TurboID screening of ApxI toxin interactants identifies host proteins involved in Actinobacillus pleuropneumoniae-induced apoptosis of immortalized porcine alveolar macrophages.

Actinobacillus pleuropneumoniae (APP) is a gram-negative pathogenic bacterium responsible for porcine contagious pleuropneumonia (PCP), which can cause porcine necrotizing and hemorrhagic pleuropneumonia. Actinobacillus pleuropneumoniae-RTX-toxin (Apx) is an APP virulence factor. APP secretes a total of four Apx toxins, among which, ApxI demonstrates strong hemolytic activity and cytotoxicity, causing lysis of porcine erythrocytes and apoptosis of porcine alveolar macrophages. However, the protein interaction network between this toxin and host cells is still poorly understood. TurboID mediates the biotinylation of endogenous proteins, thereby targeting specific proteins and local proteomes through gene fusion. We applied the TurboID enzyme-catalyzed proximity tagging method to identify and study host proteins in immortalized porcine alveolar macrophage (iPAM) cells that interact with the exotoxin ApxI of APP. His-tagged TurboID-ApxIA and TurboID recombinant proteins were expressed and purified. By mass spectrometry, 318 unique interacting proteins were identified in the TurboID ApxIA-treated group. Among them, only one membrane protein, caveolin-1 (CAV1), was identified. A co-immunoprecipitation assay confirmed that CAV1 can interact with ApxIA. In addition, overexpression and RNA interference experiments revealed that CAV1 was involved in ApxI toxin-induced apoptosis of iPAM cells. This study provided first-hand information about the proteome of iPAM cells interacting with the ApxI toxin of APP through the TurboID proximity labeling system, and identified a new host membrane protein involved in this interaction. These results lay a theoretical foundation for the clinical treatment of PCP.

Porcine Epidemic Diarrhea Virus and Its nsp14 Suppress ER Stress Induced GRP78.

Porcine epidemic diarrhea virus (PEDV), a member of the α-coronavirus genus, can cause vomiting, diarrhea, and dehydration in piglets. Neonatal piglets infected with PEDV have a mortality rate as high as 100%. PEDV has caused substantial economic losses to the pork industry. Endoplasmic reticulum (ER) stress, which can alleviate the accumulation of unfolded or misfolded proteins in ER, involves in coronavirus infection. Previous studies have indicated that ER stress could inhibit the replication of human coronaviruses, and some human coronaviruses in turn could suppress ER stress-related factors. In this study, we demonstrated that PEDV could interact with ER stress. We determined that ER stress could potently inhibit the replication of GⅠ, GⅡ-a, and GⅡ-b PEDV strains. Moreover, we found that these PEDV strains can dampen the expression of the 78 kDa glucose-regulated protein (GRP78), an ER stress marker, while GRP78 overexpression showed antiviral activity against PEDV. Among different PEDV proteins, PEDV non-structural protein 14 (nsp14) was revealed to play an essential role in the inhibition of GRP78 by PEDV, and its guanine-N7-methyltransferase domain is necessary for this role. Further studies show that both PEDV and its nsp14 negatively regulated host translation, which could account for their inhibitory effects against GRP78. In addition, we found that PEDV nsp14 could inhibit the activity of GRP78 promotor, helping suppress GRP78 transcription. Our results reveal that PEDV possesses the potential to antagonize ER stress, and suggest that ER stress and PEDV nsp14 could be the targets for developing anti-PEDV drugs.

Rearranged Diels-Alder Adducts and Prenylated Flavonoids as Potential PTP1B Inhibitors from Morus nigra.

Two novel rearranged Diels-Alder adducts, morunigrines A (1) and B (2), and four new prenylated flavonoids, morunigrols A-D (3-6), were isolated from the twigs of Morus nigra, together with four known prenylated phenolic compounds, including two flavonoids (7 and 8) and two 2-arylbenzofurans (9 and 10). Morunigrines A (1) and B (2) are a novel class of Diels-Alder adducts with unprecedented carbon skeletons featuring a rearranged chalcone-stilbene/2-arylbenzofuran core decorated with a unique methylbiphenyl moiety. The structures of the new compounds were assigned by analysis of spectroscopic data. The absolute configuration of compound 6 was determined by the measurement of specific rotation. A plausible biogenetic pathway for 1 and 2 is also proposed. Compounds 1 and 2 exhibited more potent protein tyrosine phosphatase 1B inhibitory activity with IC50 values of 1.8 ± 0.2 and 1.3 ± 0.3 µM, respectively, than that of the positive control oleanolic acid (IC50, 2.5 ± 0.1 µM).

In silico prediction of chemical reproductive toxicity using machine learning.

Reproductive toxicity is an important regulatory endpoint in health hazard assessment. Because the in vivo tests are expensive, time consuming and require a large number of animals, which must be killed, in silico approaches as the alternative strategies have been developed to assess the potential reproductive toxicity (reproductive toxicity) of chemicals. Some prediction models for reproductive toxicity have been developed, but most of them were built only based on one single endpoint such as embryo teratogenicity; therefore, these models may not provide reliable predictions for toxic chemicals with other endpoints, such as sperm reduction or gonadal dysgenesis. Here, a total of 1823 chemicals for reproductive toxicity characterized by multiple endpoints were used to develop structure-activity relationship models by six machine-learning approaches with nine molecular fingerprints. Among the models, MACCSFP-SVM model has the best performance for the external validation set (area under the curve = 0.900, classification accuracy = 0.836). The applicability domain was analyzed, and a rational boundary was found to distinguish inaccurate predictions and accurate predictions. Moreover, several structural alerts for characterizing reproductive toxicity were identified using the information gain combining substructure frequency analysis. Our results would be helpful for the prediction of the reproductive toxicity of chemicals.

Characterization of submicron particles during autumn in Beijing, China.

In this study, we performed a highly time-resolved chemical characterization of non-refractory submicron particles (NR-PM1) in Beijing by using an Aerodyne high-resolution time-of-flight aerosol mass spectrometer (HR-ToF-AMS). The results showed the average NR-PM1 mass concentration to be 56.4±58.0µg/m3, with a peak at 307.4µg/m3. Due to the high frequency of biomass burning in autumn, submicron particles significantly increased in organic content, which accounted for 51% of NR-PM1 on average. Secondary inorganic aerosols (sulfate+nitrate+ammonium) accounted for 46% of NR-PM1, of which sulfate, nitrate, and ammonium contributed 15%, 20%, and 11%, respectively. To determine the intrinsic relationships between the organic and inorganic species, we used the positive matrix factorization (PMF) model to merge the high-resolution mass spectra of the organic species and NO+ and NO2+ ions. The PMF analysis separated the mixed organic and nitrate (NO+ and NO2+) spectra into four organic factors, including hydrocarbon-like organic aerosol (HOA), oxygenated organic aerosol (OOA), cooking organic aerosol (COA), and biomass burning organic aerosol (BBOA), as well as one nitrate inorganic aerosol (NIA) factor. COA (33%) and OOA (30%) contributed the most to the total organic aerosol (OA) mass, followed by BBOA (20%) and HOA (17%). We successfully quantified the mass concentrations of the organic and inorganic nitrates by the NO+ and NO2+ ions signal in the organic and NIA factors. The organic nitrate mass varied from 0.01-6.8µg/m3, with an average of 1.0±1.1µg/m3, and organic nitrate components accounted for 10% of the total nitrate mass in this observation.

Aglaiabbrevins A-D, New Prenylated Bibenzyls from the Leaves of Aglaia abbreviata with Potent PTP1B Inhibitory Activity.

Four new prenylated bibenzyls, named aglaiabbrevins A-D (2, 4-6), were isolated from the leaves of Aglaia abbreviata, along with two known related analogues, 3,5-dihydroxy-2-[3,7-dimethyl-2(E),6-octadienyl]bibenzyl (7) and 3,5-dihydroxy-2-(3-methyl-2-butenyl)bibenzyl (8). The structures of the new compounds were elucidated on the basis of extensive spectroscopic experiments, mainly one and two dimensional (1D- and 2D)-NMR, and the absolute configuration of 5 was determined by the measurement of specific rotation. The isolated compounds were evaluated for their protein tyrosine phosphatase-1B (PTP1B) inhibitory activity. The results showed that compounds 5-7 exhibited more potent PTP1B inhibitory effects with IC50 values of 2.58±0.52, 2.44±0.35, and 2.23±0.14 µM, respectively, than the positive control oleanolic acid (IC50=2.74±0.20 µM). On the basis of the data obtained, these bibenzyls with the longer C-2 prenyl groups may be considered as potential lead compounds for the development of new anti-obesity and anti-diabetic agents. Also, the PTP1B inhibitory effects for prenylated bibenzyls are being reported for the first time.

Changing the order and ratio of substrate filling reduced CH4 and N2O emissions from the aerated constructed wetlands.

Constructed wetlands (CWs) have been used to enhance pollutant removal by filling several types of material as substrates. However, research on substrate filling order remains still limited, particularly regarding the effects of greenhouse gas (GHG) emissions. In this study, six CWs were constructed using zeolite and ferric­carbon micro-electrolysis (Fe-C) fillers to evaluate the effect of changing the filling order and ratio on pollutant removal, GHGs emissions, and associated microbial structure. The results showed that the order of substrate filling significantly impacted pollutant removal performance on CWs. Specifically, CWs filled with zeolite in the top layer exhibited superior NH4+-N removal compared to those filled in the lower layer. Moreover, the highest NH4+-N removal (95.0 % ± 1.9 %) was observed in CWs with a zeolite to Fe-C volume ratio of 8:2 (CWZe-1). Moreover, zeolite-filled at the top had lower GHGs emissions, with the lowest CH4 (0.22 ± 0.10 mg m-2 h-1) and N2O (167.03 ± 61.40 µg m-2 h-1) fluxes in the CWZe-1. In addition, it is worth noting that N2O is the major contributor to integrated global warming potential (GWP) in the six CWs, accounting for 81.7 %-90.8 %. The upper layer of CWs filled with zeolite exhibited higher abundances of nirK, nirS and nosZ genes. The order in which the substrate was filled affected the microbial community structure and the upper layer of CWs filled with zeolite had higher relative abundance of nitrifying genera (Nitrobacter, Nitrosomonas) and denitrifying genera (Zoogloea, Denitratisoma). Additionally, N2O emission was reduced by approximately 41.2 %-64.4 % when the location of the aeration of the CWs was changed from the bottom to the middle. This study showed that both the order of filling the substrate and the aeration position significantly affected the GHGs emissions from CWs, and that CWs had lower GHGs emissions when zeolites were filled in the upper layer and the aeration position was in the middle.

ARF6 promotes Streptococcus suis suilysin induced apoptosis in HBMECs.

Streptococcus suis (S. suis) is a significant zoonotic microorganism that causes a severe illness in both pigs and humans and is characterized by severe meningitis and septicemia. Suilysin (SLY), which is secreted by S. suis, plays a crucial role as a virulence factor in the disease. To date, the interaction between SLY and host cells is not fully understood. In this study, we identified the interacting proteins between SLY and human brain microvascular endothelial cells (HBMECs) using the TurboID-mediated proximity labeling method. 251 unique proteins were identified in TurboID-SLY treated group, of which six plasma membrane proteins including ARF6, GRK6, EPB41L5, DSC1, TJP2, and PNN were identified. We found that the proteins capable of interacting with SLY are ARF6 and PNN. Subsequent investigations revealed that ARF6 substantially increased the invasive ability of S. suis in HBMECs. Furthermore, ARF6 promoted SLY-induced the activation of p38 MAPK signaling pathway in HBMECs. Moreover, ARF6 promoted the apoptosis in HBMECs through the activation of p38 MAPK signaling pathway induced by SLY. Finally, we confirmed that ARF6 could increase the virulence of SLY in C57BL/6 mice. These findings offer valuable insights that contribute to a deeper understanding of the pathogenic mechanism of SLY.

Biochar and hematite amendments suppress emission of CH4 and NO2 in constructed wetlands.

Constructed wetlands (CWs) are considered a widely used cost-effective technology for pollutant removal. However, greenhouse gas emissions are a non-negligible problem in CWs. In this study, four laboratory-scale CWs were established to evaluate the effects of gravel (CWB), hematite (CWFe), biochar (CWC), and hematite + biochar (CWFe-C) as substrates on pollutants removal, greenhouse gas emissions, and associated microbial characteristics. The results showed that the biochar-amended CWs (CWC and CWFe-C) enhanced the removal efficiency of pollutants, with 92.53 % and 93.66 % of COD and 65.73 % and 64.41 % of TN removal, respectively. Both single and combined inputs of biochar and hematite significantly reduced CH4 and N2O fluxes, with the lowest average of CH4 flux obtained in CWC (5.99 ± 0.78 mg CH4 m-2 h-1) and the least N2O flux in CWFe-C (287.57 ± 44.84 µg N2O m-2 h-1). The substantial reduction of global warming potentials (GWP) was obtained in the applications of CWC (80.25 %) and CWFe-C (79.5 %) in biochar-amended CWs. The presence of biochar and hematite mitigated CH4 and N2O emissions by modifying microbial communities with higher ratios of pmoA/mcrA and nosZ genes abundances, as well as increasing the abundance of denitrifying bacteria (Dechloromona, Thauera and Azospira). This study demonstrated that biochar and the combined use of biochar and hematite could be the potential candidates as functional substrates for the efficient removal of pollutants and simultaneously reducing GWP emissions in the constructed wetlands.

Tailoring the draw solution chemistry in the integrated electro-Fenton and forward osmosis for enhancing emerging contaminants removal: Performance, DFT calculation and degradation pathway.

Integrated electro-Fenton and forward osmosis is capable to simultaneously separate emerging contaminants and degrade accumulated ones. Thus, an understanding of how draw solution chemistry in forward osmosis influences electro-Fenton is vital for maximizing overall treatment. Therefore, this study aimed to determine the transport behavior of four trace organic contaminants (TrOCs) including Diuron, Atrazine, DEET and Sulfamethoxazole under several influencing factors. Alkalic NaCl severely deteriorated degradation because of the less generation of OH caused by the interfered iron redox cycle. pH-neutral NaCl resulted in the highest reverse salt flux, namely possible largest production of active chlorine, therefore leading to the highest degradation. Compared to NaCl, Na2SO4 presented a significant lower reverse diffusion due to the larger hydrated radius of SO42- than Cl-. Meanwhile, the large consumption of OH by SO42- decreased degradation. Dissolved organic matters in the secondary effluent acted as the scavenger for OH and resulted in a degradation decline. Water extraction resulted from forward osmosis deteriorated degradation kinetics of all compounds except Sulfamethoxazole. On the other hand, Density functional theory calculations and identified intermediates contributed to propose the possible degradation pathways for each TrOC in terms of understanding TrOCs removal mechanism.

Application of alkali-heated corncobs enhanced nitrogen removal and microbial diversity in constructed wetlands for treating low C/N ratio wastewater.

Lack of carbon source is the main limiting factor in the denitrification of low C/N ratio wastewater in the constructed wetlands (CWs). Agricultural waste has been considered as a supplementary carbon source but research is still limited. To solve this problem, ferric carbon (Fe-C) + zeolite, Fe-C + gravel, and gravel were used as substrates to build CWs in this experiment, aiming to investigate the effects of different carbon sources (rice straw, corncobs, alkali-heated corncobs) on nitrogen removal performance and microbial community structure in CWs for low C/N wastewater. The results demonstrated that the microbial community and effluent nitrogen concentration of CWs were mainly influenced by the carbon source rather than the substrate. Alkali-heated corncobs significantly enhanced the removal of NO2--N, NH4+-N, NO3-N, and TN. Carbon sources addition increased microbial diversity. Alkali-heated corncobs addition significantly increased the abundance of heterotrophic denitrifying bacteria (Proteobacteria and Bacteroidota). Furthermore, alkali-heated corncobs addition increased the copy number of nirS, nosZ, and nirK genes while greenhouse gas fluxes were lower than common corncobs. In summary, alkali-heated corncobs can be considered as an effective carbon source.

Inhibition of Porcine Epidemic Diarrhea Virus by Cinchonine via Inducing Cellular Autophagy.

Porcine epidemic diarrhea virus (PEDV) could cause lethal diarrhea and dehydration in suckling piglets, which can adversely affect the development of the global swine industry. The lack of effective therapeutical and prophylactic treatment especially for PEDV variant strains underlines the importance of effective antiviral strategies, such as identification of novel antiviral agents. In the present study, the antiviral activity of cinchonine against PEDV was investigated in Vero CCL81 and LLC-PK1 cells at a non-cytotoxic concentration determined by Cell Counting Kit-8 assay in vitro. We found that cinchonine exhibited a significant suppression effect against PEDV infection and its inhibitory action was primarily focused on the early stage of PEDV replication. Moreover, we also observed that cinchonine could significantly induce autophagy by detecting the conversion of LC3-I to LC3-II by using western blot analysis. Cinchonine treatment could inhibit PEDV replication in a dose-dependent manner in Vero CCL81 cells, while this phenomenon disappeared when autophagy was attenuated by pre-treatment with autophagy inhibitor 3MA. Consequently, this study indicated that cinchonine can inhibit PEDV replication via inducing cellular autophagy and thus from the basis for successful antiviral strategies which potentially suggest the possibility of exploiting cinchonine as a novel antiviral agent.

[Effects of Hematite and Biochar Addition on Wastewater Treatment Efficiency, Greenhouse Gas Emission, and Microbial Community in Subsurface Flow Constructed Wetland].

The type and structure of the substrate in constructed wetland affects the diversity and abundance of microorganisms, thereby influencing the effect of sewage treatment. In this study, four groups of wetlands were constructed in the greenhouse:blank-constructed wetland (CW0), hematite-constructed wetland (CW1), biochar-constructed wetland (CW2), and hematite+biochar-constructed wetland (CW3), to study the differences in sewage treatment effects, greenhouse gas emissions, and microbial community structures of constructed wetland systems under different filler substrates. The results showed that the addition of hematite or biochar increased the COD removal rate of -0.12% to 1.7%. The addition of biochar increased the removal rate of NH4+-N by 22.48% and NO3--N by 6.82% and reduced the emission flux of CH4 by 83.91% and N2O by 30.81%. The addition of hematite reduced the removal rate of NH4+-N by 1.12%, increased the removal rate of NO3--N by 3.98%, and reduced the emission flux of CH4 by 33.29% and N2O by 25.2%. Adding biochar or hematite increased the relative abundances of Actinobacteria and Proteobacteria, which was beneficial to the removal of COD. The Ace, Chao, Sobs, and Shannon indexes in the substrate treated with biochar were the largest, and the Simpson index was the smallest. The treatment with hematite was the opposite, indicating that the richness and diversity of microbial communities in the treatment system with biochar was the largest. Adding hematite reduced the richness and diversity of the microbial community in the constructed wetland system. Adding biochar or hematite increased the relative abundances of Dechloromonas, Thaurea, Saccharimonadales, and other denitrifying bacteria, which was beneficial to wetland denitrification. The addition of biochar increased the abundances of nosZ, nirS, and nirK functional genes, which were conducive to the occurrence of denitrification. The addition of biochar increased the abundances of pmoA functional genes, reduced the abundance of mcrA functional genes, and inhibited the production of CH4. It also increased the abundance of methanotrophic bacteria and promoted the occurrence of the CH4 oxidation process. Although the addition of hematite increased the abundance of mcrA functional genes, Fe3+ competed with methanogens for electron donors and inhibited the production of CH4.

[Effects of Plastic Film Mulching and Biochar Application on N2O Emission from a Vegetable Field].

The aim of this research was to examine the effects of biochar addition (B0:0 t·hm-2, B20:20 t·hm-2, and B40:40 t·hm-2) and mulching (FM:film and NM:no film) on vegetables. The impact of N2O emissions in the field was based on the pepper-radish rotation vegetable field system on the farm of Southwest University, using static dark box/gas chromatography to conduct in-situ observations in the field for one year. In this experiment, a total of six treatments were set up, namely NMB0 (CK) and FMB0, NMB20 and FMB20, and NMB40 and FMB40. The results showed that FM significantly increased the content of ammonium and nitrate nitrogen in the pepper season soil (P<0.05) but had no significant effect on soil environmental factors in the radish season. Compared with that of NM, the pepper season FM increased the N2O emissions of the B0, B20, and B40 treatments by 52.87%, 52.97%, and 52.49% (P<0.05), respectively, but the radish season FM had no significant effect on N2O emissions. Biochar had no significant effect on soil environmental factors in the pepper and radish seasons. The addition of biochar in the radish season reduced N2O emissions by 28.76%-67.88% (P<0.01), and the addition of biochar in the pepper season had no significant effect on N2O emissions. Compared with that of NM, under different biochar levels, FM increased the yield of pepper by 15.85%-161.32% and increased the yield of radish by 43.97%-75.80%. Biochar significantly increased the yield of peppers and had no significant effect on the yield of radishes. Regardless of whether the film was covered or not, when the amount of biochar added was 20 t·hm-2, the yields of pepper and radish were the highest. The analysis of N2O emission intensity revealed that FM in the pepper season significantly reduced N2O emission intensity, whereas in the radish season FM and biochar significantly reduced N2O emission intensity, and both planting seasons reached the lowest N2O emission intensity under the FMB20 treatment. Therefore, mulching and applying 20 t·hm-2 biochar were the best farmland management measures for the pepper season and radish season, which could achieve high yields and the lowest N2O emissions, accomplishing a win-win for economic and environmental benefits.

Transcriptome analysis of heat resistance regulated by quorum sensing system in Glaesserella parasuis.

The ability of bacteria to resist heat shock allows them to adapt to different environments. In addition, heat shock resistance is known for their virulence. Our previous study showed that the AI-2/luxS quorum sensing system affects the growth characteristics, biofilm formation, and virulence of Glaesserella parasuis. The resistance of quorum sensing system deficient G. parasuis to heat shock was obviously weaker than that of wild type strain. However, the regulatory mechanism of this phenotype remains unclear. To illustrate the regulatory mechanism by which the quorum sensing system provides resistance to heat shock, the transcriptomes of wild type (GPS2), ΔluxS, and luxS complemented (C-luxS) strains were analyzed. Four hundred forty-four differentially expressed genes were identified in quorum sensing system deficient G. parasuis, which participated in multiple regulatory pathways. Furthermore, we found that G. parasuis regulates the expression of rseA, rpoE, rseB, degS, clpP, and htrA genes to resist heat shock via the quorum sensing system. We further confirmed that rseA and rpoE genes exerted an opposite regulatory effect on heat shock resistance. In conclusion, the findings of this study provide a novel insight into how the quorum sensing system affects the transcriptome of G. parasuis and regulates its heat shock resistance property.

Levistolide A Inhibits PEDV Replication via Inducing ROS Generation.

Porcine epidemic diarrhea virus (PEDV) variant strains adversely affect the production of pigs globally. Vaccines derived from PEDV traditional strains impart less protection against the variant strains. Moreover, sequence diversity among different PEDV variant strains is also complicated. This necessitates developing alternative antiviral strategies for defending against PEDV. This study explored a natural product, Levistolide A (LA), to possess antiviral activity against PEDV. LA was found to suppress PEDV replication in a dose-dependent manner. And the inhibitory effect of LA against PEDV was maintained in the course of time. In terms of viral RNA and protein production, LA also showed a strong inhibitory effect. In addition, LA was indicated to inhibit PEDV from attaching to the cellular membrane or penetrating the cells. Further study revealed that LA can induce the generation of reactive oxygen species (ROS), and the corresponding inhibitor, NAC, was found to antagonize the effect of LA on inhibiting PEDV replication. This illustrated that the LA-induced ROS generation played an important role in its anti-PEDV activity. LA was also identified to stimulate ER stress, which is an important consequence of ROS production and was proven to be able to inhibit PEDV replication. To conclude, this study revealed that LA can inhibit PEDV replication via inducing ROS generation.

[Treatment Effect of Corncob and Rice Straw Enhanced Subsurface Flow Constructed Wetland on Low C/N Ratio Wastewater].

The lack of carbon sources severely inhibits denitrification in wastewater with a low C/N ratio. Corncob and rice straw were chosen as supplementary carbon sources to bring into the wetland system to supplement the carbon sources needed for denitrification, and the enhancing effects of the two carbon sources on nitrogen removal from the wetland were studied. The cumulative release of carbon was in the order of rice straw[(145.17±9.44) mg·g-1]>corncob[(57.41±5.04) mg·g-1] based on the 11-day pure water extraction and release experiment, whereas the cumulative release of nitrogen was in the order of rice straw[(2.31±0.09) mg·g-1]>corncob[(0.66±0.08) mg·g-1]. The average carbon/nitrogen ratios released and accumulated by corncob and rice straw during the observation period were 94.78 and 63.64, respectively. Corncob was more suited as an additional carbon source than rice straw. COD concentrations in the effluent from the corncob and straw constructed wetlands were found to be below 50 mg·L-1 for the 58-day pilot test of subsurface flow constructed wetlands, except on days 8 to 12. The NO3--N removal rates of the corncob-added built wetlands were 93%-99% over the observation period, with good denitrification performance. In comparison, the lowest NO3--N removal rate of the constructed wetland with the addition of rice straw was only 76.8% at the late stage of operation, and the denitrification rate dropped dramatically. The control group removal rates of NO3--N were only 76.2%-77.7%, indicating a clear lack of carbon sources. The accumulation of NO2--N was also induced by a lack of carbon supply. NO2--N effluent concentrations were 2.5-6 times and 6-26 times higher in the constructed wetlands with rice straw and the control groups, respectively, than those in the wetlands constructed with corncob. The addition of corncob resulted in a more substantial reduction in NO2--N content in the constructed wetland than the addition of rice straw (P<0.05). The TN removal rates of wetlands constructed with corncob and rice straw and the control group were 83.75%-93.49%, 76.59%-78.85%, and 67.85%-72.56%, respectively, with significant differences among the three (P<0.01). Finally, pretreatment with dilute alkali heating raised the cumulative carbon release of corncob to (93.73±17.49) mg·g-1 and the carbon/nitrogen ratio to 175.8, significantly improving the carbon release performance of corncob and demonstrating that it is a suitable source of extra carbon.

TurboID Screening of the OmpP2 Protein Reveals Host Proteins Involved in Recognition and Phagocytosis of Glaesserella parasuis by iPAM Cells.

Glaesserella parasuis is a common bacterium in the porcine upper respiratory tract that causes severe Glasser's disease, which is characterized by polyarthritis, meningitis, and fibrinous polyserositis. TurboID is an enzyme that mediates the biotinylation of endogenous proteins that can fuse with proteins of interest to label protein interactors and local proteomes. To reveal the host proteins that interact with outer membrane protein P2 (OmpP2) by TurboID-mediated proximity labeling in immortalized porcine alveolar macrophage iPAM cells, 0.1 and 2.58 mg/mL His-tagged TurboID-OmpP2 and TurboID recombinant proteins were expressed and purified. By mass spectrometry, we identified 948 and 758 iPAM cell proteins that interacted with His-TurboID-OmpP2 and His-TurboID, respectively. After removal of background proteins through comparison with the TurboID-treated group, 240 unique interacting proteins were identified in the TurboID-OmpP2-treated group. Ultimately, only four membrane proteins were identified, CAV1, ARF6, PPP2R1A, and AP2M1, from these 240 host proteins. Our data indicated that CAV1, ARF6, and PPP2R1A could interact with OmpP2 of G. parasuis, as confirmed by coimmunoprecipitation assay. Finally, we found that CAV1, ARF6, and PPP2R1A were involved in the recognition and phagocytosis of G. parasuis serotype 5 by iPAM cells by using overexpression and RNA interference assays. This study provides first-hand information regarding the interaction of the iPAM cell proteomes with G. parasuis OmpP2 protein by using the TurboID proximity labeling system and identifies three novel host membrane proteins involved in the recognition and phagocytosis of G. parasuis by iPAM cells. These results provide new insight for a better understanding of Glasser's disease pathogenesis. IMPORTANCE G. parasuis can cause serious Glasser's disease, which is characterized by polyarthritis, meningitis, and fibrinous polyserositis in pigs. It can cause high morbidity and mortality in swine herds and major economic losses to the global pig industry. Understanding the mechanism of interactions between alveolar macrophages and pathogenic G. parasuis is essential for developing effective vaccines and targeted drugs against G. parasuis. To reveal the host proteins interacting with OmpP2 by TurboID-mediated proximity labeling in immortalized porcine alveolar macrophage (iPAM) cells, we identified 240 unique proteins from iPAM cells that could interact with G. parasuis OmpP2. Among them, only four membrane proteins, CAV1, ARF6, PPP2R1A, and AP2M1, were identified, and further study showed that CAV1, ARF6, and PPP2R1A are involved in the recognition and phagocytosis of G. parasuis serotype 5 by iPAM cells. This study provides new insight into proteomic interactions between hosts and pathogenic microorganisms.

Porcine circovirus type 2 infection activates NF-κB pathway and cellular inflammatory responses through circPDCD4/miR-21/PDCD4 axis in porcine kidney 15 cell.

Porcine circovirus type 2 (PCV2) causes postweaning multisystemic wasting syndrome (PMWS) as well as other PCV-associated diseases (PCVADs), which seriously affect the development of the swine industry. The association between aberrant expression of microRNA-21 (miR-21) and the pathogenesis of inflammatory diseases is already known. Also, our previous study showed elevated ssc-miR-21-5p (miR-21) levels in PCV2-infected porcine kidney 15 (PK-15) cells. However, the functional expression of miR-21 in PCV2 infection is unknown. Here, we found that the miR-21 levels were substantially upregulated in PCV2-infected cells, while the expression of PDCD4 and a PDCD4-derived circRNA (circPDCD4) were downregulated compared with the noninfected cells. The results of RNA immunoprecipitation and dual-luciferase reporter (DLR) assay revealed that circPDCD4 acted as a miR-21 sponge and PDCD4 was a miR-21 target. Functionally, we found that miR-21 overexpression induced the NF-κB pathway along with inflammation in cells exposed to PCV2. Further, the overexpression of PDCD4 or circPDCD4 downregulated miR-21 levels and subsequently, attenuated the miR-21-induced activation of inflammation and the NF-κB pathway. Thus, PCV2 activated the NF-κB pathway and cellular inflammatory responses through regulating circPDCD4, miR-21, and PDCD4 in PK-15 cells.

[Effect of Ferric-carbon Micro-electrolysis on Greenhouse Gas Emissions from Constructed Wetlands].

As the problem of global warming becomes increasingly serious, the greenhouse gas (GHG) emission reduction measures of constructed wetlands (CWs) have drawn significant attention. Ferric-carbon micro-electrolysis exhibits an excellent effect on wastewater purification as well as the potential to reduce GHG emissions. Therefore, to explore the impact of ferric-carbon micro-electrolysis on GHG emissions from intermittent aeration constructed wetlands, four kinds of different wetlands with different fillers were constructed. The four fillers were ferric-carbon micro-electrolysis filler+gravel (CW-Ⅰ), ferric-carbon micro-electrolysis filler+zeolite (CW-Ⅱ), zeolite (CW-Ⅲ), and gravel (CW-Ⅳ). Intermittent aeration technology was used to aerate the wetland systems. The results show that ferric-carbon micro-electrolysis significantly improved the nitrogen removal efficiency of the intermittent aeration constructed wetlands and reduced GHG emissions. Compared with CW-Ⅳ, the CH4 fluxes of CW-Ⅰ, CW-Ⅱ, and CW-Ⅲ decreased by 32.81% (P<0.05), 52.66% (P<0.05), and 54.50% (P<0.05), respectively. Among them, zeolite exhibited a stronger reduction effect on CH4 emissions in both the aeration and non-aeration sections. The ferric-carbon micro-electrolysis substantially reduced N2O emissions. In comparison with CW-Ⅳ, CW-, and CW-Ⅱ achieved N2O emission reduction by 30.29%-60.63% (P<0.05) and 43.10%-73.87% (P<0.05), respectively. During a typical hydraulic retention period, the comprehensive GWP caused by CH4 and N2O emitted by each group of wetland system are (85.21±6.48), (49.24±3.52), (127.97±11.44), and (137.13±11.45) g·m-2, respectively. The combined use of ferric-carbon micro-electrolysis and zeolite effectively reduces GHG emissions in constructed wetlands. Overall, ferric-carbon micro-electrolysis combined with zeolite (CW-Ⅱ) can be regarded as one of the valuable filler combination methods for constructed wetlands, which can ensure high removal efficiency of pollutants and effective GHG emission reduction in constructed wetlands.