Trypanosoma cruzi, and affects about 25 million people worldwide. N, N’-Squaramide 17 (S) is a trypanocidal compound with relevant in vivo effectiveness. Here, we produced, characterized, and evaluated cytotoxic and trypanocidal effects of macrophage-mimetic liposomes from lipids extracted of RAW 264.7 cells to release S. As results, the average hydrodynamic diameter and Zeta potential of mimetic lipid membranes containing S (MLS) was 196.5 ± 11 nm and −61.43 ± 2.3 mV, respectively. Drug entrapment efficiency was 73.35% ± 2.05%. After a 72 h treatment, MLS was observed to be active against epimastigotes in vitro (IC50 = 15.85 ± 4.82 μM) and intracellular amastigotes (IC50 = 24.92 ± 4.80 μM). Also, it induced low cytotoxicity with CC50 of 1199.50 ± 1.22 μM towards VERO cells and of 1973.97 ± 5.98 μM in RAW 264.7. MLS also induced fissures in parasite membrane with a diameter of approximately 200 nm in epimastigotes. MLS showed low cytotoxicity in mammalian cells and high trypanocidal activity revealing this nanostructure a promising candidate for the development of Chagas disease treatment.Chagas is a neglected tropical disease caused by  Chagas disease or American Trypanosomiasis is a neglected tropical disease considered endemic in twenty-one Latin American countries. Over recent decades, however, it has been increasingly detected in the United States of America, Canada, and many European countries. This is due, mainly, to population mobility between Latin America and the rest of the world and has caused more than 7000 deaths per year and over 25 million people at risk of infection . It is tN, N’-Squaramide 17 (S)  and nitroimidazole (benznidazole). However, these therapeutic options are unsatisfactory, given their limited effectiveness in preventing the chronic stage of the disease . As a nee 17 (S) .2; the pKa is 8.8 in aqueous solution, therefore, at physiological pH, both the acidic and the basic forms coexist in solution. A previous study with S showed a 67% reduction of parasitemia in the acute phase of infection in a mice model after 40 days of treatment compared to the control [S comply with Lipinski’s rule of five Ro5) as they are low molecular mass 267.4 Da); only one hydrogen bond donor; partition coefficient log P = 0.59; polar surface area values <100 Å Da; only as they There are a limited number of studies using nanotechnology seeking the treatment of Chagas disease. One barrier for the effective treatment of Chagas disease may be the fact that the parasite easily spreads throughout the body, infecting several cell types, and evades the immune system . LiposomHere, we present the preparation and characterization of mimetic lipid membranes based on lipids extracted from macrophage cells (RAW 264.7 strain) as a strategy to controlled release of the trypanocidal agent S. The nanostructures were characterized by several approaches such as hydrodynamic diameter, polydispersity index (PdI), Zeta potential, stability on the first and tenth day after synthesis (dynamic ligth scattering and electrophoretic mobility), size (dry diameter), shape (transmission electron microscopy—TEM and atomic force microscopy—AFM), chemical behavior (infrared vibration spectroscopy—IR), and entrapment efficiency (UV-Vis spectrophotometry). Finally, the nanostructures’ toxicity on mammalian cells and their trypanocidal activity (CL-Brener) were also evaluated, as well as the damage effects on the parasites using scanning electron microscopy (SEM).2 [T. cruzi (strain CL-Brener) epimastigote form was maintained at 27 °C in axenic culture RPMI-1640 containing 10 U/mL penicillin, 25 μg/mL streptomycin, 25 mM HEPES buffer, 0.03 M Hemin, and 200 mM glutamine supplemented with 10% inactivated FBS [2 in RPMI-1640, pH 7.4, supplemented with 10% FBS and 100 μg/mL gentamicin. VERO (ATCC CCL81) and RAW 264.7 cells (ATCC TIB71) were maintained in RPMI-1640 supplemented with 10% fetal bovine serum (FBS)  without phenol red at 37 °C and 5% CO2 . T. cruzated FBS . Trypoma8 cells/mL were centrifuged and washed in phosphate-buffered saline (PBS) and resuspended in deionized water for lysis. Then, cells were centrifuged and placed in deionized water-hypotonic solution to finally obtain the cell pellet and were stored at −40 °C until use. The lipids extraction was performed according to Bonatto et al. [g for 5 min for organic phase separation containing the lipids. This phase was collected and subjected to the rotary evaporator for 1 h in a water bath  at 40 °C and pressure of 200 Pa, for chloroform evaporation and consequently obtaining the formation of the lipid film.For lipids extraction from RAW 264.7 macrophage cells, about 1 × 10o et al.  with somN, N’-Squaramide 17 (MLS) at 0.032 M. The solutions were homogenized for 10 min and then submitted to the extrusion process with syringes (15 repetitions were carried out) through polycarbonate membranes with 100 nm pores (Avanti Polar Lipids). After obtaining the phospholipid films, 5 mL of ultrapure water were added to produce the following compositions: (1) Empty mimetic lipid membranes (MLV) and (2) Mimetic lipid membranes containing The MLS nanostructures were evaluated in terms of drug entrapment efficiency (EE), established as a ratio between the encapsulation (ED) over the initial quantity of the drug (ID), according to the Equation (1) below: ® Ultra 0.5 mL centrifuge filters - Millipore, Darmstadt, Germany) at 300× g for 10 min, 4 °C [® 6131, Hamburg, Germany) (2 = 0.98) and MLV absorbance was also performed to determine the background in this sample. Experiments were performed in triplicates. For the determination of EE, the S-loaded nanostructures were filtrated in 100 kDa Amicon filters  . S conce−1) were submitted to infrared vibrational spectroscopy (IR) analysis in a Perkin-Elmer model 400 IR spectrometer . The samples were mixed with potassium bromide (KBr) in an agate mortar and then introduced into pellets for reading, with a resolution of 2 cm−1, via analysis of 32 scans. Data were processed using the software OriginPro version 2018  [Mimetic membranes were lyophilized using sucrose (350 mM) as a cryoprotectant at a ratio of 1:1, 1 mL of the nanostructures , and 1 mL of sucrose . SpectraHydrodynamic diameter of the formed nanostructures was determined by dynamic light scattering (DLS) in a ZetaSizer Nano ZS  using He-Ne laser (4 mW) at 633 nm. Three measurements were performed at 25 °C under pH 7.0 and light scattering measured at an angle of 173° in automatic mode. Additionally, electrophoretic mobility measurements were obtained for the assessment of the Zeta potential in manual mode with 20 readings. Particles size (average hydrodynamic diameter), polydispersity index (PdI), as well as the Zeta potential, were acquired and processed using ZetaSizer software  . The sta2 (5 µm × 5 µm) with a resolution of 512 × 512 lines. Images were processed for XY plane correction using the software Gwyddion 2.56 verion 2018 . AFM analysis was performed on dynamic mode using a SPM-9600 equipment  with rectangular cantilevers integrated with conical tips, spring constant and a resonance frequency of about 42 N/m and 300 kHz, respectively. For the analysis, 1 µL of MLS and MLV samples were diluted 100× in ultrapure water, deposited onto freshly-cleaved mica surfaces, and air-dried in a protected environment. Analyses were performed at ∼21 °C. Up to ten images of each sample were acquired in areas of 25 µm4) for 7 min. Therefore, 10 images were acquired using an 80 kV beam voltage, and the modal dry diameter of 100 nanostructures was measured through the Image-Pro Plus software version 1.8.0  and nanostructures were evaluated according to the distribution classes [For TEM analysis (JEM-1011) , samples (MLS and MLV) were diluted at a ratio of 1:1000 in deionized water and deposited onto Formvar-coated copper grids (200 mesh). Samples were then contrasted with 1% osmium tetroxide (OsO classes .w/v) at 0.002 M (stock) and the final concentration of DMSO in the experiments never exceeded 0.25% (v/v) to not affect the cell viability [v/v). VERO cell line and RAW 264.7 macrophages were cultured in RPMI medium supplemented with 10% FBS at a density of 1 × 104 cells/wells in 96-well microplates for 48 h. After this period, cells were treated with BZ, S, MLV, or MLS serially diluted at the dosages of 800 to 6.25 μM   [The reference drug benznidazole (BZ) was dissolved in dimethyl sulfoxide (DMSO) at 5%  . Percent50) by nonlinear regression in GraphPad Prism version 7.0 .The adjusted equations were used to calculate the concentration needed to kill 50% of VERO cells and macrophage RAW 264.7 cells , incubated for further 24 h, and fluorescence was recorded as described above [4 cells/well in 24-well microplates. After 48 h, cells were infected with trypomastigotes at a ratio of 10:1 during 24 h, followed by removal of reminiscent extracellular parasites by washing with PBS. Then, cells were cultivated in the presence of BZ, S, MLV, and MLS added to the culture medium in a serial dilution of 100 to 6.25 μM for 72 h [50 values were determined by nonlinear regression in GraphPad Prism version 7.0 . Selectivity indices (SI) were calculated using the formula: SI = CC50 cytotoxicity in VERO cells/IC50 cytotoxicity in intracellular amastigotes.Treatments with BZ, S, MLV, and MLS were performed using serial dilution at 100 to 6.25 μM  and wereT. cruzi were grown at a density of 5 × 105 cells/mL in RPMI culture medium containing the compounds tested at concentrations of 7.91 µM (BZ), 6.56 µM (S), and 7.93 µM (MLS) corresponding to the half of IC50 of each compound. After 72 h these cultures were centrifuged at 1600× g for 10 min. Parasites were washed in PBS and fixed for 1 h at room temperature with 2% formaldehyde and 2.5% glutaraldehyde in sodium cacodylate buffer . Samples were then washed in 0.1 M cacodylate buffer and adhered to coverslips previously coated with poly-L-lysine . After adhesion, post-fixation was performed using a solution of 1% OsO4 containing 0.8% potassium ferrocyanide (K4Fe (CN)6). Then, parasites were dehydrated in increasing concentrations of acetone , subject to critical-point drying, metalized with gold, and observed in a scanning electron microscope (SEM)- . Sample images were visualized using a 15 kV beam voltage.Epimastigotes of p < 0.05. All analyses were performed in GraphPad Prism version 7.0 .The results were expressed as mean (± standard deviation of the mean) of three independent experiments, and one-way or two-way analysis of variance (ANOVA) were performed respectively, followed by the post- Dunnett’s test (two-tailed) comparing the treatments BZ, S, and MLV with MLS. Statistically significant differences were considered when Due to its low molecular weight 267.4 Da) and its hydrophilicity  Da and i. EvaluatIn addition to these results, MLS and MLV showed sizes (hydrodynamic dyameters) smaller than 200 nm and a PdI below 0.45 . PdI is −1, 1052 cm−1, 995 cm−1, and 1136 cm−1, which are vibrations of R–NH3, NH3+, PO2−,and POO2−, respectively. This observation indicates that there is no detectable structural change in both nanostructures [−1 and 1591 cm−1, related to the stretching vibrations C=O (carbonyls) [When comparing the IR spectra of MLS and MLV , it is nructures ,33,34. Trbonyls) ,36. ThesPrevious studies have shown that small vesicles can interact with each other and form larger liposomes during storage, showing agglomerates with sizes higher than 200 nm . For thiBefore starting the biological activity investigation of the compositions developed herein, MLV cytotoxicity was analyzed to verify its pharmaceutical “nanovehicle” potential and, thus, to validate the functionality of our nanostructure proposal. For this, MLV cytotoxicity was analyzed in VERO and RAW 264.7 cells employing doses from 50 μΜ to 800 μΜ, and evaluated after 72 h of treatment. No cytotoxic effects of MLV were observed on the cells analyzed, indicating that MLV composition has a potential for use as a diverse drug delivery system.T. cruzi are tested in epimastigote forms due to their capability to grow in axenic culture. However, as they are not found in the mammalian host, evaluations performed on these forms are only indicative of potential activity against the parasite. Therefore, a preliminary test using axenic epimastigote forms should always be complemented by a subsequent evaluation using intracellular forms (amastigotes) to assure trypanocidal activity [T. cruzi epimastigotes and amastigotes, as well as the host cells, VERO and RAW 264.7 (50 μM–800 μM) for 72 h (50 = 15.81 ± 4.63 µM and 4.76 ± 4.45 µM) and MLS (IC50 = 15.85 ± 4.82 µM and 24.92 ± 4.80 µM) induced trypanocidal activity in both forms (epimastigotes and intracellula amastigotes) of T. cruzi. BZ presented the lower IC50 on amastigotes, MLS showed a 2-fold increase in efficacy compared with S. MLV showed no trypanocidal activity  and were still less cytotoxic than BZ (CC50 = 284.44 ± 1.25 μM). S cytotoxicity has been attributed to squaric acid, and derivatives of this compound significantly increase lipophilicity and thus have their distribution both within the cell and in lysosomes [When evaluating cytotoxic activity in VERO cells, CCysosomes . The SI values found for S (56.11 for epimastigotes and 14.38 for amastigotes ) and MLS (75.68 for epimastigotes and 48.14 for amastigotes) indicate a therapeutic potential according to Drugs for Neglected Diseases initiative (DNDi) , which pThere may be a correlation between the physicochemical characteristics of the particles  and theiConversely, there is a drug internalization mechanism termed lysosomotropic–parasitotropic ,45. It cThus, after its internalization into cells under different endocytosis processes, MLS could fuse to lysosomes and parasite-containing phagolysosomes causing its action in amastigote forms. Its trypanocidal activity is likely the result of a hydrolysis process suffered by S under different pH (2 to 8), separating it into the squaric acid and two amides , as prevT. cruzi [T. brucei and Leishmania sp, in which the flagellar pocket represents the only site of endocytosis and exocytosis [Epimastigotes were incubated for 72 h with BZ, S, and MLS, and the ultrastructural changes caused by these treatments in comparison with the control (no treatment) are shown in T. cruzi . In epimT. cruzi ,50, diffocytosis .Regarding parasites treated with BZ, cell surface alterations were observed as prominences in the cell body and distortion in the flagellum B,B1. AddN, N’-Squaramide 17 nanostructured into mimetic lipid membranes (MLS) presents an enhanced trypanocidal activity with decreased cytoxicity compared to free S. MLS was able to induce structural alterations in the parasites as fissures with a diameter of approximately 200 nm in epimastigote cytoplasm membrane. In summary, the high MLS activity against parasites demonstrates its target-specific toxicity, therefore suggesting that these nanostructures are promising for application in Chagas’ disease treatment. However, to confirm this MLS potential, further in vivo studies should be performed to compare MLS and S with respect to their trypanocidal activity, cytotoxicity and bioavailability.In this work, we showed that


Pseudomonas aeruginosa. In addition to its C-terminal pore-forming domain, PyoS5 is composed of two tandemly repeated helical domains that we also identify in other pyocins. Functional analyses demonstrate that they have distinct roles in the import process. One recognizes conserved sugars projected from the surface, while the other recognizes a specific outer membrane siderophore transporter, FptA, in the case of PyoS5. Through engineering of Escherichia coli cells, we show that pyocins can be readily repurposed to kill other species. This suggests basic ground rules for the outer membrane translocation step that likely apply to many bacteriocins targeting Gram-negative bacteria.Bacteriocins are toxic polypeptides made by bacteria to kill their competitors, making them interesting as potential antibiotics. Here, we reveal unsuspected commonalities in bacteriocin uptake pathways, through molecular and cellular dissection of the import pathway for the pore-forming bacteriocin pyocin S5 (PyoS5), which targets  Pseudomonas aeruginosa in animal infection models, but its import mechanism is poorly understood. Here, using crystallography, biophysical and biochemical analyses, and live-cell imaging, we define the entry process of PyoS5 and reveal links to the transport mechanisms of other bacteriocins. In addition to its C-terminal pore-forming domain, elongated PyoS5 comprises two novel tandemly repeated kinked 3-helix bundle domains that structure-based alignments identify as key import domains in other pyocins. The central domain binds the lipid-bound common polysaccharide antigen, allowing the pyocin to accumulate on the cell surface. The N-terminal domain binds the ferric pyochelin transporter FptA while its associated disordered region binds the inner membrane protein TonB1, which together drive import of the bacteriocin across the outer membrane. Finally, we identify the minimal requirements for sensitizing Escherichia coli toward PyoS5, as well as other pyocins, and suggest that a generic pathway likely underpins the import of all TonB-dependent bacteriocins across the outer membrane of Gram-negative bacteria.Pyocin S5 (PyoS5) is a potent protein bacteriocin that eradicates the human pathogen  Pseudomonas aeruginosa and that was shown recently in animal models to be more effective at clearing lung infections than was tobramycin, the antibiotic generally used to treat P. aeruginosa in cystic fibrosis patients  to translocate through the cell envelope to deliver a cytotoxic domain, typically a pore-forming domain or a nuclease that cleaves DNA, rRNA, or tRNA  belong to a broad group of protein bacteriocins that includes colicins which kill  or tRNA . Also li or tRNA , 10. Howreversed . This chP. aeruginosa strains . The 57-kDa toxin was monomeric in solution and active against P. aeruginosa strains at subnanomolar concentrations  Size exclusion multiangle light scattering (SEC-MALS) of PyoS5 at 9.5 mg/ml (black), 4.8 mg/ml (gray), 2.4 mg/ml (red), 1.1 mg/ml (blue), and 0.6 mg/ml (yellow) shows PyoS5 is monomeric at all tested concentrations. Download FIG S1, PDF file, 0.2 MB.Characterization of purified PyoS5. (A) Coomassie-stained SDS-PAGE showing purified PyoS5, compared to molecular weight standards (Fermentas unstained protein marker). (B) Electrospray ionization mass spectrometry (ESI-MS) of full-length PyoS5 gives a mass of 57,008.00 Da, compared with a sequence-based expected mass of 58,007.98 Da. (C) PyoS5 spotted on Copyright © 2020 Behrens et al.2020Behrens et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.03230-19.2FIG S22 of 1.74. (B) Fit of the PyoS5 crystal structure into the PyoS5 SAXS envelope by Chimera shows 93% of atoms within the SAXS envelope. (C) Comparison of pore-forming domains from bacteriocins. All available structures of pore-forming domains were aligned and colored in a gradient from yellow (N terminus) to red (C terminus). The pores are shown in order of decreasing alignment quality (Q) score. All pores in the left column have immunity proteins that pass the membrane three times, and those in the right column have immunity proteins that pass the membrane four times. RMSDs, Q scores, and immunity protein topologies are shown. The following residues were aligned to PyoS5315 to 498: ColIa448 to 624 (PDB 1CII), ColE1345 to 522 (PDB 2I88), ColN188 to 486 (PDB 1A87), ColA393 to 591 (PDB 1COL), ColS4299 to 499 (PDB 3FEW), and ColB312 to 511 (PDB 1RH1). (D) Phylogenetic tree of kTHB domains with branch lengths in substitutions per site (gray) and percent protein sequence identity to the most similar crystalized kTHB (blue and red). (E) Sequence alignment of the three kTHBs that have been shown to bind CPA. Identical residues are shown in green. Download FIG S2, PDF file, 1.1 MB.SAXS data of PyoS5. (A) SAXS of PyoS5 (black) at 5.4 mg/ml shows that PyoS5 is monodispersed. The theoretical scattering curve for the crystal structure (red line) fits the experimental SAXS data well, with a χCopyright © 2020 Behrens et al.2020Behrens et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.03230-19.9TABLE S1Table S1, DOCX file, 0.03 MB.X-ray data processing, refinement, and validation statistics for PyoS5. Download Copyright © 2020 Behrens et al.2020Behrens et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the The structure of PyoS5 is composed of three ordered domains . The C-tThe other structured domains of PyoS5 are also helical bundles but of a novel fold. Domain 1 comprises residues 40 to 194, while domain 2 comprises residues 195 to 315. The core structural motif of each domain is a kinked three-helix bundle (kTHB). The two kTHB domains are structurally similar to each other  but share little sequence identity (∼12%) . Each kTRecently, White et al. reported the structure of the N-terminal domain of the nuclease pyocin PyoS2 bound to the outer membrane protein FpvAI . We founP. aeruginosa, and a C-terminal pore-forming domain. We next set out to ascribe functions to each of the domains/regions of PyoS5 that transport the pore-forming domain into P. aeruginosa cells.We conclude that PyoS5 is an elongated bacteriocin comprising a disordered region at its N terminus, two kTHB domains, which is a common structural platform for protein bacteriocins targeting 1–315, in which the pore-forming domain was removed, PyoS51–196, in which both domain 2 and the pore-forming domain were deleted, and PyoS5194–315, which only contained domain 2. The constructs were folded, as determined by circular dichroism spectroscopy, and their thermal melting temperatures largely recapitulated those found in intact PyoS5 (We expressed and purified truncations of PyoS5 that removed one or more domains/regions. These included PyoS5ct PyoS5 .10.1128/mBio.03230-19.3FIG S31–315 (black), PyoS51–196 (red), and PyoS5194–315 (gray) at RT in 20 mM NaCl and 10 mM potassium phosphate buffer (pH 7.5) show that the PyoS5 domains are helical and folded. (B) CD thermal melts of 0.1 mg/ml PyoS51–315 (black), with a melting temperature (mT) of 51.0 ± 0.1°C, PyoS51–196 (red), with a mT of 57.5 ± 0.0°C, and PyoS5194–315 (gray), with a mT of 43.7 ± 0.3°C in 20 mM NaCl and 10 mM potassium phosphate buffer (pH 7.5). Two repeats were performed; one is shown as circles with its fit as a line. Download FIG S3, PDF file, 0.05 MB.Circular dichroism (CD) spectra of PyoS5 constructs. (A) CD spectra of 0.1 mg/ml PyoS5Copyright © 2020 Behrens et al.2020Behrens et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 1–315 and PyoS5194–315 but not PyoS51–196 (in 0.2 M Na-phosphate buffer [pH 7.5]) (dK) values of 0.6 μM for PyoS51–315 and 0.3 μM for PyoS5194–315 were obtained, similar to that reported previously for intact PyoS5 binding CPA (P. aeruginosa PAO1 Δrmd were used (that do not contain CPA), no binding to PyoS5194–315 was detected  experiments. Heats of binding were observed for PyoS5pH 7.5]) . From thding CPA . When podetected . These rA sugars . SequencA sugars E and S4.10.1128/mBio.03230-19.4FIG S4FIG S4, PDF file, 0.4 MB.Conserved residues between PyoS2, PyoSD3, and PyoS5 CPA-binding domains. Residues identical in all three CPA-binding domains (red) are mapped onto PyoS5 CPA-binding kTHB domain 2 (gray). Surface and cartoon representations are shown at two different angles. Download Copyright © 2020 Behrens et al.2020Behrens et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mBio.03230-19.10TABLE S2Table S2, DOCX file, 0.03 MB.Interactions of PyoS5 with cell envelope components. Download Copyright © 2020 Behrens et al.2020Behrens et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. aeruginosa cells requires the ferric pyochelin transporter FptA, and the central region of the toxin (residues 151 to 300) has been implicated in defining this specificity . Upon the addition of ferric pyochelin to our SPR experiments, the binding of PyoS5 to FptA reduced significantly , FptA-Pch complex (red), FptA-PyoS51–299 complex (blue), and dissociated FptA (yellow). No complex of FptA, PyoS51–299, and Pch is detected. (B) SPR shows 3.4 μM FptA binding to immobilized PyoS51–315 in the presence of premixed Pch and FeCl3 at ratios of 1:0:0 (black), 1:2:8 (gray), and 1:10:40 (red) FptA:Pch:FeCl3, respectively. An excess of iron over Pch was chosen to ensure a high percentage of ferric Pch. With a 10-fold excess of Pch, no binding of FptA to PyoS51–315 is observed. Download FIG S5, PDF file, 0.07 MB.Pyochelin and PyoS5 compete for FptA binding. (A) FptA was incubated with 4-fold excess ferric pyochelin (Pch) for 45 min, and then excess Pch was removed by desalting. The native mass spectrum of this FptA sample (19 μM) in the presence of 19 μM PyoS5Copyright © 2020 Behrens et al.2020Behrens et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. aeruginosa PAO6609 are available . P. aeruginosa harbors three tonB genes, tonB1, tonB2, and tonB3 . We found that TonB1 binds PyoS51–315 with an affinity of 230 nM in SPR experiments , which they use to drive translocation across the outer membrane . It has nd tonB3 –25. PAO6ity gene . We ther deleted . It was pression . We thereriments . Moreoveeriments .10.1128/mBio.03230-19.6FIG S6P. aeruginosa PAO1 or P. aeruginosa PAO1 Δrmd from top left to bottom right. In the CPA-deficient Δrmd mutant strain, a 9-fold reduction in susceptibility but not complete resistance is observed. Download FIG S6, PDF file, 0.3 MB.PyoS5-ColIa is not TonB2 or TonB3 but CPA dependent. (A) Zones of clearance from PyoS5-ColIa did not differ between parent strain PA6609, TonB2-deficient strain K1408, TonB3-deficient strain MS231, and TonB2- and TonB3-deficient strain MS233. A 3-fold dilution series starting at 10 μM PyoS5-ColIa was added to strains grown on LB agar. (B) CPA increases susceptibility to PyoS5-ColIa. A 3-fold serial dilution starting at 10 μM spotted onto the soft agar containing Copyright © 2020 Behrens et al.2020Behrens et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. aeruginosa cell envelope. Of the two kTHB domains, domain 2 binds CPA, while domain 1 binds the ferric pyochelin transporter FptA, with a minor contribution by the disordered region, which in addition binds the inner membrane protein TonB1.In summary, through a combination of biophysical and structural approaches, we have delineated the major binding interactions of PyoS5 with the 1–315-AF488). P. aeruginosa PAO1 cells were used in these experiments since cytotoxic activity was not being monitored. PyoS51–315-AF488 readily labeled P. aeruginosa PAO1 cells (m-chlorophenylhydrazone (CCCP) with the trypsin treatment completely eradicated this remaining fluorescence, suggesting that this protected fluorescence was internalized due to the PMF (488-labeled constructs where either domain 2 was removed (PyoS51–196-AF488) or where only labeled domain 2 was added to cells (PyoS5194–315-AF488). Removal of the CPA-binding domain  decreased surface-bound fluorescence in the absence of trypsin, while the addition of trypsin still revealed internalized fluorescence (194–315-AF488 (domain 2 construct), on the other hand, labeled cells much less efficiently (likely due to its weak binding of CPA on the surface), and all of this fluorescence was trypsin sensitive, suggesting no internalization  was deleted, showed that trypsin-protected fluorophores  were no longer detected, consistent with PMF/TonB1-dependent import of PyoS5 across the outer membrane via FptA  Quantification of the average cell fluorescence observed under different conditions tested in panel A. **** indicates a P value below 0.0001 in Student’s t test, and ns indicates a P value above 0.05. Download FIG S7, PDF file, 0.2 MB.Deletion of the PyoS5 TonB-box prevents translocation. (A) Fluorescent labelling of live Copyright © 2020 Behrens et al.2020Behrens et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. aeruginosa. Thereafter, the first kTHB domain of the pyocin binds FptA in the outer membrane, which also likely acts as the translocation channel, allowing contact between the disordered TonB1 binding site of PyoS5 with TonB1 in the inner membrane and PMF-driven import of the toxin (model presented below).In summary, our fluorescence assays suggest that the import of PyoS5 occurs in two stages. Initial binding to CPA via the central kTHB domain leads to accumulation on the surface of P. aeruginosa, which reflects the array of cell envelope interactions required for import. Yet, common principles are beginning to emerge suggesting that generic import mechanisms may apply for all Gram-negative bacteria that exploit protein bacteriocins. We therefore devised a test of this hypothesis by engineering E. coli susceptibility toward PyoS5 utilizing our current understanding of its import pathway.As with most bacteriocins, pyocins are specific for a subset of strains, in this case from E. coli cells and then engineering the minimal requirements into E. coli in order for PyoS5 to be recognized and transported. A similar strategy was reported by Bosák et al., where E. coli was engineered to be susceptible to a bacteriocin specific for Yersinia kristensenii (E. coli BL21(DE3) cells. We next challenged E. coli BL21(DE3) cells expressing P. aeruginosa FptA but saw no PyoS5 killing (E. coli TonB may not recognize the TonB1 binding sites (Ton boxes) of FptA and/or PyoS5, we also expressed a chimera of E. coli TonB (TonB1–102) fused to P. aeruginosa TonB1201–342 in E. coli BL21(DE3) cells expressing FptA. In this chimera, TonB-B1, the C-terminal domain, and periplasmic regions of TonB are those from P. aeruginosa, but the transmembrane domain that associates with TonB’s partner proteins ExbB and ExbD are those from E. coli. Under these conditions, E. coli became sensitized to PyoS5-mediated killing , E. coli BL21(DE3) expressing FpvAI (B), E. coli BL21(DE3) expressing TonB-B1 (C), and E. coli BL21(DE3) expressing FpvAI and TonB-B1 (D). Zones of clearance were observed for all E. coli conditions tested when ColB PyoS5 was applied (B to D). Similarly, E. coli expressing FpvAI and TonB-B1 was susceptible to both PyoS2 and PyoS4, as was the P. aeruginosa control (A and D). Zones of clearance for PyoS5 were only observed in P. aeruginosa. Download FIG S8, PDF file, 0.3 MB.FpvAI and TonB1 constitute the minimal system for PyoS2 and PyoS4 susceptibility. (A to D) Susceptibility to 1 μM PyoS2, PyoS4, PyoS5, and ColB PyoS5 was assessed for Copyright © 2020 Behrens et al.2020Behrens et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli. They have central receptor-binding domains (kTHB domain 2 in PyoS5) and N-terminally located translocation domains (kTHB domain 1 and its associated disordered region). The confusion that has emerged in the field, that N-terminal domains of pyocins represent their receptor-binding domains, has arisen because pyocin interactions with their translocation channels  can be P. aeruginosa is by their central kTHB domains (domain 2 in PyoS5), which binds CPA embedded in the outer membrane and allows the toxin to decorate the cell surface  was supplemented with 0.4% (wt/vol) glucose, 2 mM MgSO4, and 0.1 mM CaCl2.All bacteria  were culE. coli NEB5α and BL21(DE3) strains were purchased from NEB. Antibiotics were used at the following final concentrations: 100 μg/ml ampicillin and 50 μg/ml kanamycin, and 50 μg/ml gentamicin, all from stock solutions in water; and 37 μg/ml chloramphenicol and 10 μg/ml tetracycline from stock solutions in ethanol.Genes were amplified from genomic DNA or synthesized by Genewiz. Plasmids were created by restriction enzyme digestion and ligation or whole plasmid mutagenesis. Chemically competent E. coli BL21(DE3) for 3 h at 37°C or overnight at 20°C while shaking at 120 rpm. For constructs containing the PyoS5 pore-forming domain (amino acid residues 315 to 498), the cells were cotransformed with pHB22, which carries the ImS5 immunity protein, for increased yield. The bacteria were harvested at 5,050 × g for 15 min at 10°C, resuspended in binding buffer , and sonicated on ice. They were then centrifuged at 12,500 × g for 20 min at 4°C, filtered through a 0.45-μm syringe filter, loaded onto a 5-ml HisTrap high-performance (HP) column equilibrated in binding buffer, and eluted by gradient elution using elution buffer (binding buffer plus 0.75 M imidazole). The protein was then dialyzed into size exclusion buffer  using a 12- to 14-kDa molecular weight cutoff membrane , filtered through a 0.45-μm syringe filter, and applied to a 26/60 Superdex 200 size exclusion chromatography column.PyoS5 and its derivatives, as well as ColB PyoS5, PyoS5-ColIa, PyoS2, PyoS4, and ColIa were expressed heterologously  from E. PyoS4 was expressed at 28°C in the presence of an additional copy of ImS4(pNGH243) and purified on an S200 16/60 size exclusion column.1–315 Δ2–20, PyoS5194–315, and PyoS5194–315-Cys.Mass spectrometry indicated that all bacteriocins purified without their N-terminal methionines, with the exception of PyoS56-tobacco etch virus protease (His6-TEV) at room temperature (RT) for 4.5 h. TonB1 was then purified by affinity chromatography on a HisTrap HP column and by size exclusion chromatography on a 26/60 Superdex 200 column.The TonB1 construct was purified by HisTrap HP column, as described for PyoS5, and then incubated in 300 mM NaCl and 50 mM Tris-HCl (pH 7.0) with 0.07 mg/ml HisE. coli TNE012 at 37°C while shaking at 120 rpm in LB, and upon reaching an optical density at 600 nm (OD600) of 0.6, it was induced with 0.15% (wt/vol) arabinose and supplemented with 0.15% (wt/vol) glucose. The bacteria were harvested as described for PyoS5 and resuspended in 10 mM Tris-HCl (pH 8.0) and 0.25% (wt/vol) lithium diiodosalicylic acid (LIS), sonicated as described for PyoS5, and centrifuged at 4,000 × g for 20 min at 4°C. The supernatant was collected, and the pellet was resuspended in fresh buffer and centrifuged again. Both supernatants were ultracentrifuged at 200,000 × g for 45 min at 4°C. The pellet was homogenized in 10 mM Tris-HCl (pH 8.0), 0.25% (wt/vol) LIS, and 2% (vol/vol) Triton X-100 and ultracentrifuged again. The resulting pellet was homogenized in 10 mM Tris-HCl (pH 8.0) and ultracentrifuged again. The resulting pellet was homogenized in 10 mM Tris-HCl (pH 8.0) plus 2% (wt/vol) β-OG and 5 mM ethylenediaminetetraacetic acid (EDTA) and ultracentrifuged again. FptA was purified from the supernatant by anion-exchange chromatography. A 5-ml HiTrap DEAE fast flow (FF) column was equilibrated in buffer E  and gradient eluted with buffer F (buffer E plus 1 M LiCl). This was followed by 16/60 Sephacryl 300 size exclusion chromatography in buffer E and anion-exchange chromatography on a Mono Q 4.6/100 PE column in buffer E, with gradient elution with buffer F.FptA purification was modeled after a previous BtuB purification protocol . FptA waAll protein concentrations were measured using the absorbance at 280 nm, which was converted to concentration using the sequence-based predicted molar extinction coefficient (ExPASy ProtParam). The presence of scattering impurities, such as protein aggregates, was checked for by measuring the absorbance at 320 nm. All protein masses were confirmed by denaturing electrospray ionization (ESI) mass spectrometry (MS) performed on proteins diluted in formic acid.P. aeruginosa YHP17 cells were grown to an OD600 of 0.6, and 200 μl of the culture was mixed with melted, 50°C, soft LB agar (0.75% [wt/vol] agar) and poured over an LB agar plate. Once the plate had set, 2.5 μl of each bacteriocin concentration was spotted onto the plate. The plates were left to dry and then incubated at 37°C overnight.g for 20 min, and resuspended in 10 ml of 50 mM Tris (pH 7.5), 2 mg/ml lysozyme, and 0.5 mg/ml DNase I. Cells were lysed by sonication, as described for PyoS5 isolation, the lysate was incubated for 30 min at RT, and then 0.2 mM EDTA added. An equal volume of aqueous phenol was then added and the mixture heated for 20 min at 70°C with mixing. The solution was incubated on ice for 30 min and centrifuged at 7,000 × g for 20 min, and the aqueous upper layer was extracted. Proteinase K (0.05 mg/ml) was added and the solution dialyzed overnight against 5 liters of distilled water (dH2O), followed by dialysis against 5 liters of fresh dH2O for 5 h. LPS was pelleted by ultracentrifugation for 1 h at 100,000 × g and the pellet resuspended in 10 ml dH2O. The suspension was heated at 60°C for 30 min, acetic acid was added, and the mixture was heated at 96°C for 1.5 h. Lipid A was pelleted by centrifugation at 13,500 × g for 3 min, and the supernatant, which contains the polysaccharide, was extracted with 10 ml chloroform. The aqueous phase was then lyophilized.LPS-derived polysaccharides were isolated as described previously . BrieflyNative mass spectrometry was performed in 100 mM ammonium acetate buffer, with the exception of TonB1, which was analyzed in 200 mM ammonium acetate buffer.SPR was performed on a Biacore T200 instrument. A series S sensor chip CM5 (GE Life Sciences) was docked and primed into HBS-OG buffer . This buffer was used as a running buffer for all SPR experiments.For amine coupling using the Amine Coupling kit , ligand proteins were desalted into immobilization buffer  and diluted 10-fold in 10 mM sodium acetate .For thiol coupling using the Thiol Coupling kit , ligand proteins were incubated with 10 mM dithiothreitol (DTT) for 2 h and then desalted into immobilization buffer diluted 10-fold in 10 mM sodium acetate  immediately before immobilization.Analyte proteins were desalted into HBS-OG buffer before application. The contact time for SPR was set to 120 s, the dissociation time to 600 s, and the flow rate to 30 μl/min. Lower analyte concentrations were applied first.H, ΔS, and dK are unaffected by the analyte concentration. Errors reported in the text are standard deviations of the average results from two experiments.ITC was performed using a MicroCal iTC200 instrument at 25°C in 0.2 M sodium phosphate buffer (pH 7.5). Proteins in the syringe were at a concentration of 150 μM, and polysaccharides in the cell were at a concentration of 7 mg/ml, which was estimated to be 30 μM based on a molecular weight of 10 kDa and the assumption that CPA constitutes 5% of the LPS polysaccharides. The data were fitted to a one-binding-site model in the MicroCal LLC Origin software. As the CPA concentration is estimated, the observed stoichiometry is unlikely to be correct, while ΔSAXS data were collected at the B21 beamline at Diamond Light Source proteins following in-line size exclusion chromatography on a Superdex 200 column and processed using ScÅtter and ATSAS , 33. Gui(i) Circular dichroism. Proteins were analyzed at 0.1 mg/ml in 10 mM potassium phosphate buffer (pH 7.5) and 20 mM NaCl using a Jasco J-815 spectropolarimeter. Spectra were measured between 260 nm and 190 nm at a digital integration time of 1 s and a 1-nm bandwidth. Each sample spectrum was measured in quadruplicate and averaged. Molar ellipticity was calculated by subtracting the baseline from sample spectra and dividing by the molecular weight, molar concentration, and pathlength in millimeters. Thermal melting curves for proteins were measured at 222 nm between 20°C and 86°C and 4-parameter sigmoidal melting curves were fit to the equation f = y0 + a/(1+e(x−x0)/b) using non-linear regressions in SigmaPlot to determine the melting temperature (mT), where x is the temperature, x0 is the mT, f is molar ellipticity, y0 is the minimal molar ellipticity, and b is a fitting parameter.(ii) Size exclusion multiangle light scattering. Proteins were separated in 50 mM Tris (pH 7.5) and 150 mM NaCl using a Superdex 200 10/300 GL column and detected by a Wyatt Dawn HELEOS-II 8-angle light-scattering detector and a Wyatt Optilab rEX refractive index monitor linked to a Shimadzu high-performance liquid chromatography (HPLC) system.Pyocin S5 was concentrated to 16 mg/ml in 25 ml Tris-HCl (pH 7.5) and 150 mM NaCl using a VivaSpin 20 column with a 30-kDa molecular weight cutoff (Sartorius). The crystallization screens Index (Hampton Research) and PACT, JCSG+, and Morpheus (Molecular Dimensions) were used to screen for crystals. Crystals were grown in a vapor diffusion sitting drop setup under JCSG+ screen (Molecular Dimensions) condition C7  at 18°C. The drops contained 100 nl protein and 100 nl buffer. The cryoprotectant solution was 25% glycerol, 10% (wt/vol) PEG 3000, 0.1 M sodium acetate, and 0.1 M zinc acetate (pH 4.5) for cooling the crystals in liquid nitrogen. Diffraction data were collected at beamline ID30A-3 at the European Synchrotron Radiation Facility (ESRF) at a wavelength of 0.9679 Å using an Eiger detector. We collected 225° of data with 0.15° oscillation. The transmission was 20%, and the exposure time was 0.010 s.1 space group and yielding a 98.8% complete set of indexed diffraction spots but no anomalous signal. Molecular replacement was carried out using ColIa residues 450 to 624 in Phaser and yielded electron density for the pore-forming domain of PyoS5. The lack of density for the remainder of the protein indicated that the phases, obtained from ColIa, were not sufficient to build a model for the whole protein.The raw data were analyzed in Dials, revealing a P22+. The result was additional, visible helical density beyond the pore-forming domain.Improved phases were obtained from anisotropy correction of the same data set using Staraniso in AutoProc , 38, whiRwork and Rfree upon refinement, indicating that the use of multiple TLS groups made the model worse. The refinement process was therefore continued with the whole model treated as one TLS group.Iterations of model building into the visible helical density in Coot and refinement against the complete Dials data set in Buster version 2.10.3 resulted in a model of PyoS5. The model was optimized in Coot , followeRwork was 0.212 and the Rfree was 0.272. MolProbity  Fluorescent labeling of proteins. Bacteriocins were fluorescently labeled using maleimide AF488 labels via an engineered C-terminal cysteine. To reduce the cysteine, the protein was mixed in a 1:9 ratio with DTT to yield a concentration of 10 mM DTT and incubated for 2 h at RT. To remove aggregates, the protein was centrifuged at 16,000 × g for 1.5 min and the supernatant transferred to a new tube. The supernatant was then applied to a 5-ml HiTrap desalting column and desalted into 25 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 1% (wt/vol) β-OG. The protein concentration was measured, and maleimide AF488 was immediately added in 3-fold excess. The reaction was allowed to proceed for 1 h while mixing by rotary inversion in the dark at RT. Then, the reaction was quenched by adding DTT to a final concentration of 5 mM. The solution was centrifuged and desalted as described before. The absorbance at 280 nm and 494 nm was measured using a V-550 UV-visible spectrophotometer (Jasco). Labeling efficiency was determined as described in the manufacturer’s protocol . All fluorescently labeled proteins used for microscopy were labeled with more than 95% efficiency.(ii) Fluorescent labeling of bacteria. Coverslips were cleaned by water bath sonication at 50°C for 15 min in 2% Neutracon (Decon) solution, washed in double-distilled water (ddH2O), and air dried.600 of 0.6 was reached. Six hundred microliters of this culture was used per condition. All pelleting steps were performed at 7,000 × g for 3 min at RT.Bacteria were grown overnight in LB medium. One milliliter of this overnight culture was pelleted, resuspended in 10 ml of supplemented M9 medium, and grown until an ODFor CCCP treatment, CCCP was added to a final concentration of 100 μM from a 10 mM stock in dimethyl sulfoxide (DMSO) to the bacteria before the addition of the fluorescently labeled protein. The bacteria were incubated with CCCP while mixing by rotary inversion at RT for 5 min, while all other samples were incubated without CCCP for the same time. Fluorescently labeled protein was then added to a concentration of 1 μM and the sample incubated in the dark while mixing by rotary inversion for 20 min at RT.For trypsin treatment, trypsin was added to a final concentration of 0.1 mg/ml immediately after the incubation with the fluorophore-labeled pyocin. The bacteria were incubated with or without trypsin at 30°C for 1 h at 120 rpm.Subsequently, bacteria were washed three times in supplemented M9, where each wash consisted of pelleting the bacteria, removing the supernatant, resuspending the pellet in 50 μl by repeated pipetting (10 times) with a P20 pipette, transferring the 50 μl to a new tube with 450 μl of supplemented M9, and vortexing. The bacteria were resuspended in a final volume of 30 μl. Three microliters was applied to an agar pad for microscopic analysis. Agar pads were prepared using Gene Frames (Thermo Scientific) as follows. Supplemented M9 agar (1% [wt/vol]) was prepared and 190 μl pipetted into the Gene Frame. Using a coverslip, the surface was flattened and excess agar removed. Once the agar solidified, the coverslip was removed, the bacterial suspension was added, and a new coverslip was attached to the adhesive side of the Gene Frame.All images were collected on an Oxford Nanoimager S microscope at 100-ms exposure. For every image, 200 frames were collected and averaged. Green fluorescence  was measured at 35% laser power.t tests were performed to determine P values.In ImageJ, the 200 collected frames per image were merged using the command “Z project.” Bacterial cells and background were identified in transillumination images using Trainable Weka Classifier. Regions of interest were transferred to green fluorescence images and the mean fluorescence of cells, signal, and background noise quantified. Each image contained a minimum of 15 bacterial cells. For each repeat, a minimum of six images were collected per sample, and three independent experiments were performed for each experiment. As a result, a minimum of 270 bacterial cells were quantified for each sample. Student’s Sequences were compared using NCBI BLASTn and BLASTp , MUSCLE https://www.rcsb.org/) and assigned the identifier 6THK.The data supporting the findings of the study are available from the corresponding author upon request. The crystallography data from this publication have been deposited to the PDB database (


Although a change in the microbiome structure does not necessarily indicate an upcoming critical process stage, the recorded community-level change points might be a first indication to carefully observe the process.Efforts to integrate biogas plants into bioeconomy concepts will lead to an expansion of manure-based  biogas plants, while their operation is challenging due to critical characteristics of some types of livestock manure. For a better process understanding, in this study, three manure-based small biogas plants were investigated with emphasis on microbiome diversity. Due to varying digester types, feedstocks, and process conditions, 16S rRNA gene amplicon sequencing showed differences in the taxonomic composition. Dynamic variations of each investigated biogas plant microbiome over time were analyzed by terminal restriction fragment length polymorphism (TRFLP), whereby nonmetric multidimensional scaling (NMDS) revealed two well-running systems, one of them with a high share of chicken manure, and one unstable system. By using Threshold Indicator Taxa Analysis (TITAN), community-level change points at ammonium and ammonia concentrations of 2.25 g L The analyzed BPs differed in their reactor types/configuration, their supplied feedstocks, and their general process management  . Informaded data .VS m−3 d−1 with HRTs of 68.5 ± 5.3 and 50.2 ± 3.3 days, respectively  with a volume of 1680 m³ which is insulated to maintain a temperature of approximately 30 °C. At all biogas plants, a combined heat and power unit produces 75 kW electricity and heat, whereby the electricity is fed into the grid and the heat is used for heating the fermenter and private houses. The main digester of BP 03 was operated at a mean temperature of 46.5 ± 4.1 °C  digester with a volume of 847 m³ in series with a secondary unheated digester with a volume of 1742 m³ . Overall, 654 and 450 m³ biogas can be stored in the gas-tight roof of each digester for BP 01 and BP 02, respectively. Beside these technological similarities, BP 01 and BP 02 differed in their feedstock composition: In addition to the main feedstocks liquid and solid cattle manure as well as maize and grass silage, BP 01 was fed with chicken manure , while in BP 02, horse manure was supplied  . The meaectively . Compare± 4.1 °C a and fed± 4.1 °C . Compare1.0 days . Due to Samples of the main digester content were taken monthly over one year according to Theuerl et al. , conside3) concentration was calculated as a function of the TAN concentration, the pH and the temperature, according to Hansen et al. [The following chemical analyses were conducted on all taken samples: total solids (TS), volatile solids (VS), total ammonium nitrogen (TAN), various volatile fatty acids (VFA) and pH values according to standard methods . The fren et al. . In orden et al. .Samples which were used for the taxonomic microbiome inventory were taken after a three-month lasting lead time under stable process conditions.® spin kit for soil  in combination with the Genomic DNA Clean & ConcentratorTM kit . Afterwards, 16S rRNA gene amplicon libraries were constructed by using the “16S Metagenomic Sequencing Library Preparation” protocol . The universal primer pair Pro341F/Pro805R [https://github.com/lh3/seqtk). All following processing steps were carried out within the QIIME platform which contains a broad range of different tools for high-throughput community sequencing data [In order to record the taxonomic microbiome diversity, in a first step, high-quality DNA was extracted in triplicates using the FastDNA/Pro805R  was used/Pro805R , the mer/Pro805R  and the /Pro805R . The pro/Pro805R  was useding data ,75,76. Fing data . An openTo profile the dynamic variation of the occurring microbiomes over time, one of the most commonly applied and reliable fingerprinting techniques, TRFLP, was used . As TRFL2 values with p values of 0.001.For the statistical analyses, only terminal restriction fragments (TRFs) with a relative abundance over 1% were used. All statistical analyses were carried out with the R Project for Statistical Computing  using thTo define ecological thresholds where ecosystem attributes suddenly change due to small variations in specific environmental drivers, Threshold Indicator Taxa Analysis (TITAN) is a promising method as it provides not only the detection of potential indicator taxa, but rather as it provides information for potential community-level change points ,84, wher−1 high TAN concentrations  and 80 to 400 mg L−1 (NH3) are often reported as thresholds for process inhibition [3 concentrations as they produce similar amounts of biogas as the microbiome of BP 02 . However, the VS degradation degrees and the relative residual CH4 potentials for both biogas plants are in the expected range.In general, BP 01 and BP 02 showed an overall stable process performance with low VFA concentrations , althougtrations , corresphibition ,38,39,40of BP 02 . The ext−1 . The. The−1 (3 concentrations (caused by the use of chicken manure), this study enables to answer the question whether there is a point of change where biogas microbiomes potentially differentiate from each other and (ii) by comparing well-running systems with an unstable system, this study might provide information on microbiome representatives which are specific either for well-running systems or for disturbed systems including the potential to define a respective system change point.To draw up an interim balance, the here presented study does not only provide information related to differences in biogas plant operation such as varying OLRs/HRTs or especially to the handling of potential process-critical feedstocks such as chicken manure, but it also enables the evaluation of a supposed long-lasting unstable process phase. Hence, two aspects can be addressed: (i) by comparing two well-running systems which differ in their general biogas plant operation, especially in their TAN/NHA total of 2297 OTUs were identified in the analyzed anaerobic digesters, whereas BP 01 contained 1451 OTUs, BP 02 1248 OTUs and BP 03 1510 OTUs. The taxonomic profiles showed an average of 90.2% ± 1.6% from the domain Bacteria, 8.6% ± 1.4 % from the domain Archaea and 1.2% ± 1.9% of microorganisms that have not yet been classified. The most abundant phyla were Firmicutes (58.2% ± 6.7%), Actinobacteria (12.2 ± 8.1%), Bacteroidetes (7.6% ± 6.6%), Cloacimonetes (3.8% ± 6.6%), Proteobacteria (3.3% ± 1.6%) and Euryarchaeota (8.6% ± 1.3%), which generally is in accordance with previously published studies ,57,61,93Brachybacterium (3.1%), Corynebacterium (5.6%), Fastidiosipila (4.4%), “Lachnospiraceae NK3A20” (3.2%), Paeniclostridium (3.3%), Turicibacter (3.3%), “uncultured Peptostreptococcaceae” (4.8%), “uncultured Syntrophomonadaceae” (5.4%) and Methanobrevibacter (6.3%)  into carbon dioxide (CO2) and hydrogen (H2), which are subsequently converted into methane by hydrogenotrophic archaea [Methanobrevibacter (6.3%) recorded for this BP , Actinobacteria (19.1%), Proteobacteria (3.7%), Atribacteria (1.1%), Chloroflexi (1.0%) and Euryarchaeota (7.9%) a with thr (6.3%) b. Most nsurvival . In addi archaea ,95,96. T this BP b. Taking this BP , the occtrations ,97,98.Methanosaeta (syn. Methanotrhix) [3 concentrations caused by the supply of supposedly high amounts of grass silage and chicken manure (comparing BG 01 with BP 02), and hence whether they are potential indicative taxa which can be used to elucidate points where ecosystem traits/features suddenly change , Bacteroidetes (12.3%), Cloacimonetes (11.5%), Actinobacteria (3.4%) and Euryarchaeota (10.1%) a. Comparnotrhix) , which wange see .Corynebacterium (4.7%), Paeniclostridium (5.1%), “Rikenellaceae RC9 gut group” (4.9%), Syntrophococcus (2.7%), Turicibacter (2:5%), “uncultured Lachnospiraceae” (2.1%), “uncultured Peptostreptococcaceae” (6.7%) and Methanobrevibacter (7.2%) , Actinobacteria (14.2%), Bacteroidetes (10.4%), Proteobacteria (4.7%) and Euryarchaeota (7.7%) a with thr (7.2%) b.Besides the mentioned differences, common to all three biogas plants is the occurrence of representatives from the phyla Firmicutes and Actinobacteria. While members of the phylum Firmicutes are frequently found within anaerobic digesters, members of the phylum Actinobacteria are far less recognized. Representatives of this phylum are well-known for two reasons: (i) as important pathogens causing serious diseases such as tuberculosis, leprosy or diphtheria and (ii) as important producers of drugs, particularly antibiotics, considerably reducing the impact of infectious diseases in the last century . However3, produced biogas amount) by NMDS analyses [In order to elucidate the dynamic variations of the microbial communities over time, especially for BP 03, TRFLP analyses were carried out separately for the bacterial and archaeal communities. The temporal microbiome profiles were related to the prevalent abiotic parameters , Methanomassiliicoccus (TRF470) and Methanosarcina (TRF625), while BP 02 showed a clear dominance (≥ 60 %) of the genus Methanosaeta (TRF106)  [−1 and 2.5 ± 0.4 g L−1 under the threshold values. In addition, Theuerl et al. [Methanosaeta (syn. Methanothrix) are indicators for stable process conditions if they are co-detected with members from the bacterial phyla Bacteriodetes and Cloacimonetes. For BP 02, the NMDS analysis, for example, revealed that TRF89 and TRF91  as well as TRF161 (phylum Cloacimonetes) were significantly related  to the respective microbiome structure  . Considectively) , the higl et al.  investigtructure b.In comparison with BP 01 and BP 02, very unstable process conditions over time were recorded for BP 03 see , which a3 concentration, switches corresponding to a distinct change in the microbiome structure or in particular in indicative microbiome representatives.However, in order to ensure a high process stability and efficiency with low susceptibility to disturbances , the cha3 and VFA concentrations were determined  or positively (increasing abundances) to elevated TAN/NHtermined .3 concentrations (−1 for TAN and 80–400 mg L−1 for NH3 [−1 (positive response), intersecting at 2.25 g L−1 for the TAN concentration and with 331 (negative response) and 625 mg L−1 (positive response), intersecting at 193 mg L−1 for the NH3 concentration  for the VFA concentration, at 0.12 (negative response) and 1.16 g L−1 (positive response) for the acetic acid concentration and at 0.04 (negative response) and 1.94 g L−1 (positive response) for the propionic acid concentration (−1 (VFA), 0.56 g L−1 (acetic acid) and 0.3 g L−1 (propionic acid), the points after which the sharpest changes within the microbiome structure occur. Based on the presented results, it can be assumed that well-running systems are characterized by organic acid values near the detection limit (as it was recorded for BP 02), whereby values higher than 0.75 g L−1 for total VFA concentration might be indicative of being aware of an upcoming critical process stage due to the sharp change within the microbiome structure. Although this is in contrast to Drosg [−1 indicate unfavorable process conditions, the presented results are not surprising as most chemical parameters which indicate process instabilities/disturbances  result from previous microbial activities [Considering an overload of the microbial degradation potential which is often accompanied by an acid accumulation , TITAN rntration . Both cuto Drosg , who postivities . Consequ3 concentrations after the point where the community-level change points for negative and positive responses intersect, as this indicates the point at which there is a distinct change in a particular ecosystem attribute. In contrast and with special emphasis on elevated TAN/NH3 concentrations, strongly positively responding reliable indicative taxa could be evidence for a well-adapted system with an overall stable production of sufficient amounts of biogas. However, further research is needed to verify the here presented results.Summarizing, the question remains whether community-level change points and recorded positively or negatively responding taxa might be indicators for upcoming critical process stages. Promising might be an approach which considers all reliable indicative taxa which respond negatively to, e.g., increasing VFA or elevated TAN/NH3 concentrations of 2.25 g L−1 and 193 mg L−1 or at VFA concentrations of 0.75 g L−1 were identified. These values are lower than the commonly reported thresholds for critical process stages based on chemical parameters, which supports the assumption that microorganisms show an earlier response to changing process conditions since the chemical parameters result from previous microbial activities. Specific microbiome members, particularly of the phyla Bacteroidetes and Cloacimonetes responded highly sensitively to increasing TAN/NH3 or VFA concentrations and hence can potentially be used in order to assess the process performance. However, further research with broader data sets  is needed to verify the results presented here.The microbiomes of the three manure-based small-scale biogas plants differed significantly depending on feedstocks and process conditions. However, common to all three biogas plants was the occurrence of the phylum Actinobacteria with high abundances of up to 19.1% which might be a specific characteristic of manure-fed biogas plants. Reliable values for ecological change points in terms of distinct change points for both the entire communities and specific microbiome taxa at TAN and NH


Necroptosis is a newly recognized form of programmed cell death with characteristics of both necrosis and apoptosis. The role of necroptosis in hepatic damage during sepsis is poorly understood. In this study, we investigated the occurrence of necroptosis in hepatic damage, and its contribution to hepatic damage in a piglet model of lipopolysaccharide (LPS)-induced sepsis.Two animal experiments were conducted. In trial 1, piglets were challenged with LPS and sacrificed at different time points after LPS challenge. In trial 2, piglets were pretreated with necrostatin-1, a specific inhibitor of necroptosis, prior to LPS challenge. Alterations in the hepatic structure and function, pro-inflammatory cytokine expression, and the necroptosis signaling pathway were investigated. Typical ultrastructural characteristics of cell necrosis was observed in the liver of LPS-challenged piglets.Expressions of critical components of necroptosis including kinases , mitochondrial proteins (PGAM5 and DRP1), and an intracellular damage-associated molecular pattern (HMGB1) were increased in the liver in a time-dependent manner, followed by hepatic inflammation, morphological damage, and dysfunction as manifested by elevated hepatic expression of IL-1β, IL-6 and TNF-α as well as increased serum AST and AKP activities and the AST/ALT ratio. Pretreatment with necrostatin-1 significantly reduced the expression of RIP1, RIP3 and MLKL as well as PGAM5, DRP1 and HMGB1, which subsequently led to obvious attenuation of hepatic inflammation and damage.Our study demonstrates that necroptosis occurs in the liver during sepsis and contributes to septic hepatic injury. Sepsis is a common and life-threatening illness that could lead to multiple organ failure, shock, and death as a result of an exaggerated inflammatory response to infection , 2. As aHepatic cell death is a critical event in progression of various liver diseases , 7. TradAccumulating evidence has indicated that necroptosis plays a crucial role in several forms of liver injury in mice such as alcoholic liver disease , non-alc2 pens, maintained in an environmentally controlled room, and received a standard weanling piglet diet for acclimation for 14 d with free access to feed and water. Animals were observed daily for signs of diarrhea, sickness, and abnormal behavior.Animal experimental procedures were approved by the Animal Care and Use Committee of Wuhan Polytechnic University . A total of 70 weaned, apparently healthy piglets  were purchased from Aodeng Agricultural and Animal Husbandry Technology Co., Ltd . The pigs were individually housed in 1.80 × 1.10 mEscherichia coli LPS  at 100 μg/kg body weight (BW) and then were slaughtered at 1, 2, 4, 8, 12, or 24 h after LPS challenge  at 1.0 mg/kg BW or an equivalent volume of 2% DMSO 30 min before intraperitoneal injection of LPS at 100 μg/kg BW or saline Table 2Svia jugular vein puncture, and centrifuged at 3,500 g for 10 min to collect serum. Serum was stored at –80°C until analysis. Following blood collection, the pigs were humanely euthanized with an intramuscular injection of sodium pentobarbital (80 mg/kg BW), and then the liver samples (0.5 cm3 segments) were harvested. One portion of the liver samples was fixed in fresh 4% paraformaldehyde/phosphate-buffered saline for 24 h for histological analysis. Another portion was cut into smaller pieces (about 1 mm3) and fixed with 2.5% glutaraldehyde at 4°C for ultrastructural transmission electron microscopy (TEM) analysis. The remaining liver tissue was diced into smaller pieces, snap frozen in liquid nitrogen, and stored at −80°C for further analysis.Prior to animal sacrifice, blood samples were collected into 10-mL vacuum tubes  The activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (AKP) in the serum were determined using a colorimetric method with commercial kits  according to the manufacturer’s instructions.After a 24-h fixation, liver segments (5-mm) were dehydrated with a series of increasing concentrations of ethanol, cleared with xylene, and embedded in paraffin. Cross-sections (4-μm thick) of each sample were deparaffinized, and stained with hematoxylin and eosin. Histological analysis was performed in a blinded manner by an experienced pathologist using a light microscope with a computer-assisted morphometric system .Liver samples were fixed with 2.5% glutaraldehyde, and postfixed in 1% osmium tetroxide. They were then dehydrated with ethanol and embedded in Epon 812 . Ultra-thin sections (100 nm) were cut and stained with uranyl acetate and lead citrate. Ultrastructural observation was conducted in a blinded manner by an experienced pathologist using a transmission electron microscope (TEM)  at an accelerating voltage of 200 kV and a magnification of 5,000.Frozen liver samples were weighed, homogenized in nine volumes of ice-cold saline solution, and centrifuged at 2,500 rpm for 10 min at 4°C to collect the supernatants. The protein concentrations of the supernatants were measured using the bicinchoninic acid reagent . The concentrations of tumor necrosis factor-α (TNF-α), interleukin (IL)-6 and IL-1β in the supernatants were determined using respective ELISA kits  according to the manufacturer’s protocols and expressed as pg/mg protein.#ab131552, Abcam), rabbit anti-DRP1 , rabbit anti-HMGB1 , and mouse anti-β-actin . Blots were developed using an Enhanced Chemiluminescence Western blotting kit , visualized using a Gene Genome Bioimaging System, and analyzed using GeneTools software . The relative protein abundance of target proteins were expressed as the ratio of target protein/β-actin protein.Liver samples (100-150 mg) were suspended in lysis buffer, homogenized, and centrifuged to collect the supernatants. Protein concentrations of the supernatants were measured using the bicinchoninic acid reagent . Equal amounts of hepatic proteins were loaded and separated on a polyacrylamide gel and transferred onto polyvinylidene difluoride membranes. Immunoblots were blocked for at least 1 h with 3% bovine serum albumin in TBS/Tween-20 buffer. The membranes were incubated overnight at 4°C with primary antibodies and then with the horseradish peroxidase-labelled secondary antibodies for 2 h at room temperature. Specific primary antibodies included rabbit anti-RIP1 , rabbit anti-RIP3 , rabbit anti-phosphorylated MLKL (p-MLKL) , rabbit anti-PGAM5 (® RT Kit (TaKaRa Biotechnology). Real-time PCR analysis was performed using a SYBR® Premix Ex Taq™ (Tli RNaseH Plus) qPCR kit (TaKaRa Biotechnology) on an Applied Biosystems 7500 Real-Time PCR System . The total volume of each PCR reaction was 20 μL, which contained 10.0 μL SYBR® Premix Ex TaqTM, 0.4 μL forward primer (10 μmol/L), 0.4 μL reverse primer (10 μmol/L), 0.4 μL ROX reference dye II (10×), 2.0 μL cDNA and 6.8 μL RNase-free water. After an initial denaturation at 95°C for 30 s, the reaction was cycled at 95°C for 5 s and 60°C for 34 s for 40 times. The primer pairs used are shown in -ΔΔCt method of Livak and Schmittgen  and quantified using Nano-Drop 2000 Spectrophotometer . RNA purity was assessed by determining the ratio of the absorbance at 260 and 280 nm, and the integrity was verified using agarose gel electrophoresis. Genomic DNA removal and cDNA synthesis were performed using PrimeScripthmittgen . Our resP ≤ 0.05 was considered statistically significant.In the first experiment, the data were analyzed using Duncan’s multiple comparison tests. In the second experiment, the data were analyzed using the general linear model procedures of ANOVA . Post hoc testing was conducted using Duncan’s multiple comparison tests. All data were presented as means ± standard errors of the mean. To investigate the dynamic effect of LPS on the liver, we examined hepatic histopathological changes of pigs at 1, 2, 4, 8, 12, and 24 h after LPS challenge. Compared to the control pigs (0 h), LPS-challenged pigs displayed a progressive increase in liver damage as manifested by disordered hepatic cell cord arrangement, inflammatory cell infiltration, and karyolysis, karyopyknosis, and vacuolation of hepatocytes (p < 0.05). However, LPS had no effect on serum ALT activity.To explore the dynamic effect of LPS challenge on hepatic function, we measured the activities of AST, ALT and AKP in the serum , 24. LPSp < 0.05).Hepatic inflammation contributes to hepatic damage , 24. To p < 0.05). However, LPS decreased mRNA abundance of HMGB1 at 1, 2, 4 and 24 h. Similarly, LPS elevated protein abundance of RIP1, RIP3 and phosphorylated MLKL between 2-24 h, and PGAM5 between 8-24 h and DRP1 between 4-24 h, peaking at 8, 4, 12, 12, 8 or 4 h, respectively (p < 0.05). Inconsistent with mRNA expression, protein abundance of HMGB1 was increased at 2, 4, 12 and 24 h (p < 0.05). These results, in combination with the severity of hepatic injury and inflammation, indicated that 4 h post LPS challenge is the time when the liver is experiencing dramatic changes and therefore, was chosen for analysis of the hepatic function and damage in a subsequent animal trial.To explore if necroptosis occurred concomitant with hepatic injury and inflammation in LPS-challenged pigs, we examined liver ultrastructure by TEM (p < 0.05) (p < 0.05).To further demonstrate that LPS-induced hepatic damage is partially due to necroptosis, the piglets were pretreated with Nec-1, which is a specific inhibitor of RIP1 kinase activity  and RIP1p < 0.05)  after LPS challenge , 24.Hepatic injury is closely associated with hepatic inflammation , 24. As Given the fact that necroptosis is closely related to tissue injury and inflammation , we explRip3 gene prevents ethanol-induced liver injury in mice . It is unlikely that pretreatment with Nec-1 could be applied in clinical practice, which may limit the clinical use of Nec-1. Further research is needed to determine practical and effective clinical use regimens of Nec-1 after the sepsis. Secondly, only a single dose of Nec-1 was used in this study. In future studies, multiple doses of Nec-1 should be evaluated to determine the optimal dose and therapeutic window of Nec-1 for maximal hepatic protection during sepsis.In summary, we demonstrate that LPS activates necroptosis in the liver, resulting in the damage of hepatic structure and function. Targeting necroptosis with Nec-1 alleviates LPS-induced hepatic injury. Therefore, necroptosis occurs and contributes to hepatic damage during sepsis.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Animal Care and Use Committee of Wuhan Polytechnic University.YL, C-AH, and GZ designed research. QX, JG, XL, YW, KX, XW, and HZ conducted research. YL, XL, and YW analyzed data. YL, DW, and QX wrote the paper. DW, C-AH, and GZ revised the manuscript. YL had primary responsibility for final content. All authors contributed to the article and approved the submitted version.This research was financially supported by the Project of National Natural Science Foundation of China (No. 31772615), the Project of Innovative Research Groups of the Natural Science Foundation of Hubei Province (No. 2019CFA015), and Wuhan Science and Technology Bureau (No. 2018020401011304).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Parageobacillus thermoglucosidasius. We initially compared the transformation efficiency of two strains, NBRC 107763T and TG4. We then disrupted CODH, ECH, and both enzymes in NBRC 107763T. The characterization of growth in all three disruptants under 100% CO demonstrated that both enzymes were essential for CO-dependent growth with hydrogen production in P. thermoglucosidasius. The present results will become a platform for the further metabolic engineering of this organism.The metabolic engineering of carbon monoxide (CO) oxidizers has the potential to create efficient biocatalysts to produce hydrogen and other valuable chemicals. We herein applied markerless gene deletion to CO dehydrogenase/energy-converting hydrogenase (CODH/ECH) in the thermophilic facultative anaerobe,  These mParageobacillus thermoglucosidasiusA well-known fermenter,  has been identified as a hydrogenogenic CO oxidizer, the genome of which contains one codh gene cluster comprising three genes: cooCSF for a mature CODH enzyme  and one ech gene cluster that comprises twelve genes for a mature ECH complex (AOT13_RS13410–13355)  A, both o by WGSR . Another2 from CO  (–1): 17 g tryptone, 3 g soypeptone, 4 mL glycerol, 4 g sodium pyruvate, 5 g NaCl, and 2.5 g K2HPO4 (Rkan) and three homology arms, i.e., upstream (‘up’), downstream (‘down’), and 5′ (‘start’), for each target gene in the following sequence: 5′-‘start’-Rkan-‘up’-‘down’-3′  and disruptants were cultivated in inorganic modified B medium (600), and the gas composition was analyzed by gas chromatography. All plasmids and primers used in the present study are listed in The strains NBRC 107763T and strain TG4, which we had isolated. Three different types of Escherichia coli-Geobacillus shuttle plasmids, namely, pG1C, pG2K, and pG1AK-PheB . In codh and codh–ech gene disruptions, colonies with the first crossover occurring at both ‘start’ and ‘down’ were selected  to support gene deletions at the appropriate sites, to confirm no marker insertion into the genomes, and to identify unexpected gene mutations. Consistent with the PCR results, no reads were mapped onto the loci of codh, ech, and codh–ech in Δcodh, Δech, and Δcodh–ech, respectively . In comparisons with the reference genome of DSM 2542T, six mutations were detected in WT    and S6. odh–ech) , suggest600 reached 0.001. All strains grew with similar growth rates until the OD600 reached 0.03 in the first 3 h, and then decreased to 0.02 in the next 10 h. The OD600 of WT was maintained at approximately 0.02 for the next approximately 30 h, and WT then launched CO-dependent growth via WGSR at 40 h. WT growth had a doubling time of 6.9 h until the OD600 reached 0.25, converting all available CO to H2 and CO2 by 118 h . The growth rates and OD600 of WT and the disruptants showed similar changes in the first 13 h .Parageobacillus thermoglucosidasius. Microbes Environ 35: ME20101.Adachi, Y., Inoue, M., Yoshida, T., and Sako, Y. (2020) Genetic Engineering of Carbon Monoxide-dependent Hydrogen-producing Machinery in https://doi.org/10.1264/jsme2.ME20101Supplementary Material


The combined processability, structural rigidity and easy feasibility make this polymeric membrane promising for large-scale hydrogen separations of commercial and environmental relevance.The synthesis of a polymer that combines the processability of plastics with the extreme rigidity of cross-linked organic networks is highly attractive for molecular sieving applications. However, cross-linked networks are typically insoluble or infusible, preventing them from being processed as plastics. Here, we report a solution-processable conjugated microporous thermoset with permanent pores of ~0.4 nm, prepared by a simple heating process. When employed as a two-dimensional molecular sieving membrane for hydrogen separation, the membrane exhibits ultrahigh permeability with good selectivity for H Rigidity, as seen in crosslinked polymers, and plastic processability are mutually exclusive properties. Here, the authors prepare a solution-processable conjugated microporous thermoset with permanent pores and demonstrate its application as a two-dimensional molecular sieving membrane for hydrogen separation. Membrane-based gas separation provides an energy-efficient solution to selective hydrogen separation, but performance is often limited by the trade-off between permeability and selectivity5. In the past decade, advances have been made with microporous solids possessing extremely rigid network structures and well-defined pores, such as zeolites and metal organic frameworks, from which membranes with thin permselective layers have high permeability and good selectivity9. However, industrial membranes are dominated by polymers that are fabricated by simple solution-processing techniques10. Currently, commercial gas separation membranes are fabricated from a few polymers with relatively low permeability and high selectivity11. The low gas permeability of commercial membranes requires large membrane size for sufficient production, which is a critical cost challenge for industrial applications. Thus, microporous polymers that possess rigid network structures, high gas permeability, good selectivity, and solution-processability are highly desirable for next-generation gas separation membranes.Hydrogen purification from the steam-reforming of methane, and recovery from offgas streams in a refinery or other industrial processes present a good opportunity to boost the production of clean energy13 and polymers of intrinsic microporosity (PIMs)17 are extensively studied on account of their microporosity and good solution-processability. POCs are shape-persistent organic molecules with accessible intrinsic cavities, which can be aligned during slow solvent evaporation, leading to homogenous microporous membranes assembled via van der Waals (vdWs) interactions. Cooper and co-workers prepared POC membranes for selective hydrogen separation, in which a cage molecule CC3 with a window diameter of ~0.6 nm achieved a H2 permeability of 226 barrer and a H2/N2 selectivity of 3010. Microporosity in PIMs arises from loose packing of contorted and rigid macromolecular chains, and large area membranes with moderately good mechanical strength can be readily fabricated by solution processing. However, cage molecules and linear polymer chains assembled by weak vdW forces can easily slide over one another when thermally or mechanically agitated, resulting in limited selectivity18. In addition, CO2 has a substantial plasticization effect on the membranes at high temperature, leading to further performance decay18. Therefore, there is a need to develop materials that possess chemically bonded networks for high performance gas separation, while simultaneously being solution-processable.Among microporous organic materials, porous organic cages (POCs)22. However, CMPs have extremely rigid cross-linked structures, which inevitably lead to poor processability. Developing simple and scalable processing routes is of paramount importance for their practical application24. In addition, CMPs normally possess a broad pore size distribution (10–30 Å), which are not suitable for gas separation. To simultaneously meet the demands of structural rigidity and solution-processability, we have developed a solution-processable conjugated microporous thermoset (CMT) with intrinsic pores of ~0.4 nm and a specific surface area of ~840 m2 g−1, based on a simple thermosetting process. We show that the polymeric networks based on aryl-aryl bonds can be prepared into various shapes and morphologies, driven by the substrate onto which the CMT is deposited. More importantly, 2D CMT nanosheets are solution-processed into large-size molecular sieve membranes. With contributions from both intrinsic microporosity and interlayer spacing, CMT membranes achieve a H2 permeability as high as 28280 barrer and good selective H2 separation over CO2, O2, N2, CH4, C3H6, and C3H8. In addition, CMT membrane shows no performance decay up to 700 h at 150 °C, demonstrating its remarkable stability and persistence of microporosity.Conjugated microporous polymers (CMPs) are π-conjugated networks interconnected by irreversible aryl-aryl covalent bonds. Thus, the microporosity in CMPs is persistent in the presence of water vapor, acid gases and hydrocarbons, even at elevated pressure and temperatureMw 696) was synthesized by a condensation reaction between 3,6-dibromophenanthrene-9,10-diaminium chloride  and 3,6-dibromophenanthrene-9,10-dione. The tetrabrominated planar molecule is insoluble in common organic solvents due to strong intermolecular π–π stacking. To perform structural characterization, single crystals were prepared by sublimation of the prepared crude material in a tube furnace. The structure information of 3-TBTBP crystals is provided in Supplementary Fig. 2 atmosphere  spectra  disappear after polymerization, in agreement with the FTIR measurements . Figure The sublimation of 3-TBTBP produces precursor vapor (as demonstrated in TGA), which adsorbs readily on any exposed surfaces. This was exploited to prepare 2D CMT films and nanotubes by vapor phase deposition, as demonstrated in Fig. P/P0 below 0.01, which is a typical characteristic for microporous materials. The Brunauer–Emmett–Teller (BET) surface area and pore volume are evaluated to be 840 m2 g−1 and 0.39 cm3 g−1, respectively. The adsorption isotherm fitted with non-local density functional theory model reveals that the pore-size-distribution for CMT is mainly at 0.4–0.5 nm, which agrees well with the positron annihilation lifetime spectrum (PALS) analysis. As shown in Fig. 27. In this case, the endogenous liquid-state polymerization  is able to achieve a homogeneous porous structure, because precursor molecules are polymerized in the isotropic liquid state, without randomization effects of solvents and catalysts. More importantly, the endogenous polymerization method allows the potential scale-up production of CMT due to its low cost, high yield (close to 100%) and easy processing.The porosity of CMT has been cross-verified by Ar adsorption/desorption isotherms and positron annihilation lifetime spectroscopy (PALS). As shown in Fig. 32. In addition to their large pore size distribution29, a further disadvantage of the polymerization process is the need for a highly functional substrate32. In contrast, our CMT can be prepared as uniform ultrathin sheets with controllable thickness on any arbitrary substrates. As shown in Fig. 3 dispersion displaying an obvious Tyndall effect; it remains stable even after storing under ambient conditions for two weeks, with no sign of precipitation. The intrinsic micropores in CMT, rimmed by N groups, provide interfaces for solute/solvent interactions and improve wettability. The stability of the dispersion should be contrasted with the strong tendency of exfoliated graphene or molybdenum disulfide sheets to restack and precipitate34. Fig. Interfacial polymerization strategies have previously been applied to produce films composed of cross-linked networks2 (28280 barrer), while relatively much lower permeabilities are observed for CO2 (4480 barrer), O2 (2680 barrer), N2 (2500 barrer), CH4 (2590 barrer), C3H6 (2330 barrer), and C3H8 (2260 barrer). The ideal selectivities of H2 over CO2, O2, N2, CH4, C3H6, and C3H8 are ~6.3, ~10.6, ~11.3, ~10.9, ~12.1, and ~12.5, respectively. This demonstrates the molecular sieving effect of CMT membranes. In addition, only slight variations in permeability and selectivity are observed when the membrane thickness is increased from 500 nm to 13 μm , inorganic 2D materials , as well as other organic polymeric membranes  using Polymatic algorithm37. This generates a microporous polymeric network that reproduces the experimental mass density, accessible surface area (Ar sorption) and pore size distribution of CMT well (Supplementary Table 2 and CO2 as the probe gas for the CMT model. It is found that CMT has a larger accessible volume for H2 (19.58% of membrane volume) than CO2 (16.28% of membrane volume). This suggests that CMT is able to accommodate more H2 than CO2, leading to a higher concentration gradient for H2, which facilitates transmembrane transport38. More importantly, some channels allow H2 transport but block CO2, as shown in Fig. 2 transmembrane path calculated based on accessible void space is ~710 Å, which is much shorter than that of CO2 (~3715 Å), thus contributing largely to the ultrahigh H2 permeability. The CMT membrane was further studied in H2/CO2 mixed gas separation to demonstrate its long-term stability at elevated temperature. Similar to other polymeric membranes11, the H2 permeability increases by more than 40% to 40,680 barrer when the temperature increases from 30 °C to 150 °C . This can be explained by the broadened free volume size of CMT at higher temperature (as confirmed by PALS), which facilitates diffusive transport of H2. The optimal H2/CO2 selectivity is 6.05 at 150 °C. Most importantly, CMT polymers are thermally stable and can potentially be operated up to 500 °C. Both the permeability and selectivity remain constant for 700 h at 150 °C  or similar selectivity (H2/N2 and H2/CH4)17. In addition, the rigid structure of CMT due to the conjugated C‒C connected scaffolds prevents the highly homogeneous pores from collapsing into isolated voids and allows for inner pore connectivity at elevated temperatures. Therefore, CMT membranes are unique due to the combination of three properties: they offer ultrahigh H2 permeability and solution-processability of PIMs, and yet enjoy stability and enhanced performance at elevated temperatures, typical of cross-linked rigid networks.Overall, compared with cross-linked porous solids with poor processability, CMT is solution processable and possesses interlayer channels that allow fast redistribution of the permeate gas molecules, leading to much higher H2 gas permeability with good selectivity and excellent operation at high temperatures. Although we have demonstrated only the gas separation application of CMT here, we anticipate that CMT, as well as its analogs, will also impact other applications, extending to areas such as ionic nanofiltration and water desalination.In summary, we have successfully synthesized a conjugated microporous thermoset that exhibits the processability of plastics, together with the rigidity and porosity of cross-linked microporous solids. The microporosity is persistent and stable, even at elevated temperature and pressure. CMT has a narrow pore size distribution centered at ~0.4 nm, when combined with its solution processability, large area ultrafiltration membranes of controlled thickness could be fabricated via simple filtration. Due to the coexistence of intrinsic micropores and interlayer spacing between the stacked sheets, CMT membranes exhibit ultrahigh H2OH•HCl  were added into a mixed solvent of pyridine (6.0 ml) and EtOH (60.0 ml) to make a suspension, which was heated under reflux for 24 h. The formed precipitate 3,6-dibromophenanthrene-9,10-dione dioxime was collected and washed with EtOH to remove pyridinium hydrochloride. The gray precipitate without further purification was dispersed again in 100 ml EtOH, in which a solution of SnCl2  in concentrated HCl (15 ml) was added. The mixture was stirred at 70 °C for 4 h. After cooling down to room temperature, the precipitate 3,6-DBPDA was collected and washed with water and EtOH several times, followed by drying at 80 °C. To prepare 3-TBTBP, 3,6-DBPDA  and 3,6-DBPD  were suspended in 80 ml acetic acid in a round bottom flask in Ar atmosphere under stirring. The reaction mixture was refluxed at 130 °C for 6 h after adding triethylamine (1.0 ml). The precipitate was collected by filtration, and thoroughly washed sequentially via Soxhlet extraction with ethanol, tetrahydrofuran, and N,N-dimethylformamide. After drying at 120 °C overnight in vacuum oven, 3-TBTBP was obtained  as yellow powder. Elemental analysis (%): calculated for (C28H12Br4N2): C, 48.32; H, 1.74; N, 4.02; Br, 45.92; found: C, 47.76, H, 1.97, N, 3.93, Br 45.37. MALDI-TOF mass (negative mode) m/z = 697.3 ; No solution NMR spectroscopic data were collected owning to its insolubility. Its structure was confirmed with single crystal X-ray diffraction analysis (CCDC 1909146).3,6-dibromophenanthrene-9,10-diaminium chloride  was synthesized from 3,6-dibromophenanthrene-9,10-dione . 3,6-DBPD  and NH−1 under vacuum (~35 Pa). The crude powder was heated at the first heating zone at 350 °C to promote sublimation. The second heating zone was set at 320 °C for molecular transport. 3-TBTBP single crystals were deposited at the edge of the third zone which was heated at 260 °C. Single crystals suitable for crystallography analysis can be produced in about 4 h.3-TBTBP crude powders (~100 mg) were evenly dispersed on a quartz boat, which was inserted into the quartz tube furnace. The quartz tube is connected to a supply of Ar gas and a vacuum pump. The Ar flow was adjusted to 50 ml min−1. In the tube furnace, precursors with various substrates were put in a jar equipped with a lid to minimize the disturbance of gas flow, so that only a tiny amount of precursor 3-TBTBP was evaporated out and deposited at the low temperature area of the tube furnace. Normally, samples were heated to 540 °C at a heating rate of 15 °C min−1 and held at 540 °C for 120 min to produce CMT. The synthesis details for CMT monoliths, ultrathin 2D sheets or 1D nanotubes are provided in the Supplementary Information.The thermosetting polymerization experiments were conducted in a tube furnace with an Ar gas flow of 100 ml min−1 CMT polymer dispersion in IPA led to a membrane thickness of about 500 nm. All membranes were dried at room temperature under vacuum for 24 h before use.The CMT polymer membranes were prepared by pressure-assisted filtration in a dead-end filtration cell through polypropylene , AAO disks , or nylon  membrane filters under 1 bar. The thickness of the membranes was controlled by volume and concentration of the dispersion. In a typical experiment, 20 ml of 0.025 mg mlSupplementary InformationPeer Review File