Accumulating evidence has revealed the critical role of long non-coding RNAs (lncRNAs) in cellular processes during tumor progression. As documented in cancer-related literatures, LINC00992 expression is associated with cancer progression, whereas its function in tumors including prostate cancer has not been characterized yet.Data from GEPIA database suggested LINC00992 expression in prostate cancer tissues. The expression levels of RNAs were monitored via qRT-PCR. Western blot evaluated the levels of proteins. The proliferation, apoptosis and migration of prostate cancer cells were assessed by CCK-8, EdU, TUNEL, Transwell and wound healing assays. Luciferase reporter, RNA pull down and RIP assays were applied to detect the interplays among LINC00992, miR-3935 and GOLM1.Elevated levels of LINC00992 and GOLM1 were detected in prostate cancer tissues and cells. LINC00992 exerted facilitating functions in prostate cancer cell proliferation and migration. Mechanically, LINC00992 interacted with and negatively regulated miR-3935 to elevate GOLM1 expression in prostate cancer cells. In addition, the in vitro suppressive effect of silenced LINC00992 on prostate cancer cell proliferation and migration was reversed by GOLM1 upregulation. Likewise, LINC00992 depletion restrained tumor growth in vivo was offset by enhanced GOLM1 expression.LINC00992 competitively bound with miR-3935 to elevate GOLM1 expression and therefore facilitate the oncogenic phenotypes of prostate cancer cells, implying a potential LINC00992-targeted therapy for prostate cancer. Clinically, prostate cancer manifests as a dominating cause of male-related death worldwide and is characterized as the most continually occurred tumor among men in the United States . The bigThe widely studied long noncoding RNAs (lncRNAs) are transcribed from non-protein-coding human genome and have more than 200 nt in length . LncRNAsIn past decades, the fact that lncRNAs function in tumors depending on their secondary or tertiary structures has been reported in many cancer-linked studies. For instance, in the nucleus, lncRNAs are entitled to work as molecular scaffolds or alternative splicing assistants , 15. On We conducted this research aiming to explore the function or probable mechanism of LINC00992 in prostate tumor, which might enrich the understanding in terms of prostate tumor pathology and contribute to a wider applied scope.The prostate cancer tissue samples and matched peri-tumor tissue samples were collected from 60 patients diagnosed with prostate cancer, under the approval of the Ethics Committee of the First Affiliated Hospital of Kunming Medical University. Each participant did not receive radiotherapy and chemotherapy prior to tissue collection, and signed the written informed consents before this study. All samples were snap-frozen in liquid nitrogen and then stored at -80 °C until required for further analysis.2 at 37 °C. Before using in this study, all cell lines were authenticated by STR profiling and tested for mycoplasma contamination in June 2018.The prostate epithelial cell line  and prostate cancer cells, including PC3 (CRL-1435), LNCaP (CRL-1740), C4–2 (CRL-3314), and DU145 (HTB-81), were all purchased from American Type Culture Collection  in October 2017. All cells were cultured as recommended in Dulbecco’s modified Eagle’s medium containing 10% FBS  under the condition of a cell incubator with 5% COLINC00992 shRNA or negative control shRNA, and pcDNA3.1/LINC00992, pcDNA3.1/GOLM1 or its empty control pcDNA3.1 plasmid were chemically synthesized and provided by Gene Pharma . MiR-3935 mimics, miR-3935 inhibitor and their related negative controls  were all purchased for upregulating or downregulating miR-3935 from Ribobio . In line with the directions of LipofectamineTM RNAiMAX Transfection Reagent (Thermo Fisher Scientific), transfection of these plasmids into DU145, PC3 and RWPE-1 cells was conducted and qRT-PCR checked the transfection efficiency. The sequences were as follows: sh-NC: 5′-CCGGTAGTAATTGACAACCATTATACTCGAGTATAATGGTTGTCAATTACTATTTTTG-3′, sh-LINC00992#1: 5′-CCGGATTATCCAAGAGTATTAACATCTCGAGATGTTAATACTCTTGGATAATTTTTTG-3′, sh-LINC00992#2: 5′-CCGGTGTTAGATGATCATTGAGGTGCTCGAGCACCTCAATGATCATCTAACATTTTTG-3′, sh-LINC00992#3: 5′-CCGGTTACCTAATCAGTAGATGCAGCTCGAGCTGCATCTACTGATTAGGTAATTTTTG-3′; NC-mimics: 5′-UCAGGUAGGGCUCAAACCAACC-3′, miR-3935 mimics: 5′-UGUAGAUACGAGCACCAGCCAC-3′; NC-inhibitor: 5′-CUGGCUUUAGGGUGCCACUUAG-3′, miR-3935 inhibitor: 5′-GUGGCUGGUGCUCGUAUCUACA-3′.−ΔΔCt.On the basis of the instructions of Trizol reagent , RNA extraction was executed in prostate cancer cells. After the examination of RNA purity with spectrophotometry, cDNA was obtained from above RNA with reverse transcription kit . qRT-PCR analysis was devised with the aid of a BioRad CFX96 system and SYBR green was applied for investigating the RNA levels. The internal reference for LINC00992 and mRNAs was GAPDH whereas that for miRNAs expression was U6. Relative expression was assessed based on the method of 2Protein content in cells was determined by western blot analysis. RIPA lysis buffer  was adopted for cell lysing, followed by the evaluation of the protein concentration with BCA Protein Assay Kit . 10% SDS-PAGE gel was applied for separating proteins (20 μg protein per sample) and then proteins were transferred onto 0.2 μm PVDF membranes . Antibodies including anti-GOLM1 , anti-PCNA , anti-CDK2 , anti-Cyclin D1 , anti-Bax , anti-Bcl-2 , anti-MMP2 , anti-MMP9 , anti-p-Src , anti-Src , anti-p-FAK , anti-FAK , anti-GAPDH  and anti-Tubulin , were applied to probe the membranes overnight at 4 °C. After that, the membranes were further incubated for 1 h with HRP-conjugated secondary antibody  at room temperature. ECL Substrates  was utilized for the visualization of signals, followed by exposure to X-film . The quantification of immunoblots was conducted with the aid of imageJ software , with GAPDH or Tubulin as the normalizer as needed.Fragments of full-length LINC00992 with wild-type or mutant binding sites for miR-3935 and sequences of GOLM1 3’UTR containing wild-type or mutated miR-3935 binding sites were inserted into the pmirGLO vectors  for the construction of reporters . Then, the four reporters and miR-3935 mimics or miR-3935 inhibitor (GenePharma) were co-transfected into DU145 and PC3 cells applying lipofectamine2000 (Invitrogen), as needed. Forty-eight hours later, Dual-Luciferase Reporter Assay System (Promega) was employed for the examination of the luciferase activity. GloMax® Discover Multimode Microplate Reader (Promega) assessed the ratio of Firefly/Renilla luciferase activity and the activity of Renilla was the normalized control.According to the direction for usage of Magna RIP™ RNA Binding Protein Immunoprecipitation Kit , RIP assay was strictly performed. RIP lysis buffer was firstly applied to treat the transfected DU145 and PC3 cells. Afterwards, the obtained cell lysates were processed with magnetic beads integrated with human anti-Ago2 antibodies  or anti-IgG . Following the recovery of antibody by the protein A/G beads, qRT-PCR detected the levels of LINC00992, miR-3935, and GOLM1 mRNA in the precipitates. IgG worked as the negative control for the normalization of RNA-IPs.The dispersion of LINC00992 in the prostate cancer cells was assayed as described previously . The isoIn line with the recommendation of Ribo™ FISH Kit , FISH analysis was implemented for testing the presence of LINC00992 in prostate tumor cells. Ribobio Company synthesized the LINC00992 probes, labeled by Cy3 fluorescent dye. Following the fixation by 4% paraformaldehyde and 0.5% Triton X-100 permeabilization, DU145 and PC3 cells were subsequently blocked in pre-hybridization buffer/blocking solution. Then incubation of cells with probe/hybridization buffer was later performed. Next day, after rinsing and Hoechst staining, the fluorescence was measured under a confocal laser scanning microscope .For the viability assessment in DU145, PC3 and RWPE-1 cells, CCK-8 assay was implemented as described previously . Cell viCell proliferation was examined through EdU assay as described previously , by usinTUNEL assay was carried out as described previously , for proThe application of transwell chambers with pore size 8-μm  was aimed for detecting cell migration in strict line with the instructions. Cells that were previously suspended in serum-free RPMI-1640 media were seeded into the upper chamber. RPMI-1640 medium containing 20% FBS was supplemented in lower chamber as a chemoattractant. Cells in the filters following 48 h incubation were immobilized in methanol and went through 0.1% crystal violet staining. The images of cells migrated through the filters were obtained and counted under the microscope.3 cells/well) were prepared on glass culture dishes and cultivated at 37 °C for a whole night to allow cells adhered to the plates, followed by the straight scratch made with a plastic pipette tip after cell samples reached 80% confluence. Later, cells were rinsed in PBS to clear the detached cells. Finally, the wounds at 0 and 24 h were imaged via a light microscopy .The DU145, PC3 and RWPE-1 cells  staining.Sixteen six-week-old male BALB/C athymic nude mice were commercially available from the National Laboratory Animal Center  and maintained in SPF-grade animal lab. All animal-related protocols were approved by the Animal Research Ethics Committee of the First Affiliated Hospital of Kunming Medical University. The in vivo experiment was undertaken via subcutaneous injection of 5 × 10The tumor samples collected from in vivo experiments were treated with 4% PFA, dehydrated and embedded in paraffin. Afterwards, the paraffin-embedded sections (4 μm) were prepared for IHC assay as described previously , by use P < 0.05 was considered as the threshold of significance.SPSS 17.0 statistical software  was employed in the processing of data from three biological replicates, and data were expressed as mean ± SD. Significance of difference within two groups was determined using Student’s t-test, while that among no less than two groups was tested via one-way or two-way ANOVA. LINC00992 expression pattern in prostate cancer was acquired from online GEPIA database. As a result, LINC00992 was considerably upregulated in PRAD (prostate adenocarcinoma) tissues relative to normal ones Fig. a. After http://www.csbio.sjtu.edu.cn/bioinf/lncLocator/). Result predicted that LINC00992 located mainly in cytoplasm , the binding site between GOLM1 and miR-3935 was predicted  was highly expressed in four prostate cancer cell lines relative to normal controls  and experimental data (FISH and RNA isolation of nuclear and cytoplasmic fractions). Our study for the first time uncovered that LINC00992 located mainly in the cytoplasm of prostate cancer cells. Besides, our study also completed LINC00992-modulated mechanism by disclosing the downstream target miR-3935. The direct interaction between LINC00992 and miR-3935 was also first validated in this research.In the subsequent search of mRNA targeted by miR-3935, we paid heed to Golgi Membrane Protein 1 (GOLM1). Previously, downregulation of GOLM1 inhibits glioblastoma cell proliferation and motility, and progression , 34. GOLTo sum up, all findings presented in the work contributed to a better grasp about the mechanism in prostate cancer carcinogenesis. It was elaborated that LINC00992 accelerated prostate cancer cell growth and migration via sponging miR-3935 and therefore promoting GOLM1 expression, providing new promising therapeutic biomarkers for prostate cancer. Nevertheless, current study lacks the evidence confirming the regulation of LINC00992 on tumor metastasis in vivo, which will motivate us to explore further in the future study.Additional file 1: Supplementary Figure 1. (A) qRT-PCR analysis indicated the upregulation of LINC00992 in prostate cancer tissue samples in contrast to peri-tumor samples. (B) The association of LINC00992 expression with overall survival of prostate cancer patients was analyzed via Kaplan-Meier curve. (C) The expression of proliferation-related proteins in transfected cells was evaluated via western blot. (D) The quantification of immunoblots in Fig. p < 0.01.Additional file 2: Supplementary Figure 2. (A) The efficiency of LINC00992 overexpression was assessed via qRT-PCR in RWPE-1 cells. (B-C) The proliferation ability of transfected cells was estimated via CCK-8 and EdU assays . (D) The expression of proliferation-related proteins in different groups was evaluated via western blot. (E-F) Transwell and wound healing assays were applied to analyze the migration ability of transfected cells. Scale bar = 100 μm. (G) The expression of migration-related molecular markers in different groups was analyzed via western blot. The full-length gels for western blot data in Figures S2D and S2G were presented in Supplementary Figure p < 0.01.Additional file 3: Supplementary Figure 3. (A) qRT-PCR analysis indicated the upregulation of GOLM1 in prostate cancer tissue samples in contrast to peri-tumor samples. (B-E) The immunoblots in Figs. p < 0.01.Additional file 4: Supplementary Figure 4. The full-length gel images of western blots in Fig. Additional file 5: Supplementary Figure 5. The full-length gel images of western blot data in Fig. Additional file 6: Supplementary Figure 6. The full-length images of western blots in Fig. Additional file 7: Supplementary Figure 7. The full-length images of western blot data in Supplementary Figure Additional file 8: Supplementary Figure 8. The full-length images of western blot data in Supplementary Figure Additional file 9: Supplementary Figure 9. The full-length images of western blot data in Supplementary Figure Additional file 10: Supplementary file 1. The enlarged images of picture data in Fig. Additional file 11: Supplementary file 2. The enlarged images of picture data in Figs.


Understanding the mechanisms of cataract formation is important for age-related and hereditary cataracts caused by mutations in lens protein genes. Lens proteins of the crystallin gene families α-, β-, and γ-crystallin are the most abundant proteins in the lens. Single point mutations in crystallin genes cause autosomal dominant cataracts in multigenerational families. Our previous proteomic and RNAseq studies identified genes and proteins altered in the early stages of cataract formation in mouse models. Histones H2A, H2B, and H4 increase in abundance in αA- and αB-crystallin mutant mouse lenses and in cultured cells expressing the mutant form of αA-crystallin linked with hereditary cataracts.cryaa-R49C and cryab-R120G mutant knock-in mice. We characterized the histones using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF)-mass spectrometric analysis and gel electrophoresis and characterized the lens nucleus morphology using electron microscopy (EM). The relative abundance of histone H3 protein decreased in lenses from cryaa-R49C mutant mice and the relative abundance of histone H2 increased in these lenses. Electron microscopy of nuclei from cryaa-R49C-homozygous mutant mouse lenses revealed a pronounced alteration in the distribution of heterochromatin.In this study of histones in mutant lenses, we extracted histones from adult mouse lenses from  Analysis of protein and gene expression changes in these mutant lenses found an increased expression of histone proteins and genes, suggesting cataracts and histone gene expression are related [The ocular lens consists of a single layer of lens epithelial cells on its anterior surface that overlay the lens fiber cells. The lens is encapsulated by a unique basement membrane  that surrounds the entire lens . The lenn family . Individn family . The cry related , 12.2(H2A:H2B)2. The histone amino terminal tails play a critical role in control of gene expression. Certain histone modifications are enriched in either actively transcribed or repressed chromatin regions [hist2h3c1 result in perturbed eye development and a small eye phenotype [cryaa-R49C knock-in mutant mice, we extracted histones from wild-type (WT) and cataractous mouse lenses and analyzed them using MALDI mass spectroscopy and gel electrophoresis.The chromatin core comprises four histone core proteins . Chromatin consists of a repeating unit of nucleosomes. The DNA double helix makes two turns around the octamer of four histones (H3:H4) regions . The ace regions . Histone regions . Inhibit regions . Histone regions . Mutatiohenotype . No studcryaa-R49C) and the αB-crystallin R120G mutation (cryab-R120G) on a C57Bl/6 J background were generated for this study [2 inhalation. The Institutional Animal Care and Use Committee at Washington University approved all experimental procedures using the mice.Adult het and homo knock-in mice carrying the αA-crystallin R49C mutation . The lenses were vortexed, homogenized, and incubated for 30 min at 4 °C in hypotonic lysis buffer, followed by centrifugation at 10,000×g for 10 min. The resulting supernatant was vortexed after the addition of 0.4 N H2SO4, and then incubated overnight at 4 °C. After centrifugation at 10,000×g for 10 min, the histone-containing supernatant was treated with trifluoroacetic acid, incubated overnight on ice, and then centrifuged to precipitate the histones. The resulting histone pellet was washed with ice-cold acetone, air-dried, and suspended in 80 μl deionized water to obtain the histone preparation.Histones were extracted from adult mouse lenses using a modified version of previous protocols , 19. BriThe histone extracts were dried on a SpeedVac concentrator, resuspended in 5 µl deionized water, and subjected to MALDI-TOF MS analysis. Histones extracted from mouse lenses were analyzed using reverse phase HPLC (RP-HPLC). Acid-extracted histones were run on a reverse-phase RP-300 Aquapore Octyl C8 column  with an acetonitrile gradient. A 50-µl sample loop and an Agilent Technologies HPLC system 1220 Infinity LC equipped with a variable wavelength detector with pumps, UV detector, and fraction collector were used for the HPLC. Data were collected in a Bruker ultrafleXtreme instrument and analyzed using flexAnalysis software version 3.4. The MALDI-TOF data represented the sum of 8 or 9 laser shots obtained using the LP_5-50_kDa.par linear positive mode method. Bruker calibration standard proteins were also analyzed. The peaks were identified by reference to previously published studies [Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on extracted histones was performed using 10–20% Tris–glycine gels (Life Technologies). Pre-stained molecular weight markers (Invitrogen) were used. Histones were at room temperature in electrophoresis buffer before loading on the gels. Proteins were analyzed by Coomassie blue staining and immunoblotting.cryaa-R49C-het, cryaa-R49C-homo, cryab-R120G-het, and cryab-R120G-homo mice. Electron microscopy was performed on lens sections to examine chromatin in nuclei of lens epithelial and fiber cells as described previously [Eyes were enucleated and lenses were extracted from WT, eviously .cryaa-R49C-het, cryaa-R49C-homo, cryab-R120G-het, and cryab-R120G-homo mice and was similar to bovine histones [cryaa-R49C-het lenses, we extracted histones H3 and H4 as the most abundant histone, with an H3/H4 ratio of 1.009  largely confined to the periphery; the inner nuclei consisted mostly of euchromatin. In the cryaa-R49C–het lenses, 3% of the nuclei had this type of abnormal heterochromatin distribution. There were no nuclei with this type of heterochromatin distribution in the WT group. Like the WT lenses, cryab-R120G mutant lenses (both het and homo) did not show this type of chromatin reorganization  euchromatin containing transcription factors, activated RNA polymerase, splicing machinery, and nascent RNA transcripts were all localized in the center of the nucleus. This characteristic indicated this location was the site of active gene transcription in the cryaa-R49C-homo lenses. There were a few clusters of dark heterochromatin in some of the cryaa-R49C-homo lens nuclei. The heterochromatin was also anchored to the periphery of the homo nuclei; this type of anchoring has been linked to lamin A/C and lamin B receptors. There are also models of retinal photoreceptors in which the nuclear distribution of chromatin is controlled by transcription factors [The nuclear redistribution of heterochromatin suggested a drastic change in histones occurred in the  factors .cryaa gene are associated with human cataract [cryaa-R49C homo lenses indicated an important role of this histone in the lens. Histone H3 alterations are associated with defects in lens model systems [Various mutations in the cataract . Future  systems , 17, 26.cryaa-R49C-homo lenses, due to their small size.The histone extraction procedures used in this study may not maintain the posttranslational modifications of histones (e.g. acetylation and methylation). The amount of histones extracted from individual mouse lenses is very low, particularly from the Additional file 1: Table S1. Relative intensities of histones extracted from mouse lenses .Additional file 5: Figure S3. MALDI-TOF MS analysis of histones isolated from cryab-R120G-homo mouse lenses (related to Table S1).Additional file 6: Figure S4. SDS-PAGE and immunoblotting of histones extracted from mouse lenses. (A) Coomassie blue-stained gel of histones from bovine lenses used as a control standard (lanes 1 and 2); WT mouse lenses (lane 3). Molecular weight markers (lane 4). (B) Immunoblot for the gel shown in (A) using a histone H2B antibody. Control standard (lanes 5 and 6); WT mouse lens (lane 7). (C) Coomassie blue-stained gel of histones from cryaa-R49C-het lenses (lane 8). (D) Immunoblot for the gel shown in (C) using a histone H2B antibody. (E) Coomassie blue-stained molecular weight markers on the membrane in (D). Note the increase in a band at ~ 17 kDa in the Coomassie stained gel and immunoblot in cryaa-R49C-het lenses as compared with WT. This band appears at a doublet, and both bands are present in the WT mice, although Coomassie-stained band at ~ 17 kDa did not appear in the immunoblot of WT lens histone preparation. The increase in the band intensity of the immunoblot at ~ 17 kDa in the cryaa-R49C-het mutant lenses suggested that the amount of highly modified histones increase the mutant lenses. The immunoblot analysis was performed using antibodies to histone H2B. The protein bands were visualized and quantified using an Odyssey system [y system , 27. MouAdditional file 7: Figure S5. Electron micrographs of mouse lens epithelial nuclei. Wild type, cryab-R120G het, and cryab-R120G homo lenses were analyzed. The cryab-R120G mutant lenses appear to have early stages of chromatin condensation compared with WT (Fig.  WT Fig. .


Pseudomonas syringae pv. tomato (Pst) DC3000, two T6SS gene clusters, HSI-I and HSI-II, were identified, but their functions remain unclear. We previously reported that hcp2, located in HSI-II, is involved in competition with enterobacteria and yeast. Here, we demonstrated that interbacterial competition of Pst DC3000 against several Gram-negative plant-associated bacteria requires mainly HSI-II activity. By means of a systematic approach using in-frame deletion mutants for each gene in the HSI-II cluster, we identified genes indispensable for Hcp2 expression, Hcp2 secretion and interbacterial competition ability. Deletion of PSPTO_5413 only affected growth in interbacterial competition assays but not Hcp2 secretion, which suggests that PSPTO_5413 might be a putative effector. Moreover, PSPTO_5424, encoding a putative σ54-dependent transcriptional regulator, positively regulated the expression of all three operons in HSI-II. Our discovery that the HSI-II gene cluster gives Pst DC3000 the ability to compete with other plant-associated bacteria could help in understanding a possible mechanism of how phytopathogenic bacteria maintain their ecological niches.The type VI secretion system (T6SS) is a widespread bacterial nanoweapon used for delivery of toxic proteins into cell targets and contributes to virulence, anti-inflammatory processes, and interbacterial competition. In the model phytopathogenic bacterium  Natural habitats do not always provide suitable conditions for microorganisms to survive; therefore, bacteria have evolved various secretion systems to adapt to changing environments by translocating effector proteins or DNA into the surrounding milieu or target cells . The typRhizobium leguminosarum  DC3000, a causative agent of tomato speck disease, has become one of the well-known model organisms to study plant–microbe interactions. The pathogenicity and virulence of Pst DC3000 depend on a functional type III secretion system, which delivers effectors into host cells to block the plant defense system or interfere with normal metabolism in planta  ATCC 11528, and P. s. pv. oryzae 1–6 contain two putative T6SS clusters, Hcp secretion island 1 (HSI-I) and HSI-II. The expression of an icmF (tssM) ortholog in HSI-II of Pst DC3000 has been demonstrated  supplemented with appropriate antibiotics or in hrp-inducing minimal media (HMM) substituted with different carbon sources at 28°C. E. coli strains were grown in Luria-Bertani (LB) medium at 37°C. The concentrations of antibiotics used were for gentamicin, 10 μg/mL; kanamycin, 50 μg/mL; rifampicin, 50 μg/mL; and tetracycline, 30 μg/mL.Bacterial strains and plasmids used in this study are in mobsac , phcp1p (Phcp1:uidA), pHSI-II-ip (PHSI-II-i:uidA), pHSI-II-iip (PHSI-II-ii:uidA), and pHSI-II-hcp2p (Phcp2:uidA). The resulting plasmids were then introduced into Pst DC3000, which was then grown in KBM from OD600 = 0.1 to 0.8 before cells were collected for 4-methylumbelliferyl-β-D-glucuronide (4-MUG)-based β-glucuronidase (GUS) reporter assay . Ten microliters of each mixture were spotted onto 0.22 μm nitrocellulose membranes placed on KBM plates and incubated for 2 days at 28°C. The GFP signals for each colony were recorded under a fluorescent stereomicroscope . The number of each bacterial strain in a mixed culture was counted by a method described previously  or pure culture was determined with a BacLight Bacterial Viability Kit  as described previously  antisera as primary antibodies and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG as the secondary antibody. Signals of the target proteins were detected by using Immobion Chemiluminescent HRP substrate  with a G: BOX system .Overnight cultures of different Pst DC3000 confers growth advantages over E. coli and yeast in a hcp2-dependent manner  was transformed into the selected plant-associated bacteria, including Agrobacterium tumefaciens C58, Dickeya dadantii CAS9, Pst T1, P. s. pv. syringae (Pss) 61, P. savastanoi pv. phaseolicola (Psph) 1448A, P. s. pv. tabaci (Psta) 11528, P. viridiflava, P. putida A7, and Xanthomonas euvescatoria XvT147 as well as E. coli MG1655. These competitor bacterial strains were then individually mixed with cells of the Pst DC3000 wild type and derived mutants at a 10:1 ratio. Two days after co-incubation on KBM at 28°C, changes in GFP intensity were observed under a fluorescent stereomicroscope. As shown in Pst DC3000 co-incubated, the colonies of A. tumefaciens C58 or P. viridiflava looked similar under visible or UV light in terms of GFP fluorescence signal. However, the GFP signal intensity was decreased to various degrees when the bacterial competitors D. dadantii CAS9, E. coli MG1655, Psph 1448a, Pss 61, Psta 11528, Pst T1, and X. euvesicatoria XvT147 were co-incubated with Pst DC3000 wild type and ΔHSI-I versus incubation alone or co-incubation with ΔHSI-II and ΔHSI-I/II. Unexpectedly, GFP signal was not detected in P. putida A7; however, smaller colonies could be observed when P. putida A7 was co-incubated with the Pst DC3000 wild type and ΔHSI-I, which suggests their ability to inhibit growth or spreading of P. putida A7 . To investigate how T6SS is regulated in Pst DC3000, each putative promoter in HSI-I and HSI-II clusters was transcriptionally fused to the uidA gene for a GUS reporter assay to monitor the promoter activity under different carbon sources. Consistent with a previous study  were in or flanked this region, which infers possible horizontal gene transfer events (tssM2 (PSPTO_5427 to PSPTO_5413), tssB2/C2 (PSPTO_5434 to PSPTO_5428), and hcp2 (PSPTO_5435 to PSPTO_5439) operons.In the r events , and thur events . To verir events . When cer events . These dPst DC3000 (hcp2), Δ5424 with deletion of sfa2 encoding a σ54-dependent transcriptional regulator, and Δ5427 with deletion of tssF2  and positive regulatory genes (ppkA and fha), Δ5423, representing deletion of an accessory gene orfX with unknown function, also impaired Hcp2 secretion  reduced the ability of Pst DC3000 to compete against several plant-associated Gram-negative bacteria  it is one of the two strains  with two copies of a similar T6SS cluster . In fact, records have linked higher survival of Pst to tomato seeds and host debris but not field soils  are not the targets of PSPTO_5645.T6SS effectors responsible for antibacterial activity have been identified in many bacteria, and many are encoded within the T6SS loci, which implies that their secretion strongly depends on the associated T6SS apparatus . Bioinfo strains . That weE. coli and Psph. The evidence that PSPTO_5413 plays no role in mediating Hcp2 secretion is consistent with its accessory role not related to T6SS assembly. However, constitutively expressing PSPTO_5413 in E. coli MG1655 or Psph1448a did not reveal growth inhibition (data not shown). The lack of toxicity in the target cells may suggest that additional factors(s) from Pst DC3000 may be required for toxicity of PSPTO_5413, or subcellular location of ectopically expressed PSPTO_5413 may not be the site of its mode of action. Analysis of the amino acid sequence of PSPTO_5413 revealed the presence of an EF-hand motif and a domain highly similar to the catalytic domain of the antimicrobial peptidase lysostaphin. EF-hand motifs can be found in calcium-binding proteins, acting in Ca2+ homeostasis or calcium signaling  and recombination hotspot (Rhs) domains (PSPTO_5438 and 5439) . DUF4123ph 1448a . Besides54-dependent transcriptional regulators encoded within T6SS loci. In P. aeruginosa, Sfa2 negatively regulates H2-T6SS expression, but H3-T6SS expression is Sfa3-independent  in Pst DC3000 is opposite to that of Sfa2 in P. aeruginosa. Discovery of effector proteins is important for understanding the mechanism underlying T6SS functions. Use of a vasH mutant expressing VasH under the control of an arabinose-inducible promoter revealed an effector VasX encoded outside the T6SS locus (Pst DC3000 and V. cholerae both positively control the expression of T6SS by a σ54-dependent transcriptional regulator, the same strategy may be applicable for discovery of additional T6SS effectors in Pst DC3000.Several reports have shown that the expression of genes coding for T6SS apparatus can be modulated by the σependent . In V. ccp genes . AlthougSS locus . BecausePst DC3000. From this and previous studies, we now know that instead of providing virulence activity, HSI-II-T6SS is likely used as a defensive weapon to protect Pst DC3000 against bacteria sharing the same ecological niches to ensure sufficient inoculum for successful infection when encountering appropriate host plants. At present, secretome and transcriptome analyses are undergoing, which should help us identify the effectors and regulatory components of HSI-II-T6SS for better understanding the mechanistic and biological functions of HSI-II in Pst DC3000.Using a systematic mutagenesis approach, we identified genes located in the HSI-II cluster that are required for T6SS activities, including accumulation or secretion of Hcp2, and interbacterial competition (summarized in The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.C-FC and N-CL conceived and designed the experiments. C-FC, C-YL, Y-YL, Y-HS, K-YC, and N-CL performed the experiments and analyzed the data. C-FC and N-CL wrote the manuscript.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.


Escherichia coli YdgG, was identified to function as an AI-2E. However, it’s very likely that AI-2E family members may exhibit significantly distinct functions due to low identities between them. Recently, we identified one member of this family designated as UPF0118 to represent a novel class of Na+(Li+)/H+ antiporters. In this study, we presented that UPF0118, together with its homologs, should represent an independent group of AI-2E family, designated as Na+/H+ Antiporter Group. Notably, this group shows five highly conserved motifs designated as Motifs A to E, which are not detected in the majority of AI-2E family members. Functional analysis established that polar or charged residues located in Motif A to D play a vital role in Na+(Li+)/H+ antiport activity or pH response of UPF0118. However, three basic residues located in Motif E are not involved in the function of UPF0118, although the truncation of C terminus resulted in the non-expression of this transporter. Therefore, we propose that E179-R182-K215-Q217-D251-R292-R293-E296-K298-S307 located in Motifs A to D can be used for signature functional motifs to recognize whether AI-2E family members function as Na+(Li+)/H+ antiporters. Current findings positively contribute to the knowledge of molecular mechanism of Na+, Li+ transporting and pH response of UPF0118, and the functional prediction of uncharacterized AI-2E family members.Functionally uncharacterized UPF0118 family has been re-designated as autoinducer-2 exporter (AI-2E) family since one of its members,  These antiporters play vital roles in not only mediating the circulation of various monovalent cations, mainly Na+ and K+, even Ca2+, inside and outside the cells and maintaining intracellular pH homeostasis, but also participating in many biological process including signal molecule transmission, spore development, antibiotic release, cell volume control and etc.  monovalent cation/proton antiporter-1 (CPA-1) family, such as NhaG (2+:H+ antiporter-2 (CaCA2) family, such as ChaA  CPA as NhaG ; (4) maj as NhaG , MdfA  Ca2 as ChaA ; (6) Nha as ChaA ; (7) Nha as ChaA ; (8) Nha as ChaA  and MleN as ChaA ; (9) Nha as ChaA ; (10) Nh as ChaA . The late stress . To incractivity . For exaactivity , DUF1538activity , and RDDactivity , respectactivity  and catiactivity , respectHalobacillus andaensis has been identified to represent a novel class of Na+(Li+)/H+ antiporters . Therefore, we speculate that AI-2E family members may exhibit a significant difference in function due to low identities between them, although these members are temporarily categorized into AI-2E family.In our previous study, a UPF0118 family (currently named AI-2E family) transporter from the moderately halophilic bacterium iporters . Hereby,iporters . Althoug+(Li+)/H+ antiporter, we first analyze the phylogenetic relationship between UPF0118 and its homologs and AI-2E members collected in the TCDB system. Also, we further discover the roles of polar or charged amino acid residues located in the above five motifs of UPF0118 via site directed mutagenesis. Consequently, we found out that UPF0118 and its homologs should represent an independent group designated as Na+/H+ Antiporter Group. More importantly, we propose that E179-R182-K215-Q217-D251-R292-R293-E296-K298-S307 located in Motifs A to D can be used for signature functional motifs to recognize whether AI-2E family members function as Na+(Li+)/H+ antiporters. These findings positively contribute to the understanding of molecular mechanism of Na+, Li+ transporting and pH response of UPF0118. AI-2E family includes a large number of uncharacterized members except for Escherichia coli YdgG and H. andaensis UPF0118  . Growth was ended on 24 h and then ODnm600 was evaluated. The phoA-deficient E. coli mutant DH5α was used as a host strain to test the orientation of UPF0118 in the cytoplasmic membranes as described previously (E. coli transformants.aBΔchaA)  were groaBΔchaA) . Growth aBΔchaA) . Brieflyeviously . Ampicilhttps://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome. Topological analysis was carried out via the multiple web-based softwares including HMMTOP, TMHMM, Tmpred, PredTMR, SOSUI, Phyre 2, and PredictProtein. Weblogo was created at the website http://weblogo.threeplusone.com/create.cgi.A neighbor-joining phylogenetic tree was constructed with a bootstrap analysis (1000 replications) for the stability of clusters . Proteinupf0118 using the primers listed in phoA-deficient E. coli mutant DH5α. Also, E. coli mutant DH5α transformed with the empty vector or expressing UPF0118 alone was used as the negative controls. The transformants were grown to observe the lawn color on the LB medium plate containing 0.4 mg/ml 5-bromo-4-chloro-3-indolylphosphate as the substrate, as described previously /H+ antiport activity assays.All the site-directed or C terminus-truncated variants of UPF0118 were constructed using pET22b-P-UPF0118 as a template and the corresponding primers listed in E. coli KNabc transformants by the French press method as described in our recent studies , 140 mM choline chloride, 0.5 mM dithiothreitol and 250 mM sucrose, and broken by one passage through a JG-1A French Press  at 2000 psi system pressure. Cell debris was removed by centrifugation at 5000 × g, 4°C for 10 min, and the supernatant was centrifuged at 100,000 × g for 1 h. Everted membrane vesicles were finally separated from the supernatant and re-suspended in the same buffer as above, and stored at -80°C for the following Na+(Li+)/H+ antiport assay and protein expression level analysis.Everted membrane vesicles were prepared from  studies . Cells o6-tag labeled proteins were detected using a rabbit anti-His6 tag antibody  and a goat anti-rabbit horseradish peroxidase labeled secondary antibody . Western blots were visualized by using a BeyoECL Star kit  via a Tannon-5200 multi chemiluminescent imaging system .SDS-PAGE and western blots were performed as described in our recent studies . The eve+(Li+)/H+ antiport activities were measured by using an acridine orange fluorescence dequenching protocol, as described in our recent studies  buffer. Respiration-dependent formation of ΔpH was initiated by the addition of 10 mM Tris-D-lactate. Na+(Li+)/H+ antiport activity was estimated based on its ability to dissipate the established ΔpH upon addition of final concentrations at 10 mM NaCl or LiCl. The antiport activities were expressed as the percentage ratio of dequenched fluorescence by NaCl or LiCl to the lactate-induced fluorescence quenching. The acridine orange fluorescence was monitored with excitation wavelength at 492 nm and emission wavelength at 526 nm using a Hitachi F-7000 fluorescence spectrophotometer . K0.5 values of wild-type UPF0118 or its variants for the transported cations were calculated by plotting Na+/H+ and Li+/H+ antiport activity as the respective functions of cation concentrations, followed by a non-linear regression analysis obtained with Prism 7.0, as described in our previous study  . HoweverEscherichia coli PhoA has high activity in the periplasm but low activity in the cytoplasm, and therefore the fusions with this reporter protein are usually used to judge the orientation of TMHs of transmembrane proteins  and Clostridium difficile CD630_20350 (19% identity with UPF0118)  . This clt clades .+/H+ antiporters, polar or charged residues have been established to play critical roles in cation translocation, protonation, pH response, conformational stability, electrogenesis and etc. /H+ antiporter, as the previous construct, pET22b-UPF0118 /H+ antiporter. E179D or R182K restored the complementation with E. coli KNabc under the same stress conditions, as wild-type UPF0118 (+(Li+)/H+ antiport activity. The antiport activity analysis established the important roles of side chains from E179 and R182 in the Na+(Li+)/H+ antiport activity of UPF0118 /H+ antiport activity of UPF0118. K215A offered the same growth of E. coli KNabc as that of wild-type UPF0118 /H+ antiport activity of UPF0118. More importantly, both negative charge and the length of side chain from this residue should be indispensable for the function of UPF0118.D251A or D251E failed to complement with li KNabc . Substitactivity . The wesactivity . The aboE. coli KNabc and its Na+(Li+)/H+ antiport activity (E. coli KNabc or its Na+(Li+)/H+ antiport activity (+(Li+)/H+ antiporter. Namely, in addition to positive charge, nitrogen atom or amino group in guanidyl groups of side chains may also play a vital role in the Na+(Li+)/H+ antiport activity of UPF0118. Similarly, growth tests and antiport activity assays for E296A and E296D /H+ antiport activity (+(Li+)/H+ antiport activity of UPF0118. The western blot established normal expression of the above variants in E. coli KNabc /H+ antiport activity of UPF0118, the truncated region in Motif E should determine the normal expression and even accurate localization of UPF0118 into the cytoplasmic membranes. Most of the multiple web-based softwares predicted that the last TMH of UPF0118 ends at A340 or the former residues (+(Li+)/H+ antiporter.A C terminus-truncated variant, UPF0118li KNabc  or its ali KNabc . The wes UPF0118 . UPF0118residues  whereas residues . TherefoE. coli KNabc (+(Li+)/H+ antiport activity to that of wild-type UPF0118 /H+ antiporter. It seems that polar or charged residues outside five conserved motifs may not play a determining role in the function of UPF0118 as a Na+(Li+)/H+ antiporter. This indirectly supports the possibility that conserved motifs can be used for signature functional motifs.Substitution of T39, R58, or E238 by alanine had no effect on the complementation ability of UPF0118 with li KNabc A. Also,  UPF0118 B. Howevee latter B. This sH by 0.5 B. This sli KNabc C. Since li KNabc A, T39, R+/H+ antiporter UPF0118, together with its homologs, should represent an independent group of AI-2E family. This group was proposed to designate as Na+/H+ Antiporter Group, which exhibits a distant phylogenetic relationship with the majority of AI-2E members collected in the TCDB system /H+ antiport activity or pH response of UPF0118. However, three basic residues located in Motif E are not involved in the function of UPF0118, although the truncation of C terminus resulted in the non-expression of this transporter. Therefore, we propose that E179-R182-K215-Q217-D251-R292-R293-E296-K298-S307 located within five functional motifs can be used for signature functional motifs to recognize whether AI-2E family members function as Na+(Li+)/H+ antiporters.In this study, we presented that a previously characterized NaB system . NotablyE. coli YdgG was reported to function as an AI-2 exporter /H+ antiporter /H+ antiporters. Moreover, K215 in Motif B or K298 in Motif D can’t determine the Na+(Li+)/H+ antiport activity of UPF0118, though either one plays a vital role in the response of antiport activity to pH (+/H+ antiporter if it possesses the same conserved motifs with E179(D)-R182(K)-K215(A)-Q217(N)-D251-R292-R293-E296-K298(A)-S307.UPF0118 and its homologs showed five highly conserved motifs, Motifs A to E . Notablymus YCT2 . This sutiporter . Similartiporter . In conttiporter , 7. Howety to pH , 7. Also+(Li+)/H+ antiport activity to pH (+(Li+)/H+ antiporters, except for K305 in Thermus thermophilus NapA (+(Li+)/H+ antiport activity. Also, there are two α helices and six TMHs in the topological structure of UPF0118 /H+ antiporters. Current findings not only trigger the understanding of molecular mechanism of Na+, Li+ transporting and pH response of UPF0118, but also clarify the knowledge of AI-2E family including a large number of uncharacterized members.Taken together, the results presented in this study provide a strong evidence for UPF0118 and its homologs to represent an independent group of AI-2E family, NaPublicly available datasets were analyzed in this study. This data can be found in GenBank under accession no. KY231907.JJ, LW, and QZ designed the experiments. LW and QZ constructed the subclone and variants. LW, QZ, MY, and HC performed the topological analysis of UPF0118. LW, QZ, MY, YW, SG, RZ, YS, XL, HC, LS, and LM carried out the growth tests, activity assays, or western blots. JJ wrote the manuscript. All authors checked and approved the final version of this manuscript.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.


Our study identified NLRP3 activation and IL-18R-dependent signaling as potential modulators of early liver damage in NAFLD, preceding development of histologic NASH.Non-alcoholic fatty liver disease (NAFLD) is rising in prevalence, and a better pathophysiologic understanding of the transition to its inflammatory phenotype (NASH) is key to the development of effective therapies. To evaluate the contribution of the NLRP3 inflammasome and its downstream effectors IL-1 and IL-18 in this process, we applied the true-to-life “American lifestyle-induced obesity syndrome” (ALiOS) diet mouse model. Development of obesity, fatty liver and liver damage was investigated in mice fed for 24 weeks according to the ALiOS protocol. Lipidomic changes in mouse livers were compared to human NAFLD samples. Receptor knockout mice for IL-1 and IL-18 were used to dissect the impact of downstream signals of inflammasome activity on the development of NAFLD. The ALiOS diet induced obesity and liver steatosis. The lipidomic changes closely mimicked changes in human NAFLD. A pro-inflammatory gene expression pattern in liver tissue and increased serum liver transaminases indicated early liver damage in the absence of histological evidence of NASH. Mechanistically,  Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, has become a leading cause of chronic liver disease in many regions of the world , is stilVarious risk factors have been associated with the development of NAFLD, including dietary habits, lifestyle, gut microbiome and drug intake ,9,10,11.Factors responsible for the transition of benign steatosis towards NASH remain enigmatic but may involve, among others, mitochondrial dysfunction ,13,14. IIt has been shown that the methionine–choline-deficient (MCD) diet, a widely used in vivo model of NASH in mice, induces upregulation of the NLRP3 inflammasome, and that high-fat diet (HFD) feeding can also cause inflammasome activation . InvestiTherefore, we applied the American lifestyle-induced obesity syndrome (ALiOS) model, a true-to-life mouse model of obesity. This diet reflects commonly consumed fast food. Chow provides 45% of calories from fat, including trans fats, and high-fructose corn syrup  is administered in drinking water as an equivalent for soda drinks. This diet has been shown to induce obesity, glucose intolerance, hyperinsulinemia and substantial hepatic steatosis associated with necroinflammation and a profibrogenic response in mice .Applying this model, we revisited the contribution of the NLRP3 inflammasome, i.e., its effector cytokines IL-1 and IL-18, for the development of liver damage in NAFLD. To block IL-1- and IL-18-dependent signaling, knockout animals for the respective receptors were used.Wildtype C57BL/6 mice were fed a Western diet according to the ALiOS protocol, or control diet, for 24 weeks. ALiOS-fed mice had an increase in total body weight of 32% compared to standard diet-fed mice A, and esNotably, marked hepatic accumulation of triglycerides was found D and livThus, ALiOS-fed mice developed obesity, features of metabolic syndrome and hepatic steatosis.Feeding of the ALiOS diet led to marked changes in the hepatic fatty acid composition. While the proportion of monounsaturated fatty acids increased, both di- and polyunsaturated fatty acids decreased in liver tissue A. MoreovALiOS feeding was associated with an upregulation of NLRP3, indicative of activation of this signaling pathway A. Along n = 7, each). Likewise, serum levels of IL-10, MCP and TNF-α were not different in both experimental groups. Activation of the inflammasome was confirmed by measurement of serum levels of NLRP3-associated cytokines. Serum levels of IL-1β were unaltered. In contrast, serum levels of IL-18 were increased B. IL-1α In summary, the ALiOS diet resulted in an activation of the inflammasome and was associated with an upregulation of IL-18 as well as pro-inflammatory markers in the liver.In wildtype animals, ALiOS feeding led to mild but chronic hepatocellular damage. This was evidenced by increased serum activities of ALT, indicating hepatopathy associated with NAFLD in these animals B. DespitTaken together, feeding of the ALiOS diet without simultaneous restrictions of voluntary movement induced obesity, metabolic syndrome and NAFLD in C57BL/6 wildtype mice, but not NASH.−/−Il-18r  and −/−Il-1r  mice. Body weight, visceral fat weight and serum non-esterized fatty acids (NEFA) were not different between the genotypes .NAFLD in our model was, however, accompanied by a pro-inflammatory hepatic gene expression pattern and activation of the inflammasome . It has In our animals, NLRP3 was upregulated, as well as TGF-ß1, a major pro-fibrogenic cytokine, and CXCL2. CXCL2 is produced in reaction to lipopolysaccharides by macrophages or monocytes. Monocyte markers Mcp-1 and F4/80 were also increased, indicating an inflammatory process. This is reflected by the increase in ALT.Portal macrophages are detectable in steatosis alone as the earliest change, followed by an elevated expression of pro-inflammatory cytokines such as IL-1α and TNF-alpha in early NASH. In our study, the downstream mediator of NLRP3, IL-18 but not IL-1, was particularly increased. In mice on a high-fat diet, IL-18 has previously been identified among the intermediate markers of hepatic steatosis  and simiKnockout of IL-18R did not influence the development obesity and markers of metabolic syndrome . In contIn contrast to IL-18R, the IL-1R knockout was without benefit in the ALiOS model and liver damage was not different from wildtype animals. This is in line with previous work on cholestatic liver disease, where IL-1 signaling also was of limited importance  but in cThe activation of the NLRP3 inflammasome is upstream of both IL-1 and IL-18 signaling. It has previously been reported that the knockout of this upstream target did not prevent steatosis-associated liver damage ,13. MitoThe major limitation of this study is the absence of NASH upon histopathologic evaluation. Our study thus focuses on the very early changes in steatotic liver and it remains to be determined whether IL-18R-dependent signaling also drives inflammation in established NASH. Since the true-to-life nature of the experimental model applied in this study is one of its strengths, investigation of longer time periods of feeding or introduction of an additional hit, rather than applying more artificial models such as the MCD diet, may be suitable to further explore these questions.In summary, we provide evidence that the ALiOS mouse model used in this study particularly closely mimics the pathophysiology of human NAFLD, including obesity, liver steatosis and lipidomic changes. We were able to identify IL-18-dependent signaling as a modulator of early liver damage in fatty liver, preceding development of histologic NASH. Further studies will have to elucidate to what extent IL-18R deficiency is protective in non-alcoholic steatohepatitis.−/− and Il-1r −/− mice were co-housed and kept according to the guidelines for the care and use of laboratory animals at the University Hospital Munich. Mice had free access to water. Water for ALiOS diet-fed animals was enriched with 23.1 g/L fructose and 18.9 g/L glucose reflecting consumption of soda drinks in humans. Animals were either fed with the ALiOS  or with the standard diet  for 24 weeks starting at the age of eight weeks. A total of 45% of the calories of the ALiOS diet originated from fat (control: 9% from fat) in the form of partially hydrogenated vegetable oil  and 13% polyunsaturated fatty acids (PUFAs) [Male C57BL/6NCrl mice from Charles River  and Il-18r  (PUFAs) . Animal Human liver tissue was obtained from the Biobank of the Department of General, Visceral and Transplantation Surgery, Ludwig Maximilian University, Munich, Germany, under the administration of the Human Tissue and Cell Research Foundation (HTCR) , as appr® 910 fully automated analyzer .Activity of alanine aminotransferase was quantified from fresh serum by a responsLiver tissue was embedded in paraffin after 24-h fixation in 4% formaldehyde solution or was shock-frozen in liquid nitrogen. Paraffinized blocks were cut into slices of 4 μm thickness that were mounted on SuperFrost Plus microscopic slides . After stepwise deparaffinization and rehydration, the slides were stained with hematoxylin and eosin (H&E). Staining of shock-frozen tissue with Sudan III and H&E was performed according to standard protocols. To establish the diagnosis of NASH, different well-established scoring systems were used. Both the SAF score   and the g and the supernatant was analyzed with a Response 910 system .For quantification of triglycerides (TGs) in liver, 100 mg of liver tissue was sonicated in 1 mL 5% NP40 solution, heated for 5 min at 96 °C and cooled down on ice. Afterwards, the homogenate was centrifuged for 2 min at 20,000× Hydroxyproline levels in liver tissue, serving as a read-out for liver fibrosis, were quantified according to Edwards et al. . In briev/v). The organic phase was evaporated (N2), esterified and extracted with n-hexane. Fatty acid methyl esters were separated by capillary gas chromatography with a BPX70 column from SGE at an oven temperature of 120–210 °C with an increase of 2 °C/min, H2 as the carrier gas and a flame ionization detector, and were identified by their retention time relative to standard mixtures . Quantification was performed with C15:0 as the internal standard and with Clarity Lite software . For comparison with mouse livers from our experiments, anonymized samples of human liver tissue from the Biobank of the Department of General, Visceral and Transplantation Surgery, Ludwig Maximilian University, Munich, Germany, under the administration of the HTCR [Fatty acids were extracted from mouse tissue (10 mg) according to the method of Folch . Clearedthe HTCR , were evMitochondria from mouse livers were freshly prepared and purified by Percoll™  as described . ATP syn® RNA kits  according to the manufacturer’s instructions. Complementary DNA was synthesized using reverse transcriptase SuperScript® III . Real-time PCR was performed in a SYBR® Green system  on a Realplex4 Mastercycler epgradient S . Expression was calculated according to the ΔΔCt method with GAPDH and 36B4 as the housekeeping genes and was normalized to the means of the controls. Primers were purchased at Eurofins Genomics , and the sequences were as follows:Gapdh:leftAGCGAGACCCCACTAACATCrightGGCGGAGATGATGACCCTTT36B4:leftTCTAGGACCCGAGAAGACCTrightCCCACCTTGTCTCCAGTCTTNalp3:leftAAAGCTAAGAAGGACCAGCCrightTATCCCAGCAAACCCATCCATgf-b1:leftCGCAACAACGCCATCTATGArightACTGCTTCCCGAATGTCTGACxCl2:leftTCTTGAGCTTGGTGACAAAAACrightGGCTGGAGAGCTACAAGAGGMcp-1:leftGCTGCTACTCATTCACCAGCrightCTTCTTGGGGTCAGCACAGAF4/80:leftAACCAACTTTCAAGGCCCAGrightTGCAGACTGAGTTAGGACCAmRNA was isolated from 30 mg liver tissue using Trizol reagent  according to the manufacturer’s instructions. Visceral fat (50 mg) was minced with a Polytron homogenizer  and mRNA was extracted with NucleoSpinInterleukin (IL)-1α, IL-1β, IL-6, IL-10 and IL-18, insulin, leptin and adiponectin were quantified in serum with the customized Bio-Plex Pro™ Cytokine and Diabetes Assay and the Bio-Plex 200 system from Bio-Rad according to the manufacturer’s instructions.Results from independent repeated experiments are shown as means ± standard deviation. The Shapiro–Wilk test was used to demonstrate normal distribution of fraction samples. Differences between means of two groups were validated with the t-test. Statistical testing of observed frequencies was performed by the chi-squared test or Fisher’s exact test, as appropriate. All statistic calculations were performed with the SPSS 25 software package (IBM).


N-Acylhomoserine lactones (AHLs) are used as quorum-sensing signals in Gram-negative bacteria. Many genes encoding AHL-degrading enzymes have been cloned and characterized in various microorganisms. Coagulase-negative staphylococci (CNS) are present on the skin of animals and are considered low-virulent species. The AHL-lactonase gene homologue, ahlS, was present in the genomes of the CNS strains Staphylococcus carnosus, Staphylococcus haemolyticus, Staphylococcus saprophyticus, and Staphylococcus sciuri. We cloned the candidate ahlS homologue from six CNS strains into the pBBR1MCS5 vector. AhlS from the CNS strains showed a higher degrading activity against AHLs with short acyl chains compared to those with long acyl chains. AhlS from S. sciuri was expressed and purified as a maltose-binding protein (MBP) fusion. Pseudomonas aeruginosa is an opportunistic pathogen that regulates several virulence factors such as elastase and pyocyanin by quorum-sensing systems. When MBP-AhlS was added to the culture of P. aeruginosa PAO1, pyocyanin production and elastase activity were substantially reduced compared to those in untreated PAO1. These results demonstrate that the AHL-degrading activity of AhlS from the CNS strains can inhibit quorum sensing in P. aeruginosa PAO1. N-acyl-l-homoserine lactone (AHL), is used by Gram-negative bacteria [Bacillus sp. 240B1, is the most-studied AHL-degrading enzyme [Solibacillus silvestris [Agrobacterium tumefaciens [Arthrobacter sp. [Chryseobacterium sp. [Thermaerobacter marianensis JCM 10246 [Quorum sensing is a cell-to-cell communication system used by many bacterial species depending on their population densities . One of bacteria . Once thbacteria . AHL-negbacteria ,4. Quorubacteria . AHL-degbacteria . AHL lacbacteria . AiiA-tyg enzyme . It belolvestris , AttM frefaciens , AhlD frcter sp. , and Aidrium sp. . In addiCM 10246 .Staphylococcus aureus and Staphylococcus pseudintermedius [Coagulase-negative staphylococci (CNS) are known to comprise over 30 species, which are part of the normal flora on the skin of animals . Coagulaermedius . Therefoermedius ,3. Meanwermedius . So far,Staphylococcus carnosus, Staphylococcus haemolyticus, Staphylococcus saprophyticus, and Staphylococcus sciuri were selected to evaluate their AHL-degrading activities. CNS strains were cultivated in Luria–Bertani (LB) liquid medium containing 20 μM N-hexanoyl-l-homoserine lactone (C6-HSL) or N-decanoyl-l-homoserine lactone (C10-HSL). After 4.5 and 9 h incubation, the remaining AHLs in the culture supernatant were visualized by AHL biosensors, Chromobacterium violaceum CV026 (for the short chain C6-HSL) and VIR07 (for the long chain C10-HSL), which produced the purple pigment violacein [S. sciuri ATCC 29060 and 29061 completely degraded 20 μM C6-HSL by 4.5 h incubation, suggesting higher AHL-degrading activities than those of other strains. In contrast, most C10-HSL remained by 4.5 h incubation in the supernatants of S. carnosus, S. haemolyticus, and S. saprophyticus. Lower levels of C10-HSL were detected in the culture supernatants of S. sciuri ATCC 29060 and 29061, which also had higher C6-HSL-degrading activities. After 9 h incubation, all CNS strains completely degraded 20 µM C10-HSL except for S. haemolyticus NBRC 109768. These results suggested that the CNS strains tested in this study had higher degrading activities toward C6-HSL than C10-HSL.Six strains from four CNS species, iolacein ,14. The S. carnosus NBRC 109622, S. haemolyticus NBRC 109768, and S. saprophyticus ATCC 15305 (synonym of NBRC 102446) were deposited in DDBJ/ENA/GenBank under the accession numbers BKAO01000000, BKAY01000000, and CP035294, respectively. Gene homologs encoding the AiiA-type AHL-lactonase were searched in these genome sequences. Based on the Basic Local Alignment Search Tool (BLAST) analysis [S. carnosus NBRC 109622, SHA04_07910 from S. haemolyticus NBRC 109768, and EQ030_01160 from S. saprophyticus NBRC 102446 showed similarity to the known AiiA-type AHL lactonases. Specifically, the amino acid sequences of these gene homologs showed higher similarity with AhlS from S. silvestris StLB046 [T. marianensis JCM 10246 [S. sciuri ATCC 29060 and 29061 have not been released, the ahlS gene homolog (FPV13_12710) was found in the genome of S. sciuri B9-58B (accession No. CP041879) [S. sciuri B9-58B, and the ahlS homolog genes were successfully amplified by PCR from the genomes of ATCC 29060, ATCC 29061, and StLB252. Sequencing of the three ahlS genes from S. sciuri revealed high similarity in their amino acid sequences (over 98%). The amino acid sequences of AhlS from the CNS strains were also compared to those of the known AiiA-type AHL lactonases  analysis revealed that the overexpressed protein was approximately 75 kDa in size . First, the  in size , which w in size A.P. aeruginosa is an opportunistic pathogen that uses the quorum-sensing system to regulate several virulence factors [P. aeruginosa PAO1, LasI and RhlI are responsible for the synthesis of the las and rhl signals, N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) and N-butyryl-l-homoserine lactone (C4-HSL), respectively [P. aeruginosa in response to AHL-mediated quorum sensing [P. aeruginosa that degrades tissue matrix proteins in the human host, is also regulated by the quorum-sensing system [lasI rhlI double mutant P. aeruginosa PAO-MW1  medium [ 109622T , S. haem 109768T , and S.  102446T  were obt) medium . Solid bahlS gene from the genome of S. sciuri, specific PCR primers sciseq-F (5′-TTT TAT CGG CCT GGT AAT CCC GTT TTC GGG TCG C-3′) and sciseq-R (5′-TTC ATG CCC AAG TGC CAT AGG AGT AGG GC-3′) were designed based on the sequence of FPV13_12710 from S. sciuri B9-58B. PCR was performed with the KOD FX Neo system  using the following cycling parameters: 98 °C for 10 s, 60 °C for 30 s, and 68 °C for 1.5 min for 30 cycles. PCR fragments were sequenced using a BigDye Terminator ver. 3.1 sequencing kit and an ABI 3500 Genetic Analyzer .To amplify the ahlS coding regions in the genomes of the CNS strains were amplified with the KOD FX Neo system using the following primer sets: car-F (5′-TCT GTC GAC ACC CTC AGT TGT AAT AAG CCA ATA CC-3′) and car-R (5′-TCT CTG CAG AAG TCA GAA TGA CAT AAT CCT GCT GC-3′) for ahlS from S. carnosus, hae-F (5′-TCT GTC GAC AAA GCA CAT AAA TAG AGC CTT CC) and hae-R (5′-TCT CTG CAG ACT GAT AAG TCT GAA TGG CAT ATC CC-3′) for ahlS from S. haemolyticus, sap-F (5′-TCT GTC GAC CTA AAA ATG ACA ACT TTG GAG GGC GC-3′) and sap-R (5′-TCT CTG CAG TTT ACC CAT TTC ACG CAA TAC TGC CG-3′) for ahlS from S. saprophyticus, and sci-F (5′-TCT GTC GAC TCG GTC CAT ATT CCA ACC TCA CC-3′) and sci-R (5′-TCT CTG CAG TCT GAA TGA CAT AAA CCT GCC GCT CC-3′) for ahlS from S. sciuri. The SalI and PstI restriction sites were underlined in the primer sequences. PCR was performed with the following cycling parameters: 98 °C for 10 s, 60 °C for 30 s, and 68 °C for 1 min for 30 cycles. The PCR products were digested by SalI and PstI and inserted into the same sites of a broad-host-range vector pBBR1MCS5 [E. coli DH5α and used for AHL-degradation assays.The BBR1MCS5 . The preE. coli DH5α harboring the AhlS-expressing plasmid were inoculated in fresh LB medium (1% inoculum) containing 100 μM isopropyl-β-d-thiogalactopyranoside (IPTG). C6-HSL or C10-HSL was added at a final concentration of 20 μM (for CNS strains) or 10 μM (for E. coli). After incubation at 37 °C, cells were removed by centrifugation to obtain the supernatant. The residual AHLs in the supernatant were detected using C. violaceum CV026 and VIR07. An overnight culture of CV026 or VIR07 was mixed with 25 mL LB agar medium and poured into Petri dishes. AHL samples (20 μL) were placed in paper discs , which were subsequently placed onto LB agar plates containing CV026 or VIR07. Assay plates were incubated overnight at 30 °C and the residual amounts of AHL were calculated using the relationship equations based on the size of the purple-colored zones and the known quantities of AHLs [The full-grown cultures of CNS stains or E. coli DH5α harboring the AhlS-expressing plasmid was inoculated in 4 mL fresh LB medium (1% inoculum) containing 100 μM IPTG and 10 μM C10-HSL. After incubation, the cells were removed by centrifugation at 12,000× g for 5 min, and 90 μL of the supernatant was mixed with 10 μL of 1 N HCl. After incubation for 48 h at 4 °C, 20 μL of 1 M phosphate buffer (pH 7) was added to neutralize the pH. The restored C10-HSL was detected with a VIR07 biosensor.The AHL-lactonase activity was assessed based on previously-established methods . The fulahlS gene from S. sciuri ATCC 29060 was amplified with the KOD FX Neo system using the following set of primers: MBP-F (5′-TCT GGA TCC ATG GTA AAT GTA AAT AAC CAA GG-3′) and MBP-R (5′-TCT CTG CAG TCT GAA TGA CAT AAA CCT GCC GCT CC-3′). The BamHI and PstI restriction sites were underlined in the primer sequences. PCR was performed with the following cycling parameters: 98 °C for 10 s, 60 °C for 30 s, and 68 °C for 1 min for 30 cycles. The PCR products were digested by BamHI and PstI and inserted into the same sites of a pMAL-c2X vector  to construct pMAL-ahlS.The E. coli DH5α harboring pMAL-ahlS was inoculated into 100 mL fresh LB medium containing ampicillin. After incubation at 37 °C for 2 h, the expression of MBP-AhlS was induced by adding 1 mM IPTG. After incubation for 18 h at 37 °C, cells were harvested by centrifugation and resuspended in the column buffer . Lysozyme from egg white  was added to the suspension at a final concentration of 250 μg/mL. After incubation for 4 min at 37 °C, the suspension was sonicated and centrifuged to remove cell debris. The filtrated sample was loaded on an MBPTrap affinity chromatography column  equilibrated with the column buffer and eluted with 10 mM maltose. As the negative control, MBP-LacZα fusion was expressed and purified from cells of E. coli DH5α harboring pMAL-c2X using the same method. The expression and purification of MBP fusion proteins were analyzed on 10% SDS-PAGE followed by Coomassie Brilliant Blue staining.The full-grown culture of P. aeruginosa PAO1 [2, pH 8.0). After incubation for 4 h at 37 °C with shaking at 1400 rpm, the precipitate was removed by centrifugation, and the absorbance was measured at 495 nm. The relative pyocyanin production (A520/OD600) and elastase activity (A495/OD600) of PAO1-AhlS were calculated with those of PAO1- LacZα set to 100%.osa PAO1  and its osa PAO1  were incosa PAO1  for the ahlS from S. sciuri ATCC 29060, 29061, and StLB252 have been deposited in the DDBJ/ENA/GenBank databases under the following accession number LC128625, LC128626, and LC128627, respectively.The nucleotide sequences of ahlS from the CNS strains has AHL-degrading activity. It has been reported that CNS species are part of the normal flora on the skin and are less virulent. One of the CNS species, Staphylococcus epidermidis, is a normal colonizer of the skin and can be isolated from the diseased tissue alongside P. aeruginosa [P. aeruginosa affected the biofilm formation of S. epidermidis [P. aeruginosa uses quorum sensing to regulate biofilm formation [P. aeruginosa. Based on the results from the BLAST search, AhlS was also present in the genome of S. epidermidis NCTC 4133 (accession No. LR134242). Therefore, ahlS genes are assumed to be widely distributed among CNS strains. In this study, we used the purified AhlS protein for the quorum-quenching assay and demonstrated the inhibition of virulence factors, which are regulated by quorum sensing in P. aeruginosa. CNS strains might contribute to the inhibition of the quorum-sensing system of other pathogens in the normal flora of the skin. Meanwhile, it has not been elucidated whether CNS strains produce sufficient levels of AhlS for inhibition of quorum-sensing in other bacteria in natural systems, and the cell numbers of CNS strains are abundant enough within the community to impact the quorum-sensing system of more abundant microbes. Future studies could investigate how the behavior of AHL production and degradation on the surface of skins might contribute to elucidating the complicated quorum-sensing networks in the normal flora of the skin.In summary, our work is the first to report that ruginosa . Interesdermidis ,32. Sincormation , it was


A design principle for sequence-specific DNA modifiers driven by the specific DNA recognition was proposed based on the kinetic parameters for DNA binding and modification reactions. Sequence-selective chemical modification of DNA by synthetic ligands has been a long-standing challenge in the field of chemistry. Even when the ligand consists of a sequence-specific DNA binding domain and reactive group, sequence-selective reactions by these ligands are often accompanied by off-target reactions. A basic principle to design DNA modifiers that react at specific sites exclusively governed by DNA sequence recognition remains to be established. We have previously reported selective DNA modification by a self-ligating protein tag conjugated with a DNA-binding domain, termed as a modular adaptor, and orthogonal application of modular adaptors by relying on the chemoselectivity of the protein tag. The sequence-specific crosslinking reaction by the modular adaptor is thought to proceed in two steps: the first step involves the formation of a DNA–protein complex, while in the second step, a proximity-driven intermolecular crosslinking occurs. According to this scheme, the specific crosslinking reaction of a modular adaptor would be driven by the DNA recognition process only when the dissociation rate of the DNA complex is much higher than the rate constant for the alkylation reaction. In this study, as a proof of principle, a set of combinations for modular adaptors and their substrates were utilized to evaluate the reactions. Three types of modular adaptors consisting of a single type of self-ligating tag and three types of DNA binding proteins fulfill the kinetic requirements for the reaction of the self-ligating tag with a substrate and the dissociation of the DNA–protein complex. These modular adaptors actually undergo sequence-specific crosslinking reactions exclusively driven by the recognition of a specific DNA sequence. The design principle of sequence-specific modular adaptors based on the kinetic aspects of complex formation and chemical modification is applicable for developing recognition-driven selective modifiers for proteins and other biological macromolecules. Site-specific chemical reactions that modify proteins and nucleic acids by ligands rely on specific molecular recognition of target sites and selective reactions.O2-benzylguanine (BG) modified at the given DNA sequence  (M–1 s–1) between the modular adaptor and substrate-modified DNA is represented by eqn (1) shown in kcov ≫ koff) falls into case 1.kon) of DNA-binding proteins to their specific and non-specific sites are nearly identical,kcov) to fit into the condition of case 2, kcov ≪ koff (KD) for the unmatched complex of the zinc finger motif and ODNs results in fewer cross-linking reactions of unmatched complexes. The difference in the cross-linking reactions for the matched and unmatched pairs evaluated in our previous studyOur previous study of the reaction of modular adaptors, consisting of a DNA-binding domain and a cross-link forming protein tag , suggestv ≪ koff , would bkapp) and that of the unmatched pair  were investigated with various kcov values  is two orders of magnitude higher than that of the unmatched pair . Thus, to achieve the sequence-selective crosslinking reaction, the reactivity of the cross-linking reaction should be finely tuned to meet the condition of case 2.Time-dependent changes in the yield of the cross-linking reaction Fig. S1 were sims Fig. S2. From th4 M–1 s–1),O2-benzylcytosine (BC) (∼102 M–1 s–1),–1 s–1),KD values  as shown in kapp values for the reactions of the matched pair ZF-SNAP with ODN-ZF-BG and AZ-SNAP with ODN-AZ-BG were 3.8 × 105 and 9.1 × 105 M–1 s–1, respectively  and ODN-ZF(G/C) and two orders of magnitude higher for two nucleotide substituted ODN-ZF(GG/TC) and ODN-ZF(GC/CT) than that for ODN-ZF  and ODN-ZF(G/C) were 1.6 × 103 and 2.3 × 103 M–1 s–1, respectively  and ODN-ZF(GC/CT) could not be obtained because of their very slow reactions. These results clearly demonstrated that the sequence selective cross-linking reaction of the modular adaptor consisting of CLIP-tag with the substrate BC on ODNs proceeded by discriminating single nucleotide substitution, and indicated that the selectivity of the crosslinking reaction was governed by the KD of the binding complex.Three types of modular adaptors bearing the same self-ligating protein tag but different DNA-binding domains  were tested in a sequence-selective crosslinking reaction with ODNs modified with BC . CLIP-G Three modular adaptors, ZF-CLIP, AZ-CLIP, and CLIP-G, were reacted at the designed positions on the DNA scaffold. Rectangular DNA scaffold  and S12†kapp in kcov) and the recognition process of the DNA binding domain . In our previous study, orthogonal cross-linking reactions of modular adaptors were mainly conducted by taking advantage of the chemoselectivity of protein tags  and the higher KD for the unmatched complex results in a lower rate constant for the unmatched pair  for the sequence-selective modular adaptors. Based on the simulation of the kinetic scheme in  is larger than 100. The zinc finger proteins used in this study, zif268 and AZP4, showed a nearly two orders of magnitude difference in KD for complexes with matched and unmatched DNA sequences  of ein tags .15 Thus, Table S2. In factKD in the presence of 200 mM NaCl was significantly higher for both matched and unmatched complexes  = 7.1 × 105 M–1 s–1 and kapp (200 mM NaCl) = 3.1 × 103 M–1 s–1; for the unmatched combination of ZF-CLIP and ODN-AZ-BC:  (no NaCl) = 5.3 × 103 M–1 s–1 and  (200 mM NaCl) < 10 M–1 s–1). Notably, the reactivity of the protein tag with its substrate was not influenced by the presence of NaCl   . Interes Table S6. AZ-CLIP Fig. S13. This inIn summary, we have proved a design principle for sequence-specific DNA modifiers driven by specific DNA recognition based on the kinetic parameters for the DNA binding and modification reaction. The kinetic requirements for the complex formation step of DNA and the DNA binder as well as the successive proximity-driven intermolecular chemical reaction were applied to design sequence-selective modular adaptors by using appropriate combinations of a self-ligating protein tag and sequence-specific DNA-binding protein. The modular adaptors with the same self-ligating protein tag bearing different types of sequence-specific DNA-binding proteins indeed reacted with the substrate located in the respective matched DNA sequence, showing low reactivity with substrates in the unmatched sequence. Our design principle is useful for expanding the variation of sequence-specific modular adaptors by simply conjugating various types of sequence-specific DNA binding proteinsNdeI and HindIII), and BC-GLA-NHS (S9237S) were purchased from New England Biolabs . The pCLIPf(T7)-2 vector was generously donated by New England Biolabs. Purified oligonucleotides as the staple strands for DNA origami, oligonucleotide primers for gene construction, and all other oligonucleotides were obtained from Sigma-Aldrich , Thermo Fisher Scientific , or Gene Design . Escherichia coli BL21 (DE3) competent cells were purchased from Invitrogen . A Mini Elute gel extraction kit was obtained from QIAGEN . The HiTrap SP XL cation exchange column (5 mL), HisTrap HP column (5 mL), and Sephacryl S-400 were from GE Healthcare . PrimeSTAR HS DNA polymerase, T4 DNA ligase, and E. coli DH5α competent cells were obtained from TaKaRa Bio . Ultrafree-MC-DV was obtained from Merck Millipore . A Cosmosil 5C18-MS II column (4.6 i.d. × 150 mm) and HPLC-grade acetonitrile were purchased from Nacalai Tesque . Gel electrophoresis-grade acrylamide, bis(acrylamide), phenol, and all other chemicals and reagents were purchased from Wako Chemicals  or Nacalai Tesque. Phosphate buffer (PB) was prepared by mixing 20 mM Na2HPO4 and 20 mM NaH2PO4.Single-stranded M13mp18, restriction enzymes -2 vector was amplified by PCR using the following primer pairs.CLIP-tag in the Forward primer (F_EcoRI_CLIP):5′-TAATAAGAATTCGGCGGCTCCGGCGGCTCCGACAAAGATTGCGAAA-3′Reverse primer (R_CLIP_HindIII):5′-TTATTAAAGCTTTTAATGATGGTGATGATGATGGTGATGATGGTGGGTACCATTAACCTCGAGCCCGGGG-3′EcoRI and HindIII and were purified in the same manner. These products were incubated with T4-DNA-ligase. The mixture was transferred into E. coli DH5α competent cells for amplification. The vector encoding ZF-CLIP (named pET-30a-ZF-CLIP) was purified, sequenced, and transferred into E. coli BL21(DE3) competent cells.The PCR products were separated on 1% agarose gel (TAE) and purified with a Mini Elute gel extraction kit. The PCR products and pET-30a-cys-zif268 were digested with 600 reached 0.5, and protein expression was induced with 1 mM IPTG for 24 h at 25 °C. The soluble fraction of the cell lysate containing the recombinant protein was loaded onto a HisTrap HP column equilibrated with 50 mM phosphate buffer (pH 8.0) containing 200 mM NaCl and 1 mM DTT and then eluted over an imidazole gradient. The main fractions containing the target protein were collected, loaded onto a HiTrap SP HP column equilibrated with 50 mM phosphate buffer (pH 7.0) containing 1 mM DTT, and then eluted over an NaCl gradient. The purified protein was dialyzed by using 50 mM phosphate buffer (pH 8.0) containing 1 mM DTT, 50 μM ZnCl2, and 50% glycerol and stored at –20 °C. The purity of the target protein was checked by SDS-PAGE. The major band on the gel corresponded to the calculated molecular weight with an estimated purity of over 95%  and characterized by MALDI-TOF mass spectrometry . A488-ODN-ZF: m/z calcd 11 397, observed 11 395; A488-ODN-AZ: m/z calcd 11 400, observed 11 399; A488-ODN-AP: m/z calcd 12 011, observed 12 014. ODN-AP-2BC: m/z calcd 12 290, observed 12 290, ODN-8G-AP-2BC: m/z calcd 21 577, observed 21 578, ODN-11G-ZF-BC: m/z calcd 21 160, observed 21 167, ODN-ZF-BC: m/z calcd 14 287, observed 14 287, ODN-ZF(G/T)-BC: m/z calcd 14 286, observed 14 289, ODN-ZF(G/C)-BC: m/z calcd 14 287, observed 14 282, ODN-ZF(GG/TC)-BC: m/z calcd 14 286, observed 14 288, and ODN-ZF(GG/TC)-BC: m/z calcd 14 286, observed 14 291. The method for preparing ODN-AZ-BC, ODN-AZ-BG, and ODN-24D-AZ-BC was described in a previous report.15A coupling reaction between amino-modified ODNs (100 μM) and Alexa Fluor 488 NHS ester or succinimidyl derivative of protein tag substrates (1 mM) was carried out in 100 mM phosphate buffer (pH 8.0) for 8 h at ambient temperature. The modified ODNs were purified by reversed-phase HPLC on a Cosmosil 5C18-MS II column  was incubated with ZF-CLIP (10 nM) in a buffer (pH 8.0) containing 40 mM Tris–HCl, 20 mM acetic acid, 12.5 mM MgCl2, 1 mM DTT, 1 μM ZnCl2, 0.02% Tween 20, 200 nM BSA, and 100 nM calf thymus DNA, with or without 200 mM NaCl at ambient temperature. Aliquots were collected at defined reaction times and quenched by adding SDS and formamide. The aliquots were analysed by 8 M urea PAGE and the intensities of mobility-shifted bands on the gel were analysed with a Storm 860 molecular imager . The kinetics data were fitted to a reaction model assuming first-order kinetics (eqn (1)), and then the second-order rate constants  were obtained by dividing the determined first-order rate constants by the concentration of modular adaptors.Y, k and t represent the fraction of the cross-linked product, pseudo-first-order rate constant and reaction time, respectively.The ODNs were 5′-32P-end-labeled ODN  was incubated with a modular adaptor (10 or 100 nM) in a buffer (pH 8.0) containing 40 mM Tris–HCl, 20 mM acetic acid, 12.5 mM MgCl2, 1 mM DTT, 1 μM ZnCl2, 0.02% Tween 20, 200 nM BSA, and 100 nM calf thymus DNA, with or without 200 mM NaCl at ambient temperature.To evaluate orthogonality, each 5′-2, pH 8.0) was heated at 95 °C for 1 min, annealed at 53 °C for 30 min, and held at 4 °C in a thermal cycler. The samples were purified by size-exclusion chromatography  equilibrated with a buffer (pH 8.0) containing 40 mM Tris–HCl, 20 mM acetic acid, and 12.5 mM MgCl2 in Ultrafree-MC-DV (Millipore).A solution (50 μL) containing M13mp18 single-stranded DNA  and staple DNA strands  equilibrated with the buffer used for the reaction to remove unbound modular adaptors. The fractions containing DNA origami scaffolds were utilized for AFM analyses.A DNA origami scaffold was incubated with ZF-CLIP, AZ-CLIP, and/or CLIP-G under the conditions given in the captions of the figures or tables. For example, the cross-linking reaction of each of modular adaptor was carried out for 30 min at ambient temperature with 5 nM DNA scaffold and 250 nM modular adaptor  in buffer (pH 8.0) containing 40 mM Tris–HCl, 20 mM acetic acid, 12.5 mM MgCl2. The sample was scanned in tapping mode using a fast-scanning AFM system  with a silicon nitride cantilever . At least three independent preparations of each sample were analyzed by AFM and several images were acquired from different regions of the mica surface. The total number of DNA scaffolds corresponds to the number of expected rectangular shapes possessing three cavities observed by AFM. Specific and nonspecific binding of modular adaptors was counted for only the modular adaptor bound to the perfectly folded DNA scaffold. The yield of the DNA-scaffold-assembled modular adaptor was calculated as described previouslyThe sample was deposited on a freshly cleaved mica (1.5 mm diameter) surface and adsorbed for 5 min at ambient temperature, followed by three washes with a buffer (pH 8.0) containing 40 mM Tris–HCl, 20 mM acetic acid, and 12.5 mM MgClKD)   was fitted to the reaction model assuming first-order kinetics, and then the second-order rate constants of the process were determined. These assays were carried out in a buffer (pH 8.0) containing 40 mM Tris–HCl, 20 mM acetic acid, 12.5 mM MgCl2, 1 mM DTT, 1 μM ZnCl2, 100 nM calf thymus DNA, and 0.02% Tween 20 at 25 °C with or without 200 mM NaCl.A fluorescence polarization assay by using an Infinite 200 PRO F Plex  was conducted to determine the equilibrium dissociation constant ( Fig. S11 for the  Fig. S11. The kinThe authors have no conflicts of interest directly relevant to the content of this article.Supplementary informationClick here for additional data file.