Influence of CO₂ Exposure on High-Pressure Methane and CO₂ Adsorption on Various Rank Coals: Implications for CO₂ Sequestration in Coal Seams

There exist complex interactions between coal and CO₂ during the process of CO₂ sequestration in coal seams with enhanced coalbed methane recovery (CO₂-ECBM). This work concentrated on the influence of CO₂ exposure on high-pressure methane and CO₂ (up to 10 MPa) adsorption behavior of three types of bituminous coal and one type of anthracite. The possible mechanism of the dependence of CO₂ exposure on adsorption performance of coal was also provided. The results indicate that the maximum methane adsorption capacities of various rank coals after CO₂ exposure increase by 3.45%–10.37%. However, the maximum CO₂ adsorption capacities of various rank coals decrease by 9.99%–23.93%. TG and pore structure analyses do not observe the obvious changes on the inorganic component and pore morphology of the coals after CO₂ exposure. In contrast, CO₂ exposure makes changes in surface chemistry of the coals, according to the results from FTIR analysis, which is the main reason for increases in the maximum adsorption capacity of methane and decreases in the maximum adsorption capacity of CO₂ for the coals after CO₂ exposure. The different role of CO₂ exposure on methane and CO₂ adsorption is detrimental to CO₂-ECBM. Thus, the implementation of CO₂-ECBM must take into account the influence of CO₂ exposure on the adsorption performance of the target coal seams

<i>Escherichia coli</i> and <i>Candida albicans</i> Induced Macrophage Extracellular Trap-Like Structures with Limited Microbicidal Activity

<div><p>The formation of extracellular traps (ETs) has recently been recognized as a novel defense mechanism in several types of innate immune cells. It has been suggested that these structures are toxic to microbes and contribute significantly to killing several pathogens. However, the role of ETs formed by macrophages (METs) in defense against microbes remains little known. In this study, we demonstrated that a subset of murine J774A.1 macrophage cell line (8% to 17%) and peritoneal macrophages (8.5% to 15%) form METs-like structures (METs-LS) in response to <i>Escherichia coli</i> and <i>Candida albicans</i> challenge. We found only a portion of murine METs-LS, which are released by dying macrophages, showed detectable killing effects on trapped <i>E. coli</i> but not <i>C. albicans</i>. Fluorescence and scanning electron microscopy analyses revealed that, <i>in vitro,</i> both microorganisms were entrapped in J774A.1 METs-LS composed of DNA and microbicidal proteins such as histone, myeloperoxidase and lysozyme. DNA components of both nucleus and mitochondrion origins were detectable in these structures. Additionally, METs-LS formation occurred independently of ROS produced by NADPH oxidase, and this process did not result in cell lysis. In summary, our results emphasized that microbes induced METs-LS in murine macrophage cells and that the microbicidal activity of these METs-LS differs greatly. We propose the function of METs-LS is to contain invading microbes at the infection site, thereby preventing the systemic diffusion of them, rather than significantly killing them.</p></div

METs-LS-induced cell death is independent of necrosis.

<p><b>A:</b> The J774A.1 METs-LS induced by <i>C. albicans</i> were stained with SYTOX Green and Hoechst 33342, revealing that the METs-LS are released from either viable (i) or dead (ii) macrophage cells after 120 min incubation. The arrows indicate viable METs-LS formation cells with unchanged nuclei shape, the arrowheads indicate dead MET-LS formation cells with enlarged nuclei. Scale Bars: 10 µm. These experiments were repeated independently 3 times with similar results. <b>C:</b> J774A.1 macrophages were infected with <i>E. coli</i> or <i>C. albicans</i> for 15, 30, 60, 120 and 180 min, respectively. The LDH level in the supernatant of each group was quantified. The data are presented as the means ± SD of three independent experiments.</p

METs-LS show limited killing effect on <i>E. coli</i> but not on <i>C. albicans</i>.

<p>J774A.1 macrophages or murine peritoneal macrophages were infected with <i>E. coli</i> or <i>C. albicans</i> and incubated for the indicated time points. <b>A–B:</b> The survival rates of <i>E. coli</i> (A) and <i>C. albicans</i> (B) incubated with J774A.1 macrophages. <b>C–D:</b> The survival rates of <i>E. coli</i> (C) and <i>C. albicans</i> (D) incubated with peritoneal macrophages. Experiments were performed 3 times with similar results, and a representative experiment is shown as the means ± SD (n = 3). *<i>P</i><0.05, **<i>P</i><0.01 and ***<i>P</i><0.001 compared with the control groups by two tailed Student’s t-test, respectively. <b>E–F:</b> PI staining was performed to determine the dead macrophages and dead microbes trapped in the METs-LS formed by J774A.1 cells (E) and peritoneal macrophages (F) after 120 min co-incubation. Macrophages were labeled with Hoechst 33342 (Blue). METs-LS, dead macrophages and dead microbes were stained positive by PI (Red). The solid and hollow arrowheads indicate viable and dead microbes in <i>E. coli</i> (i-ii) or <i>C. albicans</i> (iii-iv) infected groups, respectively. Only a portion of GFP- <i>E. coli</i> trapped by METs-LS released from dead macrophages was killed. Scale Bars: 10 µm. <b>G:</b> The quantification of dead <i>E. coli</i> trapped in METs-LS released from viable and dead J774A.1 macrophages and peritoneal macrophages, result is shown as the means ± SD (n = 10), ***P<0.001 compared with control group by two tailed Student’s t-test. These experiments were repeated independently 3 times with similar results.</p

Formation of METs-LS is independent of ROS produced by NADPH oxidase.

<p><b>A:</b> Fluorescence microscope determination of intracellular ROS production in PMA (positive control), <i>E. coli-</i> and <i>C. albicans</i>-stimulated J774A.1 macrophages using DCF ROS probe (Green). DNA was stained with Hoechst 33342 (Blue). The arrows indicate macrophages releasing METs-LS. <b>B:</b> The quantification of ROS production in negative, positive (PMA), <i>E. coli-</i> and <i>C. albicans-</i>infected macrophage groups. The fluorescence intensity of the ROS probe was measured using a fluorescence plate reader. The data are presented as the means ± SD of three independent experiments. *<i>P</i><0.05 and ***<i>P</i><0.001 compared with the medium-only culture control group by ANOVA with Bonferroni’s post-test. <b>C:</b> Determination of <i>E. coli</i> or <i>C. albicans</i> induced J774A.1 METs-LS in the presence of 10 µM DPI. Hoechst 33342 and SYTOX Green were added to assess METs-LS formation. <b>D:</b> Determination of intracellular ROS production in PMA stimulated J774A.1 macrophages in the presence or absence of 10 µM DPI. DNA was stained with Hoechst 33342 and intracellular ROS was determined by DCF ROS probe. Scale Bars: 10 µm. These experiments were repeated independently 3 times with similar results.</p

<i>E. coli</i> and <i>C. albicans</i> induce METs-LS formation in murine macrophage cells.

<p><b>A–B:</b> Murine J774A.1 macrophages (A) or peritoneal macrophages (B) were cultured in serum-free DMEM medium and stimulated with <i>E. coli</i> (MOI 5), <i>C. albicans</i> (MOI 1), PMA (10 µM), LPS (50 µg/ml), hydrogen peroxide (10 mM) or vehicle control at 37°C for 180 min. Hoechst 33342 (Blue) and SYTOX Green (Green) was added to stain the DNA. Fluorescence staining images merged with differential interference contrast are shown. The arrows indicate METs-LS released by macrophages in response to <i>E. coli</i> or <i>C. albicans</i>. Scale Bars: 20 µm. <b>C–D:</b> The quantification of METs-LS positive cells in murine J774A.1 macrophages (C) or peritoneal macrophages (D) stimulated with <i>E. coli</i>, <i>C. albicans</i>, LPS, PMA or hydrogen peroxide is shown as the means ± SD (n = 5). ***<i>P</i><0.001 compared with control group by two tailed Student’s t-test, respectively. These experiments were repeated independently 3 times with similar results.</p

Scanning electron microscopy (SEM) and fluorescence micrographs of microbes trapped by METs-LS.

<p>J774A.1 macrophages were seeded onto glass cover slips and incubated with <i>E. coli</i> or <i>C. albicans</i> for 180 min. <b>A–B:</b> SEM images of uninfected J774A.1 cells. <b>C–D:</b> SEM images of METs-LS induced by <i>E. coli</i>. The white arrows indicate the bacteria adhered to the surface of METs-LS. <b>E–F:</b> SEM images of METs-LS induced by <i>C. albicans</i>. The white arrows indicate the fungus trapped in the METs-LS. <b>G:</b> Fluorescence photograph of DNA (Blue) and FITC-labeled <i>E. coli</i> (Green). <b>H:</b> Fluorescence photograph of DNA (Green) and calcofluor white-stained <i>C. albicans</i> (Blue). Both microbes co-localized with METs-LS. In (G) and (H), samples were washed twice with PBS to remove unbounded microbes before analysis. These experiments were repeated independently 3 times with similar results. Scale Bars: 20 µm.</p

Characterisation of a Plancitoxin-1-Like DNase II Gene in <i>Trichinella spiralis</i>

<div><p>Background</p><p>Deoxyribonuclease II (DNase II) is a well-known acidic endonuclease that catalyses the degradation of DNA into oligonucleotides. Only one or a few genes encoding DNase II have been observed in the genomes of many species. 125 DNase II-like protein family genes were predicted in the <i>Trichinella spiralis</i> (<i>T. spiralis</i>) genome; however, none have been confirmed. DNase II is a monomeric nuclease that contains two copies of a variant HKD motif in the N- and C-termini. Of these 125 genes, only plancitoxin-1 (1095 bp, GenBank accession no. XM_003370715.1) contains the HKD motif in its C-terminus domain.</p><p>Methodology/Principal Findings</p><p>In this study, we cloned and characterised the plancitoxin-1 gene. However, the sequences of plancitoxin-1 cloned from <i>T. spiralis</i> were shorter than the predicted sequences in GenBank. Intriguingly, there were two HKD motifs in the N- and C-termini in the cloned sequences. Therefore, the gene with shorter sequences was named after plancitoxin-1-like (<i>Ts</i>-Pt, 885 bp) and has been deposited in GenBank under accession number KF984291. The recombinant protein (r<i>Ts</i>-Pt) was expressed in a prokaryotic expression system and purified by nickel affinity chromatography. Western blot analysis showed that r<i>Ts</i>-Pt was recognised by serum from <i>T. spiralis</i>-infected mice; the anti-r<i>Ts</i>-Pt serum recognised crude antigens but not ES antigens. The <i>Ts</i>-Pt gene was examined at all <i>T. spiralis</i> developmental stages by real-time quantitative PCR. Immunolocalisation analysis showed that <i>Ts</i>-Pt was distributed throughout newborn larvae (NBL), the tegument of adults (Ad) and muscle larvae (ML). As demonstrated by DNase zymography, the expressed proteins displayed cation-independent DNase activity. r<i>Ts</i>-Pt had a narrow optimum pH range in slightly acidic conditions (pH 4 and pH 5), and its optimum temperature was 25°C, 30°C, and 37°C.</p><p>Conclusions</p><p>This study indicated that <i>Ts</i>-Pt was classified as a somatic protein in different <i>T. spiralis</i> developmental stages, and demonstrated for the first time that an expressed DNase II protein from <i>T. spiralis</i> had nuclease activity.</p></div

Detection of r<i>Ts</i>-Pt nuclease activity with SDS-PAGE zymography.

<p>(A) SDS-PAGE zymography analysis of r<i>Ts</i>-Pt, after staining with ethidium bromide and exposure to UV light. (B) The same gel was stained with Coomassie Brilliant blue after zymography. Lane 1, prestained protein marker (Genview, Houston, Texas); lane 2, r<i>Ts</i>-Pt; lane 3, BSA (negative control); lane 4, DNase II (positive control).</p

Immunolocalisation of <i>Ts</i>-Pt in the different developmental stages of <i>T. spiralis</i>.

<p>Intact whole Ad1 (A and B), Ad6 (C and D), ML (E and F), and NBL (G and F) were incubated with anti-r<i>Ts</i>-Pt rabbit serum (B, D, F, and H) or normal rabbit serum (A, C, E, and G). After incubation with the Alexa Fluor 594-labeled goat anti-rabbit IgG secondary antibody, the specimens were observed under a fluorescence microscope.</p