Let-7 microRNA precursors or mRNAs, and few have addressed LIN28’s role within the nucleus. Here, we show that LIN28 displays dynamic temporal and spatial expression during murine embryo development. Maternal LIN28 expression drops upon exit from the 2-cell stage, and zygotic LIN28 protein is induced at the forming nucleolus during 4-cell to blastocyst stage development, to become dominantly expressed in the cytosol after implantation. In cultured pluripotent stem cells (PSCs), loss of LIN28 led to nucleolar stress and activation of a 2-cell/4-cell-like transcriptional program characterized by the expression of endogenous retrovirus genes. Mechanistically, LIN28 binds to small nucleolar RNAs and rRNA to maintain nucleolar integrity, and its loss leads to nucleolar phase separation defects, ribosomal stress and activation of P53 which in turn binds to and activates 2C transcription factor Dux. LIN28 also resides in a complex containing the nucleolar factor Nucleolin (NCL) and the transcriptional repressor TRIM28, and LIN28 loss leads to reduced occupancy of the NCL/TRIM28 complex on the Dux and rDNA loci, and thus de-repressed Dux and reduced rRNA expression. Lin28 knockout cells with nucleolar stress are more likely to assume a slowly cycling, translationally inert and anabolically inactive state, which is a part of previously unappreciated 2C-like transcriptional program. These findings elucidate novel roles for nucleolar LIN28 in PSCs, and a new mechanism linking 2C program and nucleolar functions in PSCs and early embryo development.LIN28 is an RNA binding protein with important roles in early embryo development, stem cell differentiation/reprogramming, tumorigenesis and metabolism. Previous studies have focused mainly on its role in the cytosol where it interacts with The online version contains supplementary material available at 10.1007/s13238-021-00864-5. Dux  cells are derived from the inner cell mass of the blastocyst of mouse early embryos and have the ability to differentiate into three germ layers and germ cells, but do not differentiate into trophectoderm and extraembryonic tissues, and therefore, are recognized as pluripotent. In contrast, embryonic blastomeres at the 2-cell stage have the potential to differentiate into all cell types of embryonic and extraembryonic tissues  at the 2-cell stage, an embryo continues to develop through the four/eight-cell, morula and blastocyst stages, and prepares itself for implantation. As embryonic cells proliferate, basic anabolic metabolism and translational processes become more active. Nucleoli, the organelles involved in translation, functionally mature from nucleolar precursor bodies (NPB) during this process , we found that LIN28A is mainly cytoplasmic in post-implantation embryos, which greatly contrasts with early pre-implantation stage embryos .in vitro correspond with pre- and post-implantation epiblast cells, we determined LIN28A localization in these two types of pluripotent stem cells in culture. We observed that when cells are in colonies, in either naïve or primed state, LIN28A localizes both in the cytoplasm and the nucleolus; however, in isolated single cells, LIN28A protein is more likely to be localized in the nucleolus in naïve state cells compared with primed state cells  knockout ES single clones derived with CRISPR/CAS9, 2) ES cells derived from Lin28 knockout mice, and 3) iPS cells derived from Lin28 knockout MEF cells . We found that Lin28a knockout induced up-regulation of 2,418 genes  in ES cells cultured in the LIF/2i naïve condition, including some 2-cell/4-cell stage marker genes Zscan4d, Duxbl1, Zfp352, Gm4340, Gm12794  and 2-cell/4-cell expressing cluster 3 (C3) genes, and decreased genes expressed in later stages   and ERV expression , and this effect was rescued by overexpressing Lin28a , demonstrating induced pluripotency needs LIN28 to repress ERV expression  and iPS cells . To further verify these findings, we used CRISPR/CAS9 to generate Lin28a knockout single clones in another ES cell line E14 , and also examined let-7 overexpression ES cells in which Lin28a level is repressed . We observed a similar trend of up-regulation of 2C/4C marker genes and MERVL-int and MT2_Mm ERVs . Together, these results demonstrate that LIN28 deficiency induces a 2C/4C-like state transcriptional program in PSCs.In order to examine how LIN28 can influence gene expression, we performed RNA-seq analysis using three different settings of wild-type and 794 Fig. , we clusne Figs.  and S2G.Lin28 knockout cell culture, more cells are induced to 2C-like cells, or alternatively if the same number of existing 2C-like cells show an increase of 2C gene expression. Using a 2C::tdTomato reporter in which a tdTomato gene is under control of a MERVL promoter  . Moreover, we examined H3K9 trimethylation which is a marker for repressed Dux genes, and found Dux and Duxf1 showed a reduced level of H3K9me3 in Lin28a knockout ES cells . Together, these results demonstrate that Lin28-deficiency induced a 2C-like transcriptional and epigenetic program in cultured PS cells mainly through upregulation of Dux.We next investigated how LIN28 regulates the 2C program in pluripotent stem cells. Recent studies have described that a pioneer transcription factor, the DUX protein, directly binds to promoters and LTR elements on 2C genes and repetitive elements, and activates their transcription in cultured PSCs , indicating disrupted phase separation of nucleolar DFC. Together these data demonstrate LIN28 interacts with RNAs in the nucleolus, and its loss leads to nucleolar abnormalities including defects in nucleolar phase separation.LIN28 interacts with microRNA is Figs. C and S5His Figs. D and S5Ire Figs. E and S5Jre Figs. , we usedLin28 knockout PSCs. As expected, we found that multiple direct p53 targets are elevated in knockout ES cells , similar to Lin28 knockout. We found that Dux and 2C gene and MERVL expression were strongly induced as well as p53 protein and its targets , recapitulating the Lin28 knockout phenotype. When the stress signaling was blocked by knocking out trp53, the activated Dux and 2C genes and MERVL were rescued . Together, these data demonstrate that the interactions between LIN28 and nucleolar proteins or ribosomal proteins maintain nucleolar/ribosomal integrity and prevent cells from nucleolar stress and p53 activation.In order to further understand how LIN28 maintains nucleolar and ribosomal integrity, we investigated the protein components of the LIN28-containing complex in the nucleus, by performing LIN28A immunoprecipitation followed by mass spectrometry with cytosolic and nuclear fractions of wild-type ES cells . LIN28A nuclear interacting partners were mostly enriched with proteins in ribosome biogenesis, rRNA processing and ribonucleoprotein complex biogenesis, etc. . Indeed, almost 10% LIN28A interacting proteins were nucleolar proteins, and a total of 64% ribosomal proteins (RPs) were pulled down by LIN28A Fig. A, includlls Fig. C. Moreovlls Fig. . Lin28 kRNA Fig. C. Correslls Fig. D and 5E,us Figs. . We thusLin28 knockout led to disappearance of the NCL-marked “ring” structure in ES cells , reminiscent of the electron microscopy results showing reduced “ring” structure above  , and they all recapitulated the activated 2C transcriptional program indicated by MERVL and Dux expression and rRNA repression as well as p53 activation .Together, these data demonstrate that LIN28, the NCL/TRIM28 complex and the 2C::tdTomato positive 2C-like cell population and analyzed its transcriptome, and found significantly reduced ribosomal protein gene and cell cycle gene categories compared with 2C::tdTomato negative cells , and found that the population with higher MERVL-int and MT2_Mm expression tends to not express proliferation marker genes Pcna and Mki67, nor ribosomal protein genes such as Rpl11 and Rps6 , consistent with the fact that loss of LIN28A give rise to more of the slowly proliferating cells. Together, these results validated that at cell population level, LIN28’s regulation on 2C-like/ES cell homeostasis is associated with the nucleolar and ribosomal mechanisms elucidated above.To validate whether the above nucleolus and ribosome-related mechanisms can explain the dynamic population homeostasis of ES and 2C-like cell cultures in the wild-type condition, we first sorted the lls Fig. A, suggesDux in cultured pluripotent stem cells. Subsequently, in order to better understand the physiological role of nucleolar LIN28 in the development of early embryos, we injected a pool of Lin28a siRNAs into fertilized single cell embryos . In the wild-type ES cell program, LIN28 in the nucleolus resides in the same complex with NCL/TRIM28 and FBL or in the liquid phase with other nucleolar RNAs/proteins and peri-nucleolar chromatin containing let-7 in both the cytosol and the nucleus through blocking DICER and DROSHA complexes, respectively  and iPS cells were cultured on 0.1% gelatin-coated plates with MEF feeder cells in N2/B27/LIF/2i media  and Neurobasal medium  containing 1× N2 and B27 supplements , 100 µmol/L non-essential amino acids , and 1,000 U/mL LIF (PEPRO TECH), 1 μmol/L PD03259010 and 3 μmol/L CHIR99021 (STEMCELL Technologies) and 100 U/mL penicillin, 100 µg/mL streptomycin . For primed state media, 20 ng/mL Activin, 10 ng/mL FGF2, and 1% KSR were added to the 1:1 DMEM/F12 and Neurobasal medium containing N2 and B27. MEF cells were cultured in high-glucose DMEM  containing 10% FBS , 100 µmol/L nonessential amino acids  and 100 U/mL penicillin, 100 µg/mL streptomycin .in vitro culture, an embryo was washed through 3 drops of M2 and then cultured in a 20 µL drop of KSOM medium (Millipore) in mineral oil (Sigma-Aldrich) at 37 °C, 5% CO2. All animal care and experimental procedures were in accordance with the Animal Research Committee guidelines of Zhejiang University.Embryos were derived from 4- to 6-week-old C57BL/6J (C57) females mated to 8-week to 3-month old C57BL/6J (C57) males. Females were superovulated by injection of 7.5 IU PMSG, followed by injection of 7.5 IU of hCG 46–48 h later. Each stage of embryos were collected at the following time periods: 20–24 h after hCG (zygotes), 31–32 h (early 2-cell), 39–40 h (mid 2-cell), 46–48 h (late 2-cell), 54–56 h (4-cell), 68–70 h (8-cell), 76–78 h (16-cell), 86–88 h (early blastocysts), 92–94 h (mid blastocysts), and 100–102 h (late blastocysts) , then cloned into the gRNA-Cas9-Puro Plasmid . ESCs were nucleofected with the plasmid containing the sgRNA and Cas9 using Lonza 4D Nucleofector. 48 h after transfection ESCs were selected with 1 μg/mL puromycin for 7 days. Clones were picked and analyzed for LIN28A expression by Western blot, and the Lin28a gRNA targeted genomic region was PCR-amplified and sequenced in Lin28a knockout clones.sgRNA was designed to target the second exon of siRNA transfections were performed in PS cells with Lipofectamine 2000 (Thermo Fisher Scientific). PS cells were seeded into 12-well plate and cultured in LIF/2i medium for overnight. The next day, 800 μL LIF/2i medium without antibiotics was added into each well. Then, the transfect mixture  and 2 μL of Lipo 2000 which was diluted in 200 μL Opti-MEM medium (Gibco)) was added into each well and incubated for 6 h at 37 °C. After incubation, the medium was exchanged for fresh complete LIF/2i medium and cells were harvested for RNA extraction approximately 48 h later. The sequences of siRNA are listed in Table S3.Lin28a pooled siRNA and non-targeting pooled siRNA (Dharmacon Germany) were resuspended in RNase free water according to the manufacturer’s instructions and stored in single-use aliquots at −80 °C. siRNAs were microinjected using an Eppendorf FemtoJet microinjector and Narishige micromanipulators. Microinjection pipettes were pulled with a Sutter P-97 pipette puller. siRNA solution (2 μL of 50 μmol/L) was loaded into the pipette and ~5 pL was injected into the cytoplasm of each zygote. A relatively consistent amount was carefully injected each time. After injection, zygotes were rinsed and cultured at 37 °C with 5% CO2. The sequences of siRNA used are listed in Table S3.The ES cells were plated on gelatinized glass coverslips on primary mouse embryonic fibroblasts. Cells were fixed with 4% PFA for 30 min followed by permeabilizing with 0.5% Triton X-100/PBS for 20 min at RT. Cells were blocked in blocking buffer  for 10 min and stained with a primary antibody for 1 h at 37 °C. After washing three times for 10 min with PBS, cells were stained with a secondary antibody for 1 h at 37 °C. Antibodies used: For LIN28A , a rabbit antibody at 1:300 and a donkey anti-rabbit Alexa Fluor 488-conjugated secondary antibody (abcam) at 1:400 were used. For Fibrillarin  and NPM1 , the mouse antibodies at 1:200 and a DyLight 594 goat anti-mouse secondary antibody (EarthOx) at 1:400 were used. Following washing for another three times with PBS, DAPI was used for nucleus staining. Cells were then imaged using Zeiss LSM880 fluorescence microscope at a 63× oil objective. For high regulation microscopy imaging, LSM800 with Airyscan module was used.2 during image acquisition. Cells were transduced with pSIN-FBL-mCherry lentivirus. FRAP experiments were performed on a ZEISS  LSM800 confocal laser scanning microscope equipped with a ZEISS Plan-APO 63×/NA1.46 oil immersion objective. Circular regions of constant size were bleached and monitored overtime for fluorescence recovery. Bleaching was once every 10 s for a total of 10 min. Fluorescence intensity data were corrected for background fluorescence, and normalized to initial intensity before bleaching using GraphPad software. Resulting FRAP curves were fitted with Four parameter logistic (4PL) curve.Mouse E14 wild-type and knockout ES cells cultured on MEF cells were grown in N2B27/LIF/2i conditions and maintained at 37 °C and with 5% COEmbryos were first fixed with 1% and 2% paraformaldehyde (PFA) in 1× PBS for 3 min sequentially, followed by treatment with 4% PFA for 30 min at room temperature (RT). Embryos were washed three times with 1× PBS, permeabilized for 15 min in PBS/0.25% Triton X-100 and blocked in blocking buffer (PBS/0.2% BSA/0.01% Tween-20) for 1 h at RT, followed by incubation overnight with primary antibodies  at 4 degree or for 1 h at 37 °C. Subsequently, embryos were washed four times for 10 min each and incubated with a secondary antibody  for 1hr at 37 °C and washed three times with PBS. Nuclei were stained with DAPI for 1 min. Embryos were observed under Zeiss LSM880 fluorescence microscope at 63× magnification with an oil immersion objective.Actin or Gapdh. For microRNA expression analysis, reverse transcription and qPCR were performed with miRNA cDNA synthesis kit and qPCR assay kit , each gene was normalized to U6. All primers used are listed in Table S2.Total RNA was isolated from ESCs and miPSCs using miRNeasy kit  according to the manufacturer’s protocol, and 1 μg RNA was reverse transcribed to cDNA with HiScript II Q RT Super Mix . Gene expression was analyzed with SYBR-Green qPCR Master mix (Bio-Rad) on Bio-Rad PCR machine (CFX-96 Touch). Each gene was normalized to 2+ and Mg2+ and placed in 0.2 Ml PCR tube, immediately transferred in liquid nitrogen and stored at −80 °C. It was hybridized with 0.5 µL oligo-dT30  and 1 μL random (1 mol/L) and 1 µL dNTP mix (10 mmol/L) in 2 μL cell lysis buffer  at 72 °C for 3 min. Then, the reaction was immediately quenched on ice. After the reaction tube was centrifuged, 2 µL was used for reverse transcription with Super Script II Reverse Transcriptase 5× first strand buffer, 0.25 µL RNase inhibitor (40 U), 0.06 µL MgCl2 (1 mol/L), 2 µL betaine (5 mol/L), and 0.5 µL Reverse Transcriptase Superscript II (Takara). Reverse transcription was carried out in the thermocycler at 42 °C for 90 min, 70 °C for 15 min, and then 4 °C for holding. Subsequently, cDNA was diluted 1:10 (v/v) with RNase free water and used for a qPCR amplification in triplicate with SYBR Green Master (Vazyme) in a final volume of 20 µL per reaction as manufacturer’s instructions. The primers used are listed in Table S2.Ten morulae were rinsed in 0.2% BSA/PBS without CaA total amount of 2 μg RNA per sample was used as input materials for the RNA sample preparation. mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Purified mRNA was fragmentated at 94 °C for 15 min by using divalent cations under elevated temperature in NEBNext first strand synthesis reaction buffer (5×). First strand cDNA was synthesized using random primer and ProtoScript II reverse transcriptase in a preheated thermal cycler as follows: 10 min at 25 °C; 15 min at 42 °C; 15 min at 70 °C. Immediately finished, second strand synthesis reaction was performed by using second strand synthesis reaction buffer (10×) and enzyme mix at 16 °C for 1 h. The library fragments were purified with QiaQuick PCR kits and elution with EB buffer, then terminal repair, A-tailing and adapter added were implemented. The products were retrieved and PCR was performed for library enrichment. The libraries were sequenced on an Illumina platform.Embryos were collected (5 embryos per sample) in 0.2 mL PCR tubes with a micro-capillary pipette and processed into cDNA with Superscript II reverse transcriptase as described described previously . 1 × 10PS cells were seeded into 6-well plates and cultured for 48 h. The cells were harvested and cell suspensions were pelleted and washed twice with PBS, and fixed in 70% ice-cold ethanol overnight at −20 °C. The samples were centrifuged at 1,100 rpm for 2 min at 4 °C. The pellets obtained were washed twice with cold PBS and treated with 100 μg/mL RNase A for 10 min at 37 °C. Cellular DNA was stained with 10 μg/mL propidium iodide (PI) for 20 min at 37 °C in darkness, and the cell cycle distribution were analyzed with flow cytometry  and FlowJo software.The PS cells were lysed with RIPA lysis buffer containing protease-inhibitors for 30 min on ice and centrifuged. Cell lysate was combined with 2.5 μg of IP antibody  per sample and incubated with rotating overnight at 4 °C. Next day, the mixture was transferred to 50 μL pre-washed magnetic beads and incubated with rotating for 3 h at room temperature. Beads were collected with a magnetic stand and washed at least 3 times, followed by addition of 40 μL 1× SDS-PAGE loading buffer to the complex and incubation at RT for 15 min with mixing. Beads were magnetically separated and the supernatant was saved for following analysis.The ES and iPS cells were lysed with RIPA lysis buffer containing protease-inhibitors for 30 min on ice and centrifuged at 13,000 rpm for 15 min, and then the supernatant was transferred to new tubes carefully. Protein concentration was quantified using BCA protein assay kit , 20 μg denatured protein samples were separated by 12% SDS-PAGE and transferred onto PVDF membranes. Blocking was performed for 1 h in 5% non-fat milk/PBS-T buffer followed by incubation overnight with primary antibodies at 4 °C. The next day, membranes were incubated with the appropriate secondary antibodies conjugated to HRP for 1 h at room temperature, and the bands were detected by ECL reagent and autoradiography.We added 9 mL fresh cell culture medium containing formaldehyde to a final concentration of 1% to cells for 10 min at 37 °C to crosslink. Then, we added 1 mL of 1.25 mol/L glycine into 9 mL of cross-link solution and incubated at room temperature for 5 min, ice-cold PBS wash cells twice. Cell pellet was resuspended with lysis buffer containing 1× Protease Inhibitor Cocktail and incubated on ice for 10 min. Afterwards, the chromatin lysate was transferred to a 1.5 mL centrifuge tube and chromatin sheared using water bath sonication with the following conditions: shear 15 cycles at 4 °C, 15 s on, 30 s off. Centrifuge and transfer supernatant to a new tube. For input, take 5 μL (1%) from the 500 μL containing sheared chromatin. Each chromatin sample was incubated with antibodies for H3K9me3 , NCL , TRIM28  or normal IgG  overnight on a rotating platform at 4 °C. The next day, the sample was incubated with protein A + G magnetic beads  for 2 h at 4 °C with rotation. The beads-antibody/chromatin complex was washed with wash buffer and resuspended with elution buffer. Then the elute DNA was treated with RNase A at 42 °C for 30 min, then treated with protease K at 60 °C for 45 min followed by heat inactivation at 95 °C for 15 min. The purified DNA was subjected to library preparation or analyzed by qPCR. The primers used are listed in Table S2.v/v) Igepal CA-630) for 10 min on ice to prepare the nuclei. Then, spin down immediately at 500 ×g for 5 min, 4 °C, to remove the supernatant. Nuclei were then incubated with TruePrep Tagment Enzyme  at 55 °C for 10 min. Immediately after the tag mentation, 1× VAHTS DNA Clean Beads  was added into the reaction to purify DNA fragments. PCR amplify the library with the following program: 72 °C for 3 min; 98 °C for 30 s; and thermocycling at 1 cycle of 98 °C for 15 s, 15 cycles of 60 °C for 30 s and 72 °C for 3 min; following by 72 °C for 5 min. Libraries were purified with the 0.6×/0.15× double sided size selection. Libraries were sequenced with the Illumina HiSeq 2500 System.50,000 cells (maximum) were harvested and lysed in 50 μL of cold lysis buffer (10 mmol/L Tris-HCl (pH 7.4), 10 mmol/L NaCl, 3 mmol/L MgCl2, 0.1% (The cytoplasmic and nuclear extracts were separated and prepared from PS cells according to a standard Thermo protocol (78833). 0.5 µg of the IP antibody  were pre-bound to 50 µL of Magnetic Protein A/G (Merck) and incubated with rotation for 30 min at room temperature. 100 µL of cytoplasmic and nuclear extracts were removed and added to each beads-anti-body complex in the presence of proteinase and RNase inhibitors, followed by incubation with rotating for 3 h at 4 °C. After washing, each immunocomplex was re-suspended in 150 µL of proteinase K buffer and incubated at 55 °C for 30 min with shaking to digest the protein. Then RNA was extracted with TRIzol for qRT-PCR.To measure protein synthesis, PS cells or mouse embryos were cultured for 1 h in 2i medium or EmbryoMax KSOM medium + amino acids (Merck Millipore) respectively, and labeling experiments were performed using Click-iT® Plus OPP Protein Synthesis Assay Kit . After labeling, the samples were fixed for 15 min at room temperature in PBS supplemented with 4% paraformaldehyde and then permeabilized in PBS supplemented with 0.25% Triton X-100 for 15 min at room temperature according to the manufacturer’s instructions. Nuclei were stained with DAPI for 2 min and the cell were observed under Zeiss LSM880 fluorescence microscope. For flow cytometry, PS cells were cultured for 1 h in 2i medium supplemented with component A  and then were fixed for 15 min in 4% PFA at room temperature.For transmission electron microscopy, PS cells cultured in one 10 cm dish were collected, cleaned from feeder cells and supplemented with 2.5% glutaraldehyde. The cell pellet was dispersed into small clusters and fixed at least 6 h at 4 °C. After that, the cell samples were treated with standard procedures. Then the slices were imaged on FEI Spirit 120 kV LaB6 Routine Cryo-EM Capable Electron Microscope.2 or 2 mmol/L MgCl2 for 2 or 3 min at 95 °C. The reaction was stopped by ethanol precipitation with 3 mol/L Na-OAc, pH 5.2. In addition, the zinc-based RNA cleavage was stopped by adding 2 µL of 0.5 mol/L EDTA pH 8.0 before ethanol precipitation. After centrifugation, the pellet was washed with 80% ethanol and then was resuspended in nuclease-free water. The sizes of generated RNA fragments were assessed by capillary electrophoresis using a PicoRNA chip on Bioanalyzer 2100  and its range is from 30 to 200 nt. RNA fragments were converted to library using VAHTS Small RNA Library Prep Kit for Illumina (NR801-01), following the manufacturer’s instructions. DNA library quality was assessed using a high sensitivity DNA chip on a Bioanalyzer 2100. Library quantification was done using a fluorometer .First, total RNA (2 µg) was subjected to the metal ion-based RNA cleavage (magnesium or zinc-ions). RNA hydrolysis was performed in the 100 mmol/L Tris-Cl buffer, pH 8.0, containing either 0.1 mmol/L ZnClhttp://hannonlab.cshl.edu/fastx_toolkit/) with the minimum quality score 20 and minimum percent of 80% bases that has a quality score larger than this cutoff value. The high-quality reads were mapped to the mm10 genome by HISAT2, a fast and sensitive spliced alignment program for mapping RNA-seq reads, with -dta paramenter . The stage-specific scores of genes expressed during mouse early embryo development were obtained by entropy-based measure   mouse ES cells. Single-cell gene expression matrix was further analyzed with Seurat v2.3.4 . We excluded the genes with expressed cell number smaller than 3 and the cells with nUMIs smaller than 500 or the expression percentages of mitochondrial genes larger than 0.2 and used 16 Principle Components (PCs) for tSNE analysis. All other bulk and single-cell RNA-seq data analyses and visualization were performed in R/Bioconductor utilizing custom R scripts.For single cell RNA-seq, we used InDrop system following the manufacturer’s protocol (OneCell). We followed the previously published pipeline . The ATAC-seq reads were aligned with the parameters: -t -q -N 1 -L 25 -X 2000 no-mixed no-discordant. The ChIP-seq reads were aligned to mm10 with the options: -t -q -N 1 -L 25. All unmapped reads and PCR duplicates were removed. The bamCoverage and bamCompare commands contained in deepTools (version 2.5.3) were adopted for downstream analysis. Using BamCoverage command with the parameters: -normalizeUsing BPM -of bigwig -binSize 100, we first normalized the raw reads signal to Bins Per Million mapped reads (BPM) signal and converted the alignment bam files to bigwig signal files. The bigwig files were imported into UCSC genome browser for visualization. To minimize the effect of chromatin structure and sequencing bias in our H3K9me3 ChIP-seq data, we corrected ChIP-seq signal using log2 ratio transformation between H3K9me3 signal and input signal by BamCompare command. The “computeMatrix” and “plotProfile” commands of deepTools were used to produce the reads density distribution plot of ATAC-seq and ChIP-seq signal in the given genomic region. For analyzing and visualizing public 5hmC-seq and MeDIP-seq data  with end-to-end mode and k = 1. Simultaneous 5′-end and 3′-end reads counting was done by bedtools v2.25.0 after conversion to *.bed fle. Final analysis was performed by calculating MethScore for single-base level quantification by using the method mentioned in  and the reported error as standard error of the mean (s.e.m). All statistics are *P < 0.05, **P < 0.01, ***P < 0.001, and were calculated by Wilcox signed rank test (for paired samples), Mann-Whitney U test (for independent samples), two-way ANOVA and t-test as described in the figure legends.All statistical analyses for next generation sequencing (NGS) data were performed with R software. The other statistical analyses were performed with GraphPad Prism software. Details of individual tests are outlined within each figure legend, including number of replication performed (Supplementary file 1 (PDF 5238 kb)Supplementary file 2 (XLSX 1229 kb)Supplementary file 3 (XLSX 13 kb)Supplementary material 4 (XLSX 9 kb)Below is the link to the electronic supplementary material.


In the crystal structure of the title compound, the cations pack as dimers connected by weak hydrogen bonds between the pyridyl nitrogen and a methyl hydrogen atom on the neighbouring cation. 17H15N2+·BF4−, were unexpectedly grown from crystallization attempts of [Pt4](BF4)2 [Smith et al. ° twist relative to the unsubstituted pyridyl group. This rotation allows for a centrosymmetric dimer of cations with weak hydrogen bonding between the pyridyl nitrogen atom and a methyl H atom on the neighbouring cation.Crystals of the title compound, C al. 2019. Comment The position of this adjacent dimer results in an N1⋯H10  distance of 2.369 Å between the pyridyl nitrogen atom and the hydrogen atom on the other pyridinium ring ° rotation between planes (comprised of the tolyl ring (C11–C16) 5) Fig. 1 ; the cen Table 1. The off Å Fig. 2. The twing Fig. 2.N-aryl structures of monosubstituted 4,4′-bipyridinium in: N-phenyl (BF4)2 4− anion and four atoms of the central pyridinium ring  in the cation are disordered over two sets of sites, with a ratio of occupancies at ca 51 and 49%. These two occupancies of the pyridinium ring form a dihedral angle of about 30°. All our attempts to improve the quality of the refinement, such as disordering of the entire cation or only some of its rings, gave us similar results.Crystal data, data collection and structure refinement details are summarized in Table 210.1107/S2414314622002486/bh4067sup1.cifCrystal structure: contains datablock(s) I. DOI: 10.1107/S2414314622002486/bh4067Isup2.hklStructure factors: contains datablock(s) I. DOI: Click here for additional data file.10.1107/S2414314622002486/bh4067Isup3.molSupporting information file. DOI: Click here for additional data file.10.1107/S2414314622002486/bh4067Isup4.cmlSupporting information file. DOI: 2156182CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Furthermore, in functional assays performed for selected metabotropic glutamate receptor subtypes, 28 did not show agonist or antagonist activity. The molecular determinants underlying the observed affinity profile of 28 were analyzed using molecular docking and molecular dynamics simulations performed for individual GluK1 and GluK3 ligand-binding domains.Kainate receptors belong to the family of glutamate receptors ion channels, which are responsible for the majority of rapid excitatory synaptic transmission in the central nervous system. The therapeutic potential of kainate receptors is still poorly understood, which is also due to the lack of potent and subunit-selective pharmacological tools. In search of selective ligands for the GluK3 kainate receptor subtype, a series of quinoxaline-2,3-dione analogues was synthesized and pharmacologically characterized at selected recombinant ionotropic glutamate receptors. Among them, compound  S)-glutamate (iGluR), the main excitatory neurotransmitter in the mammalian central nervous system (CNS), are associated with Na+, K+ and Ca2+ ion channels and are responsible for the majority of rapid excitatory neurotransmission in the CNS. The iGluR family comprises four functional classes, including N-methyl-D-aspartate receptors (NMDAR), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR), kainate receptors (KAR) and delta receptors. As with other iGluRs, kainate receptors function as tetramers (dimers of dimers), which are composed of individual subunits GluK1 to GluK5. Based on the affinity of kainic acid, the KAR subfamily is divided into low-affinity subunits GluK1–3, which can form functional homomeric or heteromeric ion channels, and high-affinity subunits GluK4–5 that form functional receptors only in combination with subunits GluK1–3 -(RS)-AMPA , GluK1: [3H]-(S)-NF608 (16.3 Ci/mmol) [3H]-kainic acid  in a total volume of 0.25 mL followed by 2 h equilibration at 4 °C. GluK1−3 assays were filtered through GF/B type glass fiber filters in microtiter plate format  and washed twice with ice-cold assay buffer on a FilterMate manifold . The filters were dried at 70 °C for 1 h, and 50 μL/well Microscint 20  was added. Radioactivity was detected with a TopCounter . GluA2 and GluK5 assays were filtered on GF/C type filters using Millipore 12-well filtration manifolds  and washed twice with ice-cold assay buffer. Filters were placed in pony vials , 3 mL of Ultima Gold scintillation fluid  was added, and DPM (disintegrations per minute) were determined using a TriCarb 2900 scintillation counter .Ligand binding affinities at recombinant rat homomeric GluA2, GluK1−3, and GluK5 were determined as previously detailed ,39,40. ICi/mmol) , GluK2,3Ki) and Hill coefficient (nH) using the one-site Ki and four-parameter logistics equations, respectively.All competition curves (n ≥ 3 per ligand) were determined in triplicate. Data were analyzed using GraphPad Prism 6  to determine ligand affinity (Q), GluK3a, and GluA2(Q)i were created. A polyclonal stable cell line expressing GluK3a was created as described previously [TM HEK293 cells followed by selecting with 20 µg/mL blasticidin  and 0.50 mg/mL geneticin in the presence of 20 µM of the AMPA receptor antagonist CNQX  until stably transfected GluK1b(Q) and GluA2(Q)i cell lines were obtained. For functional assays, cells were plated into clear-bottomed, black 96-well fluorescence culture plates , and when >70% confluent, the cells were loaded with 2 μM Fluo 4-AM calcium sensitive dye  and used for the imaging of agonist-evoked changes in intracellular Ca2+ using a FlexStation I plate reader . For IC50 experiments, cells were pre-incubated with 50 µL FLUO buffer containing (in mM): 100 choline chloride, 5 KCl, 1 MgCl2, 20 CaCl2, 10 HEPES (pH 7.4) and increasing concentrations of test compound for 20 min at room temperature to achieve equilibrium before measurement of agonist responses. The maximum compound test concentration was limited to 10 µM due to the solubility issues of all compounds in the FLUO buffer at room temperature. Changes in dye fluorescence in response to the addition of an agonist solution were then measured at 538 nm using excitation at 485 nm and emission cut-off at 515 nm. Baseline fluorescence was measured for 16 s before the addition of 50 µL agonist solution to each well of the assay plate, and fluorescence was measured for 120 to 160 s after addition of the agonists. Peak change in fluorescence was calculated as the difference between the maximal observed increase in fluorescence and pre-agonist baseline fluorescence. The agonist solutions contained 100 µM KA together with 300 µM of the KAR positive allosteric modulator BPAM344  for GluK1 and GluK3 experiments or 100 µM glutamate together with 100 µM of the AMPAR positive allosteric modulator cyclothiazide for GluA2 experiments . Responses were determined in at least triplicate at each compound concentration.For the determination of antagonistic activity of compounds at GluK1, GluK3 and GluA2 receptors, stable HEK293 cell lines expressing rat GluK1b. The Fluo-4/AM dye was obtained from Molecular Probes , and Glu was purchased from Sigma-Aldrich. The construction and basic pharmacological characterization of the stable mGlueviously .2+/Fluo-4 Assay. The mGlu2/Gqo5-HEK293 cells were cultured in Dulbecco’s Modified Eagle Medium-Glutamax-I supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, 5% dialyzed fetal bovine serum, 100 µg/mL zeocin and 1 mg/mL G418. The cells were split into poly-D-lysine-coated black 96-well plates with clear bottoms (6 × 104 cells/well). The following day, the culture medium was aspirated, and the cells were incubated in 50 µL assay buffer  supplemented with 6 mM Fluo–4/AM at 37 °C for 1 h. Then, the buffer was aspirated, the cells were washed once with 100 µL assay buffer, and then, 100 µL assay buffer was added to the cells . The 96-well plate was assayed in a FLEXStation  measuring emission (in fluorescence units (FU)) at 525 nm caused by excitation at 485 nm before and up to 90 s after the addition of 33.3 µL agonist-containing assay buffer. Antagonist activity was tested by adding the compounds on the cells in the assay buffer prior to agonist application, and Glu EC80 was used as agonist concentration. The compound was tested in duplicate both as agonist and antagonist at least three times at the cell line.Cell culture and Caq kit was purchased from Cisbio . Quisqualate and poly-D-lysine (PDL) hydrobromide were purchased from Sigma-Aldrich . Falcon clear, tissue culture-treated 96-well plates were from Corning Inc. . OptiPlate-384 white, opaque 384-well plates were from PerkinElmer, . L-AP4 and LY341495 were purchased from Tocris Bioscience . The HEK293A cells were a kind gift from Dr. Asuka Inoue . Plasmids encoding human mGluR4 and Flag-tagged human mGluR5 were kind gifts from Jesper M. Mathiesen . The plasmid encoding Gqo5 was a kind gift from Dr. B.R. Conklin .Dulbecco’s modified Eagle medium (DMEM), dialyzed fetal bovine serum (FBS), Opti-MEM, penicillin-streptomycin, Dulbecco’s phosphate-buffered saline (DPBS), Hank’s balanced salt solution (HBSS) and Lipofectamine 2000 were purchased from Thermo Fisher Scientific . An IP-One G1 Assay. HEK293A cells were cultured in DMEM with high (4.5 g/L) glucose and GlutaMAX supplemented with 10% dialyzed FBS and 100 U/mL penicillin-streptomycin at 37 °C and 5% CO2 in a humidified incubator. For measuring IP1 accumulation from mGluR4 and mGluR5, HEK293A cells were transiently transfected with Lipofectamine 2000 using a reverse transfection protocol. For the transfection of 1 mL cells, 1.6 µL of Lipofectamine 2000 was diluted in 100 µL Opti-MEM. Then, 5 min later, it was mixed with 640 ng DNA diluted in Opti-MEM. After 20–25 min the mix was added to 480,000 cells in 1 mL culture medium, and 100 µL  was distributed to each well in PDL-coated Falcon 96-well cell culture plates. For mGluR4, 1 mL of cells was transfected with 320 ng mGluR4, 80 ng EAAT3, 160 ng Gqo5 and 80 ng pcDNA3.1(+). For mGluR5, 1 mL of cells was transfected with 60 ng mGluR5, 80 ng EAAT3 and 500 ng pcDNA3.1(+). 24 h after transfection; then, cells were washed once with assay buffer  and incubated for 2 h at 37 °C to remove ambient glutamate. The assay buffer was replaced with 50 µL assay buffer or antagonist and incubated for 30 min at 37 °C. Then, 20 µL agonist diluted in assay buffer containing 40 mM  LiCl was added to each well, and the plates were incubated for 30 min at 37 °C. After stimulation, wells were emptied and 30 µL of Lysis & Detection Buffer 5 (IP-One Gq kit) was added and incubated for 30 min at room temperature. Then, 10 µL of lysate and 10 µL of detection solution were transferred to an OptiPlate-384 white, opaque 384-well plate, and the plate was incubated for 1 h at room temperature. IP1 accumulation was measured on an EnVision 2104 Multilabel Reader using a 340/60 nm excitation filter and 615/8.5 nm (donor) and 665/7.5 nm (acceptor) emission filters. HTRF ratios were calculated as the ratio of acceptor over donor emission (665 nm/615 nm) and converted to IP1 concentrations with an IP1 standard curve according to the instructions from the kit manufacturer.Cell culture and IPThe protein sequence for the human GluK3 receptor was retrieved in FASTA format from the UniProt database (accession number: Q13003). The crystal structures of GluK1-LBD in complex with antagonists belonging to the group of quinoxaline-2,3-dione derivatives were used as templates , and for each template, we employed the monomer A. GluK3-LBD homology models were constructed with Modeller 9.18  using thThe final GluK3-LBD homology model was selected based on the usefulness of the model to distinguish active from inactive compounds as well as the ability to explain the structure–activity relationship for the compounds studied. Ligands for enrichment calculations were selected from data from the literature. Active compounds constituted 10% of the total database. The enrichment plot was based on the MM-GBSA dG bind calculated for the best docking poses of each ligand. For the MM-GBSA calculations, default options of Prime MMGBSA were used, including the VSGB solvation model, OPLS4 force field and no protein flexibility .A model built on the 6SBT template using Modeller, without a conserved ligand, was found to be the best model.Docking studies were performed using the Glide from the Schrödinger Suite . All ligFor the graphical presentation of the selected docking poses with the highest docking scores, we used PyMOL software . Interachydrogen bond: max. distance 2.8 Å, donor min. angle 120°, acceptor min. angle 90°;halogen bond: max. distance 3.5 Å;salt bridges: max. distance 5.0 Å.π–π interactions “face to face”: max. distance between ring centroids 4.4 Å, max. angle 30°;π–π interactions “edge to face”: max. distance between ring centroids 5.5 Å, min. angle 60°;π–cation interactions: max. distance 6.6 Å, max. angle 30°.Due to the options implemented in the used software, NH–π and CH–π interactions are labeled in the figures as “edge to face” π–π interactions between ring centroids, and they are marked as pink dashes.Molecular dynamics simulations were performed for dimers of the GluK1–LBD and GluK3–LBD complexes with selected ligands. For GluK1, we used dimers from the 6SBT crystal structure. For GluK3, the dimers were obtained by superimposing two monomers on the GluK1 dimer. The side chains of the amino acids constituting the interface between the individual GluK3 monomers were optimized with Prime. MD simulations were performed in NAMD 2.13 using the CHARMM36m force field . The inpN--3,4-dihydroquinoxalin-1(2H)-yl)benzamide 1 as a lead structure, we have synthesized 25 new derivatives of quinoxaline-2,3-dione with various arylethynyl substituents at position 6. Compounds were evaluated in the preliminary radioligand binding studies for their affinity for recombinant rat homomeric iGluR subtypes: GluK1-GluK3, GluK5 and GluA2 receptors. This assay allowed the selection of the most potent compounds 27–29, for which antagonist properties toward individual receptor subtypes were confirmed in the intracellular Ca2+ imaging assay. Compound 29 demonstrated the highest binding affinity across the GluK1−3 and GluA2 receptors within the obtained series, while 28 showed the highest GluK3-selectivity, with a preference for GluK3 receptors of at least 400 times over the GluK1, GluK2, GluK5 and GluA2 receptors. Furthermore, 28 displayed no agonist nor antagonist activity at any of tested metabotropic glutamate receptor subtypes, which are representatives of the three mGluR families. The molecular factors underlying the observed in vitro results for 28 and 29 were examined by molecular docking and molecular dynamics methods, using the X-ray structure of GluK1-LBD (6SBT) as well as a homology model of GluK3-LBD.Taking the previously reported 28 is the first competitive GluK3 antagonist to show such a high selectivity profile among all other GluK3 antagonists. Taking into account the high potential of 28 in a further search for GluK3-selective pharmacological tools, we are going to evaluate this compound or its close analogues in more advanced in vitro and ex vivo pharmacological assays.To our knowledge, compound


Bacillus subtilis strain S4ga at population levels ranging between 0.04 and 4% on the activity of dye-decolorizing Enterococcus faecalis strain T6a1 using a dye decolorization assay. The results obtained revealed that the minority population of B. subtilis S4ga enhanced the dye-decolorizing activity of E. faecalis T6a1, resulting in a shorter lag time and longer active time of dye decolorization. These effects were related to redox potential values rather than O2 concentrations. Comparisons of the extracellular metabolites in individual incubations of E. faecalis T6a1 and B. subtilis S4ga and a co-incubation suggested a mutual relationship through the cross-feeding of specific amino acids . An analysis of intracellular primary metabolites indicated that the arginine deiminase (ADI) pathway, an ATP-producing energy-generating process, was more strongly activated in co-incubated E. faecalis T6a1 than in E. faecalis T6a1 incubated alone. These results suggest that a co-incubation with B. subtilis S4ga promoted ATP production by E. faecalis T6a1 cells and enhanced its dye-decolorizing activity.Microbes live in communities in biological wastewater treatment plants and in the intestines. However, limited information is currently available on the mechanisms by which minority bacterial populations assist other bacteria besides syntrophic relationships as well as on the microbial food web. Therefore, the present study investigated the effects of non-dye-decolorizing  Enterococcus faecalis has become an important bacterium. It has been shown to degrade azo dyes  has been used to monitor E. faecalis in microbial communities  and the oxidation-reduction potential (ORP) and analyses of intracellular and extracellular metabolites in the co-incubation and individual incubations of each strain.In the present study, we evaluated the activities of E. faecalis strain T6a1) exhibiting dye-decolorizing activity was isolated from the fingertips using a previously reported method  were isolated using the same method  liquid medium in a test tube with a lid. After a static culture at 37°C for 17 h, 30 μL of 10 g L–1 Congo red dye  was added to each test tube to achieve an initial dye concentration of 60 mg L–1. After the addition of Congo red dye, samples were statically incubated at 37°C under oxygen-containing conditions by leaving the gas phase of the test tubes in the air. The color of the liquid in the test tubes was regularly observed. Dye concentrations were assessed by measuring the absorbance of the liquid in the test tubes at a wavelength of 490 nm. To measure absorbance, liquid containing the cells and dye was centrifuged at 14,000×g for 5 min, and the supernatant was analyzed using a spectrophotometer. The dye decolorization rate was calculated from the maximum slope of the line in each dye concentration–time graph. The dye decolorization assay was performed in triplicate.Isolated strains were pre-incubated under two conditions: the incubation of each strain alone and a co-incubation of Bacillus strains examined, B. subtilis strain S4ga showed the highest acceleration of dye decolorization by E. faecalis T6a1, resulting in the complete decolorization of 60 mg L–1 Congo red to 0 mg L–1 , whereas B. subtilis S4ga was motile and had larger colonies (1.5–3 mm in diameter) after an incubation for 12 h. B. subtilis S4ga colonies were further confirmed by their Bacillus-like wrinkled appearance after an incubation for 24–48 h. Some of the colonies were analyzed by colony-PCR and sequencing.The amount of dye decolorized in the test tube was proportional to the population of dye-decolorizing ny sizes . E. faecE. faecalis T6a1 was co-incubated with non-dye-decolorizing B. subtilis S4ga and Congo red dye at 37°C. E. faecalis T6a1 incubated alone was used as a control. Each strain was inoculated in 5 mL of 10% LB liquid medium in a test tube with a lid. The dye decolorization assay was subsequently performed under oxic and anoxic conditions. Oxic conditions were created using air in the gas phase of the test tube. The initial DO concentration under oxic conditions ranged between 6 and 7 mg L–1. In contrast, anoxic conditions were created by nitrogen purging of the gas phase in the tube for 60 s. After nitrogen purging, DO in the liquid in the test tube was less than 0.5 mg L–1. The concentration of Congo red dye was set to 60 mg L–1. Immediately after the addition of the dye, DO was routinely measured using the non-contact-type DO sensor, Fibox3-trace (PreSens Precision Sensing) until the liquid in the test tube was decolorized or the incubation was terminated. The ORP value of the liquid was measured when the incubation was terminated or when the dye in the liquid was completely decolorized. Initial ORP values ranged between +155 and +175 mV. pH did not significantly change before or after the incubation, ranging between pH 7.2 and 7.6. The viable cell count as CFU was evaluated after decolorization. The dye concentration during the incubation was assessed from the RGB of routinely acquired images , which was also used for incubations. Test tubes of standard solutions were placed in a photography box  equipped with a LED backlight  at an illuminance of 2,700 lx and images of test tubes were acquired using a camera with the following settings: shutter speed, 1/100 s; aperture, f/2.8; focal length, 4.5 mm; ISO speed, 100  was prepared in a 50-mL tube. In aerobic incubations, 200 mL of 10% LB liquid medium was prepared in a 500-mL vial, and 240 μL of the B. subtilis cell suspension was added to the medium. Congo red (10 g L–1) was then added at a concentration of 60 mg L–1. Three sets of vials were shaken at 60 rpm. In microaerophilic incubations, 5 mL of 10% LB liquid medium was prepared in a 16-mL test tube with a rubber stopper, and 30 μL of the B. subtilis S4ga cell suspension was added to the medium. Congo red (10 g L–1) was added at a final concentration of 60 mg L–1. After the tube was capped with a rubber stopper, nitrogen gas was injected into the test tube with a needle for 3 s. DO was 1 mg L–1 at 5 h and thereafter remained lower than 1 mg L–1. During the incubation at 37°C, the ORP and DO of the incubation medium in the vials and test tubes were monitored for 27 h. Since it was necessary to open the caps of the vials and tubes to measure ORP, DO was assessed using a non-contact-type DO sensor before opening the vials. Unopened test tubes were opened each time to measure the ORP of the incubation solution under microaerophilic conditions. Therefore, 30 test tubes were initially prepared, and four to six test tubes were measured at each time point.Some of the (i) Incubation conditions. To investigate whether B. subtilis S4ga affected the metabolism of E. faecalis T6a1, a metabolomic analysis of the intracellular and extracellular water-soluble primary metabolites of strains T6a1 and S4ga was performed on co-incubated cultures and individual incubations of each strain. Incubations were conducted as described above (dye decolorization assay). Briefly, after a pre-incubation of each strain, the co-incubation of E. faecalis T6a1 and B. subtilis S4ga and individual incubations of each strain were performed at an initial Congo red dye concentration of 60 mg L–1 in 10% LB medium at 37°C. Incubations were terminated at 10 h during decolorization. The use of 10% LB medium reduced background amino acids and vitamins derived from tryptone and the yeast extract of the incubation medium when intracellular and extracellular metabolites were extracted.(ii) Extraction and analysis of intracellular water-soluble primary metabolites of E. faecalis T6a1. To compare the intracellular metabolites of co-incubated E. faecalis T6a1 with those of E. faecalis T6a1 incubated alone, B. subtilis S4ga cells were removed from the co-incubation solution. To achieve this, we focused on cell sizes; E. faecalis T6a1 cells were spherical with a width of approximately 0.8 μm and a length of 1.3 μm, whereas B. subtilis S4ga cells were elongated with a width of approximately 0.8 μm and a length of 2.6 μm or longer . The solution of E. faecalis T6a1 incubated alone was also filtered to ensure that the filtration procedure did not affect metabolites in E. faecalis T6a1 cells. Filtered solutions were filtered two more times to minimize contamination with B. subtilis S4ga cells. Although B. subtilis S4ga accounted for 2% of the total population after the 10-h incubation  and methionine sulfone were added as internal standards to cell suspensions at a final concentration of 10 μM. After thorough mixing and centrifugation at 20,000×g for 5 min, the supernatants of cell suspensions were collected for a metabolomic analysis. Two sets of samples were prepared for the E. faecalis T6a1 co-incubation and individual incubations.e.g., amino acids, organic acids, and coenzymes) were evaluated in intracellular extraction solutions of strain T6a1 by liquid chromatography tandem mass spectrometry  equipped with a pentafluorophenylpropyl column. The resulting intensity was normalized to the height ratio (the peak height of each metabolite divided by the peak height of 2-MES). In cases in which a small peak was overlaid with the next peak or small peaks were considered to be noise, these peaks were regarded as undetected metabolites. Metabolic pathways in E. faecalis were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (Ninety-seven water-soluble primary metabolites ((iii) Extraction and analysis of extracellular water-soluble primary metabolites from co-incubated cultures and E. faecalis T6a1 and B. subtilis S4ga incubated alone. The incubation solutions of the co-incubated culture and E. faecalis T6a1 and B. subtilis S4ga incubated alone were centrifuged at 20,000×g for 7 min, and the supernatants obtained were filtered using a 0.2-μm sterile membrane filter . In addition, 10% LB medium was used as a reference to measure the relative concentrations of amino acids from components of tryptone and the yeast extract –ATP0.2 μm-extracellular.The amount of intracellular ATP in the culture of B. subtilis S4ga in the co-incubated culture was assessed by subtracting the amount of ATP in the 0.8-μm filtration permeate containing extracellular ATP and E. faecalis T6a1 cells from the total amount of ATP in the culture medium containing the extracellular and intracellular ATP of both strain cells: ATPB.subtilis=ATPtotal–. ATP measurements were performed using BacTiter Glo ATP measurement reagent (Promega) and a luminometer  in accordance with the manufacturer’s instructions and the literature , which indicated that intracellular ATP levels varied more in co-incubated B. subtilis S4ga, even though the population of B. subtilis S4ga during the co-incubation was controlled within the same order.The co-incubation enhanced the decolorizing activity of P=0.048, A. On theed alone B. These B. subtilis S4ga to E. faecalis T6a1 on the dye-decolorizing activity of E. faecalis T6a1 by changing the amount of B. subtilis S4ga added to the same amount of E. faecalis T6a1. Effective population ratios ranged between 0.04 and 40%. No significant differences were observed in the dye decolorization rates of the co-incubated cultures within the wide range of the B. subtilis S4ga population ratios (B. subtilis S4ga (less than 1%) enhanced the dye-decolorizing activity of E. faecalis T6a1  under oxic conditions at 23 h, mainly between 3.5 and 4.2 mg L–1 DO . E. faecalis T61a incubated alone utilized all these amino acids. However, decreases in the amounts of methionine, tryptophan, phenylalanine, valine, and leucine in the co-incubated culture were not as large as those with E. faecalis T6a1 incubated alone, which may be because B. subtilis S4ga provided the co-incubated culture with these amino acids. Although B. subtilis S4ga incubated alone produced a two-fold higher amount of tyrosine than that in 10% LB medium, the co-incubation of E. faecalis T6a1 with 1% B. subtilis S4ga consumed tyrosine and arginine, both of which may be rate-limiting substrates. The amino acids produced by B. subtilis S4ga  are hydrophobic. Since hydrophobic amino acids are more permeable through the cell membrane than hydrophilic amino acids, E. faecalis T6a1 may readily acquire these amino acids. The amino acids produced by E. faecalis T6a1 were glutamine, histidine, aspartic acid, and proline (P<0.05). B. subtilis S4ga incubated alone utilized all these amino acids. The production and utilization of the amino acids produced by both strains demonstrated the cross-feeding of amino acids between E. faecalis T6a1 and B. subtilis S4ga, indicating synergistic effects on the activities of each strain.Extracellular metabolites in individual incubations of B medium . The amiE. faecalis T6a1 cells co-incubated with B. subtilis S4ga than in E. faecalis T6a1 cells incubated alone . We demonstrated that ATP levels were more than 10-fold higher in co-incubated E. faecalis T6a1 cells than in E. faecalis T6a1 cells incubated alone . From a practical viewpoint, the addition and/or maintenance of B. subtilis S4ga at 1% may be a useful index for biological textile wastewater treatments to enhance dye-decolorizing bacteria as well as for probiotics in the gut microbiota to promote beneficial bacteria, such as E. faecalis (as a probiotic promoter). Therefore, the present results provide important insights into the roles of minority bacterial populations as key players in dye decolorization and other biological processes.Few studies have shown that non-dye-decolorizing bacteria affect dye decolorization when they are present as minor populations  A Minority Population of Non-dye-decolorizing https://doi.org/10.1264/jsme2.ME21080Supplementary Material


Mutations in Nav1.5 have been associated with LQT syndrome, Brugada syndrome, and sudden arrhythmia death syndrome. Genetic studies showed that Nav1.5 mutations vary across race-ethnic groups. Here we investigated an Asian-specific mutation Nav1.5-P1090L associated with LQT syndrome. We found that Nav1.5-P1090L mutation perturbed the sodium channel function. It altered the gating process of the channel and exhibited an enhanced window current. Treatment with mexiletine reversed the depolarization shift of the steady-state inactivation produced by P1090L. Mexiletine also modified the recovery from steady-state inactivation and the development of inactivation of P1090L. It rescued the dysfunctional inactivation of P1090L and reduced the P1090L channel’s availability.The voltage-gated sodium channel Na Long QT syndrome (LQTS) is a heart rhythm disorder and increases risk for life-threatening sudden cardiac arrest . It is av1.5, encoded by SCN5A, plays a critical role in the fast depolarization of the cardiac action potential duration (APD) (v1.5 linked with long QT syndrome type 3 (LQTS3) affected channel’s function and remodeled ventricular APD  . Geneticular APD . Some Napatients . Althougv1.5-P1090L associated with LQTS. The Nav1.5 mutation P1090L was first reported to be linked with familial long QT syndrome in Japanese population  and mutation P1090L. The cells were reseeded on coverslips in 6-well plates containing Dulbecco modified Eagle’s medium  supplemented with 10% fetal bovine serum (Invitrogen), 100 U/mL penicillin, and 100 μg/ml streptomycin at 37°C under 5% CO2. After growth to ∼80% confluence, cells were transiently transfected with Nav1.5 cDNA plasmids. The pRc/CMV plasmid containing the sequence of the human Nav1.5 channel was kindly provided by Dr Alfred L. George Jr . Single-point mutation of P1090L in pRc/CMV-hNav1.5 was introduced with standard PCR techniques. The mutation was introduced to the template plasmid using primers in a PCR protocol. The PCR cycles were initiated at 98°C for 1.5 min, followed by 25 amplification cycles. Each amplification cycle consisted of 98°C (20 s), 60°C (30 s), and 72°C (5 min). The cycles were finished with an extension step at 72°C for 15 min, followed by 4°C for 30 min. The template plasmid was removed using DpnI , and Stbl2 competent cells  were transformed with the PCR product. Plasmids were isolated from Stbl2 cells with resulting colonies using QIAprep Spin Miniprep Kit . The mutation confirmed by the DNA sequencing was used for subsequent transfection.HEK-293T cell lines  were used to express human Nav1.5 WT or P1090L mutation and 0.25 μg of a plasmid encoding enhanced green fluorescent protein (GFP) using Lipofectamine 3000 reagent  according to the manufacturer’s protocol. The mixture was then added to the culture dish and the cells were incubated at 37°C for 24 h before the electrophysiology studies were conducted. A coverslip with HEK-293T cells was placed in a recording chamber containing bath solution on the stage of a fluorescence microscope , and the transfected cells identified by the fluorescent signal emitted from GFP were used for electrophysiological measurements. Patch clamp experiments were conducted 24–48 h after transfection.The HEK-293T cells were transiently transfected with 2 μg of the cDNA encoding Na2, 2 mM CaCl2, 10 mM HEPES, adjusted to pH 7.4 with NaOH. The intracellular solution contained 120 mM CsF, 15 mM CsCl, 20 mM NaCl, 2 mM EGTA and 5 mM HEPES, adjusted to pH 7.3 with CsOH. All measurements were performed at 22 ± 3°C. Pipettes had 2–4 MΩ access resistance.Patch clamp measurements were performed in whole-cell configuration using an Axopatch 200B amplifier controlled by pClamp11 software through an Axon Digidata 1550B system . The extracellular solution contained 130 mM NaCl, 5 mM KCl, 1 mM MgClmax = 1/(1 + exp(V-V1/2)/k), where G/Gmax is the relative conductance normalized by the maximal conductance, V1/2 is the potential of half activation, V is test pulse, and k is the Boltzmann coefficient. Steady-state inactivation curves were fitted by the Boltzmann equation: I/Imax = 1/(1 + exp(V-V1/2)/k), where I/Imax is the relative conductance normalized by the maximal conductance, V1/2 is half-maximal inactivation, V is test pulse, and k is the Boltzmann coefficient.The activation G-V curves were fitted by the Boltzmann equation, as previously explained : G/Gmax dev), where y0 is the offset and A is amplitude. The double exponential functions of the form y = A1(1-exp[-t/τ1])+A2(1-exp[-t/τ2]) were fitted to recovery curves to determine time constants , A1 and A2 are the fractions of fast and slow inactivating components, and τ1 and τ2 are their time constants Ʈrec,f and Ʈrec,s respectively.Exponential functions of the form y = y0+A(1-exp[-t/τ]) were fitted to development curves to determine time constant , where %i,max is the maximal percentage of channel inhibition by blocker B, h is the Hill coefficient, and IC50 is the concentration of mexiletine required for 50% inhibition.Dose-responses of channel inhibition by mexiletine were fitted by the Hill equation, as previously described : %i = %i1/2, Ʈdev, Ʈrec,f, Ʈrec,s) were determined from each individual cell and used for comparison with t-test. We repeated transfection experiments 3 or 4 times for each group cell, and 2 to 3 sodium currents were recorded in each independent culture. p < 0.05 was considered to indicate a statistically significant difference.All data were presented as the mean ± SEM. Significance between means was determined by Student’s t-test. Electrophysiological parameters  at position 1090 is highly conserved across vertebrate species (The P1090 is located in the intracellular loop between the DII-DIII domain of the Na channel . Nav1.5  species .v1.5 wild-type (WT) and P1090L mutation in transiently transfected HEK293T cells were studied. We did not observe that P1090L altered the current amplitudes compared with WT , and shifted voltage dependence of steady-state inactivation to depolarized direction   . The dep, n = 8) . This la, n = 8) .dev) , and decreased fast recovery time constant   and slow recovery time constant    , suggest, n = 8) .v1.5 mutations and modulate sodium channel function  with little effect on the channel activation  , and increased Ʈrec,s of P1090L  (We observed that the treatment with mexiletine shifted the steady-state inactivation of P1090L currents by around 10 mV to hyperpolarizing direction (−88.2 ± 1.7 mV iletine) . The lariletine) . In addiiletine) . The druiletine) . These rv1.5 mutation P1090L linked with LQTS perturbed the process of steady-state inactivation of the channel and generated an enhanced window current that is associated with a gain-of-function mechanism. Treatment with mexiletine rescued the dysfunctional inactivation of the mutation, and decreased the larger window sodium current mediated by P1090L.In the present study, we showed that Nav1.5 conducts inward sodium currents initiating the cardiac action potential  that eliminates the larger window current would reduce the occurrence of arrhythmic events in patients. In the present study, we found that mexiletine reduced the larger window current mediated by the Nav1.5-P1090L mutation. Mechanism-based inhibition of the larger window current by a therapeutic drug would be expected to decrease arrhythmic events in Nav1.5-P1090L patients.Recent studies on the Nav1.5-P1090L were focused on case reports or genetic analysis, and there was not yet a report on clinical applications of mexiletine in P1090L patients. Like the P1090L, another LQTS mutation Nav1.5-V411M also generates a larger sodium window current associated with the gain-of-function mechanism . The subv1.5-P1090L. As cardiomyocytes have distinct electrophysiological properties, the in vitro method could not capture full spectrum of functional changes of the LQTS-linked mutations. Meanwhile, studies showed that Nav1.5 mutation and other channel are regulators of gene transcriptional networks in cardiomyocytes (One limitation of the present study is that we used heterologous expression system to study the race-specific mutation Namyocytes , furthermyocytes .v1.5 mutation P1090L linked with LQTS, and showed that mexiletine effectively reduced the enhanced window current generated by P1090L. The results provide insights into the mechanisms by which Nav1.5 mutation causes arrhythmias, and might potentially enable a mechanism-based approach to the treatment of LQTS.In summary, we characterized electrophysiological properties of Na


The as-obtained g-C3N4QDs were successfully applied in the determination of tetracycline (TC) with a good linear relationship in the range of 0.23–202.70 μM. The proposed fluorescent sensor shows excellent stability, good repeatability, high selectivity and outstanding sensitivity to TC with a low detection limit of 0.19 μM. The fluorescence quenching mechanism of g-C3N4QDs with TC was mainly governed by static quenching and the inner filter effect. The method was successfully applied to monitor TC in tap water and milk powder samples.Enhanced blue fluorescent carbon nitride quantum dots (g-C 3N4QDs were synthesized by a simple solvothermal “tailoring” process from bulk g-C3N4 which have a “strong quenching” behaviour in the presence of TC. The proposed fluorescent sensor has been successfully applied to detect TC in actual samples.The g-C Usually, g-C3N4 is prepared by the high-temperature pyrolysis of nitrogen-rich precursors such as melamine, cyanamide, and dicyandiamide.12 However, the synthetic bulk g-C3N4 materials generally have low specific surface area, poor luminescence performance, and insolubility in most solvents, which limit their practical applications. Therefore, it is rewarding to search for solutions to overcome the shortcomings and limitations of bulk g-C3N4 materials. Quantum dots (QDs) as quasi-zero dimensional nanomaterials have attracted wide attention because of quantum effects, and possess exotic features, such as large specific surface area, good water solubility, and unique optical and electronic properties superior to those of large particles. Currently, researchers have developed various types of QDs including metallic QDs,13 nonmetallic QDs,14 and composite QDs15 among others and applied them in electroluminescence devices, solar cells, photocatalysis, imaging and sensing based on their extraordinary optical properties: electrochemiluminescence, phosphorescence, and fluorescence.16 Thus, exploration of g-C3N4QDs may provide the promising properties and applications of g-C3N4 materials. As a result, a series of fluorescent g-C3N4QDs have been fabricated and used as promising fluorescent sensor materials in recent years.17–19 For instance, K. Patir et al. applied g-C3N4QDs as a photoluminescent sensor for Hg2+ detection.19 Up to now, several routes such as hydrothermal, solvothermal, microwave and chemical etching, which are mainly categorized into top-down or bottom-up approaches, have been employed to synthesize QDs.16,20 However, the present methods of g-C3N4QDs whether top-down or bottom-up synthesis are usually complicated and difficult to control. Therefore, developing highly efficient and facile methods to synthesize g-C3N4QDs is of great importance. In this paper, we report a facile and environmentally friendly solvothermal “tailoring” method to synthesize fluorescent g-C3N4QDs. Moreover, the application of g-C3N4QDs still needs further exploration.Since its re-discovery in 1990s, graphite carbon nitride  is one of the major broad-spectrum antibiotics, which can inhibit a wide variety of bacteria, and has been extensively used in human therapy, animal disease control and agricultural feed additives because of its excellent therapeutic effect and low cost.3N4QDs with low costs, water solubility and bright blue fluorescence via a facile and environmentally friendly solvothermal “tailoring” method. The prepared g-C3N4QD material displayed good stability, reproducibility, high selectivity and sensitivity for TC determination on the basis of the fluorescence quenching method , oxytetracycline (OTC), doxycycline hydrochloride (DTC), ciprofloxacin (CIP), amoxicillin (AMX), sulfadiazine (SDZ), and chloramphenicol (CAM) were purchased from Aladdin Industrial Corporation. All reagents were of analytical grade and used as received without further purification.2.23N4 was synthesized by a simple thermal polymerization of melamine according to our previous work.28 Typically, 5 g of melamine was heated to 500 °C in a tube furnace at a ramping rate of 10 °C min−1 and kept at this temperature for 2 h and then continued to heat up to 520 °C at the same heating rate and kept for another 2 h. After naturally cooling to room temperature, the obtained yellow product was ground into a homogeneous powder.Firstly, the bulk g-C2.33N4QDs were prepared by a solvothermal method according to ref. 3N4 (0.10 g) was mixed with ethylene glycol (15 mL) and ammonia (15 mL). The mixture was transferred into a Teflon-sealed autoclave and kept at 180 °C for 12 h. The resultant product was cooled to room temperature and filtered with a 0.22 μm membrane. Finally, the obtained filtrate containing highly dispersed g-C3N4QDs was stored at 4 °C before use.The g-C2.4α radiation . A transmission electron microscope JEM-2100F (Japan JEOL Ltd.) was used to record transmission electron micrographs (TEM) at an acceleration voltage of 200 kV. The XPS measurements were carried out on an ESCALAB 250Xi spectrometer  with a pass energy of 30 eV and 100 W. The absolute quantum yield is measured with a C11347-11 Quantaurus-QY absolute quantum yield measurement instrument . Fourier transform infrared spectra (FT-IR) were collected with a Thermo Scientific Nicolet 380 FT-IR spectrometer with a resolution of 4 cm−1. The ultraviolet-visible (UV-Vis) absorption spectra were measured on a TU-1901 UV-Vis spectrophotometer (UV). The photoluminescence (PL) spectra were recorded on a Shimazu RF-5301 PC fluorescence spectrophotometer.The X-ray diffraction (XRD) patterns were obtained with an X-ray diffractometer  using Cu K2.53N4QDs was measured using an absolute PL quantum yield spectrometer with an integrating sphere  under excitation with a 150 W xenon light source at 325 nm. The test principle and method were referred to ref. 30The photoluminescence absolute quantum yield (QY) of the prepared g-C2.63N4QD solution and then diluted with deionized water to 5 mL . After being stirred thoroughly and placed for 15 min at room temperature, the fluorescence intensity of the mixture was collected under an excitation wavelength of 325 nm. To explore the selectivity, PL of the solution containing g-C3N4QDs and other substances  was detected using the same method. Moreover, PL intensities of the mixture of g-C3N4QDs and TC in the presence of other substances were also recorded under identical conditions.0.5 mL of TC solution with various standard concentrations was mixed with 0.5 mL of g-C2.7The tap water samples were obtained from our lab, filtered through 0.22 μm membrane filters for further analysis and added with a series of different concentration levels of TC. After that, the solutions were analyzed with the same proposed method.32,33 Firstly, 2 g of milk powder was diluted to 20 mL with ultrapure water. Then 2 mL of 10% trichloroacetic acid  was added into the sample solution and sonicated for 30 min to precipitate proteins and dissolve other organics in the matrix. After that, the mixture was centrifuged at 8000 rpm for 10 minutes to separate the precipitate. Thereafter, the supernatant was filtered with a 0.22 μm filter membrane to remove lipids, and the filtrate was taken for further analysis.Milk powder samples were obtained from the local supermarket. The procedure for pretreating actual samples was performed on the basis of the reported works with little modification as follows.3.3.13N4QDs. As presented in 3N4QDs are well mono-dispersed and have a relatively uniform spherical shape. The particle sizes are mostly in the range of 2–6 nm with a narrow distribution, and the average particle size is approximately 3.5 nm. 3N4QDs, and the spacing of the lattice fringe is 0.24 nm corresponding to the (100) plane of g-C3N4.343N4 and g-C3N4QDs. As shown, the diffraction peaks of g-C3N4QDs are in good agreement with those of bulk g-C3N4, indicating that they have the same basic crystal structure. The strong peak at 27.5° is corresponding to the (002) plane, and a relatively weak diffraction peak at 13.1° is attributed to the (100) plane. These two characteristic diffraction peaks reflect the inter-planar stacking of the aromatic ring structure and the in-planar tri-s-triazine unit packing motif, respectively.35 Moreover, compared with bulk g-C3N4, the relative intensity of the (100) peak for g-C3N4QDs is much weaker, indicating that the in plane repeated unit structure may be damaged to a certain extent after solvothermal treatment.36TEM and high resolution TEM were employed to observe the morphology of g-C3N4 and g-C3N4QDs are presented in −1 belonging to the out of plane breathing vibration modes of heptazine heterocyclic rings.37,38 Several intense bands in the range of 1200–1650 cm−1 are also detected for both bulk g-C3N4 and g-C3N4QDs, which are attributed to typical stretching vibrations of tri-s-triazine units. The results indicated that the basic surface functional structures of g-C3N4 are not changed in the process of solvothermal “tailoring”. Moreover, the broad bands in the 3000–3500 cm−1 region assigned to stretching vibrations of N–H (−3100 cm−1) and O–H (−3300 cm−1) of adsorbed water are observed for the two samples.11 Nevertheless, the intensity of the N–H and O–H peaks for g-C3N4QDs is much stronger than that of g-C3N4. And these hydrophilic groups are probably beneficial to increase the water solubility of g-C3N4QDs. In addition, compared with bulk g-C3N4, two new peaks at ca. 1731 and 1032 cm−1 appeared, which are ascribed to C PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbd" format="PBM" resolution="300" x-size="8" y-size="10" xml:space="preserve">00000000000000000000000000000000111111110000000011111111000000000000000000000000</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="13.200000pt" height="16.000000pt" viewBox="0 0 13.200000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 440 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z M0 280 l0 -40 320 0 320 0 0 40 0 40 -320 0 -320 0 0 -40z"/></g></svg>O stretching vibration and C–O vibration in the C–O–C group, respectively.39,40 It seems possible that oxygen atoms may be introduced into g-C3N4QDs.The FT-IR spectra of bulk g-C3N4QDs. The survey scan of the XPS spectrum displays three peaks located at 284.8, 398.7 and 532.8 eV (3N4QDs possess two types of nitrogen species: N–(C)3 (399.7 eV) and C–NC (398.7 eV).1,41 The deconvolution of the C 1s spectrum (2 CC or sp3 C–C), C–NHx , N–CN, and C–O.12,42–44 For the O 1s spectrum , and then decreases slowly. In the presence of TC, the quenching efficiency [(F0 − F)/F0] displays almost the same change trend. In view of that the neutral ultrapure water system is environmentally friendly and readily available, using the ultrapure water (pH 7–8) as the solvent is also researched, and the results showed that the fluorescence intensity and quenching efficiency can reach the maximum in the ultrapure water system. Further studies reveal that the PL intensity can remain unaffected by ion strength when adding NaCl into the ultrapure water system with the salt concentration increasing from 0 to 500 mM  and y = 0.00224x + 0.0930 (R2 = 0.9988), where y and x are the quenching efficiency [(F0 − F)/F0] and the concentration of TC, respectively. The limit of detection (LOD) of 0.19 μM is calculated according to 3 times the standard deviation of the blank measurements divided by the slope of the standard curve (LOD = 3SD/s). For comparison, some of the reported fluorescence sensors based on other materials for TC detection are displayed in Under the optimized experimental conditions, the analytical performance of g-C02.70 μM . The cor+, Mg2+, Ba2+, K+, Cu2+, and Fe3+) and common antibiotics . As described in 3N4QDs is distinctly quenched only by TC, OTC and DTC, which belong to tetracycline antibiotics (TCs). Among them, the quenched efficiency of TC is the highest. However, another tetracycline antibiotic CTC produces a fluorescence enhancement phenomenon. What's more, except TCs, other above mentioned components show negligible fluorescent quenching. Moreover, the experiments of adding the mixtures of 200 μM TC and 400 μM of the above mentioned components were performed. The interference effects of other coexisting metal ions and antibiotics except TCs on the fluorescence quenching of TC can be ignored  are summarized in Table S2.61 The G value was calculated by formula xq is the questionable value and x̄ is the average value. As shown in Table S2,G values of the two sets are 1.28 and 1.24, which are both lower than the critical value G0.05,6 of 1.89.62 Therefore, the outliers of 9.31 and 158.22 μM should be retained. After checking the outliers, the t test was used to determine the systematic error of the test data. Formula t. If t ≥ tα,f, where α = 1 − 0.95 = 0.05 and f = n − 1, it means that there is a significant difference between the average measured value and true value. Otherwise, there is no significant difference. The calculated t values of the two sets of data are 0.59 and 2.24 respectively, which are lower than the t0.05,5 of 2.571.62 Hence, there is no significant difference between the average measured value and true value. Moreover, the RSDs of the two sets of data are 3.30% and 1.10%, respectively, indicating that the precision is satisfactory. Above all, the described fluorescence sensor method is feasible for TC detection within the range of the two calibration curves with high accuracy and precision.Statistical evaluation was employed to check the feasibility of the proposed fluorescence sensor method for TC detection. TC standard solutions of 9.01 and 157.66 μM were prepared and determined by the presented method in six parallel tests. The measured values, mean values and standard deviations  or fluorescence resonance energy transfer (FRET). FRET is a non-radiative energy transfer caused by vibration collision within a limited distance of 10 nm between the donor and acceptor, which will shorten the fluorescence lifetime of the donor,66 whereas the IFE is a radiation energy transfer and not limited by distance, which is induced by the formation of the ground-state complex. Therefore, for the IFE, the absorption spectra change and the fluorescence lifetime remains constant of the fluorescent substance with the quencher.65 As shown in 3N4QDs. Moreover, combined with the front results of fluorescence lifetime analysis, we deduced that the fluorescence quenching of g-C3N4QDs with TC is attributed to the IFE, not FRET.Furthermore, in order to get more information about the mechanism of fluorescence quenching, we analyze the fluorescence spectra of g-Carefully . The exc4.3N4QD solution with excellent fluorescence properties and water-solubility by a simple, low cost and environmental-friendly solvothermal “tailoring” method. The resultant g-C3N4QDs have a “strong quenching” behavior in the presence of TC, which is mainly controlled by a static quenching process because of the IFE mechanism. Based on this, we developed a rapid fluorescent sensor to detect TC in a highly selective and sensitive manner with a wide detection range and low detection limit. The proposed g-C3N4QDs have promising application prospects in the monitoring of TC in real samples.In summary, we have successfully prepared a g-CThere are no conflicts to declare.RA-011-D1RA04272F-s001