE3 undermines autophagy-mediated immunity by the specific degradation of CsATG8 family proteins in a CsGAPC1-dependent manner. CsATG8 degradation is largely rescued by treatment with an inhibitor of the late autophagic pathway, E64d. Furthermore, ectopic expression of CsATG8s enhances Phytophthora resistance. Collectively, these results suggest that SDE3-CsGAPC interactions modulate CsATG8-mediated autophagy to enhance Las progression in citrus.Abbreviations: ACP: asian citrus psyllid; ACD2: ACCELERATED CELL DEATH 2; ATG: autophagy related; Ca. Liberibacter: Candidatus Liberibacter; CaMV: cauliflower mosaic virus; CMV: cucumber mosaic virus; Cs: Citrus sinensis; EV: empty vector; GAPC: cytosolic glyceraldehyde-3-phosphate dehydrogenase; HLB: huanglongbing; H2O2: hydrogen peroxide; Las: liberibacter asiaticus; Laf: liberibacter africanus; Lam: liberibacter americanus; Pst: Pseudomonas syringae pv. tomato; PVX: potato virus X; ROS: reactive oxygen species; SDE3: sec-delivered effector 3; TEM: transmission electron microscopy; VIVE : virus-induced virulence effector; WT: wild-type; Xcc: Xanthomonas citri subsp. citri.

Investigation of membrane fouling mechanism of intracellular organic matter during ultrafiltration.

This study investigated the ultrafiltration (UF) membrane fouling mechanism of intracellular organic matter (IOM) from Chlorella vulgaris (CV) and Microcystis aeruginosa (MA). Both CV- and MA-IOM caused severe membrane fouling during UF; however, there were significant differences in the membrane fouling by these two materials. Neutral hydrophilic (N-HPI) compounds were the organics that caused the most severe membrane fouling during CV-IOM filtration, whereas the MA-IOM membrane fouling was induced by mainly hydrophobic (HPO) organics. From an analysis based on Derjaguin-Landau-Verwey-Overbeek theory, it was found that the interaction energy between the membrane and foulants in the later stage of filtration was the major factor determining the efficiency of filtration for both CV-IOM and MA-IOM. The TPI organics in CV-IOM fouled the membrane to a more severe degree during the initial filtration flux; however, when the membrane surface was covered with CV-IOM foulants, the N-HPI fraction of CV-IOM caused the most severe membrane fouling because its attractive energy with the membrane was the highest. For MA-IOM, regardless of the initial filtration flux or the late stage of filtration, the HPO organics fouled the membrane to the greatest extent. An analysis of modified filtration models revealed that cake layer formation played a more important role than other fouling mechanisms during the filtration of CV-IOM and MA-IOM. This study provides a significant understanding of the membrane fouling mechanism of IOM and is beneficial for developing some strategies for membrane fouling control when treating MA and CV algae-laden waters.

Reversible and irreversible membrane fouling in hollow-fiber UF membranes filtering surface water: effects of ozone/powdered activated carbon treatment.

This study investigated the reversible and irreversible membrane fouling behavior of micro polluted water by ozone/powdered activated carbon (PAC)/ultrafiltration treatment. The results indicated that PAC mainly adsorbed low-molecular weight organics and reduced the irreversible fouling resistance in ultrafiltration, while there existed a threshold PAC dosage for total and reversible fouling resistance alleviation. Ozone at low doses exerted little effect on membrane fouling alleviation, while higher doses controlled total and reversible fouling by reducing macromolecular biopolymers and humic-like substances. Combined ozone and PAC pretreatment had greater effects on both reversible and irreversible fouling reduction than individual PAC and ozone treatment, demonstrating synergistic effects in the reduction of organic content in the feed water, including macromolecular biopolymers, humic-like, low-molecular weight neutral and building blocks. Backwashing and chemical cleaning analysis revealed that biopolymers and humic-like substances were the main organics that caused hydraulic reversible fouling, whereas low-molecular organics of building blocks and neutral, as well as humic-like substances were the main components that caused hydraulic irreversible fouling. Combined ozone and PAC treatment not only improved the backwashing efficiency but also reduced the membrane fouling during backwashing, as well as reversible and irreversible fouling. The cake layer formation and standard pore blocking were the major mechanisms for ultrafiltration membrane fouling, of which standard pore blocking exerted more important effects in the membrane fouling formation and alleviation by individual and combined PAC and ozone treatment.

Establishment of a novel virus-induced virulence effector assay for the identification of virulence effectors of plant pathogens using a PVX-based expression vector.

Plant pathogens deliver virulence effectors into plant cells to modulate plant immunity and facilitate infection. Although species-specific virulence effector screening approaches have been developed for several pathogens, these assays do not apply to pathogens that cannot be cultured and/or transformed outside of their hosts. Here, we established a rapid and parallel screening assay, called the virus-induced virulence effector (VIVE) assay, to identify putative effectors in various plant pathogens, including unculturable pathogens, using a virus-based expression vector. The VIVE assay uses the potato virus X (PVX) vector to transiently express candidate effector genes of various bacterial and fungal pathogens into Nicotiana benthamiana leaves. Using the VIVE assay, we successfully identified Avh148 as a potential virulence effector of Phytophthora sojae. Plants infected with PVX carrying Avh148 showed strong viral symptoms and high-level Avh148 and viral RNA accumulation. Analysis of P. sojae Avh148 deletion mutants and soybean hairy roots overexpressing Avh148 revealed that Avh148 is required for full pathogen virulence. In addition, the VIVE assay was optimized in N. benthamiana plants at different developmental stages across a range of Agrobacterium cell densities. Overall, we identified six novel virulence effectors from seven pathogens, thus demonstrating the broad effectiveness of the VIVE assay in plant pathology research.

Primary colorectal diffuse large B-cell lymphoma initially presenting with pleural effusion: report of one case and review of literature.

Gastrointestinal (GI) diffuse large B-cell lymphoma (DLBCL) is one of the frequently reported histologic subtypes of non-Hodgkin lymphoma (NHL) that occur in the GI tract. However, the presentation of quite different clinical manifestations, morphologic characteristics, immunophenotypes, and molecular biologic features is challenging for its diagnosis. Herein, we describe a rare case of primary colorectal DLBCL that occurred in a 59-year-old immunocompetent Chinese female who attended our respiratory clinic for the third time with an asymptomatic pleural effusion and pleural thickening. In her clinical setting, there was no history of trauma or travel, and no evidence of infections, connective tissue diseases, or malignancies such as pleural mesothelioma. Lymphoma was highly suspected for the enlargement of systemic lymph nodes and the multiple polypoid appearance in the rectum found by endoscopy examination. In a repeated colonoscopy, immunohistochemical and molecular features of the multiple "polyps" allowed diagnosis of colorectal diffuse large B-cell NHL. To our knowledge, this is the first case of a verified diagnosis of pleural effusion associated with a primary colorectal DLBCL. The purpose of this report is to alert clinicians that when we evaluate the causes of unexplained pleural effusion, lymphoma should be considered, particularly when the available examination data cannot be corroborated by clinical manifestations.

Descending necrotizing mediastinitis caused by Streptococcus constellatus in an immunocompetent patient: case report and review of the literature.

Descending necrotizing mediastinitis is a severe infection of the mediastinum. This syndrome manifests as fever and chest pain following cough and sputum production. A 49-year-old woman presented with fever and a 14-day history of pneumonia. CT showed mediastinal abscesses with a giant calcified mediastinal lymph node (21 × 18 mm) and pneumonia. Bronchoscopy by EBUS-TBNA under general anesthesia was performed. The pathogen found in the puncture culture was Streptococcus constellatus, and antibiotics (mezlocillin/sulbactam 3.375 IVGTT q8h) was administered. A proximal right main bronchial neoplasm, suspected lung cancer, was found and conformed to inflammatory granuloma. A total of 22 months post-discharge the patient was clinically stable. We also conducted a review of the literature for all Streptococcus constellatus descending necrotizing mediastinitis infections between 2011 and 2017.

Comparison of the Abundance and Community Structure of N-Cycling Bacteria in Paddy Rhizosphere Soil under Different Rice Cultivation Patterns.

Eco-agricultural systems aim to reduce the use of chemical fertilizers in order to improve sustainable production and maintain a healthy ecosystem. The aim of this study was to explore the effects of rice-frog farming on the bacterial community and N-cycling microbes in paddy rhizosphere soil. This experiment involved three rice cultivation patterns: Conventionally cultivated rice (CR), green rice-frog farming (GR), and organic rice-frog farming (OR). The rice yield, paddy soil enzyme activities, physicochemical variables and bacterial and N-cycling bacterial abundances were quantitatively analyzed. Rice-frog cultivations significantly increased soil protease, nitrate and reductase activity. Additionally, the nirS gene copy number and the relative abundance of denitrifying bacteria also increased, however urease activity and the relative abundance of nitrifying bacteria significantly decreased. The bacterial community richness and diversity of OR soil was significantly higher than that of the GR or CR soil. Nitrogen use efficiency (NUE) of GR was highest. The N-cycling bacterial community was positively correlated with the total carbon (TC), total nitrogren (TN) and carbon to nitrogen (C:N) ratio. The present work strengthens our current understanding of the soil bacterial community structure and its functions under rice-frog farming. The present work also provides certain theoretical support for the selection of rational rice cultivation patterns.

Overexpression of Napsin A resensitizes drug-resistant lung cancer A549 cells to gefitinib by inhibiting EMT.

Lung cancer is one of the most common malignant tumors and also the leading cause of cancer-related deaths in the world. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI), such as gefitinib, have been used in the therapy of lung cancer. However, the acquisition of drug resistance is a major limitation in the clinical efficiency of EGFR-TKIs. Epithelial-mesenchymal transition (EMT) has been demonstrated to be an underlying mechanism of acquired resistance. A previous study has reported that Napsin A expression can inhibit EMT in lung cancer cells. The present study therefore investigated the effect of Napsin A on the sensitivity of EGFR-TKI-resistant lung cancer cells. First, a drug-resistant lung cancer cell line was generated using the EGFR-TKI gefitinib on A549 cells (termed here A549-GFT). EMT was demonstrated to be induced in the drug resistant A549-GFT cells, evidenced by reduced E-cadherin expression and increased Vimentin expression compared with control A549 cells. Next, Napsin A was overexpressed in the cells by transfection of the Napsin A-expression vector, PLJM1-Napsin A. Western blot analysis confirmed that the protein expression levels of Napsin A were significantly elevated in the Napsin A-overexpressing cells. Cell proliferation and apoptosis assays were performed to evaluate the effect of Napsin A overexpression on resistant A549 cells. The results of MTT assay demonstrated that Napsin A overexpression inhibited the proliferation of A549 and drug-resistant A549-GFT cells and that the proliferation of Napsin A-overexpressing A549-GFT cells was significantly inhibited by gefitinib treatment compared with control A549-GFT cells. The results from the Annexin V/propidium iodide double staining apoptosis assay indicated that Napsin A overexpression enhanced gefitinib-induced apoptosis in A549-GFT cells. Additionally, EMT was reversed following Napsin A expression in A549-GFT cells, as evidenced by the restoration of E-cadherin and downregulation of Vimentin expression. Further investigation demonstrated that Napsin A overexpression resulted in inhibition of focal adhesion kinase, a critical factor in integrin signaling, in the resistant A549-GFT cells. These data suggested that Napsin A resensitized the drug-resistant A549-GFT cells to gefitinib, possibly by reversing EMT via integrin signaling inhibition. Therefore, Napsin A combined with a TKI may be a more effective treatment strategy for lung cancer.

Napsin A is negatively associated with EMT­mediated EGFR­TKI resistance in lung cancer cells.

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR­TKI) have been used as a standard therapy for patients with lung cancer with EGFR­activating mutations. Epithelial­mesenchymal transition (EMT) has been reported to be associated with the development of EGFR­TKI resistance, which limits the clinical efficacy of EGFR­TKI. Therefore, investigating the resistance­associated mechanism is required in order to elucidate an effective therapeutic approach to enhance the sensitivity of lung cancer to EGFR­TKI. In the present study, EGFR­TKI erlotinib­sensitive H358, H322 and H441 lung cancer cells, erlotinib­moderately sensitive A549 cells, and erlotinib­insensitive HCC827 cells with EGFR­mutation (exon 19 deletion) were used to detect the mRNA and protein expression of the EMT­associated proteins E­cadherin and vimentin, and napsin A, by reverse transcription­quantitative polymerase chain reaction analysis and western blotting. It was observed that the E­cadherin expression level in erlotinib­sensitive cells was increased compared with the moderately sensitive A549 cells and HCC827 cells; however, vimentin exhibited opposite expression, suggesting a correlation between EMT and erlotinib sensitivity in lung cancer cells. The napsin A expression level was observed to be positively associated with erlotinib sensitivity. In addition, napsin A highly­expressingH322 cells were used and napsin A­silenced cells were constructed using small interfering RNA (siRNA) technology, and were induced by transforming growth factor (TGF)­ßl. It was observed that TGF­ßl partially induced the alterations in E­cadherin and vimentin expression and the occurrence of EMT in napsin A highly­expressing cells, while TGF­ßl significantly induced EMT via downregulation of E­cadherin and upregulation of vimentin in napsin A­silenced cells; cell proliferation and apoptosis assays demonstrated that TGF­ßl induced marked resistance to erlotinib in napsin A­silenced cells compared with napsin A­expression cells. These data indicated that napsin A expression may inhibit TGF­ßl­induced EMT and was negatively associated with EMT­mediated erlotinib resistance, suggesting that napsin A expression may improve the sensitivity of lung cancer cells to EGFR­TKI through the inhibition of EMT.

[Culture supernatants of lymphocytes from different lymphoid tissues induce transdifferentiation of Caco2 cells into M-like cells].

OBJECTIVE: To establish the microfold (M)-like cell model in vitro and identify M-like cells through detecting the capacity of transporting fluorescent beads and the levels of the associated genes, and to observe the effects of lymphocyte culture supernatants stimulated by concanavalin A (Con A) from different lymphoid tissues on the differentiation of Caco2 cells into M-like cells. METHODS: The isolated lymphocytes of Peyer's patch (PP), mesenteric lymph node (MLN) and spleen (Sp) were incubated with 3 µg/mL Con A for 3 days. The culture supernatants were collected and co-cultured with Caco2 cells. The fluorescent bead suspension was added into the upper compartment of the Transwell™ inserts, and then basolateral solutions were then sampled and analyzed. The number of transported fluorescent beads was measured by flow cytometry. The expressions of M-like cells-associated genes, such as chemokine (C-C motif) ligand 20 (CCL20), claudin4 (CLDN4), tumor necrosis factor receptor superfamily member 9 (TNFRSF9), and Spi-B were detected by reverse transcription PCR. RESULTS: Compared with blank control group, the number of fluorescent beads transported by induced Caco2 cells and the levels of CCL20, CLDN4, TNFRSF9 and Spi-B mRNAs significantly increased in induced Caco2 cells treated with the culture supernatants of lymphocytes from PP, MLN and Sp. After Con A stimulation, the number of fluorescent beads transported by induced Caco2 cells and the levels of CCL20, CLDN4, TNFRSF9 and Spi-B mRNAs were higher than those in the unstimulated group. CONCLUSION: The lymphocyte culture supernatants stimulated or unstimulated by Con A can induce the transdifferentiation of Caco2 cells into M-like cells.

[Effect of qidong huoxue decoction on inflammatory factors and TLR4 mRNA Expression in acute lung injury rats].

OBJECTIVE: To explore the effect of qidong huoxue decoction (QHD) on inflammatory factors and Toll-like receptor (TLR4) mRNA expressions in acute lung injury (ALI) rats. METHODS: Totally 50 healthy male SD rats were randomly divided into the blank control group, the lipopolysaccharide (LPS) model group, low, middle, high dose QHD groups according to body weight, 10 rats in each group. Rats in low, middle, high dose QHD groups were intragastrically administered with QHD at 4, 8, and 16 mL/kg 24, 12 h before modeling and 12 h after modeling, respectively. Normal saline was intragastrically administered to rats in the blank control group and the LPS model group. An ALI rat model was established using intratracheal instillation of LPS. Rats were killed after 24-h modeling. Then the bronchoalveolar lavage fluid was prepared. Contents of TNF-α, IL-1ß, and L-10 were detected using ELISA. TLR4 mRNA expressions were determined byreal time PCR. RESULTS: Compared with the blank control group, contents of TNF-α, IL-1ß , and IL-10 increased (P <0. 01), TLR4 mRNA expressions also increased in the LPS model group (all P <0. 01). Compared with the LPS model group, contents of TNF-α and IL-1ß decreased (P <0. 05, P <0. 01), IL-10 levels increased (P <0. 01) , TLR4 mRNA expressions were also reduced (P <0. 01), in high and middle dose QHD groups. Compared with the high dose QHD group, con- tents of TNF-α and IL-1ß increased in middle and low dose QHD groups (P <0. 05); IL-10 levels decreased (P <0. 05) in the low dose QHD group(P <0. 05), TLR4 mRNA expressions also increased in the low dose QHD group (P <0. 05). Compared with the middle dose QHD group, IL-10 levels was reduced, but TLR4 mRNA expressions increased in the low dose QHD group (P <0. 05). CONCLUSIONS: QHD had the protective effect on LPS induced ALI rats. Its mechanism might be associated with inhibiting TLR4 mRNA expressions, leading to decreased pro-inflammatory cytokines such as TNF-α and IL-ß, elevated anti-inflammatory cytokine IL-10, and thereby, correcting unbalanced inflammation.

Organic nitrogen components in soils from southeast China.

OBJECTIVE: To investigate the amounts of extractable organic nitrogen (EON), and the relationships between EON and total extractable nitrogen (TEN), especially the amino acids (AAs) adsorbed by soils, and a series of other hydrolyzed soil nitrogen indices in typical land use soil types from southeast China. Under traditional agricultural planting conditions, the functions of EON, especially AAs in the rhizosphere and in bulk soil zones were also investigated. METHODS: Pot experiments were conducted using plants of pakchoi (Brassica chinensis L.) and rice (Oryza sativa L.). In the rhizosphere and bulk soil zone studies, organic nitrogen components were extracted with either distilled water, 0.5 mol/L K2SO4 or acid hydrolysis. RESULTS: K2SO4-EON constituted more than 30% of TEN pools. K2SO4-extractable AAs accounted for 25% of EON pools and nearly 10% of TEN pools in rhizosphere soils. Overall, both K2SO4-EON and extractable AAs contents had positive correlations with TEN pools. CONCLUSIONS: EON represented a major component of TEN pools in garden and paddy soils under traditional planting conditions. Although only a small proportion of the EON was present in the form of water-extractable and K2SO4-extractable AAs, the release of AAs from soil exchangeable sites might be an important source of organic nitrogen (N) for plant growth. Our findings suggest that the content of most organic forms of N was significantly greater in rhizosphere than in bulk soil zone samples. However, it was also apparent that the TEN pool content was lower in rhizosphere than in bulk soil samples without added N.

Regression modeling of streamflow, baseflow, and runoff using geographic information systems.

Regression models for predicting total streamflow (TSF), baseflow (TBF), and storm runoff (TRO) are needed for water resource planning and management. This study used 54 streams with >20 years of streamflow gaging station records during the period October 1971 to September 2001 in Pennsylvania and partitioned TSF into TBF and TRO. TBF was considered a surrogate of groundwater recharge for basins. Regression models for predicting basin-wide TSF, TBF, and TRO were developed under three scenarios that varied in regression variables used for model development. Regression variables representing basin geomorphological, geological, soil, and climatic characteristics were estimated using geographic information systems. All regression models for TSF, TBF, and TRO had R(2) values >0.94 and reasonable prediction errors. The two best TSF models developed under scenarios 1 and 2 had similar absolute prediction errors. The same was true for the two best TBF models. Therefore, any one of the two best TSF and TBF models could be used for respective flow prediction depending on variable availability. The TRO model developed under scenario 1 had smaller absolute prediction errors than that developed under scenario 2. Simplified Area-alone models developed under scenario 3 might be used when variables for using best models are not available, but had lower R(2) values and higher or more variable prediction errors than the best models.