Here, we investigate the function of the CBX8 CD in vitro and in vivo. We find that the CD is in fact a major driver of CBX8 chromatin association and determine that this is driven by both histone and previously unrecognized DNA binding activity. We characterize the structural basis of histone and DNA binding and determine how they integrate on multiple levels. Notably, we find that the chromatin environment is critical in determining the ultimate function of the CD in CBX8 association.Polycomb repressive complex 1 (PRC1) is critical for mediating gene repression during development and adult stem cell maintenance. Five CBX proteins, CBX2,4,6,7,8, form mutually exclusive PRC1 complexes and are thought to play a role in the association of PRC1 with chromatin. Specifically, the N-terminal chromodomain (CD) in the CBX proteins is thought to mediate specific targeting to methylated histones. For CBX8, however, the chromodomain has demonstrated weak affinity and specificity for methylated histones  Eukaryotic DNA is packaged into the cell nucleus in the form of chromatin. At its most basic level, chromatin is made up of repeats of nucleosome particles, which consist of ∼147 base-pairs (bp) of DNA wrapped around an octamer of histones H2A, H2B, H3 and H4. The N-terminus of each histone (and the C-terminus of H2A) protrude from the nucleosome core and are collectively known as the histone tails. These tails can be heavily post-translationally modified, catalyzed by a group of proteins known as ‘writers’. The modification of histone tails can alter chromatin structure directly and/or recruit or regulate chromatin associated proteins contributing to transcriptional changes  domain, in the Polycomb (dPc) subunit (in vitro (d>500 μM) in these studies and no measurable specificity for H3K27me3 peptides (The Polycomb Group (PcG) proteins, which are critical for lineage specification and adult stem cell maintenance , form tw subunit . There a subunit , that fo subunit . The mamin vitro ,16. Of tpeptides . Meanwhipeptides , making peptides . Specifipeptides  and is opeptides . Inhibitpeptides .in vitro histone tail binding is weak, and that nucleosome association in vitro is driven by DNA binding, we find that both DNA and H3K27me3 binding contribute to robust chromatin association in vivo, highlighting that the chromatin context is critical in determining ultimate reader domain function. These studies not only provide novel insight into reader domain and PRC1 function but will pave the way for the development of CBX8 inhibitors.Here, we demonstrate that CBX8 association with chromatin is largely driven by the CD. Notably, we find that this is mediated through a combination of H3K27me3 binding, and a previously unrecognized interaction with DNA. We investigate the structural basis of both of these interactions, defining the root of moderate specificity for H3K27me3, and how histone and DNA binding integrate on multiple levels. Notably, despite the fact that ® (Clontech) and the pGSTag vector . WT CBX8 gene was obtained from the Mammalian Gene Consortium (ID#4121509). The ΔCD (aa 77–389) was generated. QuikChange (Stravagene) was used to generate full length CBX8 W32A mutant. Additional full length CBX8 mutant constructs  were purchased from GenScript and amplified. All CBX8 genes were cloned into the tet-inducible conditional lentiviral vectors TetO-FUW (a gift from Rudolf Jaenisch Addgene plasmid # 20323 (® (Clontech). Infusion reactions were transformed in Stbl3 Competent cells (Invitrogen), DNA was isolated, and precipitated with 2 volumes ethanol and 0.1 volume sodium acetate for lentiviral transduction. CBX8 and control CRISPR guide RNAs were cloned into px459 v2.0 vector . GST-CB # 20323 ) containd #62988 ). EZH2 gd #62988 ). Guide Escherichia coli cells. Cells were grown in LB-medium or M9-minimal media supplemented with 15N-NH4Cl or 15N-NH4Cl and 13C-glucose. For unlabeled protein, cells were grown shaking at 215 rpm at 37°C until an OD ∼1.0 was reached and induced with 1 mM IPTG for 16–18 h overnight. For isotopically-enriched protein, cells were grown in LB-medium until an OD ∼1.0, spun down at 4000 rpm for 10 min, and resuspended in M9-medium (4 l LB cells per 1 l M9) supplemented with either 15N-NH4Cl or 15N-NH4Cl/13C-glucose. The cells were allowed to recover in M9 media for 1 h shaking at 18°C and induced with 1.0 mM IPTG for 16–18 h overnight. Cells were subsequently collected by centrifugation at 6000 rpm for 20 min, frozen in N2(l) and stored at –80°C.The recombinant CBX8 CD was expressed in BL21 (DE3) (New England Biolabs) 15N-CBX8 CD and 15N/13C-CBX8 CD were used in a final NMR buffer containing 100 mM NaCl and 40 mM phosphate buffer (pH 6.8). For EMSAs, the unlabeled CBX8 CD was used in a final buffer containing 25 mM phosphate buffer (pH 6.8), 25 mM NaCl, 1 mM EDTA, 1 mM DTT.For purification, cells were resuspended in a buffer containing 100 mM NaCl, 25 mM Tris (pH 7.5) supplemented with DNase I and lysozyme. Cells were then lysed using an Emulsiflex homogenizer (Avestin) or by sonication, and lysate cleared by centrifugation at 15 000 rpm for 1 h at 4°C. The soluble supernatant was incubated with glutathione agarose resin (ThermoFisher Scientific) rotating at 4°C for 1 h. The GST-tagged CBX8 CD was washed extensively with a high salt buffer containing 1 M NaCl and 25 mM Tris (pH 7.5) before elution with a buffer containing 150 mM NaCl, 25 mM Tris (pH 7.5) and 50 mM reduced glutathione. The GST-CBX8 CD was concentrated using a 3000 MWCO centrifugation filter unit to 2 ml and cleaved with TEV protease for 3 h at 25°C. The cleaved CBX8 CD was further purified using a combination of cation-exchange and size exclusion chromatography . For NMR studies, 2O to a final concentration of 20 mM and pH adjusted to ∼7.0 with NaOH. For peptide pulldown experiments, peptides were resuspended to a final concentration of 1 μg/μl in 10% dimethyl sulfoxide (DMSO). Ethylcysteine alkylation of H3K27C peptide was performed as previously described (The unmodified H3 (1–44) and H3K27C (23–34) peptides were synthesized by GenScript. The H3K9me3 (1–21), H3K27me3 (23–34), Biotinylated H3 (21–44) and H3K27me3 (21–44) peptides were obtained from AnaSpec. For NMR studies, peptides were resuspended in Hescribed . BrieflyThe single stranded DNA oligonucleotides (5′-GCGTTTAAGCG-3′ and 5′ CGCTTAAACGC-3′) were obtained from Integrated DNA technologies. For annealing, oligonucleotides were resuspended in 100 mM NaCl and 40 mM phosphate buffer (pH 6.8), heated to 90°C for 5 min and cooled overnight. The annealed DNA oligonucleotides were further purified by S75 size-exclusion chromatography in a buffer containing 100 mM NaCl and 40 mM phosphate buffer (pH 6.8) and concentrated to 3.2 mM in a 3000 MWCO centrifugal concentrator.Unmodified human histones H2A.1, H2B.1, H3.2 and H4 (T71C) were transformed into Rosetta2 (DE3) or BL21 (DE3) (New England Biolabs) and expressed in LB media. Cells were induced at OD ∼0.4 with 0.2 mM IPTG for histone H4 or 0.4 mM IPTG for histones H2A, H2B, and H3 for 3–4 h. The histones were extracted from inclusion bodies and purified by anion and cation exchange chromatography) .Unmodified NCPs octamers were made largely following the protocol in . In shorUnmodified nucleosome core particles (NCPs) were reconstituted using the unmodified octamers and the Widom 601 DNA (147 bp) through a desalting method . Unmodif15N/13C-CBX8 CD sample on a Bruker Avance II 500 MHz spectrometer with a 5mm triple resonance probe at 25°C. The triple resonance experiments were processed in NMRpipe CACB experiments were collected on a 0.275 mM  NMRpipe  and furt NMRpipe . Initial NMRpipe  and furt NMRpipe .15N-HSQC spectra on 0.05–0.10 mM 15N-CBX8 CD in the apo state and upon addition of increasing molar ratios of the respective ligands. Titrations experiments were performed at 25°C (with the exception of H3Kc27me3 that was performed at 16°C) on a Bruker Avance II 800 MHz NMR spectrometer equipped with a 5 mm triple resonance cryoprobe or a Unity Inova 600 MHz Oxford AS600 spectrometer equipped with a 5 mm triple resonance probe. The data was processed using NMRPipe (Kd), GraphPad PRISM was used for nonlinear least-squares analysis and the data fit to a single-site binding model accounting for ligand depletion using the equation:δ is the normalized chemical shift change and Δδmax is the normalized chemical shift change at saturation, calculated as:δ is the chemical shift in parts per million (ppm).Titrations experiments were performed by collecting  NMRPipe  and furt NMRPipe . To deteKd values were determined by averaging the individual Kd values for all resonances significantly perturbed in the titration experiments and reported as the average and standard deviation. A resonance was considered significantly perturbed if the Δδ value was greater than the average plus one standard deviation after trimming 10% of residues (five resonances) with the largest Δδ value. Individual resonances with Kd values greater than two standard deviations from the mean of the global Kd were removed from the analysis. If 50% or more of the individual residue Kd values were greater than two standard deviations from the global mean Kd, than the global Kd was reported as lower limit (e.g. >0.8 mM).Global 2. For generation of CBX8 and control CRISPR knockout lines, 200 000 T98G cells were plated in six-well 24 h prior to transfection. The respective vector (3.3 μg) was co-transfected with 13 μl of Fugene 6 (Promega). Media was changed 24 h post-transfection. Transfected cells underwent puromycin selection (2 μg/ml) for 3 days, 48 h post-transfection. EZH2 knockout lines were transduced with the MSCV_Cas9_puro vector  , 10% fetal bovine serum , 1% glutagro (Corning), 1% penicillin/streptomycin (Corning), 1% sodium pyruvate (Corning). GBM T98G cells were cultured in Eagle's Modified Essential Media, 10% FBS, 1% non-essential amino acids (Corning), 1% glutagro (Corning), 1% penicillin/streptomycin (Corning) and 1% sodium pyruvate (Corning). Hs68 cells were cultured in Dubecco's Modified Essential Media, 10% fetal bovine serum (JR Scientific), 1% penicillin/streptomycin (Corning) and 1% glutagro (Corning). All cells were grown at 37°C and 5% CO #65655)  followedHEK293T cells were co-transfected with either TetOFUW constructs or pLenti CMV rtTA3 Hygro (w785-1) (a gift from Eric Campeau Addgene plasmid # 26730) and viral packaging vectors (pMD2.G and psPAX2). Viral supernatant was harvested and concentrated by ultracentrifugation at 17 300 rpm for 2 h, 72 h following transfection. Virus was resuspended in PBS and TetOFUW and rtTA were co-added to T98G and Hs68 cells and spun at room temperature for 1 h at 200 × g. Forty-eight hours after infection, cells were selected with puromycin (2 μg/ml) and hygromycin (200 μg/ml) for a week to generate stable cell lines. The doxycycline was added every 48 h at 1 μg/ml to induce expression.2, 25 mM KCl, 0.05 mM EDTA, 10% glycerol, 0.1% NP40) with protease inhibitor cocktail for 10 min at 4°C. Nuclei were pelleted at 5500 × g for 5 min at 4°C. The nuclei pellet was resuspended in 500 μl of mRIPA 0 mM NaCl  and incubated on ice for ∼10 min. Chromatin was pelleted at 6000 × g for 5 min at 4°C. Supernatant was collected, and the pellet was resuspended in mRIPA with 100 mM NaCl. This was repeated up to 500 mM NaCl. 4× SDS loading dye with beta-mercaptoethanol (BME) and immunoblot was performed. Immunoblots were quantitated using ImageJ  using PRISM GraphPad. For the EZH2i SSEs, cells were treated for 48 h with 1 μM GSK343  or DMSO prior to being harvested.Protocol was performed as previously described . Brieflyg ImageJ  to calcuT98G cell lines were harvested and washed with PBS. Cell membranes were lysed with buffer A with protease inhibitor cocktail for 15 min on ice. Nuclei were pelleted at 1000 × g for 5 min at 4°C. The nuclei pellets were resuspended in IP buffer  and lysed on ice for 20 min. Chromatin was pelleted and supernatant was collected. Lysate was pre-cleared with 10 μl of Pierce™ Protein A/G Magnetic beads (ThermoFisher) overnight. Two hundred micrograms (200 μg) of lysate was incubated with 2 μg of V5 antibody  for 2 h at 4°C. Following, 10 μl of Pierce™ Protein A/G Magnetic beads equilibrated in IP buffer were added for 1 h. IPs were washed 3× with IP buffer and resuspended in 4× SDS loading dye with BME and immunoblot was performed.T98G mutant cell lines were washed with PBS and harvested with Buffer A. Cells were incubated in Buffer A for 15 min on ice. Nuclei were pelleted at 1000 × g for 5 min at 4°C. The nuclei were re-suspended in IP buffer and lysed for 20 min on ice. Chromatin was pelleted and supernatant was collected. Biotinylated H3 (21–44) and H3K27me3 (21–44) peptides  were pre-bound to streptavidin agarose resin (TriLink) for 1 h at 4°C. Following incubation, lysate  was added and incubated for 2 h at 4°C in peptide pulldown buffer . Pulldowns were washed three times for 5 min with peptide pulldown buffer. Samples were resuspended in 4× SDS loading dye with BME and immunoblot was performed.® 800CW or IRDye® 680 (LI-COR) secondary antibody. Blots were imaged on the Licor Odyessy®. Primary antibodies used: CBX8 , V5 , V5 , EZH2 , H3 , H3K27me3 , CBX4 , CBX7 , CBX2 .Lysates were boiled and loaded on a 4–12% SDS-page gel (Invitrogen). Gels were transferred to PDVF membranes (Millipore) and incubated in 5% bovine serum albumin (BSA) in PBS-t (PBS with 0.1% Tween-20) prior to primary antibody. Blots were incubated at 4°C overnight in primary antibody. Blots were washed with PBS-t and incubated for an hour at room temperature in goat anti-rabbit or mouse conjugated to IRDyeLinear 601 DNA or reconstituted biotinylated mononucleosomes (Epicypher®) were incubated with CBX8 chromodomain in EMSA buffer  at varying molar ratios for 15 min on ice. Samples (10 μl) containing 0.5 pmol nucleosome/0.25 pmol DNA, varying concentrations of chromodomain and 2× sucrose loading dye were loaded onto a prewarmed 6% (59:1) non-denaturing acrylamide gel and run in 0.4× TBE (pH 8.3) at 100 V on ice for 1 h. Gels were stained with ethidium bromide for 5 min and imaged on BioRad ChemiDoc™.® (Invitrogen) and RNA was isolated according to manufacturer's instructions. Isolated RNA was further purified using the RNeasy® kit (Qiagen). Library construction  and sequencing were carried out by Beijing Genomics Institute (BGI). The total RNA samples were enriched for mRNA by targeting polyadenylated (poly(A)) using oligo (dT) magnetic beads. Isolated mRNA was resuspended in fragmentation buffer and sonicated into short fragments of ∼200 bp. mRNA was reverse transcribed into a single strand of causing random hexamer-primers. The second strand of cDNA was synthesized using DNA polymerase and the double stranded cDNA was purified with magnetic beads. End reparation and 3′-end Adenine addition were performed subsequently. Thereafter, sequencing adaptors were ligated to the fragments and the fragments were enriched by PCR amplification. Finally, the library products were sequenced on the BGISeq500. The quality of the reads was evaluated with FastQC. Adapters were trimmed using Trimmomatic via the Galaxy web interface  and RNA was isolated according to manufacturer's instructions. Isolated RNA was further purified using the RNeasy® kit (Qiagen). Two micrograms (2 μg) of isolated RNA was converted to cDNA using Verso™ cDNA synthesis kit (ThermoFisher). cDNA was diluted ∼4-fold with water and qRT-PCR was performed with maxima SYBR green master mix (Thermo) and run on the BioRad CFX Connect™ thermo cycler. Six biological replicates were performed in technical triplicate. Data was normalized to the primer efficiency and B2M expression (Cells were plated at a density of 187 000 cells in 60 mm tissue culture plates and treated with 1 μg/ml of doxycycline for 72 h. Cells were harvested with Trizolpression .3) two times for 20 min at RT. Samples, including input, were treated with RNase A at 37°C for 30 min followed by proteinase K for 3 h at 55°C. Samples were un-crosslinked overnight at 65°C. Phenol chloroform followed by isopropanol precipitation were used to extract and isolate the DNA. DNA was resuspended in 20 μl TE for qPCR analysis. Antibodies used for IP were V5 , CBX8 , IgG . qPCR was performed on the isolated ChIP DNA. qPCR was performed using maxima SYBR green master mix (Thermo) and run on the BioRad CFX Connect™ thermo cycler. Three biological replicates were performed in technical triplicate. Enrichment was determined by percent input.Two million Hs68 cells were harvested from 10 cm plates and washed with PBS. The cells were re-suspended in CiA Fix buffer  and crosslinked with 1% formaldehyde for 10 min at room temperature. Crosslinking was quenched with 0.125 M glycine for 5 min at 4°C. Following, cells were washed once with PBS then resuspended in CiA NP Rinse buffer 1  for 10 min. Cells were pelleted at 1200 × g for 5 min at 4°C. The supernatant was removed and the cells were resuspended in CiA NP rinse buffer 2 . Cells were collected by centrifugation, 1200 × g for 5 min. Supernatant was removed and the cells were washed twice with shearing buffer . Following, cells were resuspended in shearing buffer and sonicated for 7 min with the probe (Branson 250). Lysate was centrifuged at 21 000 × g for 15 min to remove debris. Supernatant was collected and pre-cleared overnight with Pierce™ Protein A/G magnetic beads (ThermoFisher). For immunoprecipitation, cell lysate was divided in half and incubated with 1 μg of antibody and Pierce™ Protein A/G magnetic beads for 3 h. Ten percent (10%) input was saved for downstream analysis. The IPs were washed two times for 3 min at RT with IP buffer  followed by a DOC , 1 mM EDTA) wash and a 1× TE wash. Protein was eluted from beads  or Gene Expression Omnibus (GEO) as BED files ,44. Peakin vitro for H3K27me3 and H3K9me3 peptides with indeterminate specificity  with H3K27me3 peaks . Ad. Adin vis Figure , reveales Figure . In conts Figure  (47). Ths Figure . This suTo further assess chromatin association of CBX8, we investigated its interaction with bulk chromatin using a sequential salt extraction (SSE) assay, in which relative binding is determined by the salt concentration required to elute proteins from chromatin . We initin vivo and that robust chromatin association is largely dependent on the CD.To evaluate the contribution of the CBX8 CD to this association, we deleted the corresponding amino acids 1–76 and re-expressed either this CD deletion (ΔCD) or wild-type (WT) CBX8 in a GBM T98G CBX8 knockout (sgCBX8) cell line using a doxycycline-inducible system. Importantly, both wild-type and ΔCD expressed CBX8 are incorporated normally into the PRC1 complex Figure . Using tin vitro studies demonstrated weak affinity of the CD for both H3K9me3 and H3K27me3 peptides. Because binding was beyond the limit of detection for the technique used, specificity was not distinguishable (1H–15N-heteronuclear single quantum coherence (HSQC) spectrum of 15N-CD CACB triple-resonance experiments , H3K9me3 (residues 1–21), or H3K27me3 (residues 23–34). Addition of increasing concentrations of the H3K9me3 and H3K27me3 peptides resulted in significant chemical shift perturbations (CSPs) in the CD spectrum, suggesting an interaction with both peptides  were calculated for H3K9me3 and H3K27me3 by fitting normalized CSPs (Δδ) to a one-site binding model accounting for ligand depletion (see methods for details). This yielded a Kd = 0.7 mM for H3K27me3 and Kd = 2.0 mM for H3K9me3  was not bound by the CD as has been seen with a subset of other reader domains .,49.in vi domains , indepen domains . Togethe15N-CD resulted in significant CSPs for a subset of CD resonances . Addition of increasing concentrations of unmodified NCPs resulted in significant CSPs in a subset of residues indicating binding, accompanied by global intensity decreases, which are expected due to the size of the complex Figure . Analysi15N-CD pre-bound to the 11 bp DNA. This resulted in significant CSPs indicating that the DNA-bound CD can interact with the H3K27me3 peptide  or together (3×RA). The V5-tagged mutants were re-expressed in the T98G CBX8 knockout cell line earlier described and incorporated into the PRC1 complex as observed by immunoprecipitation . Two maociation  and inteociation . NotablyThere are two major implications from these results regarding the mechanism of reader domain mediated chromatin association. The first is the importance of DNA binding by canonical histone reader domains. Several studies have now demonstrated that histone reader domains, including chromodomains, bromodomains, PWWP domains, Tudor domains and PHD fingers, can interact with nucleic acids and that this binding is often much stronger than for histone peptides . Though in vitro, this binding interaction contributes significantly to the affinity of CBX8 for chromatin in vivo. This could be in part related to the increased effective concentration of repressive modifications in large regions of heterochromatin. For example HP1α is retained at H3K9me3-containing nucleosome arrays upon array compaction, due to both multivalent binding and probability of rebinding   . CBX pro. RNA-seq data can be accessed on the NCBI GEO, accession number GSE123689. All data is available upon reasonable request.NMR data can be accessed on the Biological Magnetic Resonance Bank under accession number 27616 (BMRB, Supplementary DataClick here for additional data file.


To assess the effect of cathodic pH on H2O2 yield, the MEC was tested with an anion exchange membrane (AEM) and a cation exchange membrane (CEM), respectively. The maximum current density reached 0.94–0.96 A/m2 in the MEC at applied voltage of 0.35–1.9 V, regardless of membranes. The highest H2O2 conversion efficiency was only 7.2 ± 0.09% for the CEM-MEC. This low conversion would be due to further H2O2 reduction to H2O on the cathode or H2O2 decomposition in bulk liquid. This low H2O2 conversion indicates that large-scale MECs are not ideal for production of concentrated H2O2 but could be useful for a sustainable in-situ oxidation process in wastewater treatment.A pilot-scale dual-chamber microbial electrolysis cell (MEC) equipped with a carbon gas-diffusion cathode was evaluated for H Because of the dual benefits, MECs have gained tremendous attention in the last decade ,2. Severoduction  to deplo2O2-producing MECs can give significant profits over other MECs due to high cost and demand of H2O2 [2O2 from organic waste or wastewater can be used as an in-situ oxidant in wastewater treatment, improving the sustainability of wastewater management. Similar to a conventional MEC system, H2O2-producing MECs comprise of two chambers separated by an ion exchange membrane. A solution containing dissolved organic matter is fed to the anode chamber where anode-respiring bacteria (ARB) such as Geobacter sp., Pseudomonas sp., Shewanella sp., etc. oxidize the organics and use the anode as the electron sink [2O2 at the cathode surface by the two-electron pathway shown in Eq. H of H2O2 . In addiron sink . The ele2O2-MECs at the lab scale, investigating H2O2 conversion efficiency, reactor design, electrode materials, and so on [2O2-MECs, but scale-up tests are essential to demonstrate performance and benefits of the MECs; however, no large-scale MECs for H2O2 generation have been conducted yet.All studies to date have examined Hnd so on . These l2O2 production. The pilot MEC was featured with anode modulation for provision of high surface area for biofilm formation and passive oxygen diffusion to a non-Pt carbon cathode. To evaluate the effect of catholyte pH on H2O2 yield, the pilot-scale MEC fed with acetate medium was run using an anion exchange membrane (AEM) and a cation exchange membrane (CEM), respectively, as electrode separator. Performance of the MEC was summarized, focusing on electrode potential, current density, pH, and H2O2 yield.This study is the first pilot-scale MEC (110 L) experiment for H22.12/m3 anode. The carbon fibers were pretreated with nitric acid (1 N), acetone (1 N), and ethanol (1 N), and finally washed with tap water before use [fore use . Perista2O2 producing MECs. Precious-metal-free carbon cathodes are preferred for H2O2 production [2O2  was used as the cathode  in this study. Passive diffusion of O2 from atmosphere through the CGC, means no energy requirement for oxygen supply to the cathode in the MEC. An anion exchange membrane (AEM)  having a surface area of 0.5 m2 was used for the MEC, which was later replaced with a cation exchange membrane (CEM)  for comparison.Due to advantages of high conductivity, low cost, long-term stability and low catalytic activity of H2.22·2H2O (20 mM) and Na2S·9H2O (77 mM) were added to acetate medium (1 mL per L). The pH in acetate medium was constant at 7.3 ± 0.1. The cathode chamber was filled with tap water. The AEM-MEC had been run in batch mode (∼4 months) until a peak current density of ∼0.9 A/m2 (∼0.45 A) was repeatedly observed in the MEC. Then, experimental data was collected in the batch pilot MEC. AEM was replaced with CEM later, and the CEM-MEC was operated for comparison experiments.The pilot MEC equipped with AEM was inoculated with effluent from lab-scale MECs (3.5 L of anolyte) operated with acetate medium, and was fed with 20 mM acetate medium . The med2O2 concentration, and anolyte COD concentration, approximately 10 mL of liquid samples were taken.To monitor voltage and electrode potentials, a saturated calomel electrode (SCE) , USA) was used as the reference electrode placed in the anode chamber; here, electrode potentials were reported against SCE reference electrode. The anode modules and the cathode were connected to a data logging system  with copper wires . The pow2.32O2 concentration was determined spectrophotometrically with vanadate, according to the literature [2O2 conversion efficiency was calculated using Eq. 2O2 generated (2 mol e−/mol H2O2) F is Faraday’s number , V is the catholyte volume (10 L), C is the concentration of H2O2 measured, and Q is the cumulative coulombs during operation (C).Hterature  and the COD measurement was carried out spectrophotometrically using the dichromate method . Chemica33.12 (0.47–0.48 A) during the experiments. This current density is much lower than ∼10 A/m2 in MECs fed with acetate medium, although the enrichment procedure and inoculum used in this pilot was the same to our lab scale MECs showing ∼10 A/m2 [Geobacter in the biofilm anode of the pilot MEC, we only used the effluent from lab-scale MECs, instead of recycle activated sludge or anaerobic digestion sludge. Despite long acclimation for ∼4 months, the maximum current density was less than 1 A/m2. This result means that biomass density would be very small in the biofilm anode, although five anode modules were installed to provide large surface area to ARB’s biofilm formation. This suggests the significance of inoculation or bio-augmentation in large-scale MECs.The peak current density was 0.94–0.96 A/m∼10 A/m2 . The siganode) in the AEM-MEC and the CEM-MEC was kept relatively stable at −0.3 and −0.5 V (vs SCE) during operation. In comparison, significant polarization was observed for the cathode potential (Ecathode), ranging from −2.0 to −2.4 V (applied voltage 1.6–2.0 V) and from −1.4 to −2.0 V (applied voltage 1.0– 1.6 V) in the AEM-MEC and the CEM-MEC, respectively (see Supporting information). The applied voltage is relatively higher than literature values from 0.2 to 1.3 V [cathode  were found, probably due to water evaporation in the cathode chamber, leading to poor contact with the cathode and consequently substantial cathodic polarization in the MEC. To overcome this operational challenge, fresh tap water was manually added to the cathode chamber. The anode potential . As shown in 2 dissolution. In addition, ion exchange membranes can be swallowed during operation of MECs, providing small headspace in the anode. Then, biogas in the anode might be built up, accelerating membrane expansion and possibly deteriorating ion transport due to a gap between membrane and anolyte (increase of ohmic resistance). Alternatively, partial circulation of alkaline catholyte to the anode can readily neutralize acidic anolyte, but it will decrease H2O2 recovery. Catholyte circulation can be an effective solution to neutralize acidic anolyte if produced H2O2 is directly utilized to oxidize reduced forms of contaminants  in the anode chamber.In both AEM-MEC and CEM-MEC, the anolyte pH was gradually decreased with time; the final pH was ∼6.5 after 15–20 d of batch operation (Supporting information). This acidic pH can significantly inhibit ARB’s metabolism and decrease current density , which ihe anode ,15,25; m3.32O2, but H2O2 production (2O2 MECs [2O2 concentration was only 9.0 ± 0.38 mg/L (p = 0.007) in 20d operation, and H2O2 conversion efficiency was extremely low at 0.35 ± 0.05% (p = 0.050). For the CEM-MEC in 15d operation, the cumulative H2O2 concentration was 98.48 ± 1.6 mg/L (p = 0.007) with H2O2 conversion efficiency of 7.2 ± 0.09% (p = 0.006).The MEC designed for passive air diffusion to the non-Pt carbon cathode successfully produced Hoduction  was very2O2 MECs ,13. In t2O2 conversion efficiency approached to 100% in a catholyte continuous flow electrolysis cell as hydraulic retention time in a cathode chamber was changed from 10 to 0.6 min ; the electrostatic repulsion between the peroxide species and the cathode can attenuate the H2O2 loss on the cathode [An abiotic test using a small electrolysis cell showed that H 0.6 min . This su cathode .Fig. 4Sm42O2 production in the pilot MEC equipped with the non-Pt cathode for passive air diffusion. The non-Pt carbon cathode successfully produced H2O2 without intensive air supply, but the maximum cumulative H2O2 concentration was only 98 mg/L in 20d of operation with 7.2% of conversion efficiency, indicating significant losses of H2O2 in either further reduction on the cathode or decomposition in bulk liquid. It is challenging to stop spontaneous, substantial H2O2 losses in the pilot MEC, and hence using H2O2-MECs as in-situ oxidation will be more practical than H2O2 synthesis.This study first assessed HNone.


A functional link between surface A2BR and adenosine deaminase (ADA) was found in parietal cells, but whether this connection is a general feature of gastric mucosa cells is unknown. Here we examine whether A2BR is expressed at the membrane of histamine-producing enterochromaffin-like (ECL) cells, the major endocrine cell type in the oxyntic mucosa, and if so, whether it has a vicinity relationship with ADA. We used a highly homogeneous population of rabbit ECL cells (size 7.5–10 µm) after purification by elutriation centrifugation. The surface expression of A2BR and ADA proteins was assessed by flow cytometry and confocal microscopy. Our findings demonstrate that A2BR and ADA are partially coexpressed at the gastric ECL cell surface and that A2BR is functional, with regard to binding of adenosine analogs and adenylate cyclase activation. The physiological relevance of A2BR and ADA association in regulating histamine release is yet to be explained.Adenosine is readily available to the glandular epithelium of the stomach. Formed continuously in intracellular and extracellular locations, it is notably produced from ATP released in enteric cotransmission. Adenosine analogs modulate chloride secretion in gastric glands and activate acid secretion in isolated parietal cells through A Adenosine (Ado) is a regulatory metabolite that modulates a broad spectrum of processes in many cells of the gastrointestinal tract ,2. Ado i1, A2A, A2B and A3), the A2B adenosine receptor (A2BR) still remains the most enigmatic subtype because of the relatively low potency of Ado at this receptor  5′-N-ethyl-carboxamido-adenosine (NECA). As shown in 3H]NECA to membranes revealed that equilibrium was rapidly reached. 2 = 0.88), giving a Kd of 103.8 ± 0.3 µM. NECA is a nonspecific agonist for Ado receptors. However, the fact that data fit to a unique binding site and that the obtained Kd value is in the range of those described for A2BR in many experimental models 5′-N-ethyl-carboxamido-adenosine (NECA) and [3H]cAMP were obtained from GE Healthcare Life Sciences , and ATP, NECA and CCh were obtained from Sigma-Aldrich Química S.L. .NECA (850 nCi/nmol) with 0.5 mg membrane protein/mL in buffer A at room temperature. Dissociation was induced by addding unlabeled NECA after an association period of 40 min. At the times specified, free and membrane-bound radioligands were quickly separated by vacuum filtration through Whatman GF/B filters previously soaked in 0.3% polyethylenimine, pH 10, for 2 h. The filters were sequentially bathed in 6 mL of buffer A and placed in 10 mL of a scintillation cocktail for radioactivity measurement. The experiment was repeated three times and the assays were performed in duplicate.Binding assays were carried out as described by Casadó and co-workers . Associa2Cl and 1 mM DTT, the reaction was initiated by adding 200 μM ATP and was stopped 10 min later. The samples were then centrifuged at 12,000 g for 2 min, and 50 μL supernatants were taken for cAMP determination. To each well of a 96-well microplate, 50 μL of [3H]cAMP (54 Ci/mmol) and 100 μL of 0.3 mg/mL bovine adrenal PKA were added in a total volume of 250 μL. A standard curve of cAMP was included in each plate. Plates were incubated at 4 °C for 150 min and then freed and PKA-bound [3H]cAMP were separated by vacuum filtration through Whatman GF/B filters using a Skatron Micro 96 Harvester. Filters were washed twice with 3 mL of buffer A and placed afterwards in 3 mL of a scintillation cocktail for radioactivity measurement. The experiment was repeated three times and the assays were performed in quadruplicate.Adenylate cyclase activity was determined in cell membranes by quantifying the cAMP generated from ATP using a competitive protein binding procedure , as desc2+ concentration was recorded in single cells using a multiple excitation microfluorescence system , as described in a previous work [v/v), cells were seeded (1 × 106–2 × 106 cells) on Matrigel-precoated coverslips and cultured for 1 h at 37 °C in 5% CO2 atmosphere in DMEM:F12  supplemented with 15 mM HEPES, pH 7.4, 2 mM l-glutamine, 10 nM hydrocortisone, 1 mg/mL insulin, 0.1 mg/mL gentamicin, 0.5 μg/mL transferrin, 5 μg/mL sodium selenite, 10 mM glucose, 5 μg/mL geneticin and 2 mg/mL BSA. Then, cells were washed and loaded with 5 μM fura-2/AM for 60 min at 37 °C. Coverslips were placed in a superperfusion chamber of a Nikon Diaphot microscope thermostatically controlled at 30 °C and the cells were excited alternatively at 340, 360 and 380 nm to monitor the fluorescence emitted at 510 nm. Control and calibration procedures as well as the algorithms used to calculate the cytosolic Ca2+ concentration are described previously [The cytosolic Caous work . After teviously .t-test using GraphPad Prism . Differences were considered significant at a value of p ≤ 0.05.All values are given as the mean ± SEM of 3–4 independent experiments. Differences between two groups were assessed by unpaired Student’s two-tailed


The unique two-dimensional structure of N-doped graphene nanosheets, can not only accommodate the volume changes during lithium intercalation/extraction processes and suppress the particles aggregation but also act as an electronically conductive matrix to improve the electrochemical performance of Fe3O4 anode, especially the rate capability. What's more, by etching Fe3O4@G to remove the iron-based oxide template, porous N-doped graphene composites (NGCs) were prepared and presented abundant pore structure with high specific surface area, delivering a specific capacitance of 172 F·g−1 at 0.5 A·g−1. In this way, Fe2O3 was both template and activator to adjust the pore size of graphene. And the effect of specific surface area and pore size tuned by the Fe2O3 activator were also revealed.Metal oxides and carbonaceous composites are both promising materials for electrochemical energy conversion and storage devices, such as secondary rechargeable batteries, fuel cells and electrochemical capacitors. In this study, Fe Derived from Fe2O3 and liquid polyacrylonitrile (LPAN), the Fe3O4@G can not only present enhanced conductivity but also accommodate the volume expansion of Fe3O4. In addition, after etching by hydrochloric acid (HCl) to remove the metal oxides template, porous N-doped graphene composites (NGCs) were obtained. Furthermore, the controllable preparation of porous graphene materials by template activated method was established. This approach takes some advantages. Firstly, Fe2O3 was not only raw material to transform to Fe3O4, but also a template and an activating agent to adjust the pore size of graphene in which we call it a template activated method. Secondly, as previously reported  and LPAN (2 g) were mixed and stirred in ethanol for 4 h. Then the mixture was preoxidated in air at 220°C for 3 h and carbonized in an argon atmosphere at a series of temperature  respectively for 4 h to prepared Fe3O4@G and N-doped graphene nanosheets (G). The as-prepared samples were named as Fe3O4@G-X, which X represented the carbonization temperature. As comparison, pure LPAN was also preoxidated in air at 220°C for 3 h and annealed in an argon atmosphere at 600°C. The product was named as G.Fe3O4@G-600, Fe3O4@G-700, Fe3O4@G-800 and Fe3O4@G-900) were treated with HCl solution (4 mol·L−1) for 48 h and repeatedly washed by de-ionized water. Finally, the products were dried in a vacuum oven at 90°C for 3 h. The final product was referred to as NGC600, NGC700, NGC800, or NGC900, corresponding to the carbonization temperature of 600, 700, 800, or 900°C. As comparison, pure LPAN was also cured in air at 220°C for 3 h and carbonized in an argon atmosphere at 700°C. The product was named as G700.The as-prepared samples  was used to calculate the mass fraction of graphene.The morphology and structure of the samples were characterized by field emission scanning electron microscope  and a Tecnai G2 transmission electron microscope . The crystalline structures were obtained by a D8 advance X-ray diffraction spectrometer  using Cu Kα radiation. X-ray photoelectron spectroscopy (XPS) was carried out on the ESCAlab220iXL electron spectrometer from VG scientific using 300-W Al Kα radiation. Raman spectra were performed at the room temperature . The specific surface area and pore size distributions of the samples were measured by BELSORP-MAX with N2O3, G, Fe3O4@Gs), 10 wt% carbon black (CB) and 10 wt% polyvinylidene fluoride (PVDF) dispersed in N-methyl pyrrolidinone (NMP), were pasted on copper foil. Then the coated Cu foil was dried at 100°C for 12 h and then cut into pieces with a diameter (φ) of 14 mm. The loading amount of active material was ~0.8 mg·cm−2. The electrochemical performances of the samples were tested using 2,032 coin-type cells, Celgard 2400 separator, 1 mol·L−1 LiPF6/EC:EMC:DMC (1:1:1 by volume) electrolyte, and Li-foil as the counter electrode in an Ar-filled glove box  with oxygen and moisture contents of <0.1 ppm. Galvanostatic charge-discharge measurements were performed on a LAND-CT2001A battery test system (China) in a voltage range of 0.01–3.0 V (vs. Li+/Li) at various current densities. Cyclic voltammetry (CV) was evaluated at 0.1 mV·s−1 on a Solartron analytic 1470E cell test system in the range of 0.01–3.0 V. Electrochemical impedance spectroscopy (EIS) was conducted on a Solartron Impedance analyzer 1260A at an AC voltage of 10 mV amplitude from 100 kHz to 0.01 Hz.Mixtures, which consisted of 80 wt% active materials , chronopotentiometry (CP), and electrochemical impedance spectroscopy (EIS) were performed by electrochemical workstation (CHI 670 C) at room temperature. Cycle performance was tested by the LAND CT2001A instrument. The EIS was conducted using a sinusoidal signal of 5 mV over the frequency range from 100 kHz to 0.01 Hz.Electrochemical performances were estimated by symmetric electrode-type coin cells. The fabrication of working electrodes was described as follow: the mixture of active materials, carbon black additive and PTFE emulsion (with a mass ratio of 85:10:5) were added in ethanol solvent. After a full stirring, the as-prepared slurry was coated on the nickel foam (φ14 mm) and dried at 80°C for 6 h in a vacuum oven. The loading amount of active material was 0.5–0.8 mg·cmThe corresponding specific capacitance is calculated by the following equation:  further reacted with graphene, etching the graphene to increase the specific area as well as pore size. After treated with HCl solution, the FeOx was removed, and NGC could be obtained.As shown in Figure 2O3 sample, the TG curve was almost horizontal, indicating that thermal decomposition reactions didn't take place inside the Fe2O3 under 1,000°C except for the release of a little water. In the TG curve of pure LPAN, thermal decomposition reaction inside LPAN took place all the time because of the inherent nature of the organics. Before estimating by TG, Fe2O3/LPAN precursor was heated at 220°C in air for 3 h, to make the crosslinking reaction occurred inside the LPAN. There was a big mass break from 500 to 700°C in the mixtures. And the weight of samples didn't change until the temperature reaches 750°C, which indicated the reduction reactions inside the Fe2O3/LPAN precursors completed. As the Fe3O4@G was the intermediate of the reduction reactions, so the following carbothermal temperature will range from 500 to 700°C. LPAN is the oligomer of chained acrylonitrile and converts to graphene after carbonization . But if the temperature is 700°C, the other reduced phases including FeO (41.8°) (Equation 3) and Fe (44.8°) (Equation 4) were produced and the content of Fe3O4 will decrease accordingly. The equations of carbothermal reaction and activated reaction were given as followed  and Fe2O3 (162.7 mAh·g−1), the Fe3O4@G-600 delivered a capacity of 453.6 mAh·g−1 after 50 cycles. Fe3O4@G-600 delivered a high specific capacity of 1,023 mAh·g−1 at the first discharge process with a reversible specific capacity of 726.8 mAh·g−1, where the initial coulombic efficiency was around 71%. The relatively low initial coulombic efficiency resulted from the irreversible capacity loss for the formation of SEI and decomposition of the electrolyte    . It is obvious that the semicircle for Fe3O4@G-600 was smallest among Fe3O4@Gs, suggesting Fe3O4@G-600 presented the smallest charge transfer resistance (Rct)  by etching the FeOx.According to the result of XRD in Figure −1 corresponded to the D and G bands of graphene, respectively. There was a broad peak of 2D band of carbon at around 2,600–3,100 cm−1  equation and the pore size distribution was estimated from the desorption branch of N2 isotherms by the Barrett–Joyner–Halenda (BJH) method  to 505.41 m2·g−1 (NGC700), and then decreased to 372.02 m2·g−1 (NGC800) and 220.69 m2·g−1 (NGC900). NGC600 presented a small specific area, for Fe2O3 mainly transform to Fe3O4 at 600°C , thinning of the channel and resulting in the increase of surface area. Then the specific area of NGC800 and NGC900 decreased because the quantities of tunnels diminished resulting from the further activation of Fe2O3. Schematic diagram of the activation of porous carbon by FeOx was presented in Figure From Figures C Figure . The reaThe morphology and structure of NGC700 were estimated by SEM and TEM. As shown in Figure 2C-C (284.6 eV), N-sp2C (285.4 eV), and N-sp3C (288.4 eV). Most of the carbon atoms form π-π conjugated system, for the main peak at 284.6 eV corresponding to sp2 carbon. N 1s , chronopotentiometry (CP) and electrochemical impedance spectroscopy (EIS) were measured in a two-electrode system by coin-type cells. In Figures −1 for other NGCs were also estimated, and the calculation correlation of specific capacitances with scan rates for the composites were shown in Figure −1 at the current density of 0.5, 1, 2, 5, and 10 A·g−1, respectively. Generally, the capacitance of materials showed correlation with not only specific surface area but also the pore distribution, conductivity, wettability, etc. The improvement of specific capacitance from NGC600 to NGC700 could be ascribed to its larger specific surface area and conductivity as discussed above. The decrease in capacitance from NGC700 to NGC800 could blame for the same reason. It was worth noting that NGC900 presented higher specific capacitance but lower specific surface area than NGC800. Because high temperature was favor for composites forming high conductivity and more defects. It was consistent with the test results of Raman and BET. After 4,000 cycles, the specific capacitance of NGC700 kept at a stable level and delivered a capacity of 150 F·g−1 at the current density of 0.5 A·g−1, indicating excellent cycle performance (Figure Chronopotentiometry at the current density from 0.5 to 10 A·ge Figure .Electrochemical impedance spectroscopy further verified our speculation above. As shown in the Nyquist plot, there was a partial semicircle at high frequency and a vertical straight at the low frequency in each plot. In the high-frequency area, the partial semicircle corresponding to electrode resistances were 2.202, 1.579, 0.730, and 0.350 Ω (Table 3O4@G from Fe2O3/LPAN precursors showed optimal cycle stability and rate capability among these synthetic conditions, which delivered a capacity of 355.6 mAh·g−1 at the high current density of 5 A·g−1 after 50 cycles. What's more, NGCs were prepared by removing the FeOx template form Fe3O4@G, which presented high specific surface area and abundant pore structure. Ascribed to the activated template FeOx, it was reduced with graphene during carbonization, resulting in not only an increase in mesoporous and micropores but an increase in pore size. As a result, NGC700 performed higher specific capacitance and electrochemical stability, delivering a specific capacitance of 172 F·g−1 at 0.5 A·g−1 current density after 4,000 cycles for supercapacitor. Prepared Fe3O4@G by one step carbothermal reduction method and NGC by activated template method in one approach, should be very worthy for consideration. Significantly, the raw materials Fe2O3 is abundant in the earth, so the low cost will guarantee the prospect of the products promising for the next generation LIB and other application in energy storage.In summary, the prepared FeHM and JL contributed conception and design of the study. HM, XY and JH did the experiments, organized the database and performed the statistical analysis. HM and XY wrote the first draft of manuscript. HM and QZ revised the manuscript. All authors contributed to manuscript revision, read and approved the submitted version.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.


Abiotic flux of H2 can be very low in these environments, and there is a gap in our knowledge about the origin of CH4 in these habitats. In the hyperthermophile Methanocaldococcus jannaschii, growth yields increased as H2 flux, growth rates, and CH4 production rates decreased. The same trend was observed increasingly with interspecies H2 transfer between M. jannaschii and the hyperthermophilic H2 producer Thermococcus paralvinellae. With decreasing H2 availability, isotopic fractionation of carbon during methanogenesis increased, resulting in isotopically more negative CH4 with a concomitant decrease in H2-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression and increase in F420-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression. The significance of our research is in understanding the nature of hyperthermophilic interspecies H2 transfer and identifying biogeochemical and molecular markers for assessing the physiological state of methanogens and possible source of CH4 in natural environments.Hyperthermophilic methanogens and H 2 limited in hot subseafloor environments, and their survival may be due in part to physiological adaptations to low H2 conditions and interspecies H2 transfer. The hyperthermophilic methanogen Methanocaldococcus jannaschii was grown in monoculture at high (80 to 83 μM) and low (15 to 27 μM) aqueous H2 concentrations and in coculture with the hyperthermophilic H2 producer Thermococcus paralvinellae. The purpose was to measure changes in growth and CH4 production kinetics, CH4 fractionation, and gene expression in M. jannaschii with changes in H2 flux. Growth and cell-specific CH4 production rates of M. jannaschii decreased with decreasing H2 availability and decreased further in coculture. However, cell yield (cells produced per mole of CH4 produced) increased 6-fold when M. jannaschii was grown in coculture rather than monoculture. Relative to high H2 concentrations, isotopic fractionation of CO2 to CH4 (εCO2-CH4) was 16‰ larger for cultures grown at low H2 concentrations and 45‰ and 56‰ larger for M. jannaschii growth in coculture on maltose and formate, respectively. Gene expression analyses showed H2-dependent methylene-tetrahydromethanopterin (H4MPT) dehydrogenase expression decreased and coenzyme F420-dependent methylene-H4MPT dehydrogenase expression increased with decreasing H2 availability and in coculture growth. In coculture, gene expression decreased for membrane-bound ATP synthase and hydrogenase. The results suggest that H2 availability significantly affects the CH4 and biomass production and CH4 fractionation by hyperthermophilic methanogens in their native habitats.Hyperthermophilic methanogens are often HIMPORTANCE Hyperthermophilic methanogens and H2-producing heterotrophs are collocated in high-temperature subseafloor environments, such as petroleum reservoirs, mid-ocean ridge flanks, and hydrothermal vents. Abiotic flux of H2 can be very low in these environments, and there is a gap in our knowledge about the origin of CH4 in these habitats. In the hyperthermophile Methanocaldococcus jannaschii, growth yields increased as H2 flux, growth rates, and CH4 production rates decreased. The same trend was observed increasingly with interspecies H2 transfer between M. jannaschii and the hyperthermophilic H2 producer Thermococcus paralvinellae. With decreasing H2 availability, isotopic fractionation of carbon during methanogenesis increased, resulting in isotopically more negative CH4 with a concomitant decrease in H2-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression and increase in F420-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression. The significance of our research is in understanding the nature of hyperthermophilic interspecies H2 transfer and identifying biogeochemical and molecular markers for assessing the physiological state of methanogens and possible source of CH4 in natural environments. Both shThermococcus and thermophilic methanogens are collocated in hydrothermal vents  increases when cultures are shifted from H2-replete to H2-limited growth conditions and if YCH4 remains high or increases further during interspecies H2 transfer. This study also examined if interspecies H2 transfer stimulates the growth rate or cell yield of T. paralvinellae or ameliorates its H2 inhibition relative to its growth in monoculture. Furthermore, isotopic carbon fractionation was examined to determine if CH4 is isotopically lighter when H2 flux is reduced, as previously observed in moderately thermophilic methanogens  when grown on 80 to 83 μM H2 to 0.50 ± 0.09 h−1 when grown on 15 to 27 μM H2 . Attempts to grow M. jannaschii in coculture with T. paralvinellae in the chemostat when either maltose or formate was the energy source, with and without stirring and gas sparging of the medium with CO2 and N2, were unsuccessful. This was likely due to the open reactor that permits gas to flow out of the reactor without any gas pressure increase. Coculture growth was readily established in sealed bottles that contained 1 atm of gas pressure at room temperature. At 82°C, the gas pressure in the bottle was 1.2 atm, which slowed H2 efflux from the growth medium to the headspace. Therefore, the cocultures were grown in sealed bottles with the same volume of medium and headspace as the chemostat. The growth rates of M. jannaschii decreased further when it was grown in coculture with T. paralvinellae to 0.12 ± 0.01 h−1 and 0.22 ± 0.03 h−1 when T. paralvinellae was grown on maltose and formate, respectively .A summary of the growth conditions is provided in 27 μM H2 . Cell coectively . Relativ4 production rate decreased 3.6-fold when M. jannaschii was grown on 15 to 27 μM H2 (139 ± 8 fmol cell−1 h−1) relative to growth on 80 to 83 μM H2 (496 ± 21 fmol cell−1 h−1) (−1 h−1) and on formate (24.8 ± 4.3 fmol cell−1 h−1) (YCH4) for M. jannaschii grown in coculture were significantly higher when grown on maltose (9.1 ± 1.9 [×1012] cells per mol CH4) and formate (13.5 ± 2.0 [×1012] cells per mol CH4) than growth yields in monoculture on 15 to 27 μM H2 (2.1 ± 0.2 [×1012] cells per mol CH4) and 80 to 83 μM H2 (1.5 ± 0.1 [×1012] cells per mol CH4) .The cell-specific CHl−1 h−1) . The ratl−1 h−1) . Howevermol CH4) . SummariT. paralvinellae when it was grown with or without M. jannaschii or with a change in carbon source  relative to growth in coculture (1.05 ± 0.15 pmol cell−1 h−1)  when grown in monoculture (0.60 ± 0.18 pmol cell−1 h−1) but not when grown in coculture  than for monoculture growth on maltose (0.9 ± 0.1 fmol cell−1 h−1) (Table S3). A summary of the growth and metabolite production kinetics data for T. paralvinellae is available in the supplemental material . There was no growth of M. jannaschii when it was incubated in monoculture in medium supplemented with only 0.01% yeast extract or 0.1% sodium formate and 0.01% yeast extract with N2:CO2 in the headspace. These additions also did not stimulate the growth of M. jannaschii in monoculture when an H2:CO2 headspace was provided.There was no change in the specific growth rate or maximum cell concentration of ectively . Furtherl−1 h−1) . Howeveroculture . The cel13CCO2) values were −24.4 to −21.6‰ in the coculture bottles and −33.3 to −28.2‰ in the chemostat  increased from 28.5 to 29.3‰ during high H2 growth to 43.7 to 45.9‰ during low H2 growth   and t-distributed stochastic neighbor embedding (t-SNE)  results. Pairwise comparisons of M. jannaschii grown in monoculture on high and low H2 showed up to 12 genes to be differentially expressed  with 1 gene downregulated and 11 genes upregulated during growth on low H2 relative to growth on high H2 (Table S4). Under low-H2 conditions, F420-dependent methylene-tetrahydromethanopterin (H4MPT) dehydrogenase  gene expression increased 3.5-fold    for M. jannaschii grown in monoculture on high and low H2 in the chemostat. The genes that code for a GTP binding protein (MJ_RS01180), bacteriohemerythrin (MJ_RS03980), radical SAM protein (MJ_RS04390), a signal recognition particle (MJ_RS05550), a transcriptional regulator (MJ_RS06225), and four hypothetical proteins were upregulated on low H2, while a gene that codes for a histone (MJ_RS04990) was upregulated on high H2 (Table S4).RNA-Seq mapped 1,866 transcripts to the 3.5-fold . There wT. paralvinellae genome and 1.5% mapped to the M. jannaschii genome. For cocultures grown on formate, 67% of the reads mapped unambiguously to the T. paralvinellae genome and 29% mapped to the M. jannaschii genome. These proportions generally matched the proportions of T. paralvinellae and M. jannaschii cells in each coculture type based on cell concentration estimates . Merged pairwise comparisons of M. jannaschii gene expression for cultures grown in monoculture and M. jannaschii grown in coculture with T. paralvinellae showed up to 338 genes to be differentially expressed  with 146 upregulated genes and 192 downregulated genes when grown in coculture relative to growth in monoculture on high and low H2 (Table S5). However, we cannot rule out the possibility that some of these gene expression changes are caused by the switch from the chemostat to bottles.For cocultures grown on maltose, 97% of the reads mapped unambiguously to the 420-dependent methylene-H4MPT dehydrogenase  gene expression was upregulated 4.3-fold in coculture relative to that of M. jannaschii grown under monoculture conditions  were both downregulated 2.1-fold in M. jannaschii grown in coculture relative to that of M. jannaschii grown in monoculture (M. jannaschii genes that code for a V-type ATP synthase (MJ_RS01130 to MJ_RS01165 and MJ_RS03255) were downregulated when cultures were grown in coculture relative to expression in M. jannaschii grown in monoculture  increased when the cultures were H2 limited  slow growth but efficient metabolism and high yields when resources are scarce, and (ii) fast growth but inefficient metabolism and low yields upon rich resources. The rate-yield trade-off is suggested to be integral to evolution and the coexistence of species  decreased while expression of cofactor F420-dependent methylene-H4MPT (mtd) increased when growth was H2 limited relative to that of H2-replete growth  yeast extract , 1 μM Na2WO4·2H2O, 0.26 μM (NH4)2Fe(SO4)2·6H2O, and 0.25 μM (NH4)2Ni(SO4)2·6H2O. The primary carbon and energy source added for T. paralvinellae was either 0.5% (wt vol−1) maltose (Sigma) or 0.1% (wt vol−1) sodium formate (Fluka). All media were pH balanced to 6.00 ± 0.05 and reduced with 0.025% (wt vol−1) each of cysteine-HCl and Na2S·9H2O before inoculation. To test if M. jannaschii can use formate or yeast extract for growth in the absence of H2, or if they stimulate growth in the presence of H2, M. jannaschii was incubated in monoculture on the base medium amended with 0.1% formate and 0.01% yeast extract or 0.01% yeast extract only as described above, each in serum bottles with 1 additional atm (100 kPa) of either H2:CO2 (80%:20%) or N2:CO2 (80%:20%) added to the headspace at room temperature prior to incubation.DSM 2661  and TherSM 27261  were purdium 282 . For theM. jannaschii was grown in monoculture at 82°C and under high and low H2 concentrations in a chemostat to measure its growth and CH4 production kinetics and to generate biomass for gene expression analysis. A 2-liter bioreactor  with gas flow, temperature (±0.1°C), and pH  controls was used with 1.5 liters of growth medium. The medium was maintained at pH 6.0 ± 0.1 by the automatic addition of 0.25 mM HCl. For high-H2 conditions, the bioreactor was gassed with a mixture of CO2 (20.5 ml min−1) and H2 (132 ml min−1). For low-H2 conditions, the bioreactor was gassed with a mixture of CO2 (20.5 ml min−1), N2 (130 ml of gas min−1), and H2 (2.5 ml min−1). Pure gases were blended using a mass flow controller (Matheson Tri-Gas) and added to the bioreactor through a single submerged fritted bubbler . The reactor is an open system and remains at ambient gas pressure. It was stirred at 150 to 180 rpm using a four-blade open impeller (6-cm diameter) with a glass shaft and Teflon blades. Aqueous H2 and CH4 concentrations were measured before and after inoculation by drawing 25 ml of medium from the bottom of the bioreactor directly into anoxic 60-ml serum bottles and measuring the headspace gas. H2 was measured using a gas chromatograph fitted with a thermal conductivity detector (Shimadzu GC-8A) and a 60/80 Carboxen 1000 column . CH4 was measured using a gas chromatograph fitted with a flame ionization detector (Shimadzu GC-17A) and a 5A 80/100 molecular sieve column . The aqueous H2 concentrations in the bioreactor prior to inoculation were 80 to 83 μM for the high-H2 condition and 15 to 27 μM for the low-H2 condition  was determined by plotting cell concentration against time and fitting a logarithmic curve to the growth data. M. jannaschii was grown in batch reactor mode until the culture reached mid-logarithmic growth phase, and then the bioreactor was switched to chemostat mode by pumping sterile growth medium into the bioreactor from a sealed 12-liter reservoir that was degassed with N2 through a submerged glass tube and heated to 75°C. Simultaneously and at the same rate, spent growth medium was pumped out of the bioreactor using a dual-channel peristaltic pump. The H2 and CH4 concentrations in the headspace of the bioreactor were measured using gas chromatography as described above. At high and low H2 concentrations, cells were grown in the reactor at low enough cell concentrations such that there was excess H2 in the headspace and the cells were not H2 limited .The media were inoculated with 50 to 100 ml of a logarithmic-growth-phase culture of M. jannaschii was stable in the chemostat after three volume replacements of the medium within the reactor  and was monitored for an additional ∼0.5 volume replacements to obtain kinetic data. The CH4 production rate per cell (q) was calculated from the sum of the CH4 concentration in the headspace times the gas flow rate and the CH4 concentration in the medium times the medium dilution rate , which was normalized by the total cell concentration in the reactor. The cell yield per mole of CH4 produced (YCH4) was calculated by dividing the cell production rate (dilution rate times cell concentration) by the CH4 production rate. The complete contents of the bioreactor then were drained into ice-cooled centrifuge bottles, spun in a centrifuge at 10,000 × g and 4°C for 60 min, resuspended in 1 ml of TRIzol (Invitrogen), and frozen at −80°C until processed. Chemostats were run in triplicate for both conditions.Growth of M. jannaschii and T. paralvinellae were grown in coculture at 82°C in 2-liter gas-tight flasks  containing 1.5 liters of medium with ambient pressure of N2:CO2 (80%:20%) in the headspace at room temperature without agitation and either maltose or formate as the energy source  of M. jannaschii and T. paralvinellae were determined by plotting cell concentration against time and fitting a logarithmic curve to the growth data. The total amounts of CH4 and H2 in the bottles were determined by gas chromatography. The concentrations of formate, acetate, butyrate, isovalerate, and 2-methylbutyrate were measured from aliquots of syringe-filtered (0.2-μm pore size) spent medium from each coculture and T. paralvinellae monoculture incubation at various time points  using ultra-high-pressure liquid chromatography (UHPLC) as previously described (YCH4) were determined from the linear slope of the number of methanogen cells per bottle plotted against the amount of CH4 per bottle  as previously described  were determined from the linear slope of T. paralvinellae cell concentration plotted against acetate, formate, or H2 concentration. When the cocultures reached late logarithmic growth phase, the cells were harvested for transcriptome analysis as described above . Cocultures grown on maltose were grown in triplicate, while cocultures grown on formate were grown in quadruplicate.The growth rates (escribed . Methanor bottle . The ratescribed . SimilaroT) and end (fT) of each chemostat run, 20 ml of chemostat headspace was transferred in triplicate into evacuated vials (Labco Exetainer). M. jannaschii also was grown in monoculture in 245-ml serum bottles containing 100 ml of medium and 1 additional atm (100 kPa) of H2:CO2 (80%:20%) added to the headspace at room temperature prior to incubation. M. jannaschii was also grown in coculture with T. paralvinellae in 245-ml serum bottles containing 100 ml of either 0.5% maltose medium or 0.1% sodium formate medium as described above. The isotopic signatures of CH4 were determined using a gas chromatography-combustion-isotope ratio mass spectrometer  equipped with a GS-CarbonPlot column . Isotopic signatures were determined using external CH4 standards of known isotopic signatures (−57.40 ± 0.06‰) that were obtained from Arndt Schimmelmann (Indiana University). The error of the analysis was determined from external standards, and the standard deviation of multiple injections was 0.3‰. At oT and fT of the chemostat runs and the serum bottles, triplicate samples of dissolved inorganic carbon (DIC) were drawn from the growth medium. Each DIC sample (either 0.8 or 1.0 ml) was syringe filtered (0.2 μm pore size) and injected into prepared vials (Labco Exetainer) that had been flushed with He and contained 100 μl of phosphoric acid. Samples were analyzed by GasBench-IRMS. DIC standards were prepared in concentrations from 0.5 to 7.0 mM using KHCO3 and Li2CO3 of known isotopic composition . The error of analysis was determined from external standards, and the standard deviation of multiple injections was 0.3‰. The δ13CCO2 value was calculated from the δ13CDIC value using the relationship of Mook et al.  relative to the VPDB (Vienna Pee Dee Belemnite) standard:Rsample is the 13C/12C ratio of the sample and Rstandard is 0.0112372. The ε notation is used to express isotope fractionation factors in per mille (‰):Carbon isotopic compositions are presented as δRCO2 is the 13C/12C ratio of the initial CO2 and RCH4 is the 13C/12C ratio of the CH4 produced. The propagated error of the fractionation factors was 0.4‰, except in the case of the M. jannaschii monoculture.The fractionation factor, α, is defined as the ratio between the isotopic ratio in the substrate and product:M. jannaschii monoculture serum bottles was extensively drawn down, substantially altering the 13C signature of the remaining reactant. The fractionation factor was therefore calculated by setting the initial CO2 isotopic signature equal to that in serum bottles without cells (−26.1 ± 0.8‰) and reacting it stepwise under different fractionation factors. To obtain final isotopic compositions that match the remaining CO2 (+18.9 and +15.5‰) and the final accumulated product, CH4 (−32.9‰ and −34.2‰), fractionation factors of 22.1 ± 1.3‰ and 23.0 ± 1.3‰ were required in the two different experiments.The inorganic carbon in the M. jannaschii and T. paralvinellae genomes using BBSplit from the BBMap package (https://sourceforge.net/projects/bbmap/). BBSplit is an aligner tool that bins sequencing reads by mapping them to multiple references simultaneously and separates the reads that map to multiple references to a special “ambiguous” file for each of them. For further analyses, we removed all ambiguously mapped reads to both genomes and worked with only the reads that unambiguously map to the M. jannaschii genome. Two to 5% of the reads were lost in this step.Total RNA was extracted from 13 cell pellets from each growth condition  using a M. jannaschii were aligned to the M. jannaschii genome and sorted using the STAR aligner, version 2.5.1b  in R (version 3.3 [http://www.r-project.org]) and on a Galaxy platform using DEBrowser  using the pheatmap package. The heatmap color scale represents the z-score, which is the number of standard deviations the mean score of the treatment is from the mean score of the entire population.Genes that were differentially expressed were identified using DESeq2 in the Bioconductor software framework (EBrowser –55. RelaGSE112986.The count files and raw sequences are available in the NCBI Gene Expression Omnibus (GEO) database under accession no.


Characterization by P-XRD, FE-SEM, and TEM confirm Fe3O4 has a spherical crystalline structure with an average diameter of 15 nm, which after functionalization with BTCA, increases to 20 nm. Functionalization also enhances the surface area and surface charge of the material, confirmed by BET and zeta potential analyses, respectively. The dye adsorption capacity of Fe3O4@BTCA has been investigated for three common dyes; Congo red (C.R), Methylene blue (M.B), and Crystal violet (C.V). The adsorption studies show that the material rapidly and selectively adsorbs C.R dye with very high adsorption capacity (630 mg/g), which is attributed to strong H-bonding ability of BTCA with C.R dye as indicated by adsorption mechanism study. The material also shows excellent recyclability without any considerable loss of adsorption capacity. Adsorption isotherm and kinetic studies suggest that the adsorption occurs by the Langmuir adsorption model following pseudo-second-order adsorption kinetics.In this study, the new material Fe There are many techniques for wastewater treatment, for instance, coagulation, electrocoagulation, chlorination, ozonation, flotation, chemical oxidation, filtration, membrane separation, adsorption, and ultrafiltration17. Among various reported techniques, the adsorption is the most convenient and well-established technique because of high efficiency, simplicity, and minimum energy requirement24.Organic dyes are one of the significant contributors to water pollution caused by the discharge of effluent from various industries such as textile, plastic, printing, photographic, paper-pulp, paint, and leather28. Among various metal oxides, Fe3O4 nanoparticles (NPs) are drawing considerable attention due to their unique properties such as excellent biocompatibility, low synthetic cost, ease of functionalization, and high magnetic susceptibility leading to easy magnetic recovery34. Owing to the negatively charged surface, bare Fe3O4 NPs have been shown as dye absorbents in many reports37; however, its applicability is limited due to low adsorption capacity and poor selectivity and recyclability. Chaudhary et al. have reported the use of Fe3O4 NPs for adsorption of acridine orange dye, Coomassie brilliant blue R-200 dye, and Congo red dye37. Jia et al. developed 3D hierarchical porous Fe3O4 NPs, which have been used for C.R dye adsorption, however, with low adsorption capacity of 39.10 mg/g25. Similarly, hydrothermal preparation of Fe3O4 NPs and its application for C.R dye removal from aqueous solution, with adsorption capacity of 28.46 mg/g, has been reported by Wang et al.35.In recent times, porous metal oxides have been recognized as excellent solid adsorbents for wastewater treatment owing to their high surface area and the presence of sufficient active surface sites3O4 surface by introducing organic chelate groups42 and metal oxide47. For example, polyaniline (PANI) and carbon nanotube (CNT) functionalized Fe3O4 NPs were reported by Zhao et al., which showed a moderate value of adsorption capacity (417.38 mg/g)40. Wang et al. reported polyethyleneimine/graphene oxide/Fe3O4 based nanocomposite material with 574.7 mg/g adsorption capacity38.Besides electrostatic interactions as applicable to the cases mentioned above, the adsorption of dye is also actively facilitated by hydrogen bonding, which depends on functional groups and active coordination sites present on the solid surface. Thus, to achieve higher adsorption capacity, many researchers have focused their efforts on the functionalization of the Fe3O4 nanoparticles for rapid and selective adsorption of a ubiquitous industrial pollutant Congo red dye. Though there are many reports on surface functionalization with carboxylate groups50, BTCA group, containing four carboxylate groups at 1, 2, 4 and 5- positions of the benzene ring as functionalization agent, is expected to significantly improve the adsorption capacity for selective dyes with better recyclability as illustrated in Fig. 3O4 surface will cause a firm anchorage of BTCA on Fe3O4 surface. The remaining two –COO− groups, formed under synthetic conditions, can form a strong H-bonding with a primary amine-containing dye, like Congo red, which will lead to high adsorption capacity. To best of our knowledge, it is the first report where BTCA has been employed as a surface functionalization ligand for any material.In the present work, we have explored a multi carboxylate organic ligand 1,2,4,5-Benzentetracarboxylic acid (BTCA) for surface functionalization of Fe3O4 NPs (Fe3O4@BTCA), which rapidly and selectively adsorbs a primary amine-containing Congo red dye from its aqueous solution. The adsorption studies show that Fe3O4@BTCA material exhibits significantly higher adsorption capacity and can be reused in multiple cycles without undergoing any considerable loss in adsorption performance. Mechanisms for selective adsorption and adsorption kinetics have also been described.The present report discusses the synthesis and characterization of BTCA functionalized Fe3O4 and subsequent immobilization of BTCA groups to obtain Fe3O4@BTCA is depicted in Fig. 3O4 and Fe3O4@BTCA materials  compared to Fe3O4 (1.07%) indicates more water adsorbed on Fe3O4@BTCA due to stronger hydrogen bonding facilitated by the presence of free carboxylate groups. The second region showing 7.01% weight loss in Fe3O4@BTCA compared to 1.51% in Fe3O4 indicates the presence of higher organic contents (BTCA) in the case of former. As expected, both materials show similar thermal decomposition behavior beyond 500 °C. Overall, a comparison of three decomposition segments in both materials hints a successful grafting of BTCA group on to the surface of Fe3O4. Presence of BTCA group on to the surface of Fe3O4 in Fe3O4@BTCA is also supported by its EDX spectrum, where peak corresponding to carbon can be clearly seen , which supports that both materials have a pure Fe3O4 phase with a spinel structure. As evident from the diffraction pattern, immobilization of the BTCA group does not change the Fe3O4 phase. Using Scherrer equation on PXRD line broadening, the calculated average particle size of Fe3O4 is found to be 12 nm. The (311) plane mean diffraction peak at 2θ = 35.49 was used to determine the particle size because of its intense nature.The structural information was verified by the powder X-ray diffraction (PXRD) technique. The powder X-ray diffraction patterns of both materials are illustrated in Fig. 3O4, Fe3O4@BTCA, and Congo Red dye loaded species (Fe3O4@BTCA@C.R) are shown in Fig. 3O4 and Fe3O4@BTCA  range of 0.05–1  compared to that of Fe3O4 (12.41 nm). This indicates that the immobilization of BTCA occurs on the outer surface of Fe3O4. The enhanced surface area of Fe3O4@BTCA (72.35 m2/g) compared to that of Fe3O4 (46.66 m2/g) can be attributed to a higher dispersity of Fe3O4@BTCA NPs due to negatively charged surface repulsion.The porous structures of as-synthesized Fe1 Figure , and by 1 Figure . The por3O4 and Fe3O4@BTCA show that the former, having −7.21 mV potential, possess significantly lesser negative surface charge than the later having −17.8 mV. Observation of the higher negative surface charge in the case of Fe3O4@BTCA is understandable as it bears BTCA groups with a number of free carboxylate groups covering the surface. For Fe3O4@BTCA, information about the higher surface area from BET and the idea of greater negative surface charge from surface charge analysis together support our assumption of obtaining better adsorption efficiency by immobilization of BTCA groups.Surface charge analyses by zeta-potential calculations of Fe3O4@BTCA bears free carboxylate groups, reprotonation and de-protonation are expected to affect its surface charge. Therefore, the surface charge analysis of Fe3O4@BTCA was carried out at various pH, and the calculated zeta-potentials at different pH are shown in Fig. 3O4@BTCA surface, as discussed later.As the surface of Fe3O4@BTCA to adsorb dyes on to its surface, leading to the color removal of aqueous dye solutions was investigated towards C.R (Congo red), M.O (Methyl Orange), and C.V  by UV-Vis absorption spectroscopy. Figure 3O4@BTCA for 15 minutes. It is evident that Fe3O4@BTCA does selective adsorption of C.R dye leading to 97% color removal , and pH conditions (2–10). The resulting graph and bar diagram with corresponding values are given in Fig. The ability of Feval Fig. . Howeverval Fig. . UV-Vis val Fig.  in ESI. 3O4@BTCA, using FT-IR and UV-Vis spectroscopy, we also ensured the formation of Fe3O4@BTCA@C.R species containing adsorbed C.R dye on the surface of Fe3O4@BTCA. Fe3O4@BTCA@C.R was obtained by stirring 50 mg of Fe3O4@BTCA in 100 ppm aqueous solution of C.R dye for 15 minutes, followed by its magnetic separation, washing and drying at 70 °C for 24 h. The UV-Vis absorption spectrum of this material shows two weak peaks at 498 and 338 nm , adsorption isotherm studies were performed through batch experiments. Prior to the adsorption study, a calibration plot  through vigorous shaking for 15 minutes. After completion of adsorption, the equilibrium concentration of C.R dye in each magnetically separated filtrate was calculated by comparing its UV-Vis spectrum with the calibration plot. The equilibrium adsorption capacity (qe) for each batch adsorption can be calculated using Eq. 61.Ci and Ce are initial and equilibrium concentrations (mg/L) of C.R dye, respectively. V is the volume of C.R solution (50 × 10−3L), W is the amount of Fe3O4@BTCA (5 × 10−3g). The adsorption capacity of Fe3O4@BTCA for C.R dye can be estimated from the plot of qe and Ce values obtained at a different concentration of C.R dye , and KL (L/mg) is Langmuir equilibrium constant, which is related to adsorption energy.Adsorption behavior of C.R dye on the surface of Fe64.Kf and n are Freundlich constants related to adsorption capacity and adsorption intensity, respectively.The possibility of multilayer adsorption is allowed in the Freundlich adsorption model, and its adsorption isotherm is given in Eq. 2 = 0.9837 for Langmuir and 0.8722 for Freundlich). Other parameters  for both adsorption isotherms are given in Table From Fig. 65.In order to understand the adsorption kinetics, the order of the adsorption process was ascertained by fitting the experimental data into the pseudo-first-order and pseudo-second-order equations Eqs.  and 565.qt) towards C.R dye was calculated by UV-Vis spectroscopy at different time intervals (t) taking 20 ppm initial aqueous solution of C.R dye with 5 mg Fe3O4@BTCA. With increasing time , the obtained values of qt are plotted in Fig. qe (192 mg/g) is achieved in 15 min and remains constant with further increase in time interval. Upon fitting the value of qe (192 mg/g) and that of qt obtained at a different time interval in straight line Eqs. 2 = 0.9757) was obtained for pseudo-second-order adsorption , the presence of BTCA groups on the Fe3O4 surface provides lots of free carboxylate groups (–COO−) covering the surface of Fe3O4. During interaction of Fe3O4@BTCA and C.R dye, the free carboxylate groups facilitate the adsorption process through formation of a strong H-bond with free –NH2 groups of C.R dye as depicted in Fig. 3O4@BTCA@C.R. The involvement of H-bond in the formation of Fe3O4@BTCA@C.R is supported by the fact that the asymmetric C-O stretching of carboxylate groups in Fe3O4@BTCA at 1581 cm−1 almost disappears in Fe3O4@BTCA@C.R  on Fe3O4@BTCA were re-protonated to convert them all to corresponding carboxylic acid (-COOH), whose H-bond acceptor ability is much less than the carboxylate groups. When acidified Fe3O4@BTCA was subjected to C.R dye adsorption under similar conditions, the observed percentage of color removal was 33% only  and can be reused for multiple adsorption-desorption cycles without undergoing any considerable loss in its performance. Adsorption isotherm and kinetics studies reveal that the adsorption of C.R dye on the Fe3O4@BTCA surface takes place by the Langmuir adsorption model and follows pseudo-second-order adsorption kinetics.A new FeThe high adsorption capacity, selectivity and better recyclability of the material can be attributed to surface functionalization ligand BTCA, which, owing to its four uniquely positioned carboxylate group, offers strong surface encourage and participates in the formation of a strong H-bonding only with Congo red dye, containing a primary amine group. This report opens up new possibilities for 1,2,4,5-Benzenetetracarboxylic acid to be explored for surface functionalization of other materials to achieve improved targeted applications.3.6H2O (98%), FeCl2.4H2O (99%) and 1,2,4,5-Benzenetetracarboxylic acid (BTCA) were purchased from Sigma Aldrich. Ammonia solution (25%), HCl, Congo Red (C.R), Methyl Orange (M.O), and Crystal Violet (C.V) were purchased from Spectrochem. pH buffer tablets were purchased from SRL.All reagents and chemical used were of analytical standards. In all experiments, double distilled-deionized water was used. FeCl3O4 nanoparticles were synthesized by chemical co-precipitation method51. Initially, FeCl3.6H2O  and FeCl2.4H2O  were dissolved in 400 mL deionized water and kept at room temperature for 15 min with vigorous stirring under a nitrogen atmosphere. After that, 10 mL of ammonia solution (25%) was added drop by drop into the salt solution, and stirring was continued for another 2 h at 80 °C. The solution was cooled, and solid black particles were magnetically separated and repeatedly washed with Millipore water. The particles were finally dried in a vacuum oven at 70 °C for overnight.Fe3O4 was taken in a 500 mL round bottom flask containing 200 mL of deionized water. To this suspension, 2 g BTCA (3.905 mmol) and 5 mL aqueous NH4OH solution were added until the pH reached 11.0. The suspension was refluxed for 24 h in a nitrogen atmosphere with constant stirring. The resulting Fe3O4@BTCA material was separated through magnate and washed thoroughly with deionized water thrice to remove unreacted BTCA. The residue was dried under vacuum at 70 °C for 24 h to give brown colored Fe3O4@BTCA material.In a direct functionalization procedure, 1 g of Fe−1) were collected on a Perkin-Elmer GX spectrophotometer (U.S.A) using KBr disks. The zeta potentials were recorded using a Malvern instrument . Scanning electron microscope (SEM) (Leo series 1430 VP) equipped with INCA was used to determine the morphologies of samples. Transmission electron microscopy (TEM) was performed using a JEOL JEM 2100 microscope. Thermogravimetric analysis (TGA) was done using Mettler Toledo TGA/DSC 1 analyzer. Powder X-ray diffraction (XRD) measurements were conducted on Rigaku smart lab automated multipurpose X-ray diffractometer system with CuKα1 radiation (λ = 1.540593 Å) in the 2θ range of 20–70° at scanning speed of 3° per minute with 0.01° scan step size. The Brunauer− Emmett−Teller (BET) surface area analysis was conducted on an Autosorb iQ, version 1.11 (Quantachrome Instruments).Electronic spectra of suspension were recorded on Varian UV−visible spectrophotometer (Carry 100 Bio). FT-IR spectra  measurements for Congo red dye were done by taking 5 mg of Fe3O4@BTCA material in ten batches of 50 ml aqueous dye solution of different concentration (10 ppm to 100 ppm). Each batch containing a suspension of 5 mg of Fe3O4@BTCA in aqueous dye solution was stirred at room temperature for 15 min (pH of dye solutions ~7.4), after which, the concentration of unabsorbed dye was determined from UV-Vis spectrum of the filtrate. All the filtrations were done using a strong magnet. All the adsorption experiments were repeated thrice to remove the experimental error.Batch adsorption experiments were carried out in an aqueous dye solution to determine several adsorption parameters. Adsorption capacity  was allowed on 5 mg FeSupplementary information.


The planar structure of 10 was determined from spectroscopic analyses, and its relative and absolute configurations were established from density functional theory (DFT) calculations of the GIAO NMR shielding tensors, and advanced Marfey’s analysis of the N-MeLeu residue, respectively. Compounds 1 and 3 showed moderate inhibition of β-secretase 1 (BACE1), whereas 1–9 exhibited moderate to potent inhibition of growth of human glioma (U251) cells. Compounds 1–2 and 4–7 were also active against human pancreatic carcinoma (Panc-1) cells.Several known sesquiterpenoid quinones and quinols ( Tethya crypta by Bergmann and Feeney in the early 1950s + .White, amorphous solid; 10) in 0.5 mL 6N HCl in a capped reaction vial was stirred for 24 h at 110 °C. Solvent was removed by streaming N2 over the reaction mixture and the residue was analyzed by ESIMS in negative mode to confirm the presence of signals at m/z 247 and 249. To the dried hydrolysate in the vial were added a solution of 500 μg  1-fluoro-2,4-dinitrophenyl-5-l-leucinamide (L-FDLA) in acetone (160 μL), H2O (100 μL) and 1N solution of NaHCO3 (50 μL). The contents in the vial were warmed to 40 °C for 1 h, cooled to room temperature, and analyzed by LC-MS , 2.1 × 50 mm, 1.8 µm particle size; eluted with acetonitrile and water (+0.1% formic acid in each) at 0.4 mL/min flow using the following gradient: 20–50% acetonitrile (0–10 min), 50–100% acetonitrile (10–12 min), 100% acetonitrile (12–16 min); detection at 340 nm). The LC-MS data was compared to the data obtained from similar protocols on the standard amino acids DL-N-methylleucine and L-N-methylleucine, which were obtained from the amino acid standards collection in the Williams laboratory. The retention times for the LL-adduct and the DL-adduct were 9.2 min and 10.3 min, respectively. A solution of 200 μg  of kauamide . Statistical comparisons of the computed shifts with the experimental data were carried out using the applet provided by Goodman [Conformers within 5 kcal/mol of the lowest energy conformer were searched using the Monte Carlo multiple minimum (MCMM) method and the OPLS-2005 force field in MacroModel   and an inhibitor standard  used as a positive control) were also tested in triplicates. An initial test concentration of 30 μg/mL was used for all compounds, with subsequent dose response curves determined for any compound that showed greater than 50% inhibition at that initial test concentration. The chemiluminescence signal was read using a Fluostar Optima spectrophotometer . Data were analyzed using GraphPad Prism . BACE1 activity was calculated as a percent of the positive control using a nonlinear regression analysis function that corresponded to a best one-fit model. Test concentrations and percent inhibition for the inactives are as follows: 4 (17% inhibition at 75 μM), 5 (5% inhibition at 73 μM), 6 (0% inhibition at 67 μM), 7 (44% inhibition at 80 μM), 8 (17% inhibition at 77 μM), 9 (0% inhibition at 84 μM), 10 (15% inhibition at 83 μM).The HitHunter BACE1 enzyme fragment complementation chemiluminescence assay was purchased from DiscoveRx . The proteolytic cleavage of amyloid precursor protein was assayed as described by Naqvi . Test co2 at 37 °C for 72 h. Then the medium with compounds were replaced with 1 × dye binding solution prepared according to the manufacturer’s instruction  and incubated with 5% CO2 at 37 °C for 60 min. After that, cell viability data were collected with a Multimode Plate Reader  according to the manufacturer’s instruction. CC50 curves were generated using GraphPad Prism .Human glioblastoma (U251MG) cell lines were maintained in RPMI 1640 Medium  supplemented with 10% premium fetal bovine serum  and 100 U/mL penicillin and 100 µg/mL streptomycin . A day prior to treatment, cancer cells were seeded at 4000 cells per well into a 96-well tissue culture plate. Twenty hours post seeding, the serially diluted compounds were added to the cells for the cytotoxicity assay, and co-incubated with 5% CO2 for 72 h. Then 40 µL MTS dye  was added to each well and incubated with 5% CO2 at 37 °C for 90 min. Cell viability data were collected with a Modulus Microplate Reader  and CC50 curves were generated using GraphPad Prism .Human pancreatic carcinoma (Panc-1) cell lines were maintained in DMEM media supplemented with 10% premium fetal bovine serum and 50 U/mL penicillin and 50 µg/mL streptomycin. One day before treatment, cancer cells were seeded at 5000 cells per well into a 96-well tissue culture plate. Twenty-four hours post seeding, the serially diluted compounds were added to the cells for the cytotoxicity assay, and incubated at 37 °C with 5% CO