Rectal cancer is one of the most lethal of gastrointestinal malignancies. Metabonomics has gradually developed as a convenient, inexpensive and non-destructive technique for the study of cancers.A total of 150 tissue samples from 25 rectal cancer patients were analyzed by liquid chromatography–mass spectrometry (LC–MS), and 6 tissue samples were collected from each patient . The differential metabolites of tumor tissues and 5 cm from the tumor  were first selected. The differential metabolites between tumor tissues and normal tissues were regrouped by hierarchical clustering analysis, and further selected by discriminant analysis according to the regrouping of clustering results. The potential safe margin of clinical T(cT)1,cT2 stage rectal cancer and cT3,cT4 stage rectal cancer at the metabolomic level was further identified by observing the changes in the level of differential metabolites within the samples from group 1 to group 6.We found 22 specific metabolites to distinguish tumor tissue and normal tissue. The most significant changes in metabolite levels were observed at 0.5 cm  and 2.0 cm  from the tumor, while the changes in the tissues afterwards showed a stable trend.There are differential metabolites between tumor tissues and normal tissues in rectal cancer. Based on our limited sample size, the safe distal incision margin for rectal cancer surgery in metabolites may be 0.5 cm in patients with cT1 and cT2 stage rectal cancer and 2.0 cm in patients with cT3 and cT4 stage rectal cancer. Rectal cancer is the third most prevalent malignant gastrointestinal tumor, and is a serious threat to human health. There are more than 2.2 million new cases of rectal cancer worldwide every year, which has become an important topic in cancer research –3. The 5The present study was approved by the Ethics Committee of the First Hospital of Jilin University , and all patients provided written informed consent. In this experiment, 150 rectal tissue samples were obtained from 25 rectal cancer patients  who attended the Department of Gastrocolorectal and Anal Surgery, Baiquan First Hospital of Jilin University from July 2020 to November 2020. Inclusion criteria: (1) patients with solitary rectal cancer without distant metastases; (2) no previous oncologic history; (3) this was the first diagnosis; (4) no significant acute inflammatory disease. Exclusion criteria: (1) hepatic, renal, cardiopulmonary insufficiency; (2) pregnancy, female patients during lactation; (3) long-term alcohol and drug addiction; (4) long-term use of steroid hormones or nonsteroidal anti-inflammatory drugs; (4) chronic inflammatory diseases such as ulcerative colitis; (5) acute inflammatory reactions within the last 2 weeks; (6) major stress reactions such as major surgery, major changes or trauma within the last 2 weeks; (7) hematologic diseases such as leukemia; (8) infectious diseases.In each case, a fresh rectal cancer surgical sample was incised along the longitudinal axis of the intestinal canal and washed with saline to remove the intestinal contents and mucus on the surface of the intestinal mucosa. The washed sample was spread and laid flat on a plate, vascular forceps were clamped on the margin of the distal intestinal canal of the tumor, and the intestinal canal was torn flat with moderate force to facilitate resection. The length of the rectal intestinal canal was measured, and 0.5 cm diameter of whole intestinal wall tissue was cut at tumor tissue, 0.5 cm from tumor, 1 cm from tumor, 2.0 cm from tumor, 3 cm from tumor, and 5 cm from tumor. The samples were placed in an EP tube and immediately stored in a -80 °C refrigerator. The frozen tissue sample was allowed to thaw for 20 min at room temperature prior to analysis. The 10 mg of sample added 200 µl of water, and 800 µl of methanol:acetonitrile  was vortexed for 30 s, sonicated for 10 min (ice bath) and placed in a -20 °C refrigerator for 1 h. Subsequently, the samples were centrifuged at 13,500 × g/min for 15 min at 4 °C, and the supernatant was removed in a vacuum dryer until it evaporated. Then, 100 µl of acetonitrile:water  was added again, followed by vortexing for 30 s, sonicating for 5 min (ice bath), centrifuging at 13,500 × g/min for 15 min at 4 °C, and collecting the supernatants for LC–MS analysis. The quality control (QC) group consisted of all 150 samples, and each QC sample was inserted evenly into 10 experimental samples to assess the accuracy of the experiment. To ensure the accuracy and authenticity of the experimental data, the automatic calibration uses the chromatographic data system (CDS), which was used every fifth experimental sample.LC–MS analysis was performed using the AB Sciex TripleTOF 5600 system (Sciex) and Exion Ultra Performance Liquid Chromatography (UHPLC) system  according to the manufacturer's protocols. The samples were mixed using a Vortex 3, Germany IKA; the centrifuge was a 4 °C low temperature centrifuge H165R from Xiangyi Centrifuge. Experimental reagents included acetonitrile , formic acid , methanol , deionized water , Positive Calibration Solution(AB Sciex) and Negative Calibration Solution(AB Sciex). Liquid chromatography was performed on an Exion UHPLC System. The column was an ACQUTIY UPLC HSS T3  maintained at 35 °C during separation. The mobile phases in positive ion mode were 0.1% formic acid in (A) water and (B) acetonitrile; the mobile phases in negative ion mode were (A) water and (B) acetonitrile. Samples were eluted with 98% A and 2% B for the first 0.5 min, a gradient from 2% B to 95% B over 10.5 min, followed by 95% B for 4 min. Then, the percentage of B was dropped to 2% within 0.1 min and maintained for 5 min. The flow rate was constant at 350 µl/min.Mass spectrometry was performed using the AB Sciex TripleTOF 5600 system, which was fitted with an electrospray ionization source operating in positive and negative ion modes. Mass spectrometry conditions: the ion source level mass spectrometry parameters are as follows: Positive mode, IonSpray voltage (V): 5500; Temperature (℃): 550; Gas 1 (psi): 55; Gas 2 (psi): 55; Curtain gas (psi): 30; Declustering potential (DP): 100; Collision energy (CE): 10. Negative mode, IonSpray voltage (V): -4500; temperature (°C): 550; gas 1 (psi): 55; gas 2 (psi): 55; curtain gas (psi): 30; declustering potential (DP): -100; collision energy (CE): -10. The ion source secondary mass spectrometry parameters are as follows: Positive mode, IonSpray Voltage (V):5500;Temperature (℃):550;Gas 1 (psi):55;Gas 2 (psi):55; Curtain gas (psi): 30; Declustering Potential (DP):100; Collision Energy (CE):35;Collision Energy Spread (CES):15;Ion Release Delay (IRD):67;Ion Release Width (IRW):25;Negative mode, IonSpray Voltage (V):-4500;Temperature (℃):550;Gas 1 (psi):55;Gas 2 (psi):55; Curtain gas (psi): 30; Declustering Potential (DP):-100; Collision Energy (CE):-35;Collision Energy Spread (CES):15; Ion Release Delay (IRD): 67; Ion Release Width (IRW): 25.P < 0.05 was considered to indicate a statistically significant difference), partial least squares discriminant analysis (PLS-DA), support vector machine discriminant analysis (SVMDA) and hierarchical clustering analysis to identify metabolites. SPSS 20.0 software (IBM Corp.) was used to generate receiver operating characteristic (ROC) curves. The mass-to-charge ratio (M/S) of the metabolites was imported into the Human Mass Spectrometry Database (HMDB) to find their material structure. The mean levels of the metabolites within 6 groups  were plotted as a scatterplot to observe the trend of the levels of these metabolites with distance from the incision margin.The LC–MS data were acquired using TF 1.7.1 software (AB Sciex LLC) and processed using PeakView software 2.2 (AB Sciex LLC). Normalization, scaling, noise filtering and peak alignment were performed using MarkerView software 1.3.1 (AB Sciex LLC). MetaboAnalyst 5.0 software was used to perform the principal component analysis (PCA), T test  of metabolites in the tumor tissue Fig. a c and 5Comparison of the tumor tissue and 5 cm from the tumor  indicated significant differences for 458 metabolites in the positive ion mode Fig. a and 764http://www.hmdb.ca), the following 22 metabolites met the criteria of the present study: Ethyl 3-Hydroxy Triester, Ditriglyceride, 7-Dehydrocholine, Acetamido phenylalamine, Triglyceride Monocelate, 24-O-B-D-Glycoside, Triglyceride, N-Ethanolamine, Propyl carnitine, Fatty Acetyl, Fatty Alcohol, 3,4-Dihydrooxyphenylpropanol, 4,5 Epoxy amide, 4-hydroxybutadibenzoic acid, 3'-phosphoamine sulfate 5'-phosphate, 1-O-β-D-glucosol-2,1-acetylglycerol phosphate, 1,2,3-triacetylglycerol,Amphetamine nucleoside monophosphate, adenosine acid, N-acetyl-α amino acid, and N-acrylamide  subgroups in the positive ion mode Fig. a and in Hierarchical clustering analysis was performed for these 22 metabolites in 30 samples from 5 patients with cT1 and cT2 stage tumors. This analysis separated these 30 samples from group 1–6 into subgroup 1 and subgroup 2 as shown in the top of Fig. These 11 differentiating metabolites are shown in Table Therefore, this procedure had a sensitivity of 80.0% and a specificity of 84.0%. Figure Hierarchical clustering analysis was performed for these 22 metabolites in 120 samples from 20 patients with cT3 and cT4 stage tumors. This analysis separated these 120 samples from group 1–6 into subgroup 1 and subgroup 2 as shown in the top of Fig. These 7 differentiating metabolites are shown in Table Therefore, this procedure had a sensitivity of 91.6% and a specificity of 85.0%. Figure Rectal cancer is the third most common malignancy, and the distal margin distance is associated with local recurrence of the tumor, while there are conflicting opinions on the safe distal margin distance for low rectal cancer , 21–23.TSeveral previous metabolomic studies on rectal cancer have found changes in fatty acid metabolism, amino acid metabolism and glucose metabolism in patients with rectal cancer . For exaThere are differential metabolites, with levels varying with distance from the tumor, between tumor tissues and normal tissues in rectal cancer. In patients with cT1 and cT2 stage rectal cancer, the most significant changes in metabolite levels were observed at 0.5 cm from the tumor, while the changes in the tissues afterwards showed a stable trend. In patients with cT3 and cT4 stage rectal cancer, the most significant changes in metabolite levels were observed at 2.0 cm from the tumor, while the changes in the tissues afterwards showed a stable trend. Based on our limited sample size in our study, the safe distal incision margin for rectal cancer surgery in metabolites may be 0.5 cm in patients with stage cT1 and cT2 stage rectal cancer and 2.0 cm in patients with cT3 and cT4 stage rectal cancer. However, this conclusion needs to be further confirmed by studies that contain larger sample sizes in the fulture.


Intensive agriculture systems have paved the way for a growing human population. However, the abundant use of mineral fertilizers and pesticides may negatively impact nutrient cycles and biodiversity. One potential alternative is to harness beneficial relationships between plants and plant-associated rhizobacteria to increase nutrient-use efficiency and provide pathogen resistance. Plant-associated microbiota profiling can be achieved using high-throughput 16S rRNA gene amplicon sequencing. However, interrogation of these data is limited by confident taxonomic classifications at high taxonomic resolution (genus- or species level) with the commonly applied universal reference databases. High-throughput full-length 16S rRNA gene sequencing combined with automated taxonomy assignment (AutoTax) can be used to create amplicon sequence variant resolved ecosystems-specific reference databases that are superior to the traditional universal reference databases. This approach was used here to create a custom reference database for bacteria and archaea based on 987,353 full-length 16S rRNA genes from Askov and Cologne soils. We evaluated the performance of the database using short-read amplicon data and found that it resulted in the increased genus- and species-level classification compared to commonly use universal reference databases. The custom database was utilized to evaluate the ecosystem-specific primer bias and taxonomic resolution of amplicon primers targeting the V5–V7 region of the 16S rRNA gene commonly used within the plant microbiome field. Finally, we demonstrate the benefits of custom ecosystem-specific databases through the analysis of V5–V7 amplicon data to identify new plant-associated microbes for two legumes and two cereal species. A growing world population necessitates continued improvements in agricultural output to ensure food security. Current agricultural practices may involve excessive applications of mineral fertilizers and pesticides, which need to be reduced and supplemented with more sustainable solutions   and two cereal (Hordeum vulgare and Zea mays) species.Here, we demonstrate how ecosystem-specific reference databases can be used for exploratory studies to increase the taxonomic resolution in soil microbial profiling studies, leading to new insight. For this, we sequenced 987,353 full-length 16S rRNA genes from two soils commonly used for plant microbiome research: Askov soil, which has been used for plant and soil studies for more than 125 years . The soil was sampled in 2018 from 0- to 20-cm soil depth in three replicate plots  in the B4 field. This field is part of the Askov Long-Term Experiment established in 1894, and the plots sampled for this study have been kept without manure and fertilizer application since then. The soil is a light sandy loam with 11% clay, 13% silt, and 76% sand and classifies as Alfisol . The soil grows a four-course rotation of winter wheat, silage maize, spring barley, and a grass-clover mixture used for cutting. The addition of lime every 4–5 years keeps soil pH in the range of 5.5–6.5    and Zea maise cv. W22 (Zm) were received from the Crop Science Centre, University of Cambridge.Seeds of 2O, and germinated on wet filter paper for 2–5 days. Seedlings were aseptically transferred to sterile pots  that had been filled with ~350 g Askov soil . Three pots were set up for each plant species. Plants were grown in a growth chamber under controlled conditions: 16/8 h day/night, 75% humidity, 22°C (day), and 18°C (night). Watering throughout the experiment was with sterile H2O only.Seeds were surface sterilized in a 1:20 bleach solution for 5–15 min, washed five times in sterile H2O, and roots were transferred to a new Falcon tube. The initial Falcon tube was centrifuged , and most supernatants were removed. The soil pellet was resuspended in the remaining liquid (ca. 2 ml) using a cut P1000 tip; 300 μl of this suspension was transferred to a Lysis matrix E tube  forming the rhizosphere fraction. The root material was washed three times in sterile H2O for 30 s, once in detergent (1× TE + 0.1% Triton X-100) for 2 min, once in 80% ethanol for 30 s, once in 3% bleach for 30 s and finally five times in sterile H2O. The surface sterilized root material was then transferred to Lysis matrix E tubes forming the endosphere fraction. For Lj and Mt, nodules were removed from sterilized root material and collected in separate Lysis matrix E tubes forming the nodule fraction. Details about all samples can be found in After 3 weeks of growth, at which point mature nitrogen-fixing nodules had formed on the legumes, plants were removed from pots, and roots separated from shoots. Root material was vortexed for 30 s in 50 ml Falcon tubes containing 35 ml sterile HConcentrations of DNA were measured using a Qubit 3.0 fluorometer (Thermo Fisher Scientific), and quality was determined using Agilent 2200 Tapestation (Agilent Technologies). AMPure XP beads were used for DNA cleanup in accordance with manufacturer's protocol except for the washing step, where 80% ethanol was used. All commercial kits were used according to manufacturer's protocol unless otherwise stated.DNA was extracted using the FastDNA Spin Kit for Soil (MP Bio) with the homogenization performed using a Precellys tissue lyser (Bertin Instruments) for 2 × 30 s at 6,000 rpm or using the RNeasy PowerSoil Total RNA Kit with the PowerSoil DNA Elution Kit (Qiagen). Quality was determined by tapestation using a genomic DNA ScreenTape. Concentrations were measured using Qubit™ dsDNA HS Assay Kit.Lj, Mt, Hv, and Zm grown in Askov soil for 3 weeks and Lj rhizosphere grown in Cologne soil for 3 weeks  or 20 μl (archaea) of 5× Q-solution (Qiagen), 0.5 μl of 5 U/μl Taq polymerase (Qiagen), 100 ng of template DNA, and nuclease-free water to 100 μl. The reaction was incubated with an initial denaturation at 94°C for 3 min followed by 2 cycles of denaturation at 94°C for 30 s, annealing at 56°C (bacteria) or 54°C (archaea) for 30 s, and extension at 72°C for 3 min, and then a final extension at 72°C for 5 min. The sample was purified using 0.6× AMPure XP beads and eluted in 21-μl nuclease-free water.Adaptors containing sample barcodes, unique molecular identifiers (UMI), and defined primer binding sites were added to each end of the bacterial 16S rRNA genes by PCR. The reaction contained 10 μl of 10× PCR Buffer (Qiagen), 2 μl of 10 mM dNTP (Qiagen), 5 μl of 10 μM f16S_pcr1_fw, 5 μl of 10 μM f16S_pcr1_rv, 4 μl (bacteria) or 8 μl (archaea) of 25 mM MgClThe following versions of the f16S_pcr1_fw forward primers were used: Askov rhizosphere (fw1), Askov bulk soil (fw2), Cologne rhizosphere (fw3), and Cologne bulk soil (fw4). The f16S_pcr1_rv1 reverse primer was used for all samples.2, 44 μl nuclease-free water, and 1 μl 2U/μl Phusion HF DNA polymerase (NEB). The reaction was incubated with an initial denaturation at 98°C for 30 s followed by 15 (bacteria) or 20 (archaea) cycles of denaturation at 98°C for 10 s, annealing at 62°C for 30 s, and extension at 72°C for 1 min, followed by a final extension at 72°C for 5 min. DNA was eluted in 11 μl nuclease-free water. Quality was determined using a D5000 ScreenTape and concentrations were measured using Qubit™ dsDNA HS Assay Kit. Libraries for the different sample barcodes were pooled with an equal amount of DNA from each sample.The adaptor-annealed 16S rRNA gene amplicons were amplified using PCR to obtain enough product for quantification and sequencing. The reaction contained 19 μl of adaptor annealed sample, 20 μl of 5× Phusion HF (New England Biolabs), 2 μl of 10 mM dNTP, 5 μl of 10 μM f16S_pcr2_fw, 5 μl of 10 μM f16S_pcr2_rv, 4 μl of 25 mM MgCl2, 0.5 μl of 5 U/μl Taq polymerase (Qiagen) and 10 μl diluted adaptor annealed amplicon product. The reaction was initiated by denaturation at 94°C for 3 min, followed by 20 cycles of denaturation at 94°C for 30 s, annealing at 62°C for 30 s and extension at 72°C for 2 min, followed by a final extension at 72°C for 5 min. The PCR product was purified using 0.6× AMPure XP beads with elution into 21 μl nuclease-free water. The product quality and concentration were analyzed on a D5000 screen tape and with the Qubit dsDNA HS Assay Kit, respectively.Adaptor annealed amplicon libraries were diluted to ~200,000 molecules/μl and amplified by PCR to obtain clonal copies of each uniquely tagged amplicon molecule. The PCR reaction contained 63.5 μl nuclease-free water, 10 μl of 10× PCR buffer (Qiagen), 2 μl of 10 mM dNTP, 5 μl of 10 μM f16S_pcr2_fw, 5 μl of 10 μM f16S_pcr2_rv, 4 μl of 25 mM MgClA Nextera library preparation kit (Illumina) was used to prepare a paired-end read-tag sequencing library from the clonal library using a customized protocol. A tagmentation reaction was prepared with 100 ng of the clonal library in 22.5 μl nuclease-free water, 25 μl tagment DNA buffer (Illumina), and 2.5 μl tagment DNA enzyme (Illumina). The reaction was incubated at 55°C for 5 min. The product was immediately diluted to 100 μl and purified using 0.6× AMPure XP beads with elution into 42 μl nuclease-free water.The tagmentation products were PCR amplified using two separate PCRs (A and B). PCR A selectively amplified fragments containing the 5′ termini of the amplicons and PCR B selectively amplified fragments containing the 3′ termini. The reactions contained 20 μl purified tagmentation product, 5 μl N504 Nextera adaptor (Illumina), 5 μl of 10 μM f16S_readtag_fw (PCR A) or f16S_readtag_rv (PCR B), 5 μl PCR primer cocktail (Illumina), 10 μl of 5× Phusion HF buffer (NEB), 1 μl of 10 mM dNTP, 3.5 μl nuclease-free water, and 0.5 μl of 2U/μl Phusion HF DNA polymerase (NEB). The following PCR program was used: Initial elongation at 72°C for 3 min, initial denaturation at 98°C for 30 s, and 10 cycles of denaturation at 98°C for 10 s, annealing at 60°C for 30 s and elongation at 72°C for 3 min, followed by a final extension at 72°C for 5 min. The raw read-tag libraries were purified using 1.0× AMPure XP beads with elution into 21-μl nuclease-free water.seq = Peak molarity [pmol/l] * (−0.0124*(peak size [bp] – 215 bp) + 10.332) . For fractions between 950 and 1,250 bp, the entire aliquot was used for pooling (40–50 μl). The pooled aliquots were then purified using 1.0× AMPure XP beads with elution into 11 μl nuclease-free water. The quality and concentration of the coverage-optimized read-tag libraries were analyzed on D1000 screen tapes and with the Qubit dsDNA HS Assay Kit, respectively.To ensure even sequencing coverage across the length of the 16S rRNA gene amplicons, the size distribution of the read-tag libraries was optimized , 1.25 μl NEBNext End Repair Enzyme Mix (New England Biolabs), and nuclease-free water to 25 μl. The reaction was incubated at 20°C for 30 min. The end-repair reaction was purified using 1.0× AMPure XP beads and eluted into 10 μl nuclease-free water.The end-repaired sample was circularized in an intramolecular blunt end ligation reaction containing 150 μl nuclease-free water, 20 μl of 50% (w/w) PEG 4000 solution (Thermo Fisher Scientific), 20 μl of 10× T4 DNA ligase buffer (NEB), 8 μl of T4 DNA ligase (NEB), and 2 μl of the end-repaired clonal library. The reaction was incubated at 16°C for 60 min. The circularized products were purified using 1.0× AMPure XP beads and eluted in 10 μl nuclease-free water.The junction sequence, which contains both UMI tags, was amplified by PCR in a reaction containing 8 μl of circularized clonal library, 5 μl of 10× PCR buffer (Qiagen), 1 μl of 10 mM dNTP mix, 2.5 μl of 10 μM f16S_linktag_fw, 2.5 μl of f16S_linktag_rv, 2 μl (bacteria) or 4 μl (archaea) 25 mM MgCl2, 0 μl (bacteria) or 10 μl (archaea) 5 × Q-solution (Qiagen), 0.25 μl 5 u/μl Taq polymerase (Qiagen), and nuclease-free water to 50 μl. The PCR reaction was initiated by denaturation at 94°C for 3 min, followed by 20 cycles of denaturation at 94°C for 20 s, annealing at 56°C (bacteria) or 54°C (archaea) for 20 s, and extension at 72°C for 20 s, followed by a final extension at 72°C for 3 min. The PCR product was purified using 1.0 × AMPure XP beads and elution into 12 μl nuclease-free water. The quality and concentration of the linked-tag libraries were analyzed on D1000 ScreenTape and with the Qubit dsDNA HS Assay Kit, respectively.The coverage normalized read-tag libraries A and B were diluted to 0.9 ng/μl. The linked-tag library was diluted to 0.2 ng/μl. The libraries were pooled by combining 4.6 μl read-tag library A, 4.6 μl read-tag library B, and 0.8 μl linked-tag library.The libraries were paired-end (1 × 240 bp and 1 × 25 bp) sequenced on a HiSeq 2500 instrument (Illumina) using on-board clustering and rapid run mode with a HiSeq PE Rapid Cluster Kit v2 (Illumina) and HiSeq Rapid SBS Kit v2, 200 cycles (Illumina). The SBS reagents were supplemented with 9.5 ml Incorporation Master Mix, 9.5 ml Cleavage Reagent Mix, and 7 ml Universal Scan Mix to enable sequencing of 265 cycles. The HiSeq was running HiSeq Control Software v.2.2.68 (Illumina) and Real Time analysis v.1.18.66.3 (Illumina). The libraries were prepared and loaded on the HiSeq using the standard procedures  with the following changes. A volume of 10 μl library pool was denatured by adding 10 μl of 0.1 N NaOH solution, mixing well by pipetting, and incubating for 5 min at 25°C. The denatured library pool was diluted by adding 980 μl of cold Hybridization Buffer (Illumina); 400 μl of the denatured and diluted library pool was mixed with 20 μl of denatured and diluted 10 pM PhiX control v3 library (Illumina) and stored on ice until loading. Custom read2 primer mix was prepared by mixing 25 μl of 100 μM f16S_read2_fw and 25 μl of 100 μM f16S_read2_rv in a conical tube (15 ml) and diluting with 4,950 μl Hybridization Buffer . When the paired-end reagent rack was loaded on the HiSeq, the Illumina primer mix in position nr. 16 was replaced with the custom read2 primer mix prepared above. When setting up the HiSeq run in the control software, the standard procedure was followed except for the following steps: For the “Recipe Screen”, the following options were chosen: Index type options = No Index; Read 1 cycles = 240; Read 2 cycles = 25. After sequencing, bcl2fastq v.2.17.1.14 (Illumina) was used to generate fastq files from bcl files using standard settings .de novo assembled into the synthetic long-read rRNA gene sequences as previously described  were loaded into ARB, and the AutoTax generated taxonomy was added to the sequences (AutoTax: output/tax_complete.csv) after being concatenated in excel. All bacterial or archaeal sequences were hereafter exported from ARB as a fasta alignment using the positional variability by parsimony filter ssuref:bacteria or ssuref:archaea. Phylogenetic trees were constructed using FastTree v.2.1.11  on the Illumina MiSeq platform.A two-step PCR protocol was used to amplify the 16S rRNA V5-V7 region and index samples using the 799F (5′-AACMGGATTAGATACCCKG-3′) and 1192R (5′-ACGTCATCCCCACCTTCC-3′) primers as described previously  for the many uncultured environmental taxa, which are not yet taxonomically classified at all seven ranks , we applied synthetic long-read sequencing to obtain near full-length bacterial and archaeal 16S rRNA genes using DNA from bulk soil and rhizosphere samples of plants grown in the two soils. Approximately the same number of sequences were obtained for each soil, resulting in a total of 987,353 full-length 16S rRNA gene sequences after primer and quality filtering. These sequences were subsequently processed using AutoTax  at the different taxonomic ranks ; Table 1cosystem . This wacosystem . The asscosystem .Hordeum vulgare, Zea mays, Medicago truncatula, and Lotus japonicus grown in Askov soil to the AsCoM database as well as commonly applied universal reference databases and calculated the percentage of ASVs with high-identity (>99%) references in the databases  recovered from atabases . The V5–atabases .For bulk soil and rhizosphere samples, the AsCoM database  has more high-identity reference sequences (>99% identity) than the universal databases GreenGenes 16S v.13.5   of the ASVs from bulk soil and rhizosphere samples was classified at the genus level using AsCoM, representing a significant improvement over SILVA (up to 40%). The 15% of ASVs that were not assigned genus-level taxonomy by AsCoM can be explained by the relatively low phylogenetic signal contained within the V5–V7 amplicons  belonging to the family Roseiflexaceae. ASVs from this genus were enriched in the rhizosphere of all plant species except H. vulgare and should, therefore, be a target for further investigations. Other well-represented genera included Flavobacterium (95 ASVs), Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium (48 ASVs), Rhizobacter (37 ASVs), and Devosia (36 ASVs).Different species or strains within the same genus can have different host preferences  probes provides opportunities to visualize the morphology and spatial arrangement of individual species in complex samples and can be combined with Raman microspectroscopy to elucidate their activity and metabolic traits in situ  to each end of the original 16S rRNA gene template molecule before any PCR amplification steps allows the filtering of true biological sequences from chimera already in the synthetic long-read assembly .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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Lung cancer is the malignancy most commonly seen worldwide. Emerging evidences indicated that lncRNAs may serve as a prognosis marker and play important role in NSCLC tumor biology. In this work, we analyzed the prognosis value of RP11-10A14.5 using TCGA and GEPIA database and expression profiles using PCR and FISH assay. The biological roles of RP11-10A14.5 in cell growth and invasion were determined by in vitro and in vivo experiments. Expression of RP11-10A14.5 is correlated with increased clinical stage and poor survival prognosis. In vitro experiments revealed that RP11-10A14.5 was widely expressed in lung cancer cell lines and mainly distributed in the cytoplasm and enhanced the growth, invasion and migration ability of NSCLC cell lines. Immunofluorescence assay suggested that RP11-10A14.5 may promote EMT by downregulating E-cadherin and upregulating N-cadherin and Vimentin. Flow cytometry results suggested that RP11-10A14.5 did not significantly affect cell cycle function, but could significantly inhibit apoptosis which may further enhance metastasis cell survival. In conclusion, RP11-10A14.5 is associated with clinical stage and poor survival outcome, may serve as a diagnosis and prognosis predictor for LUAD. Further, RP11-10A14.5 could promote LUAD cell growth and metastasis.The online version contains supplementary material available at 10.1007/s12672-022-00493-2. Lung cancer is one of the most common type of cancers in the world . GloballLong non-coding RNAs (lncRNAs) are a class of functional RNA molecules with transcripts longer than 200 nt (nucleotide units), which cannot encode proteins . Many evIn this study, to identify a potential prognosis biomarker for LUAD, we first identified differentially expressed lncRNAs in LUAD using TCGA and our LUAD cohort. Among these lncRNAs, the expression of FAM83A-AS1 and RP11-10A14.5 was the top 2 lncRNAs significantly positively correlated with clinical stage and associated with poor survival outcomes. FAM83A-AS1 has been reported in previously study that aggravated the malignant development of esophageal cancer by binding to miR-495-3p . The rol2 concentration.Human lung cancer cell lines  were cultured with RPMI-1640 or DMEM medium containing 10% fetal bovine serum (Gibco) and 1% penicillin–streptomycin (Gibco). All the cell lines were incubated in 37 °C incubator with 5% COHuman lung cancer and paracancerous tissues were obtained from The First Affiliated Hospital of Guangzhou Medical University. The protocols used in human sample were conducted in according to the Ethical Review Committees of The First Affiliated Hospital of Guangzhou Medical University (2018-82). The written informed consent was provided from each individual.https://rnaidesigner.thermofisher.com/rnaiexpress/), an online siRNA design website provided by Thermo. The designed siRNA sequence was synthesized by GenePharma .For RP11-10A14.5 overexpression, the full-length of RP11-10A14.5 was amplified by Phanta Master Mix  using primer , and then inserted into the vector pCDH-CMV-MCS-EF1-CopGFP. The siRNA sequence targeting RP11-10A14.5 was designed by BLOCK-iT™ RNAi Designer . microRNA expression was determined by miRNA 1st Strand cDNA Synthesis Kit by stem-loop and miRNA Universal SYBR qPCR Master Mix (Vazyme Biotech). All primer used in this study were showed in Table 1.Total RNA was extracted using TRIzol reagent (Invitrogen) and the RNA concertation was determined by NanoDrop 2000. Then RNA was reverse transcribed to cDNA using a PrimeScript™ RT reagent kit  according to manufacturer’s instructions. RP11-10A14.5 expression was determined by SYBR Green Master Mix  on Biorad . 2The cellular distribution of RP11-10A14.5 was detected using FISH assay. H1299 cells were fixed using 4% formaldehyde and then hybridized with probe labeled with digoxigenin , and incubated at 37 °C overnight. Subsequently, cells were incubated with anti-digoxigenin-AP  for 30 min then incubated with diluted CSPD solution overnight and imaged using a microscope.H1299 cells were harvested 48 h after transfection by trypsinization and supernatant was removed. Then, cells were stained with Annexin V-APC and 7-AAD by using the AnnexinV-APC7-AAD apoptosis kit  following the manufacturer’s instruction then analyzed by flow cytometry . For cell cycle assay, H1299 cells were stained with PI by using the cell cycle and apoptosis analysis kit  following the manufacturer’s instruction, and the stained cells were analyzed using flow cytometry . The distribution of cells in G0/G1, S, and G2/M phase were calculated using FlowJo V10.Cell invasion assays was performed using a 24-well Transwell chamber  coated with Matrigel (Corning). Cells (1 × 105) were added to the upper layer of chamber and 800 µl of 10% FBS medium was added to the lower layer of chamber. After 24 h of incubation, cells in the upper part of the chamber were wided away using a cotton swab. Cells that had invaded the lower chamber were fixed with 4% paraformaldehyde (PFA) for 30 min and stained with 1% crystal violet.Cells were seeded into the wells of 96-well plates (1000 cells/well) containing 100 ml of RPMI-1640 medium. Each well was added with 10 µl MTT solution, and the 96-well plate was incubated for 1 h at 37 °C. Then, the supernatants of each well were removed and 150 μl of dimethyl sulfoxide (DMSO) solution was added into each well and incubated for 15 min at 37 °C. Finally, the optical density (OD) value at 570 nm of each well was measured using microplate reader.Cells were seeded into each well of the 24-well plate. A 200 μl pipette tip was used to scrape the cells and thereby creating a cell-free area among the cell monolayer. The change of the cell-free area was measured to evaluate the ability of the cell migration. The 24-well plate was incubated at 37 °C. Pictures were taken at 0, 12 and 24 h.Cells were fixed using 4% paraformaldehyde for 10 min at room temperature, then a permeabilization protocol was applied. The slides were then incubated with primary antibodies of E-Cadherin , N-Cadherin  and Vimentin . After incubating with second antibodies of AF594-labeled anti-Mouse IgG (1:200) or AF647-labeled anti-Rabbit IgG (1:200), slides were incubated with DAPI (1:1000) for nuclear staining. The slides were imaged using a microscopy.Cell lysis was attained using RIPA buffer  containing protease and phosphatase inhibitors  then concentration was determined using standard bicinchoninic acid (BCA) test  following manufacturer guideline. Proteins were separated on 12% SDS-PAGE gel and then transferred to PVDF membrane which was blocked using 5% nonfat milk, and incubated with primary antibodies overnight at 4 °C and then secondary antibody next day. The protein bands were detected using an ECL kit . The antibodies used were as follow: BAX , BAK , CASPASE-3 , GAPDH , goat anti-rabbit IgG .The “limma” data package in R software was used to determine the differentially expressed gene between high and low RP11-10A14.5 expression groups . An adjuGene set enrichment analysis (GSEA) was performed using the R package “Cluster Profiler”  to identhttp://www.mircode.org/). We used the animal transcription factor database  predicting the possible binding transcription factors of RP11-10A14.5, as well as the transcription factors that may regulate the target protein.We predicted the target miRNAs of RP11-10A14.5 and the possible miRNAs bound to the target proteins using online database miRcode (http://gepia.cancer-pku.cn/) [Survival analysis for DEGs in LUAD was performed using GEPIA online database (pku.cn/) . We diviH1299 cells  were subcutaneously transplanted into the backs of 8–10 weeks female nude mice for 21 days. At 7 days after inoculation with WT H1299 cells, RP11-10A14.5 siRNA or scramble siRNA (5 nmol/kg) were intratumorally injected every 3 days for addition 2 weeks. Tumor growth was measured using calipers, and the tumor volume was calculated as V = (width)2 × length/2. All mice were sacrificed at day 22 for the collection of their lung and tumors.R (4.0.2) software was used to perform all statistical analyses. A two-tailed student t-test was used for comparison between two groups. Univariate Cox regression analysis was used to evaluate to analyze the association between clinicopathologic parameters and survival time. A two-sided P < 0.05 was considered statistically significant in all the statistical analysis.We first identified differentially expressed lncRNA between tumor tissues and normal tissues using the TCGA LUAD cohort. Based on the cutoff criteria of |logFC|> 2 and adjusted P value < 0.05, we acquired a total of 175 differentially expressed lncRNAs of LUAD, 101 of which were differentially up-regulated lncRNAs and 74 were differentially down-regulated lncRNAs  was constructed. The expression levels of RP11-10A14.5 in the H1299-RP11OV cell line were significantly upregulated compared to the control group, which was confirmed by RT-qPCR  were identified as DEGs between the high RP11-10A14.5 and low RP11-10A14.5 groups, as shown in the volcano plot


Arachis hypogaea L., AABB) is an allotetraploid hybrid between two diploid peanuts, A. duranensis (AA genome) and A. ipaensis (BB genome). Miniature inverted-repeat transposable elements (MITEs), some of which are known as active nonautonomous DNA transposons with high copy numbers, play important roles in genome evolution and diversification. AhMITE1, a member of the MITE family of transposons, but information on the peanut genomes is still limited. Here, we analyzed AhMITE1, AuMITE1 and ApMITE1 in the cultivated (A. hypogaea) and two wild peanut (A. duranensis and A. ipaensis) genomes.The cultivated peanut (AhMITE1, AuMITE1 and ApMITE1 family members, respectively. These three family members exhibited highly conserved TIR sequences, and insertions preferentially occurred within 2 kb upstream and downstream of gene-coding and AT-rich regions. Phylogenetic and pairwise nucleotide diversity analysis showed that AhMITE1 and ApMITE1 family members have undergone one round of amplification bursts during the evolution of the peanut genome. PCR analyses were performed in 23 peanut varieties and demonstrated that AhMITE1 is an active transposon and that hybridization or chemical mutagenesis can promote the mobilization of AhMITE1.The cultivated and the two wild peanut genomes harbored 142, 14 and 21 AhMITE1, AuMITE1 and ApMITE1 family members were identified based on local BLAST search with MAK between the cultivated and the two wild peanut genomes. The phylogenetic, nucleotide diversity and variation copy numbers of AhMITE1, AuMITE1 and ApMITE1 members provides opportunities for investigating their roles during peanut evolution. These findings will contribute to knowledge on diversity of AhMITE1, provide information about the potential impact on the gene expression and promote the development of DNA markers in peanut.The online version contains supplementary material available at 10.1186/s12864-022-08732-0. Arachis hypogaea L.), also known as groundnut, is an allotetraploid (2n = 4x = 40) resulting from the hybridization of two wild diploids A. duranensis (AA genome) and A. ipaensis (BB genome). Peanut is an important oil crop with global production of 53.64 Mt (with shells) from an area of 31.57 Mha [The cultivated peanut (oid 2n = x = 40 reDs (Dissociation) was the first transposable element discovered by McClintock in maize [TEs are major components of many plant and animal genomes and have been found in virtually all species investigated to date , 7. Ds  rice mutant [ZmNAC111 is associated with seedling drought tolerance [ZmNAC111 promoter region was correlates with lower ZmNAC111 expression in maize [stowaway-like MITE embedded in the 3'-UTR of the agronomically important gene Ghd2 directly represses its protein synthesis, affecting grain number, plant height, and heading date in rice [ahFAD2B gene caused a frameshift, resulting in the high-oleate phenotype in Mycogen-Flavo and M2-225 mutants [AhMITE1 in a gene for fatty-acid desaturase possessed excision activity [AhMITE1 markers were developed and used for mapping [Miniature inverted-repeat terminal elements (MITEs) are short nonautonomous class II transposons that do not encode transposase –18. MITEcterized –23. MITEcterized . MITEs pcterized , as theyanalysis , 27. Tane mutant . In maizolerance . Previouin maize . A stowa in rice . In peanactivity . Subsequ mapping –34. WithAhMITE1. We identified the numbers of AhMITE1 copies in cultivated peanut and its wild ancestors A. duranensis (AA genome) and A. ipaensis (BB genome). Following the genomic sequence, phylogenetic tree analyses of AhMITE1 indicated that these elements clustered into five subfamilies. The genomic distribution and preferences of insertional loci were also investigated. The results improve understanding of the potential impact of AhMITE1 on differentiation in cultivated and wild peanut species.In this study, we used peanut sequencing data to determine the characteristics of AhMITE1 family in the genomes of peanut, we performed a BLASTN analysis of genome to identify AhMITE1 in the reference genome of A. hypogaea cv. Tifrunner and the two wild genomes of A. duranensis (AA genome) and A. ipaensis (BB genome). A previous study showed that the 205 bp AhMITE1 includes a 9 bp target duplication (TSD) and 25 bp terminal inverted repeats (TIRs) [AhMITE1 harboring the TSD and TIR sequences in the cultivated peanut genome were identified, and these copies shared over 90% similarity  , 31. FinAhMITE1 elements were also identified in the two wild genomes. Fourteen and 21 AuMITE1 (AuMITE1_1 to AuMITE1_14) and ApMITE1 (ApMITE1_1 to ApMITE1_21) elements were obtained from the A. duranensis and A. ipaensis genomes, respectively  occurred 2 kb upstream and downstream of gene-coding regions, and 25 (17.61%) and 29 events (20.42%) were located in intergenic and intron regions, respectively  and 52.0% (133 of 256) of ectively , 35. InvA. duranensis and A. ipaensis genomes, we found that the locations of insertion events were similar to those in A. hypogaea. In the A. duranensis genome, 2 (14.29%) and 3 (21.43%) insertion events occurred in introns and 2 kb upstream or downstream of gene-coding regions, respectively  and 6 (28.57%) insertion events were located in introns and 2 kb upstream or downstream of gene-coding regions, respectively . Clade I was the largest branch with 120 members. Clade I and Clade II comprised 120 AhMITE1 members and only one member, respectively, and were two subgroups from one branch , and found that AhMITEs insertion into the promoter of Arahy.7UQ7HQ  and Arahy.8H8JUA  would reduce the genes expression level compared with no AhMITEs insertion  and A. ipaensis (21) genomes. DNA methylation, generally considered to be a heritable epigenetic modification that functions in silencing TEs, plays roles in the maintenance of genome stability, genetic imprinting and the regulation of gene expression [AhMITE1, AuMITE1 and ApMITE1 members maintained transpositional activation after the differentiation of cultivated and wild peanut.Genome-wide analysis revealed that different copy numbers of pression –41. Methpression . Thus, tAhMITE1 members preferentially inserted within 2 kb upstream or downstream of the gene-coding and genetic regions, and no insertions occurred in exons of the gene regions. Previous studies have also demonstrated that the insertion of both mJing and mPing elements were preferentially in the flanking region of the gene [AhMITE1 elements were preferentially located at the ends of chromosome arms rather than in the central regions of chromosomes. Bertioli et al. (2019) demonstrated that gene densities are highest in distal chromosome regions and DNA transposons are more frequent in euchromatic arms in peanut [AhMITE1 elements preferentially inserted into the promoters of gene-coding regions and were located in the distal chromosome regions. As for MITEs, the density of genes is higher in distal regions of chromosomes.The the gene . In addin peanut . Taken tAhMITE1 elements may reflect the amplification characteristics during peanut evolution. The pairwise nucleotide diversity of AhMITE1, AuMITE1 and ApMITE1 members in cultivated and wild peanut revealed the occurrence of different amplification pattern bursts during peanut evolutionary history, which is similar to patterns observed in the MITE families in rice [AuMITE1 is lower than that of AhMITE1 and ApMITE1 members indicating that AuMITE1 amplification occurred before AhMITE1 arose during peanut genome evolution. Therefore, both phylogenetic and pairwise nucleotide difference analyses demonstrated that AhMITE1, AhMITE1 and AhMITE1 families have experienced one-time burst expansion during peanut evolution. In Supplementary Fig. AuMITE1 and 21 ApMITE1 members were clustered into two Clades, 14 AuMITE1s were clustered into one Clade and 20 ApMITE1s were clustered into another Clade , high-oleic acid varieties , and mutant lines  were used. In addition, seeds of the wild peanut species two wild diploids A. duranensis and A. ipaensis were obtained from the USDA or NCSU germplasm collections. Peanut seeds were germinated in distilled water and then planted in pots filled with matrix media. All peanut plants were grown in a greenhouse, and the growth conditions were set as 16 h/8 h of light/dark at 24 °C. For the field trials, peanut plants were grown in a field in Qingdao , China.To validate A. hypogaea cv. Tifrunner) and two wild peanut (A. duranensis and A. ipaensis) reference genome sequences were downloaded from the peanut genome database . A local BLAST search with MAK was performed using the 205 bp AhMITE1 as a query [–71 and the similarity > 90.00%. The target sequences were discarded if they did not satisfythese conditions. In total, 142, 14 and 21 full-length AhMITE1, AuMITE1 and ApMITE1 members were discovered, respectively. Information about all of the AhMITE1, AuMITE1 and ApMITE1 members is provided in Supplementary Tables Both the cultivated  using TBtools ) [All vanced)) .AhMITE1, AuMITE1 and ApMITE1 members was performed using CLUSTALW with default parameters [AhMITE1, AuMITE1 and ApMITE1. Nine bp and 21 bp TSD and TIR sequences of AhMITE1, AuMITE1 and ApMITE1 were depicted using WebLogo 3.0, respectively [A multiple sequence alignment of rameters . TSD andectively .AhMITE1, AuMITE1 and ApMITE1 members, 142 AhMITE1, 14 AuMITE1 and 21 ApMITE1 insertion sites were aligned with the A. hypogaea, A. duranensis and A. ipaensis genomes (https://peanutbase.org/gbrowse_peanut1.0), respectively. Based on each distance between an insertion site and the annotated gene, the position of insertion was categorized as being in the 3’-UTR, 5’-UTR, exon or intron, upstream or downstream of the annotated gene coding region. If the distance between the insertion site and the annotated gene exceeded 8 kb (> 8 kb), the insertion site was categorized as being in an intergenic region.To analyze the insertion preferences of AhMITE1, 14 AuMITE1 and 21 ApMITE1 members were extracted from the genomes of A. hypogaea, A. duranensis and A. ipaensis, respectively. In addition, 142, 12 and 21 of sequences 205 bp in length were randomly selected with TBtools (Sequence Toolkit/Fasta Tools/Fasta Extract (Basic)) from the A. hypogaea, A. duranensis and A. ipaensis genomes, respectively [Sample 100 bp upstream and downstream sequences with TSDs of 142 ectively . A PERL AhMITE1, AuMITE1 and ApMITE1 members in the peanut genome, we calculated the pairwise nucleotide diversity for each species. Pairwise nucleotide diversity among AhMITE1, AuMITE1 and ApMITE1 elements in the A. hypogaea, A. duranensis and A. ipaensis genomes was calculated using a PERL script and MEGA [To investigate the amplification of and MEGA . If therAhMITE1, 14 AuMITE1 and 21 ApMITE1 members from A. hypogaea, A. duranensis and A. ipaensis genomes were aligned using MUSCLE, respectively [In total, 142 ectively . The aliectively , and visectively .AhMITE1 insertions in the cultivated peanut genomes, PCR primers were designed based on the flanking sequences of the AhMITE1 insertion. PCR amplifications were performed using 10 ~ 50 ng peanut genomic DNA in a 5 μL reaction volumes containing 0.2 µL of LA Taq® DNA polymerase (1 U), 0.2 µL of dNTP (200 mM), 1 µL of 10 × PCR buffer and 1 µL each of forward and reverse primers (30 μM). The PCR protocol was performed with an initial denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 50 °C for 30 s and 72 °C for 45 s, and a final extension at 72 °C for 10 min. The products were separated using polyacrylamide gel electrophoresis (PAGE) in 1% (w/v) agarose gels to distinguish the presence or absence polymorphisms of AhMITE1 insertions in the cultivated peanut genomes.To validate the 12–18 primer. RT-PCR was performed using a CFX96 real-time system  and the peanut expressing gene U6 was used as the internal control to normalize the gene expression data. Relative expression levels were calculated from three biological replicates. qRT-PCR was performed with a CFX96 real-time system . All reactions were performed with three biological replicates. Statistical analysis and sample comparisons were performed using the relative quantification method (2−ΔΔCT).Total RNA from the leaves was extracted using TRIzol reagent (Thermo Fisher Scientific) and purified using a Qiagen RNeasy Kit  according to the manufacturer’s instructions. First-strand cDNAs were synthesized using SuperScript RT Kit (Thermo Fisher Scientific) with an oligo(dT)The primers used in this study are listed in Supplementary Table Additional file 1:Supplementary fig 1. Additional file 2: Supplementary fig 2.Additional file 3: Supplementary fig 3.Additional file 4: Supplementary fig 4.Additional file 5: Supplementary fig 5.Additional file 6: Supplementary fig 6.Additional file 7: Supplementary fig 7.Additional file 8: Supplementary fig 8.Additional file 9: Supplementary fig 9.Additional file 10: Supplementary fig 10.Additional file 11: Supplementary table 1.Additional file 12: Supplementary table 2. Additional file 13: Supplementary table 3.Additional file 14: Supplementary table 4. Additional file 15: Supplementary table 5. Additional file 16: Supplementary table 6. Additional file 17: Supplementary table 7. Additional file 18: Supplementary table 8.Additional file 19: Supplementary table 9. Additional file 20: Supplementary table 10.Additional file 21: Supplementary table 11.Additional file 22: Supplementary table 12.


The pioneering experiments of Anfinsen on the oxidative folding of RNase have been revisited discovering some details, which update the statement of his dogma and shed new light on the leading role of the correct disulfide in the attainment of the native structure. CD analysis, mass spectrometry, fluorescence spectroscopy and enzyme activity indicate that native disulfides drive the formation of the secondary and tertiary structures that cannot be entirely formed in their absence. This opposes a common opinion that these structures are first formed and then stabilized by the native disulfides. Our results also indicate that a spontaneous re-oxidation of a reduced RNase cannot produce a complete recovery of activity, as described by many textbooks; this can be obtained only in the presence of a reshuffling solution such as GSH/GSSG. Anfinsen dogma” ,132+ andFor sake of simplicity in the present work, we will continue to use the term auto-oxidation or spontaneous oxidation (used by Anfinsen and many other authors and textbooks) instead of the more correct “metal catalyzed oxidation”.2+ did not overlap the one of the native enzyme, indicating lower amounts of beta sheets and turns , we also replicated this procedure obtaining a new reduced RNase . The full oxidation of this reduced protein is again slow (4.5 day at 25 °C)  and CD aFurthermore, the replica of a famous experiment of re-oxidation of rRNase II at extreme low concentrations (1.8 µM), capable of recovering 100% of the original activity in 1 h, as described by Anfinsen , gave diThe results claimed by Anfinsen for a spontaneous complete recovery of activity generated the belief that the reduced RNase spontaneously forms all its native structure and that proper disulfides can be formed later as stabilizers. This is what is described in many textbooks ,2,3. HowThe recovery of activity, obtained using different reduction procedures, prompted us to investigate if different reductive conditions may generate structurally different rRNases . CD spectra suggest that rRNase I and rRNase II display not negligible differences in secondary structure possibly responsible for the observed different restoration of activity A. For exThe rRNase I displays a much more fluorescence emission compared to the rRNase II confirming also different tertiary structures B. Anfinsen used 8 M urea and a relevant amount of β-ME (about 0.6 M) to reduce all the four disulfides in RNase . The preThe incubation of native RNase with DTT causes a progressive and complete reduction of the four disulfides even in the absence of urea A. DuringA comparison of the CD spectra of the rRNase I and II, obtained in the presence of urea, with rRNase III, obtained in absence of urea , suggests very similar reduced structures showing a relevant lack of secondary structures more evident for beta-sheets compared to the native form . TherefoSurprisingly, the reduction of only one disulfide is able to generate some not negligible structural changes by lowering the amount of beta structures and reducing the activity to 74% . Other p+1 monisot. = 2401.166 m/z, corresponding to the fragment 40–61 of RNase (CKPVNTFVHESLADVQAVCSQK) with a non-native disulfide bridge, i.e., Cys40-Cys58   A–C. Both164 m/z) D.This finding is a strong indication that one of the two free cysteines, originated by DTT reduction of a single disulfide, interchanges with another distant disulfide and this becomes possible only if relevant secondary and tertiary structural changes occurred.Only a few experimental studies about the partial reduction of RNase have been performed, but they support our results. In particular, the study of Li and co-workers  identifiA further experiment, never done in the past, was designed to observe the evolution of the structure and activity of the reduced RNase, when incubated avoiding the disulfide formation . The rRNase IV was incubated at pH 7.4 in the presence of 0.2 mM DTT. Either after one hour or after 24 h, no appreciable recovery of activity was observed. CD spectra also indicate a relevant absence of alpha structures (~−70%), and an evident but not dramatic lack of beta structures (~−30%) .We believe this result to be important as, for the first time, it suggests that without disulfides a reduced RNase is completely unable to re-form native-like secondary structures.a beautiful example of how an entirely acceptable conclusion can be reached that is entirely wrong because of the paucity of knowledge at that particular time” [In 1955, Anfinsen described a curious behavior of RNase. Apparently, prolonged incubation of this enzyme in 8 M urea at 5 °C did not produce any loss of activity . This rear time”   indicate that this enzyme displays a different secondary structure with a lower amount of beta structures and a progressive loss of activity . In partIn conclusion, the well accepted assumption that RNase refolds spontaneously into correct secondary and tertiary structures and that disulfides consolidate such natural architecture must be completely refused. An opposite scenario it seems more reasonable, i.e., that the native disulfide formation is the necessary requirement to assemble the polypeptide into complete and correct alpha and beta structures. Refined CD analyses, not disposable sixty years ago, advise for this conclusion. Obviously, CD spectra do not fulfill any information on the tertiary structure, but some indications can be obtained by the changes of the fluorescence emission of the six tyrosines present in RNase. For example, the relevant increase in fluorescence emission at 300 nm (compared to the one of the native enzyme) observed in all the re-oxidized RNases under different conditions, points for different tertiary structures beside the above described different secondary structure. More precisely, it appears that in these non-native structures, tyrosines are internalized in a more hydrophobic environment compared to the native enzyme . AlternaThe reshuffling procedure, necessary for a complete recovery of activity indicates that some uncorrected disulfides are first formed with only partial development of secondary structures. The continuous breaking and reformation of these disulfides, promoted by catalytic amount of reducing agent, or by a mixture of GSH/GSSG, is the mechanism needed for the formation of all correct disulfides. This event is crucial and precedes the definitive folding into all secondary and correct tertiary structures . The theIn conclusion, this study describes new details of the in vitro oxidative pathway described many years ago by Anfinsen, but we are aware that the in vivo process can proceed in different ways. The recent discovery of an ultra-rapid glutathionylation of Cys95 ,32 when Ribonuclease A (RNase) from bovine pancreas , bromopyruvic acid, copper sulfate, dithiothreitol (DTT), ethylendiamminotetreaacetic acid (EDTA), 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), L-glutathione (GSH), oxidized glutathione (GSSG), β-mercaptoethanol (β-ME), ribonuclease A activity kit and all other reagents were purchased from Sigma-Aldrich . The GSH solutions were prepared immediately before use and the GSSG concentration was less than 1% as assayed by standard analytical procedures.All organic solvents were of LC-MS grade. Acetonitrile (ACN), methanol (MeOH), formic acid (FA) and water were from Merck . Trypsin (Gold MS Grade) was from Promega .−1 cm−1 at 280 nm [RNase concentration was evaluated by an extinction coefficient of 9440 Mt 280 nm .M TNBS− = 14,100 M−1 cm−1), 25 °C [RNase reduction was carried out with a protein concentration of 0.13 mM in 10 mM sodium phosphate buffer pH 7.4, 8 M urea, 1 mM EDTA and 10 mM DTT at 37 °C for 120 min. The reduced protein was passed through Sephadex G-25 column (1 × 20 cm) equilibrated in 10 mM sodium phosphate buffer pH 7.4 at 25 °C (through the manuscript the reduced protein after this process is indicated as rRNase I), or G-25 equilibrated in 0.1 M acetic acid at 25 °C (through the manuscript the reduced protein after this process is indicated as rRNase II). The number of -SH/mole of the eluted protein was titrated with DTNB at pH 8.0 (ε), 25 °C .M TNBS− = 14,100 M−1 cm−1), 25 °C. Alternatively, reduction of RNase 0.1 mM (or 0.2 mM), in absence of denaturant, was carried out in 0.1 M sodium phosphate buffer pH 7.4, 1 mM EDTA and 100 mM DTT at 37 °C for different times. After reduction, the excess of DTT was removed by passing through a Sephadex G-25 column (1 × 20 cm) equilibrated with 10 mM sodium phosphate buffer pH 7.4 at 25 °C . The number of -SH/mole of the eluted protein was titrated with DTNB at pH 8.0  for the preparation of rRNase I omitting the Sephadex G-25 chromatography. The rRNase IV was diluted to a final concentration of 2 µM in 10 mM sodium phosphate buffer pH 7.4, 1 mM EDTA, 0.2 mM DTT and 0.05 M urea. The solution was incubated at 37 °C for 1 and 24 h before the CD analyses and enzyme activity assays. 2+ 10 µM at 37 °C; (iv). rRNase I (14 µM) in 20 mM Tris-HCl buffer pH 8.5, Cu2+ 0.3 µM at 25 °C; (v). rRNase I (14 µM) in 20 mM Tris-HCl buffer pH 8.5, 11 µM β-ME, and Cu2+ 0.3 µM at 25 °C; (vi). rRNase I (14 µM) in 20 mM Tris-HCl buffer pH 8.5, Cu2+ 1 µM at 25 °C; (vii). rRNase I (14 µM) in 20 mM Tris-HCl buffer pH 8.5, Cu2+ 10 µM at 25 °C; (viii). rRNase II (14 µM) in 20 mM Tris-HCl buffer pH 8.5 at 37 °C; (ix). rRNase II (14 µM) in 20 mM Tris-HCl buffer pH 8.5 at 25 °C; (x). rRNase II (1.8 µM) in 20 mM Tris-HCl buffer pH 8.5 at 37 °C; (xi). rRNase II (1.8 µM) in 20 mM Tris-HCl buffer pH 8.5 at 25 °C; (xii). rRNase II (14 µM) in 20 mM Tris-HCl buffer pH 8.5, Cu2+ 0.3 µM at 25 °C; (xiii). rRNase II (14 µM) in 20 mM Tris-HCl buffer pH 8.5, Cu2+ 1 µM at 25 °C; (xiv). rRNase III (14 µM) in 20 mM Tris-HCl buffer pH 8.5 at 25 °C; (xv). rRNase III (14 µM) in 20 mM Tris-HCl buffer pH 8.5 at 37 °C. At different times, the number of -SH/mole were determined using DTNB as titrant, and aliquots were taken to perform CD, fluorescence and activities analyses. The re-oxidation of rRNase I, rRNase II and rRNase III was carried out in sixteen different manners: (i). rRNase I (14 µM) in 20 mM Tris-HCl buffer pH 8.5 at 37 °C; (ii). rRNase I (14 µM) in 20 mM Tris-HCl buffer pH 8.5 at 25 °C; (iii). rRNase I (9.2 µM) in 20 mM Tris-HCl buffer pH 8.5, CuFinally, (xvi). the oxidation of rRNase II using the mixture GSH/GSSG was performed according to the following procedure: rRNase II (2 µM) was incubated with GSH (2 mM) and GSSG (0.4 mM) in 50 mM sodium phosphate buffer pH 7.5, 5 mM EDTA at 30 °C. At different times an aliquot was analyzed for enzymatic activity.w/v), and 0.2 mM EDTA in 50 mM sodium acetate buffer pH 5.0. Only for rRNase IV, the concentrations of EDTA, urea and DTT were 40 µM, 2 mM and 8 µM, respectively. For the measurements of native RNase in 6 M, 7 M, and 8 M urea, the RNA was dissolved in the reaction buffer with 6 M, 7 M, or 8 M urea, respectively. The recovered activity was monitored spectrophotometrically in continuous at 300 nm, 25 °C. The data were reported as percentage of the ratio between sample and native RNase. Only in the case of native RNase at different urea concentrations, data were reported as normalized absorbance ratio between sample and native RNase.Activities of native RNase, native RNase at different urea concentrations, rRNase I and II, rRNase III with different amount of disulfide bonds, rRNase IV with 1 mM EDTA, 0.2 mM DTT and 0.05 M urea, and re-oxidized rRNase (i-xvi as reported above) were assayed by the ribonuclease A detection kit  which uses RNA as substrate . The subCD spectra of native RNase, native RNase in 0.03 M urea, rRNase I, rRNase II, rRNase III, and rRNase IV in 0.03 M urea, 625 µM EDTA, 0.125 mM DTT were performed in 10 mM sodium phosphate buffer pH 7.4, at 25 °C, and in all cases with a protein concentration of 1.25 µM using a spectropolarimeter Jasco J-1500 . The setting panel was: slit 2 nm, sensibility 20 mdeg, resolution 0.5 nm, and range values 190–260 nm using a quartz cuvette of 0.5 cm light path. CD spectra of native RNase and rRNase in the presence of 0.03 M urea cannot be extended below 200 nm due to the interference of urea. CD spectra for all the samples of re-oxidized rRNase I and re-oxidized rRNase II were acquired in the same manner. The analyses of CD spectra were performed using DichroWeb  and BeStAll the fluorescence analyses of native, reduced RNases (I–III) and re-oxidized rRNases (I and II) were performed on a Fluoromax-4 Horiba spectrofluorometer with an asymmetric quartz cuvette of 1 × 0.4 cm path length at 25 °C.The fluorescence emission spectra of native, reduced RNases (I–III) and re-oxidized rRNases (I and II), in all cases 1.25 µM, were recorded in 10 mM sodium phosphate buffer pH 7.4 between 290 and 350 nm, using the following parameters: excitation wavelength of 275 nm and slits 5–8 nm.w/w) ratio with respect to the protein content. Enzymatic digestion was stopped by addition of 1 μL of 100% FA, and then 1μg of total protein content per sample was used for MS analyses.The rRNase III at three different times of reduction , was diluted to a final concentration of 1.25 μM in 10 mM sodium phosphate buffer with 1 mM bromopyruvic acid that alkylates residual protein cysteines within 1–2 s. Then, the samples were lyophilized. The samples (50 μg each one) were submitted to an overnight digestion at 3 °C using a porcine trypsin gold  in 1:50 (v/v) as eluent A and ACN/FA  as eluent B in the following gradient elution: (a) 5% B (2 min), (b) from 5% to 60% B (30 min), (c) from 60% B to 99% (10 min), (d) 99% B (10 min), (e) from 99% to 5% B (2 min), and (f) 5% B (10 min) at a flow rate of 0.3 μL/min. The injection volume was 5 μL corresponding to 1 μg of total protein content per sample. Peptide trapping and concentration were obtained by loading the sample for 5 min into the Acclaim PepMap 100 nano-trap cartridge operating at 10 μL/min in eluent A. Chromatographic separations were performed at 35 °C. The Orbitrap Elite instrument was operating in positive ionization mode, performing MS/MS fragmentation by higher energy collisional dissociation (HCD) of the five most intense signals of each spectrum, measured at a 60,000 resolution in 150–2000 m/z acquisition range, in data-dependent scan (DDS) mode. The minimum signal was set to 500.0, the isolation width to 2 m/z, the default charge state to +2, and the activation Q to 0.25 MS/MS spectra acquisition was performed in the Orbitrap at 60,000 resolution.Nano-HPLC/nano-ESI-Orbitrap Elite analyses were performed on an UltiMate 3000 RSLCnano System coupled to an Orbitrap Elite MS detector with an EASY-Spray nano-ESI source . EASY-Spray column 15 cm × 50 μm ID, PepMap C18  (Thermo Fisher Scientific), was used for bottom-up analyses, in coupling to an Acclaim PepMap 100 cartridge  (Thermo Fisher Scientific). Bottom-up nano-HPLC-MS/MS analyses were performed using aqueous solution of FA . Data were obtained from three independent experiments performed in different days by the same operators using the same instruments. The graphic and results visualization were obtained using GraphPad Prism software v5.0 . Crystal structure of native RNase is derived by PDB id: 1FS3 , the str


Therefore, carboncapture from CO2 emissions of industrial processes is increasinglyimportant to halt and prevent the disruptive consequences of globalwarming. Covalent organic frameworks (COFs) as porous nanomaterialshave been shown to selectively adsorb CO2 in high quantitiesand with high CO2/N2 selectivity. Interactionswith amines are recognized to selectively adsorb CO2 andhelp capture it from exhaust emissions. Herein, a novel COF (Me3TFB-(NH2)2BD), which was not accessiblevia a direct condensation reaction, was synthetized by dynamic linkerexchange starting with Me3TFB-BD. Despite the linker exchange,the porosity of the COF was largely maintained, resulting in a highBET surface area of 1624 ± 89 m2/g. The CO2 and N2 adsorption isotherms at 273 and 295 K were studiedto determine the performance in carbon capture at flue gas conditions.Me3TFB-(NH2)2BD adsorbs 1.12 ±0.26 and 0.72 ± 0.07 mmol/g of CO2 at 1 bar and 273and 295 K, respectively. The COF shows a high CO2/N2 IAST selectivity under flue gas conditions . The interaction of the aromatic amine groupswith CO2 is based on physisorption, which is expected tomake the regeneration of the material energy efficient.CO Since then, COFs have gained increasingly more interestand a huge variety of different materials and applications have beenreported.5 Their permanent porosity and channel-like structurelead to a high surface area, which is of interest for, among others,energy storage,6 chemical sensing,7 photocatalysis,9 andgas separation.10 Usually, two multifunctionalbuilding blocks are linked by dynamic covalent chemistry (DCC).12 DCC refers to reversible reactions carried out under thermodynamicreaction conditions, which enables error correction within the reaction,leading to crystalline frameworks with long-range order. Additionally,the exchange of building blocks (linker exchange) in already existing,crystalline frameworks opens the possibility to synthesize COFs withimproved crystallinity13 or make COFs accessiblethat were not accessible via condensation of the respective buildingblocks.15 Qian et al. reported the first COF-to-COFlinker exchange by replacing 1,4-phenylenediamine (PA) with benzidine(BD) in an imine-linked COF, which yielded an almost complete transformation.16 The linker-exchange strategy was applied toachieve, among others, different COF linkages,20 changes in the pore hierarchy and size by different linker symmetries,23 the transformation of linear polymers into COFs25 or 3D into 2D COFs,26 and the introductionof functional groups into the framework.27Porous materials are a class of solid,permanently porous materialswith pore sizes of a few to several hundred nanometers. These porescan be classified into macropores (>50 nm), mesopores (2–50nm), and micropores (<2 nm).2 plays an importantrole in climate change. Strategies to mitigate global warming includedthe reduction of CO2 emissions via carbon capture and storage.The separation of CO2 from N2—and theassociated CO2/N2 selectivity—play acrucial role in such strategies. In more detail, CO2 captureis industrially performed by amine solutions that react with it.29 However, in this case the CO2 covalently binds, whichmeans that the regeneration process requires a lot of energy. Alternatively,porous materials as outlined above are promising candidates for carboncapture because their large surface area enables them to store largequantities of CO2. To store CO2, it must firstbe—ideally selectively—separated from a gas mixture,for example, from the atmosphere or flue gas. For direct air capture,400 ppm of CO2 in N2 is relevant because thisis the current atmospheric CO2 concentration.31 Coal flue gas contains 10–25 vol % CO2.32 Capturing the CO2 and separatingit from the other gases before the flue gas gets diluted by the atmospherecould help to reduce CO2 emissions into the atmosphere.31 In this context, the porosity of COFs is advantageous to store largequantities of gas.34Being a primary greenhouse gas, CO2/N2 selectivity can be calculatedout of pure component isotherms based on the ideal adsorption solutiontheory (IAST), which was developed by Myers and Prausnitz.35 To simulate the adsorption behavior of gas mixtures,the pure component isotherms must be measured at the same temperatureand on the same adsorbent. The basis of IAST is analogous to Raoult’slaw for vapor–liquid equilibrium.35 For IAST, the adsorbed phase is assumed to behave like an idealsolution that is in an equilibrium with the bulk adsorptive. It isassumed that the adsorbent is thermodynamically inert, that the Gibbsdefinition of adsorption applies, and that the surface area is universallyaccessible and temperature-invariant. It must be noted that IAST isonly of limited validity for polar adsorptives or mixtures, in whichone component strongly adsorbs and the other one weakly. Other limitationsare heterogeneous adsorbents and low loadings as they can lead topoor predictions of mixture adsorption. In more detail, for example,low loadings can lead to inaccurate spreading pressures, falsifyingthe selectivity calculation. If the fitted isotherms do not accuratelyreproduce the Henry constant for the specific adsorbate, the IASTcalculation will predict incorrect selectivity values.36 Nevertheless, the simplicity of these calculationsmakes IAST a widely used method for selectivity determination andprovides good results for screening the applicability of porous materialsin gas separation. The selectivity S of a binarygas mixture in IAST is defined as the ratio of the mole fractionsin the adsorbed state (q) over the mole fractionsof the bulk phase (p) of components 1 and 2 .They have been shown to store CO2 in large quantities (upto 5.19 mmol/g at 273 K and 1 bar) with good CO2/N2 selectivity values (63 at 298 K and 1 bar in a 0.1/0.9 mixture).45 Post-synthetic functionalization can further enhance the selectivity.41 The well-known amine–CO2 interaction29 makes the amine-functionalized benzidine linker 3,3′-diaminobenzidine((NH2)2BD) interesting for CO2/N2 separation. For example, this linker was employed to synthesizea benzimidazole-linked COF (IISERP-COF3),46 a semi-crystalline covalent organic polymer,47 and benzimidazole POPs for CO2 adsorption.43 Besides benzimidazole-linked porous materials, imine-linked materialshave also been investigated, which often have CO2/N2 IAST selectivity values in the range of 10–30 withoutadditional pore–wall functionalization that would then be presentinside the pores.49 To the best of our knowledge,the highest CO2/N2 IAST selectivity value sofar (185.8 at 273 K for 15% CO2) was reported by Mahatoet al. for a covalent triazine framework.50 Recently, Yaghi and co-workers studied the incorporation of aliphaticamine groups into a tetrahydroquinoline COF.51 They found high CO2 adsorptions of 0.304 mmol/g at 0.4mbar and 25 °C, which are relevant conditions for direct aircapture, and that 50% humidity even enhances the CO2 uptaketo 0.393 mmol/g at 298 K and 1 bar. Based on solid-state NMR, theyshowed that carbamates were formed during the adsorption, thereforeconfirming chemisorption interactions.Several different porousmaterials have been investigated for suchCO2 to achieve high adsorption quantitiesand high selectivity values for carbon capture. The nitrogen-CO2 interaction—especially the one of amine-CO2—has shown to be beneficial in terms of adsorption and selectivityand can be used for the rational design of novel materials for carboncapture. However, the chemical properties, e.g., the pKa value, are dependent on the nature of the nitrogen atom.Aliphatic amines, as used by Yaghi and co-workers,51 can interact more strongly with CO2 comparedto aromatic amines, leading to chemisorption. Since chemisorptionrequires higher energies for the regeneration of the material thanphysisorption, we decided to use aromatic amines, which are directlylinked to the COF backbone. In a classical condensation reaction,these additional amine groups are expected to interfere with the frameworkformation, so we used the dynamic linker-exchange strategy to obtainMe3TFB-(NH2)2BD). After the characterizationof the novel COF, we further studied the CO2/N2 adsorption and selectivity under flue gas conditions for gas separation.Thus, functional groupsare important for the interaction of porousmaterials with CO52 All other solvents, lab supplies, and glacialacetic acid (AR) were purchased from commercial sources and used withoutfurther purifications.3,3′-Diaminobenzidine was purchased from TCI Europe N.V., benzidine (98%) was purchasedform Abcr, and all chemicals were used without further purification.Mesitylene  was purchased from Fisher Scientific,1,4-dioxane (99%) was purchased from Acros Organics B.V.B.A., and2,5,6-trimethyl-1,3,5-benzenetricarboxaldehyde was synthesized before.1H and 13C{1H} cross-polarization magic angle spinning (CPMAS) solid-stateNMR (ssNMR) spectra were recorded on a Bruker AVANCE III HD spectrometerat 700.13 MHz (16.4 T) and 176 MHz, respectively. Solid-state NMRsamples were packed into 4 mm zirconia rotors and spun at MAS frequenciesof 11 and 14 kHz at 298 K. The 13C CPMAS spectra were recordedby using a CP pulse sequence. SPINAL64 decoupling was applied on protonswith a decoupling strength of 104 kHz. The 13C CPMAS spectrawere obtained with a recycle delay of 3 s, and the strength of theCP contact pulses of 0.08 kHz related to a contact time of 3 ms unlessstated differently. The 13C ssNMR spectra were referencedwith respect to adamantane . The spectrawere analyzed using MestReNova (version 14.1.0).–1.FT-IR spectrawere obtained on a Bruker Tensor 27 spectrometer with an attenuatedtotal reflection accessory called Platinum. The samples were appliedas powder on top of the crystal. A total of 64 scans were performedwith a resolution of 4 cmPowder X-raydiffraction measurements were recorded with a PhilipsX’pert-PRO at 40 kV and 40 mA from 4–40°  and from 1.5–10°. X-rayswere generated by a Cu anode Kα (1.54 λ) radiation.–5 forthe slope and intercept, respectively. The pore-size distributioncurves were obtained from the adsorption branches using the method“HS-2D-NLDFT, Carb Cyl Pores (ZTC) N2@77K”. An optimumbetween Goodness of Fit and smoothness of the pore size distributionwas aimed for. The average of three different COF batches was usedto determine the BET surface areas. Nitrogen and carbon dioxide adsorptionmeasurements were performed at 295 K with absolute pressure dosingand an equilibration time of 20 s between 3 and 1200 mbar. The freespace values were determined after the adsorption measurement. Nitrogenmeasurements at 273 K were performed under the same conditions asthose at 295 K. Carbon dioxide measurements at 273 K were performedby increment dosing up to p/p0 = 0.03 with an increment of 0.13384 mmol/g and an equilibrationtime of 20 s. The gases were considered as ideal gases under all thesecircumstances. Isotherm cycling was performed without a degassingstep in between. At 273 K, the adsorption branch was measured up to p/p0 = 0.03 with an incrementof 0.15 mmol/g, and the desorption branch was measured down to p/p0 = 0.0003 with an incrementof 0.15 mmol/g. At 295 K, the adsorption branch was measured between3 and 1050 mbar, and the desorption branch was measured between 1050and 12.5 mbar.Nitrogen adsorption–desorption measurements were performedon a MicroActive for Tristar II Plus 3030 at 77.350 K. Before themeasurement, the samples were outgassed at 120 °C overnight.Surface areas were calculated from the adsorption data using Brunauer–Emmet–Teller(BET) methods and Rouquerol criteria. The fitting parameters are 2.643± 0.142 and 4.29 ± 0.16 × 102/N2 selectivitieswere predicted basedon the ideal adsorption solution theory (IAST)36 by GraphIAST53 at 1 bar and a compositionof 0.15/0.85. The isotherms were fitted with the interpolator model.The COThermogravimetric analysis was performed on a PerkinElmer STA 6000.The sample was heated to 30 °C, the temperature was held for1 min, and afterward, the sample was heated with 10.00 °C/minto 700 °C in a nitrogen atmosphere (20 mL/min).55 The PBE functionalbased on the generalized gradient approximation was chosen to accountfor the exchange–correlation energy.56 A plane-wave basis set in combination with the projected augmentedwave (PAW) method was used to describe the valence electrons and thevalence–core interactions, respectively.57 The kinetic energy cut-off of the plane wave basis setwas set to 500 eV. Gaussian smearing of the population of partialoccupancies with a width of 0.05 eV was used during iterative diagonalizationof the Kohn–Sham Hamiltonian. The threshold for energy convergencefor each iteration was set to 10–5 eV. Geometrieswere assumed to be converged when forces on each atom were less than0.05 eV/Å. The Brillouin zone integration and k-point sampling were done with a Gamma-centered 1 × 1 ×8 and 2 × 2 × 4 grid points for the eclipsed and staggeredunit cells, respectively. The Van der Waals (vdW) interactions wereincluded by using Grimme’s DFT-D3(BJ) method as implementedin VASP.58 Simulated XRD patterns wereobtained by using VESTA (version 3.4.8).59 Coordinates of all crystal structures are provided as separate files.All DFT calculations for COFstructures were performed by using the Vienna Ab Initio SimulationPackage .3TFB-COF  and (NH2)2BD  were addedto a 50 mL round-bottom flask (RBF), together with a stirring rod,and were dissolved in 10 mL of 4:1 v/v 1,4-dioxane:mesitylene mixture.The mixture was heated to 70 °C for 5 min. Water (0.42 mL) andglacial acetic acid (0.56 mL) were added. The reaction mixture wasstirred at 70 °C for 3 days. Afterward, the reaction was cooledto RT and the precipitate was collected via Büchner filtration.The solid was dispersed in dimethylformamide (DMF), stirred at 90°C for 30 min, and collected via Büchner filtration. Thesesteps were repeated with DMF , ethanol , acetone , and hexane . After the final Büchner filtration, the COFs were driedovernight at 120 °C in a regular oven. Brown powder  was obtained. After drying, the COFs were kept ina glovebox for storage.Me3TFB-COF.The synthesis was repeated in totalthree times: twice with the amounts specified and once scaled-up to200 mg of Me3TFB-(NH2)2BD was carried out accordingto our previously published synthesisprocedure.52The direct condensation ofMe3TFB) and3,3′-diaminobenzidine ((NH2)2BD) werereacted in a 1:4 v/v mixture of mesitylene:1,4-dioxane. Acetic acidwas added as a catalyst for the reaction. The reaction mixture washeated to 70 °C for 3 days before the obtained precipitate wasisolated by Büchner filtration and subjected to the extensivewashing procedure from Dichtel and co-workers.60 Afterward, the obtained powder was dried at 120 °Covernight in a regular oven before the characterization was carriedout. The results of Fourier-transform IR spectroscopy (FT-IR), powderX-ray diffraction (PXRD) and N2 sorption analysis indicatethe formation of an amorphous polymer without crystallinity and porosity.In an attempt to directly synthesizethe amine-functionalized COF,2,4,6-trimethylbenzene-1,3,5-tricarbaldehyde (Me3TFB-BD COF was synthesized by condensationof Me3TFB and benzidine (BD) according to a previouslypublished procedure.52 Afterward, Me3TFB-BD was dispersed in a 1:4 v/v mixture of mesitylene:1,4-dioxanetogether with acetic acid and water to achieve an equilibrium betweenformed and broken imine-bonds. Additionally, (NH2)2BD was added, which can exchange with benzidine  has/have been formed.Theoretically, the linker-exchangereaction can lead to two differentbond formations, namely, imine bonds with unreacted primary aminesas le bonds 1. Both b3TFB-(NH2)2BD was measured and compared to theone of Me3TFB-BD. In short, the C=N imine band at1627 cm–1 indicates the formation of imine bonds. A broad band is visible between 3600and 3000 cm–1, which can be assigned to the N–Hstretch, confirming the linker exchange. The band at 1693 cm–1 that can be assigned to C=O stretching is significantly smaller,indicating a low amount of aldehyde groups likely present only atthe periphery of the 2D sheets.63 However, FT-IR cannot be usedto unambiguously assign the bond formation because the respectivebands 52 are too close to each other.To study the linker exchange,the FT-IR spectrum of Me3TFB-(NH2)2BD display several diffraction peaks, which areclearly baseline-separatedfrom each other at 3.5, 6.0, 6.9, 9.2, 12.0, 12.5, and 15.2°,and a broad peak at 24° . Pawley refinement was performed in triplicate , using the space group P6/m, which corresponds to a hexagonal, eclipsed stackingstructure. The refined unit cell dimensions are a = 29.58 ± 0.05 Å and c = 3.75 ±0.08 Å with wpR = 3.44 ± 0.46%and pR = 2.31 ± 0.26%. The unit celldimensions are close to those of Me3TFB-BD COF.The PXRD patterns of Mek at 24° 1C. The pSupporting Information. Next, the PXRD diffractograms were modeled forboth computed structures. The simulated PXRD diffractions of the eclipsedpattern  match well with theexperimental PXRD result, indicating an eclipsed stacking conformationfor Me3TFB-(NH2)2BD ;further details can be found in the pattern S24 match3TFB-(NH2)2BDCOF, which could not beobtained via the classical one-step condensation reaction. Thereby,the linker-exchange strategy provides a powerful tool to obtain previouslyinaccessible COFs with high crystallinity even if the functional groupon the linker molecule can interfere with the bond formation.These PXRD results confirm the formationof a crystalline Me13C cross-polarizationmagic angle spinning solid-state NMR (13C CPMAS ssNMR)was conducted. The spectrum of the newly obtained Me3TFB-(NH2)2BD sample as well as its CP build-up curve weremeasured . In comparisonwith spectra of the small model compound 2-phenylbenzimidazole  and Me3TFB-BD,65 the linkage was found to consist of imine bonds(3TFB-(NH2)2BD shows a signal at 162 ppm, clearly indicatingimine-bond formation. Additionally, the CP build-up curve of the imineCOF (Me3TFB-BD) matches with the CP build-up curve of Me3TFB-(NH2)2BD, while 2-phenylbenzimidazoleshows a significant slower build-up  as wellas 120 ppm (C-H) indicates the formation of a partlyexchanged material.To unambiguously assign the linkage, ne bonds2. In morne bonds2A. The sbuild-up 2B. This 1H-NMR was attempted, butthe COF was toostable to be completely broken down to monomers. In NMR digestion,it is aimed to depolymerize a material. Here, the COF was attemptedto depolymerize into its monomers by using DCl in D2O indeuterated DMSO-d6, before analyzing theresulting solution by 1H-NMR. Compared to Me3TFB-BD, which could be digested under the same conditions,66 no clear solution of digested Me3TFB-(NH2)2BD could be obtained, but a dispersionwas obtained. Thus, the post-synthetic linker exchange seems to enhancethe chemical stability in acidic media. To further support this, Me3TFB-(NH2)2BD was immersed in 1 M ofHCl for 5 days, and upon re-isolation and drying at 120 °C overnight,the PXRD pattern was recorded again. The COF could be isolated fromthe vial used for the stability test, showing that the material wasnot completely amorphized. The disappearance of the PXRD diffractionsindicates that the crystallinity was not retained . Therefore, Me3TFB-(NH2)2BD shows an enhanced acid stability over Me3TFB-BDbut cannot yet be considered as stable under these conditions.Digestion 3TFB-(NH2)2BD COF shows an excellentthermal stability up to 438 °C .The thermal stability was investigated using thermogravimetricanalysis (TGA). The threshold for thermal stability was determinedat the position where the COF lost maximum 5% of its initial weight.Me2 sorption measurements werecarriedout to determine the BET surface area. All three samples of Me3TFB-(NH2)2BD show almost identical adsorption–desorptionisotherms, confirming the good repeatability of the synthesis . The pore volumeat p/p0 = 0.95 was measuredto be 0.90 ± 0.05 cm3/g.As a next step, Nynthesis 3A. The ih 2.7 nm 3B. The f3TFB-(NH2)2BD can potentially be used in CO2 captureand storage,N2 and CO2 sorption experiments at 273 and 295K have been conducted . The aromatic amine groups on the (NH2)2BDlinker are expected to act as potential adsorption sides for CO2, which should lead to higher adsorption quantities comparedto the Me3TFB-BD.66 The N2 adsorption isotherms of Me3TFB-(NH2)2BD are linear, and the CO2 isotherms showa significant increase at low pressures and resemble a Langmuir isothermwithout reaching the plateau up to 1 bar. At 1 bar, CO2 adsorption capacities of 1.12 ± 0.26 mmol/g at 273 K and 0.72± 0.07 mmol/g at 295 K are found 2BD has a modest adsorptioncapacity. As explained in the 51 They reportedon approximately 2.1 mmol/g CO2 at 1 bar at 298 K. It isnoted that the difference between COF-609 and Me3TFB-(NH2)2BD is not only the type of amine, i.e., aliphaticvs aromatic, but also the number of primary and secondary amine groupsthat can interact with CO2. COF-609 consists of two primary,two secondary, and one tertiary amine per repeating unit, while Me3TFB-(NH2)2BD contains only two aromaticamine groups per repeating unit, assuming a 100% conversion duringlinker exchange. These differences are believed to contribute to thedifferences in CO2 adsorption. Furthermore, Yaghi’sCOF-609 contains additional triazine moieties that can contributeto CO2 adsorption,50 and theiraliphatic amines form carbamates with CO2, leading to chemisorbedgas. Chemisorption interactions, which cover covalent bond formation,are energetically stronger than physisorption interactions, whichare mainly based on Van der Waals interactions.1To study whether Meonducted 4A. The ure found 1. In com2 at 1 bar was measuredto be 0.12 ± 0.02 mmol/g at 273 K and 0.07 ± 0.02 mmol/gat 295 K and thus much lower compared to CO2 adsorption.For direct air capture, CO2 adsorption at 0.04 mbar isrelevant. Since the measured CO2 isotherm is not recordedat such low pressures, the first data point at approximately 4 mbarwas used to evaluate the material for direct air capture applications.At 4 mbar, Me3TFB-(NH2)2BD adsorbed0.0222 ± 0.0004 mmol/g CO2 at 273 K, which is lessthan that reported by Das et al. (∼0.3 mmol/g at 273 K and4 mbar).69 Moreover, with 0.304 mmol/gat 273 K at an even lower partial pressure of 0.4 mbar, Yaghi andco-workers reported a higher adsorption capacity.51 This shows that the material is not suitable for directair capture.Moreover, the quantity of adsorbedN2 was measured to be 0.342 ±0.046 mmol/g at 273 K and 0.183 ± 0.025 mmol/g at 295 K, respectively.69Therefore, we looked into the adsorption capacityat 150 mbar sincethis is relevant at coal flue gas conditions. At this pressure, thequantity of adsorbed COectively1. The qu3TFB-(NH2)2BD, the reversibilityof CO2 adsorptionwas investigated over five adsorption–desorption cycles . From the adsorption–desorptionisotherm, it can be seen that the desorption occurred without hysteresisexcept for the first run, indicating the reversibility of the isotherm.It was also shown that the adsorption–desorption cycles at273 K have a comparable adsorption (1.4 mmol/g vs 1.3 mmol/g) duringthe first adsorption cycle compared to the other four cycles, whichfurther supports the reversibility of adsorption. The reversibilityof the adsorption–desorption isotherm is also given at 295K . To further study the COF–CO2 interaction, the COF was subjected to CO2 adsorptionat 273 K. Without any additional treatment, the sample was then measuredby FT-IR and ssNMR immediately afterward to probe the formation ofcarbamates, which would indicate chemisorption of CO2.Both the FT-IR spectrum  andthe ssNMR spectrum  did notshow any difference compared to those of the as-synthesized Me3TFB-(NH2)2BD, and no carbamate bandsor signals could be observed. In comparison to Yaghi’s COF-609where the aliphatic amines groups were found to chemisorb CO2, the adsorption–desorption cycles and the lack of carbamatesignals in the FT-IR and NMR spectra of Me3TFB-(NH2)2BD after exposure to CO2 indicatethat the CO2–COF interaction is based on physisorptionand that the CO2 adsorption–desorption is reversible.In terms of applications, these results suggest that the materialcan be regenerated at relatively low cost in an energy-efficient way.To study the interaction involvedin Me53 softwareat coal flue gas conditions. First, the entire selectivity value rangewas scanned between 0.05 and 1.0 bar and mole fractions ranging from0.05 to 0.95. The mole fraction of 0.15 CO2/N2 was used to investigate the CO2/N2 IAST selectivityfor flue gas separation. IAST selectivity values of 83 ± 11 at273 K and 47 ± 11 at 295 K were found in a mixture of 0.15/0.85CO2/N2 at 1 bar (3TFB-(NH2)2BD is promising for gas separation applications.Here, it is useful to compare our results with that of recent literature.Das et al. studied an imine-based network and determined IAST CO2/N2 selectivities of 211 at 273 K and 100 at 298K.69 These values were validated by breakthroughexperiments, leading to a selectivity of 125 at 298 K, which is evenhigher than predicted by IAST. The quantities of adsorbed CO2 are 3.3 mmol/g at 273 K, and 2.4 mmol/g at 298 K. Another porousimine-based network, studied by Popp et al., adsorbs 1.8 and 1.2 mmol/gCO2 at 273 and 298 K, respectively, with an IAST selectivityvalue of 31 at 298 K and 1 bar.68 Huanget al. synthesized an imine-linked porphyrin COF, of which the channelwalls were post-synthetically functionalized with carboxyl groupsto enhance selectivity.33 The CO2 adsorption was reported to be 3.95 and 1.73 mmol/g at 273 and 298K, respectively, and the CO2/N2 selectivityof a 15% mixture of CO2/N2 at 298 K and 1 barwas 77, an enhancement of more than a factor of 9 compared to theunfunctionalized pristine COF. These examples show that channel–wallpostfunctionalization49 can significantly enhance theCO2/N2 selectivity, while our current approachusing post-functionalization via linker exchange can lead to comparablygood results. The adsorbed quantities significantly increased fromMe3TFB-BD to Me3TFB-(NH2)2BD, not only indicating the successful linker exchange but also showingthat the aromatic amine groups of the (NH2)2BD linker interacts with CO2.The isotherms were used to calculate the IAST selectivity withour recently developed GraphIASTat 1 bar 1. These 3TFB-(NH2)2BD, whichis not accessible with classical direct condensation reactions, wassynthesized by the linker-exchange strategy in a facile two-step synthesis.The linkage could unambiguously be assigned to imine bonds based onthe chemical shift and CP build-up curves in ssNMR, and the crystallinityand porosity were confirmed by PXRD and physisorption experiments.Upon the exchange of Me3TFB-BD to Me3TFB-(NH2)2BD, a BET surface area of 1624 ± 89 m2/g was found, which shows that the linker exchange maintainsmost of the COF porosity.A novel COF Me3TFB-(NH2)2BD wasstudied for gas separation applications of CO2 and N2. We found modest CO2 adsorption quantities andhigh CO2/N2 selectivity values based on IASTcalculations under coal flue gas conditions (83 ± 11 at 273 K/1bar and 47 ± 11 at 295 K/1 bar). The enhanced CO2 uptakein comparison with the pristine Me3TFB-BD COF is causedby the aromatic amine groups of the COF linker. While aliphatic aminegroups bind CO2 by chemisorption as shown by Yaghi andco-workers, the adsorption–desorption cycles of Me3TFB-(NH2)2BD have shown that the interactionof aromatic amine groups with CO2 are based on physisorption.As physisorption is mainly based on weak Van der Waals interactions,we therefore anticipate that the regeneration of Me3TFB-(NH2)2BD would require less energy, making it an interestingcandidate for carbon capture.Furthermore, Me