This study evaluated the feasibility of performing molecular-genetic imaging by using the new radiotracer (18F-TFB) for the hNIS gene, to track dendritic cell (DC) migration in live mice. A murine dendritic cell line (DC2.4) co-expressing the hNIS and effluc genes (DC/NF) was established. To confirm the functional cellular expression of both effluc and NIS in the inoculated DC/NF cells by bio-medical imaging, combined bioluminescence imaging (BLI) and 18F-TFB positron emission tomography/computed tomography (PET/CT) imaging was performed after intramuscular injection with parental DCs and DC/NF cells. For DC-tracking, parental DCs or DC/NF cells were injected in the left or right mouse footpad, respectively, and 18F-TFB PET/CT and BLI were performed to monitor these cells in live mice.Recently, 18F-TFB PET/CT imaging showed higher radiotracer activity in the draining popliteal lymph nodes (DPLNs) in DC/NF injection sites than those in DC injection sites on day 2. BLI also showed DC/NF cell migration to the DPLNs on day 2 after the injection.In vivo PET/CT and BLI showed a clear signal in DC/NF injection sites but not in parental DC injection sites. The signal intensity in DC/NF cells was correlated with time. In vivo 18F-TFB PET/CT imaging of the NIS gene and optical imaging of the effluc gene in live mice. These data support the feasibility of using 18F-TFB as a substrate for hNIS reporter gene imaging to track the migration of DCs to the lymph nodes in live animals. The use of 18F-TFB may facilitate enhanced PET imaging of the hNIS reporter gene in small animals and humans in future studies.Migration of DCs to the lymph nodes was successfully monitored using The online version of this article (doi:10.1186/s13550-017-0280-5) contains supplementary material, which is available to authorized users. Tumor antigen-loaded dendritic cells (DCs) are used as a cancer vaccine to prevent tumors development and to eradicate existing tumors. DCs strongly express histocompatibility molecules (MHC class I and II molecules), co-stimulatory molecules (B7), and adhesion molecules . Antigen-presenting DCs stimulate helper and killer T cells in vivo –6. HowevSeveral non-invasive imaging techniques including gamma scintigraphy with In-111 , magneti18F-tetrafluoroborate (TFB) by using current 18F-fluoride production and purification methods [18F-TFB uptake in normal tissues expressing NIS such as the thyroid, stomach, and salivary glands, and the inhibition of 18F-TFB uptake by perchlorate. They also showed that 18F-TFB was an effective hNIS probe in hNIS-transfected colon carcinoma cell line, HCT116 [In a recent study, Jauregui-Osoro et al. labeled  methods . They de, HCT116 .18F-TFB for the hNIS gene in live mice. DC2.4, an immortalized DC cell line [18F-TFB PET/CT imaging were performed to assess the initial distribution and subsequent migration of infused DCs toward the draining lymph nodes in live mice.In the present study, we investigated the feasibility of performing molecular-genetic imaging with the new radiotracer ell line , 20. For2 atmosphere. DC2.4 cells were retrovirally transduced to express both the effluc and Thy1.1 genes [The murine dendritic cell line, DC2.4, was cultured in RPMI1640 medium supplemented with 10% fetal bovine serum , 0.05 mM β-mercaptoethanol , 1% non-essential amino acids , and 1% penicillin-streptomycin (Gibco) at 37 °C in 5% CO.1 genes . Thy1.1-18F-TFB was synthesized using a labeling protocol developed previously [18F-fluoride (12–18 GBq) trapped in a QMA cartridge was eluted using 1.5 mol/L HCl into a reactor containing sodium tetrafluoroborate. The reaction mixture was heated to 120 °C for 10 min, cooled to 25 °C, passed through a silver ion-loaded cation exchange cartridge and two Sep-Pak Light Alumina N, and sterile filtered.eviously . BrieflyLabeling was monitored by performing thin-layer chromatography (TLC) with alumina TLC strips, fluorescent indicator , and methanol as the mobile phase. The strips were scanned using Bio-scan 2000 radio TLC scanner. Presence of TFB at Rf: 0.60–0.65 was determined by scraping alumina from the TLC plate.18F-TFB, and 10 μmol/L sodium iodide (specific activity 740 MBq/mmol) at 37 °C for 30 min. After incubation, the cells were washed twice with ice-cold bHBSS buffer as quickly as possible and were lysed using 500 μL 2% SDS. Radioactivity was measured using Packard Cobra II gamma-counter .Indicated number of cells was plated in 24-well plates. After 1 day, the cells were incubated with 500 μL Hank’s balanced salt solution (HBSS) containing 0.5% bovine serum albumin (bHBSS), 3.7 kBq carrier-free 4 cells/well. After 1 day, 10 μL CCK-8 solution was added to each well, and the plates were incubated at 37 °C for 3 h. Absorbance was measured at 450 nm by using BMG Lab Tech microplate reader.Cell proliferation was determined using cell counting kit (CCK)-8 . Briefly, parental DC2.4 cells and DC/NF cells were plated in 96-well plates at a density of 1 × 10Parental DC2.4 and DC/NF cells were stained with PE-conjugated CD54, CD86, H-2Kb (MHC class I), and I-A/I-E (MHC class II), and APC-conjugated CD205 (DEC-205) at 4 °C for 30 min. The cells were washed twice with 0.1% BSA/PBS and analyzed using FACS Accuri C6 flow cytometer (BD Biosciences). Isotype-matched monoclonal antibodies were used as controls.Study 1: 18F-TFB was injected into the via tail vein of mice (n = 5) and PET/CT images were acquired at 30 min after the injection.Study 2: Animal experiments were conducted as depicted in Fig. 7 each) were intramuscularly injected into the left and right hind thighs of mice (n = 5), respectively. BLI and 18F-TFB PET/CT imaging were performed on days 1 and 4 after the injection.Study 3: Migration of DCs toward lymphoid organs was monitored by performing animal experiments, as depicted in Fig. 6 DCs/mouse) were subcutaneously injected into the left and right hind footpads of mice (n = 5), respectively, and BLI and 18F-TFB PET/CT images were obtained on day 2 after the injection.For PET/CT imaging, a 20 min (tumor imaging) scan was performed using the Triumph II PET/CT system . CT scans were performed using an X-ray detector  immediately after acquisition of the PET images. The PET images were reconstructed using 3D-OSEM iterative image reconstruction, and the CT images were reconstructed using filtered back-projections. All mice were anesthetized using 1–2% isoflurane gas during imaging. The PET images were co-registered with anatomical CT images by using a 3D image visualization and analysis software VIVID . To measure uptake (counts) for volumes of interest (VOIs), VOIs from each image were manually segmented from co-registered CT images by using VIVID and PMOD software . Uptake in the region of interest (ROI) was measured using PMOD 3.5 software.d-luciferin . Grayscale photographic images and bioluminescent color images were superimposed using LIVINGIMAGE software  and IGOR Image Analysis FX software . Bioluminescent signals are expressed as photons per cm2 per second per steradian (P/cm2/s/sr).BLI was performed using IVIS Lumina III imaging system (PerkinElmer) 10 min after an intraperitoneal injection of t test, with Graph Pad Prism version 5 (GraphPad software Inc.). Differences with p values less than 0.05 were considered statistically significant.All data are expressed as mean ± standard deviation, of at least three independent experiments. Statistical significance was determined using an unpaired Student’s 18F-TFB, an hNIS imaging agent, as a novel imaging agent for PET/CT imaging . After reaction completion, the solution was column filtrated for Na18F to remove the unreacted radioactive Na18F. After purification, radiochemical purity of 18F-TFB was found to be over 90% . 18F-TFB uptake increased in a cell number-dependent manner in DC/NF cells, but not in parental DCs  a well-established specific inhibitor of 18F-TFB uptake by NIS. In DC/NF cells, 18F-TFB uptake was 4.1-fold and 3.2-fold-higher than that in parental DCs and KClO4-treated DC/NF cells, respectively (R2 = 0.8274).Rapid accumulation of DCs Fig. . Uptake 18F-TFB in mice to demonstrate the imaging agents. Mice received 18F-TFB via tail vein injection, and PET imaging was performed at 30 min after the injection. Uptake of 18F-TFB was observed NIS-expressing organs such as the thyroid and stomach at 30 min after the injection.Next, we monitored the in vivo distribution of intravenously injected The effects of cellular labeling by virus infection in the immune cells confirm the cell proliferation and phenotype marker expression levels in parental DCs and DC/NF cells. The cell viability assay (CCK-8) indicated no significant difference in the proliferation of parental DCs and DC/NF cells Fig. a. Moreov18F-TFB PET/CT imaging was performed after the intra-muscular injection of parental DCs and DC/NF cells to confirm the functional cellular expression of both effluc and NIS in the inoculated DC/NF cells by bio-medical imaging , parental DC and DC/NF cells were subcutaneously injected in to the respective footpads, as described in Additional file 18F-TFB uptake was more intense in DC/NF cell-injected footpads and DPLNs of DC/NF cell-injected footpads than in parental DC-injected footpads and DPLNs of paternal DC-injected footpads  and 99mTc-pertechnetate, which emit γ-rays, have been used for imaging endogenous and exogenous hNIS. However, imaging with these radioiodides is associated with limitations such as reduced resolution and relatively long half-life of these radionuclides. Single-photon emission computed tomography (SPECT) by using 99mTc-pertechnetate was used for the in vivo tracking of NIS-expressing cell. Fruhwirth et al. detected NIS-expressing tumor cells by performing SPECT with 99mTc-pertechnetate in a preclinical metastasis model [99mTc-pertechnetate provides high-resolution images. However, PET imaging offers better image resolution and detection sensitivity than SPECT. Furthermore, PET imaging allows quantitative analysis. Recent studies have used 124I for dose planning in radionuclide therapy and immune cell tracking studies [124I also is associated with some drawbacks. PET imaging with 124I is not as efficient as expected, because of its complex decay scheme, with a low abundance of positrons (23%) and emission of high-energy γ photons. The high-energy γ photons have an energy close to 511 keV energy window and compete in abundance with positron decay . These may interfere with annihilation photons leading to increased background noise and poor image quality. Moreover, 124I has a long half-life (4.18 days), which cause a high absorbed dose of radiation and availability of 124I is limited due to its complex production.Traditionally, radioiodides , high positron yield (97%), low positron energy (Emax = 0.634 MeV), and easy accessibility due to production using a medical cyclotron. The utility of the new radiotracer, 18F-TFB, to visualize the thyroid and other NIS-expressing organs has been previously described in a preclinical study assessing PET/CT images of rodents [18F-TFB PET/CT imaging, we successfully demonstrated the proliferation of DC/NF cells in mice from days 1 to 4 after their intramuscular injection. Furthermore, in vivo 18F-TFB PET/CT imaging as well as BLI showed the migration of DCs to DPLNs of DC/NF cell-injected footpads on day 2 after their injection. Results of 18F-TFB PET/CT imaging obtained in the present study are similar to those obtained of 124I PET/CT imaging for tracking DC/NF cells in our previous study [18F-TFB PET/CT images than in 124I PET/CT images. These results first demonstrated the use of 18F-TFB PET/CT for immune cells in addition to thyroid tissues and exogenous cancer cell expression, suggesting that NIS combined with 18F-TFB PET imaging is a good tool for cell tracking.In contrast, an ideal PET tracer would be based on  rodents . Thus, ous study . HoweverIn the present study, we demonstrated that nuclear medicine imaging with the NIS gene is a feasible technique for tracking the migration of DCs to lymphoid organs in live mice. Because in vivo imaging with the NIS gene is hampered by both high background noise and signals emitted from normal tissues and non-specific organs, it is difficult to visualize the initial distribution and low population of infused DCs. To overcome these hurdles for tracking DCs with the NIS gene, we combined in vivo nuclear medicine imaging with BLI based on the effluc gene, which allows tracking the early distribution of DCs and verifying the feasibility of the NIS gene as a reporter for tracking the migration of DCs to lymphoid organs. Thus, an effluc reporter gene was additionally adopted for tracking the migration of DCs and dual reporter DCs expressing both the NIS and effluc genes were successfully established using a viral vector system expressing the reporter genes.18F-TFB could be a valuable approach in experiments for improving the migration efficiency and antitumor efficacy of DCs.In vivo DC migration efficiency is unsatisfactory  when DCs are administered through an intradermal injection, which is the most common method used in DC-based immunotherapy. In the present study, in vivo DC migration efficiency was approximately 1% on day 2. The most commonly used method to improve the migration of DCs is the pre-injection of pro-inflammatory cytokines that helps provide a suitable inflammatory microenvironment for the migration of DCs through lymphatic vessels . Pre-inj18F-TFB is a promising PET imaging probe for hNIS, the 18F-TFB synthesis method used at present involving isotopic exchange provides 18F-TFB with a suboptimal radiochemical yield and specific activity. The specific activity of 18F-TFB was found to be approximately 1 GBq/μmol in a previous study [18F-TFB with high specific activity and sufficient yield [18F-TFB synthesis method which was achieved via direct radiofluorination of boron trifluoride using an automated synthesis system [18F-TFB was achieved. The increased specific activity of 18F-TFB may allow for enhanced PET imaging of the hNIS reporter gene in future human and animal studies.Although us study , which wnt yield . Jiang es system . Using t18F-TFB PET imaging of the NIS gene and optical imaging of the effluc gene. Together, these results highlight the potential of 18F-TFB as a new NIS imaging probe for cell tracking. Further studies should be performed to use this probe for tracking other immune cells such as cytotoxic T lymphocytes and natural killer cells.In conclusion, we successfully monitored the proliferation of DCs and their migration to lymph nodes in live mice by performing Additional file 1: Figure S1.18F-TFB PET/CT imaging was performed on day 1 or 4 after the injection of DC and DC/NF cells into the right and left thighs of mice, respectively. Figure S2. Schematic diagram for the in vivo monitoring of reporter DC/NF cell migration to the DPLNs. In vivo BLI and 18F-TFB PET/CT imaging were performed on day 2 after the injection of DC and DC/NF cells in the right and left footpads of mice, respectively. Figure S3. Synthesis and characterization of 18F-TFB. (A) Schematic representation of 18F-TFB synthesis. (B) Chromatograms of radio-TLC to monitor the radiolabeling reaction. (PPTX 1047 kb) Schematic diagram for the in vivo monitoring of reporter DC/NF cells after their intramuscular injection. Briefly, combined BLI and


Using microarray-based miRNA profiling in K562 cells, a model of chronic myeloid leukemia (CML), we found that the oncoprotein BCR-ABL1 regulates the expression of miR-21, an “onco-microRNA”, found to be overexpressed in several cancers. This effect relies on the presence of two STAT binding sites on the promoter of miR-21, and on the phosphorylation status of STAT5, a transcription factor activated by the kinase activity of BCR-ABL1. Mir-21 regulates the expression of PDCD4 (programmed cell death protein 4), a tumor suppressor identified through a proteomics approach. The phosphoSTAT5 — miR-21 — PDCD4 pathway was active in CML primary CD34 Among other consequences, activated or inhibited transcription factors induce important modifications of the CML cells gene expression pattern that could impact cell cycle control, apoptosis and genetic instability, leading to the expansion of the oncogene-transformed cells and to the acquisition of potentially harmful 2 as the most over-expressed protein in imatinib-treated cells. We showed that miR-21 can bind to PDCD4 3'UTR and decrease its expression. The STAT5 - miR-21 - PDCD4 pathway was conserved in CML primary CD34+ cells, and to some extent in acute myeloid leukemia (AML) models as well; the known functions of miR-21 and PDCD4 suggest that their regulation by BCR-ABL1 could participate in the antileukemic response triggered by tyrosine kinase inhibitors.Here, we studied the effects of a clinically relevant concentration of imatinib, a tyrosine-kinase inhibitor (TKI) that blocks BCR-ABL1, on the BCR-ABL1+ cells . Fi. Fi+ celmiR-21, pri-miR-21, that was quantified by RT-qPCR on RNA extracted from the nuclear fractions of treated- or non-treated cells : the BCR-ABL1+ cells expressed higher levels (x2) of miR-21 than the BCR-ABL1- cells  concern factors of the Signal transducer and activator of transcription (STAT) family  directed against the two isoforms STAT5A/B [the miR-21 promoter was reduced in STAT5-shRNA cells , a semi-quantitative proteomic technique. The results allowed the identification (by 2 distinct peptides or more) and the relative quantification of 1137 different proteins . Since it has been demonstrated in several models that PDCD4 is a characterized target of miR-21 [In order to gain insight into miRNA-dependent protein regulations, the proteome of control- or imatinib-treated K562 cells was analyzed by iTRAQ , as revealed by sequencing  and an anti-miR-21 oligonucleotide , or with a control LNA. The efficacies of these treatments were checked using cells co-transfected with the miR-21 “sensor” plasmid (not shown). Anti-miR-21 LNA transfected K562 cells expressed a higher level of PDCD4 protein than the control LNA- or miR-21 mimic-transfected cells Figure . The expquencing . The 9 tquencing , confirmPDCD4 by miR-21: this is suggested by luciferase activity measurements in K562 cells transfected with a reporter plasmid containing a 554 bp fragment of the PDCD4 3’UTR placed downstream of a Renilla luciferase coding sequence; co-transfection of these cells with the anti-miR-21 LNA induced an increase of the luciferase activity, revealing a regulatory effect of miR-21 on the 3’UTR of PDCD4 , in particular in the subtypes harboring the internal tandem duplication (ITD) mutations of the tyrosine kinase FLT3 . In paras Figure , and decy Figure . The TKIy Figure . MOLM13 y Figure . Taken t+ cells of TKI-resistant patients [+ CML cells [miR-21. The two STAT binding sites we worked on were previously identified [miR-21 promoter.MiR-21 is overexpressed in almost all types of human cancers, including leukemia, and has been associated with the regulation of proliferation, growth, invasion and chemoresistance. Its anti-apoptotic properties were previously confirmed in K562 cells , 29. Mirpatients  and in tpatients ; it mighML cells . Using vML cells , 25. TheML cells , 14 and ML cells , 19 in tentified , 14 as p+, TKI induce a rapid and complete dephosphorylation of STAT5. The effect on STAT3 is much less pronounced, a difference that could rely on the ability of BCR-ABL1 to directly phosphorylate STAT5 [In CML CD34te STAT5 . This rete STAT5 , 21, in te STAT5 . Intereste STAT5 , 27. Tak+ cell line K562 and the FLT3-ITD+ AML cell lines (MOLM13 and MV4.11) were obtained from the DSMZ (German Collection of Microorganisms and Cell Cultures). They were grown at 37°C / 5% CO2 in RPMI medium (Invitrogen) supplemented with 10% foetal calf serum and penicillin/streptomycin, and split every 2-3 days.The BCR-ABL1Etablissement Français du Sang recommendations. Mononuclear cells were obtained by Ficoll density gradient centrifugation. They were labeled with the StemSep™ Human CD34 Positive Selection Cocktail (Stemcell Technologies) and isolated on MS column (Miltenyi Biotec). CD34+ cells were kept for 2-3 days in StemAlpha A medium (StemAlpha) in the presence of Flt3-ligand (50ng/ml), thrombopoietin (50ng/ml) and stem cell factor (50ng/ml). Cytokines were not included during the course of imatinib treatments.Cytapheresis samples from four CML patients were obtained at diagnosis according to the When prepared for microarray hybridization, RNAs were extracted from Trizol homogenates using a modified protocol: after chloroform extraction and phases separation, 1.5 volumes of 100% ethanol were added to the upper aqueous phase. The mix was deposited onto a RNeasy column (Qiagen), that was further processed according to manufacturer instructions. Cyanine-3 (Cy3) labeled miRNAs were prepared from 0.2 μg RNA using the miRNA complete labeling kit version 2.2 (Agilent Technologies). Labeled miRNAs were hybridized overnight at 55°C onto Human miRNA Microarray V2 (Agilent Technologies) slides. The slides were then washed as recommended by the manufacturer, and scanned on an Agilent G2565CA scanner, at 5 μm resolution and using the 20-bit scan mode. Images were processed with Feature Extraction (version 10.7). Data were quantile-normalized for inter-array comparisons and analyzed using the “BRB-Array Tools” package, version 4.2.0 . Genes tFor RT-qPCR experiments, RNAs were prepared from Trizol homogenates by precipitation from the aqueous phase following the supplier's recommendations. For RT-qPCR of miR-21 and miR-21*, RNA (200ng) were polyadenylated and reverse-transcribed using the High Specificity miRNA First Strand cDNA synthesis kit (Agilent). After reverse transcription, the PCR reaction was analyzed by real time quantitative PCR (RT-qPCR) using a universal reverse primer (GACGAGCTGCCTCAGTC) and either a miR-21 (TAGCTTATCAGACTGATGTTGA), a miR-21* (ACAGCCCATCGACTGGTGTTG) or a U6 (ATTGGAACGATACAGAGAAGATTAG) forward primer. The cycle thresholds (CT) obtained for miR-21 and miR-21* were normalized using U6 as endogenous gene. For RT-qPCR of PDCD4 and pri-miR-21, the Transcriptor First Strand cDNA synthesis kit (Roche) was used for reverse transcription. The sequences of the primers were TTTTGTTTTGCTTGGGAGGA and AGCAGACAGTCAGGCAGGAT (for pri-miR-21); GAAGGTTGCTGGATAGGCGA and TTGGTAGTCCCCTTCCTTTCC (for PDCD4); TGGAGGGTGTCCGCAATGTT and GAAGGCCTTGACCTTTTCAG . Both types of qPCR (miR-21 and PDCD4/pri-miR-21) were performed with the GoTaq qPCR Master Mix (Promega), using a Biorad CFX96 Real-Time PCR system.® High-Fidelity DNA Polymerase (New England Biolabs). For the miR-21 sensor plasmid construction, two 42-mer oligonucleotides (CCGCTCGAGTCAACATCAGTCTGATAAGCTACTAT CATCAACATCAGTCTGATAAGCTAGCGGCCGCATT CTTAT and ATAAGAATGCGGCCGCTAGCTTATCAGACTGATGTTGATGATAGTAGCTTATCAGACTGATG TTGACTCGAGCGG) were annealed to form a double-stranded adapter containing two miR-21-binding sequences, and that was cloned at the XhoI/NotI of the psiCheck2TM (Promega) downstream of the Renilla luciferase cds. The same strategy was used to construct the miR-21* sensor plasmid with the oligonucleotides CCGCTCGAGACAGCCCATCGACTGGTGTTGCTATCAACAGCCCATCGACTGGTGTTGGCGGCCGCATTCTTAT and ATAAGAATGCGGCCGCCAACACCAGTCGATGGGCTGTTGATAGCAACACCAGTCGATGGGCTGTCTCGAGCGG). The miR-21 promoter . The sequence corresponding to the miR-21 seed-binding site (ATAAGCTA) was deleted from the PDCD4 3'UTR as described earlier. The STAT5- and control-shRNA lentiviral vectors were a gift of Dr. I. Dusanter-Fourt, Institut Cochin, Paris, France). For the ChIP experiments, the coding sequences of human STAT3 (NM_139276.2) and STAT5B (NM_012448.3) were PCR amplified from K562 cDNA and cloned in phase with a 3X FLAG, under the control of a MND promoter . Lentiviruses were prepared and titrated by the Vect'UBfacility, Bordeaux, France.The PCR steps were done using Q5a et al. ) was PCR+ cells and AML cell lines, cells were transduced at a MOI of 5 with the miR-21 promoter lentivirus and used thereafter without selection or sorting.For reporter analysis , cells were split the day before transfection at 200.000 cells/ml, then seeded in 96-well white plates (Dutscher). They were transfected with 400ng of plasmid using the Viafect reagent (Promega). For miR-21 promoter studies (pGL4.10 transfections), 20ng of the CMV-Renilla luciferase pGL4.75 plasmid (Promega) was added in order assess and take into account the transfection efficiency. The Firefly and Renilla luciferase activities luminescence were measured 24h post-transfection using the DualGlo-Luciferase Reporter Assay System (Promega). For STAT5 knock-down experiments (STAT5- or control-shRNA lentiviruses), cells were transduced at a multiplicity of infection (MOI) of 5, then sorted 48 hours later according to the expression of GFP. For miR-21 promoter activity measurements in CD34For miR-21 knock down, cells were transfected with 0.1 μM of either a control or an anti-miR-21 LNA oligonucleotide (Exiqon), using the transfection reagent Lipofectamine RNAi max (Thermofisher).50 were calculated using GraphPad software.To monitor cell growth, transfected or transduced cells were seeded in 96-well white plates (5000 cells per well). The ATP contents of the wells were assessed every day using the Cell Titer Glo reagent (Promega). For the imatinib sensitivity studies, serial dilutions of imatinib were added to the wells directly after the seeding. ATP contents were measured 48h later. The ICi.e. nuclease) version. K562 cells were transfected with the plasmid by Amaxa nucleofection, and 24 hours later puromycin (2μg/ml) was added to the culture medium. After 48 hours of selection, live cells were diluted in methylcellulose-containing medium (1000 cells per ml). After 10 days, colonies were picked up and transferred into individual wells containing liquid medium. Mutations in the miR-21 gene were detected by cloning (into the pCR2.1-TOPO® plasmid (Invitrogen)) of PCR products obtained using primers CTAGCATGTACTCTGGTTTCAACAGA and CACAAAAGACTCTAAGTGCCACCA on cells lysates. Twelve distinct plasmids obtained from each cell clone were sequenced. The quantification of miR-21 expression was performed by RT-qPCR as described earlier.Two oligonucleotides (CACCGGTCTGATAAGC TACCCGACA and AAACTGTCGGGTAGCTTATCA GACC) were annealed to form a double-stranded adapter that was cloned at the BbsI of the pSpCas9n(BB)-2A-Puro plasmid . The D10 nickase Cas9 cloned into this plasmid was replaced by the wild-type . Samples (50μg) were denatured by incubating 5 min at 95°C in reducing sample buffer, loaded on 10% SDS-PAGE gels. After migration, proteins were transferred onto a PVDF membrane. The antibodies  were used at a 1/1000 dilution in TBST/5% nonfat dry milk. The horseradish peroxidase-conjugated secondary antibodies  were used at the dilution of 1/10000 and detected using Western Lighting Plus-ECL (Perkin Elmer).Cells were pre-treated with 10μM Na6) were seeded at the concentration of 500.000 cells/ml and either treated with 1μM imatinib for 24h or left untreated. After centrifugation, they were washed twice with PBS and the pellets were dissolved in 100 ul of urea (8M), thiourea (2M), Chaps (2%), Hepes (50mM), pH7.5. The undissolved materials were removed by centrifugation. Proteins were precipitated in TCA 15% (v/v), washed twice with acetone and suspended in urea (7M), thiourea (2M), Chaps (4%). Supernatant protein concentration was assessed by Bradford. A hundred μg was digested with trypsin and labeled with iTRAQ 8-plex (AB SCIEX) according to the manufacturer's instructions. Briefly, samples were reduced with TCEP and alkylated with MMTS before being digested overnight with trypsin. The labeled samples were pooled and subsequently purified on SCX chromatography, desalted on a C18 cartridge and eluted peptides were submitted to the 3100 OFFGEL Fractionator (Agilent Technologies) with the OFFGEL High Res Kit pH 3-10 (Agilent Technologies) immobilized pH gradient (IPG) DryStrips. The peptides were separated according to the manufacturer's instructions. After separation, the 24 fractions were acidified with acetic acid 0.1% (v/v) and subsequently analyzed by LC-MS/MS with a nano LC system coupled ton an LTQ Orbitrap XL (ThermoFinnigan) as previously described [K562 cells (5.10escribed . Ten micData were searched by SEQUEST through Proteome Discoverer 1.3 (Thermo Fisher Scientific Inc.) against Homo sapiens Complete Proteome Set (Uniprot version 2011-01: 58009 entries). Spectra from peptides higher than 5000 Da or lower than 350 Da were rejected. The search parameters were as follows: mass accuracy of the monoisotopic peptide precursor and peptide fragments was set to 10 ppm and 0.5 Da respectively. Only b- and y-ions were considered for mass calculation. Oxidation of methionines (+16 Da) was considered as variable modification and cysteine alkylation with MMTS (+46 Da) as fixed modification. iTRAQ 8-plex labeling of lysine and peptide N-terminal were also considered as fixed modification. Two missed trypsin cleavages were allowed. Peptide validation was performed using Percolator algorithm  and only® M2 antibody or normal rabbit IgG as a negative control), DNA-antibody bound complexes isolation, DNA cleanup and qPCR.K562 cells were transfected with the control plasmid, the FLAG-tagged-STAT3 plasmid or the FLAG-tagged-STAT5 plasmid. The expression of the 3X-FLAG tagged proteins waere checked by western-blot. The Pierce Agarose ChIP Kit (Thermo Fisher) was used according to the manufacturer's instructions for the steps of cross-linking, DNA digestion, immunoprecipitation (using 6 μg of a Sigma monoclonal anti-FLAGRT-qPCR datas and luciferase measurements were analyzed by the GraphPad software. When shown on graphs, error bars represent S.E. of the number of determination. Two-tailed Student t tests were used for statistical analysis.


Drosophila, we used the P{ry+t7.2= PZ}Dl05151 P-element insertion in the Drosophila melanogaster genome that causes a dominant visible phenotype: the presence of characteristic extra-veins in the fly wings. This extra-vein phenotype presents variable expressivity and incomplete penetrance. The insert is a PZ element located 680 bp upstream from the ATG of the Delta (Dl) gene, encoding the Notch ligand involved in wing vein development, and acts as a null allele. In the G2 offspring from a cross between the heterozygous transgenic stock and wild-type flies, we observed the transmission of the extra-vein phenotype to wild-type flies without the transgene, independently of gender and across many generations. This is a “paramutation-like” example in the fly: the heritable transmission of a phenotypic change not linked to a classical genetic mutation. A “paramutagenic” allele in heterozygotes transmits the phenotype of the heterozygotes to the wild-type allele (“paramutant”) in a stable manner through generations. Distinct from paramutation events so far described in Drosophila, here we deal with a dominant effect on a single gene involving variable hereditary signals.The hereditary transmission of a phenotype independent from DNA sequence implies epigenetic effects. Paramutation is a heritable epigenetic phenomenon observed in plants and animals. To investigate paramutation in  Changes in gene expression arise because of genetic and environmental variation in living organisms . The extbooster 1 (b1) locus, DNA methylation of an upstream repeated motif is associated to the establishment of paramutation . In aiewed in ,9. A difnsertion . This phicroRNAs . More phicroRNAs ,12. Thesbithorax complex  = PZ}Dl05151 insertion was confirmed through the perfect excision of the transgene by crossing Dl05151/TM3, ryRK, Sb (hereafter TM3) females with males carrying the Δ2–3 transposase [Dl05151/P{ry[+t7.2= Delta2-3}99B males were crossed individually to Ly/TM3, ry-, Sb females and 134 excisions over TM3, ry-, Sb stocks were established and analyzed. Seventy independent excisions were obtained and they were all homozygous lethal. To test whether the excised chromosomes carry a secondary lethal, 11 excised stocks were crossed to the Dl Df(3R)BSC850 deficiency. Of these 11 stocks, seven were viable when hemizygous against the Dl deficiency. Using the oligonucleotides shown in Dl05151 insert  Dl05151, ry-/Dl*, ry-  heterozygotes present an extra-vein phenotype with highly variable expressivity as in the parental Dl05151/TM3 stock  flies with different degrees of extra-veins , possibly because the phenotype is more penetrant in females than in males carrying the insert. Detailed data for each individual male are reported in In order to obtain wild-type flies without the  females  and maleins Figs  and 5. Wed above . A repreing gaps , extra-ving gaps , extra-ving gaps  and smaling gaps . This werequency ; S3 Fig.requency . The pennificant . The penry- chromosome that has been in contact with the Dl05151 transgene (indicated as Dl*) has been “paramutated”, leading to the same phenotype as that caused by the insertion. This result is reminiscent of paramutation at the mouse Kit locus [Kittm1Alf/+ heterozygotes exhibited the white feet and white tail phenotype of heterozygous parents carrying a lacZ insertion in the Kit locus (Kittm1Alf). Both events call for the transmission of a visible character through epigenetic mechanisms modifying gene expression.The it locus , where hDl05151/Dl+ G2 ry+ offspring carrying the Dl05151 insertion shows a stronger phenotype than its parents  [veinlet (ve) [N with many other gene products are known [Dl expression on the homologous chromosome through a transmissible system that potentiates itself throughout generations producing a very variable wing vein phenotype.It has long been known that a decrease in ed veins ,26. In oormation . Reductihibition  and contid (rho)  and veinlet (ve) . Interacre known ,42,43. OSo far, many cases of paramutation and transvection observed in a variety of organisms involve the activity of ncRNAs ,45. DireDl* chromosome to become paramutagenic, the extra-vein phenotype was followed through 12 generations of the Dl*/Dl+ stocks from three independent G0 males. The full penetrance and high expressivity of the paramutant phenotype in the G12 females and males . TraDrosophila melanogaster model system, almost devoid of DNA methylation [Here we show that the hylation ,31,33 (ahylation ), can behylation .ry506 stock utilized was a kind gift of Anna Digilio . The P{ry+t7.2= PZ}Dl05151 stock is Bloomington stock n. 11651. Flies showed a decrease in the expressivity of the extra-vein phenotype through the span of a few years, but the same stocks ordered at different times within the last year show the same variation in penetrance and a high variation in the expressivity of the phenotype. Df(3R)BSC850 is w1118; Df(3R)BSC850/TM6C, Sb1cu1 (Bloomington stock n. 27922). DlX and DlRevF10 are Bloomington stocks n. 60336 and n. 6300, respectively.Flies were grown at 23–25°C in standard cornmeal medium with melassa as a source of sugar. The +t7.2 = PZ}Dl05151 P-element, 20 Dl05151/TM3, ryRK, Sb females were crossed to ten y1w1; Ki1P{ry+t7.2= Delta2-3}99B (Bloomington stock n. 4368) males and 134 stocks with ry- eyes  were established by crossing individually each Dl05151/Ki1P{ry+t7.2= Delta2-3}99B male to Ly/TM3, ryRK, Sb females. DlΔ05151/TM3, ryRK, Sb stocks were all homozygous lethal. The Dl deficiency Df(3R)BSC850 was used to obtain hemizygous Dl05151-excised animals. Seven out of eleven DlΔ05151/Df(3R)BSC850 stocks analyzed were viable. To test for wing rescue, DlΔ05151/TM3, ryRK, Sb males were outcrossed to ry females and wings of the DlΔ05151/ry- progeny analyzed. The w1118; kar2, ry506, Vno/TM3, ryRK, Sb, delta2-3 and Ly1, {FRT80B, ry+}, kar2, ry506TM3, ryRK, Sb stocks were kind gifts of Pascal Heitzler .For the phenotypic analysis of the excisions of the P{ryDl05151/TM3, ryRK, Sb males were crossed to ry-/ry- females and 1–20 females or males of the G1 Dl05151/ry- progeny were crossed to ry-/ry- flies. The G2 progeny analyzed is shown in Dl*/Dl+ and in the ry-/ry- flies were compared using simple χ2 tests. To obtain the G3 balanced with the TM2, ry- balancer, G1 Dl05151/Dl* females and males were crossed to TM2, ry-/MKRS flies. The resulting G2 Dl*/TM2, ry- females and males were inbred. G1 and G2 data are available upon request.For the paramutation crosses, five individual Wings that looked damaged were discarded. Wings were dissected from the adults using fine Dumont forceps, immersed in 100% Ethanol and mounted in Euparal (BioQuip). Images were acquired thanks to the SPIBOC imaging facility of the Institut Sophia Agrobiotech on a LEICA (Switzerland) MZFLIII stereomicroscope using the ZEN software (ZEISS).003, 5’-CGGAGTCTTCTCCTTTTCACG-3’; 004, 5’-AAATAAACACCCATCCGGTTGAAG-3’; 006, 5’-TAATAGCACACTTCGGCACG-3’; 011, CAATCATATCGCTGTCTCACTCAG-3’. The cycles were: one minute denaturation at 95°C followed by 35 cycles of 15 sec denaturation at 95°, 15 sec annealing at 57°C and 30 sec extension at 72°C. Reactions ended with 5 min extension at 72°C.DNAs were extracted with phenol and ethanol precipitated. Single fly DNA was resuspended in 20 μl of 10 mM Tris pH7.5, 1 mM EDTA (TE). Ten nanograms of DNA were used for each PCR. For PCR reactions, the MyTaq  was used in 50 μl reactions according to instructions. Oligonucleotides were used at a 10 mM dilution and their sequences are as follows: S1 FigDl05151/TM3 female were stained for lacZ expression by X-gal coloration. lacZ is expressed in the gut (A), in the brain and in the antenna and eye disk (B), in the wing disk (C), and in the leg disks (D).Imaginal disks of a (PDF)Click here for additional data file.S2 FigDlX/TM3, Sb1 female. (B) Right wing of a DlX/TM3, Sb1 male. (C) Right wing of a DlRevF10, e*, SerRX82,P{ry+t7.2= neoFRT}82B/TM6B, Tb1 female. (D) Right wing of a DlRevF10, e*, SerRX82,P{ry+t7.2= neoFRT}82B/TM6B, Tb1 male. These wings show extra-veins very similar to those of Dl05151 heterozygotes .(A) Right wing of a (PDF)Click here for additional data file.S3 Figry-/ry- stock, extra-veins in the L4/L5 inter-vein region (A) or small dots (B and C) are observed. Many flies have wild-type wings (D).In a small percentage of flies of the (PDF)Click here for additional data file.S4 FigDl05151/TM3 males were crossed to homozygous ry-/ry- females and heterozygous Dl05151/Dl+, ry- females and males were backcrossed to ry-/ry- flies. In the G2 progeny, a certain number of paramutant Dl*/Dl+ flies with extra-veins in their wings were found. (B) Heterozygous Dl05151/TM3 males were crossed to homozygous ry-/ry- females and heterozygous Dl05151/ry- females and males were crossed to flies carrying the TM2 and MKRS balancers in order to mark the chromosomes. Dl*/TM2 females and males were crossed to each other and the G3 progeny analyzed.(A) Heterozygous (PDF)Click here for additional data file.S1 TableDl05151/TM3 stock. L+R EV: extra veins in both left and right wings. L EV: extra-vein only in the left wing. R EV: extra-vein only in the right wing. WT: absence of extra-veins. TOT: total number of flies. TOT EV: total number of flies with at least one extra-vein. % EV: percent of total extra-veins. Females show higher penetrance and expressivity than males.Amount of females and males with extra-veins in the (PDF)Click here for additional data file.S2 TableFemales and males with extra-veins were counted separately. As shown in the “parents” column, in some cases, more than one cross was carried out with G1 flies showing different phenotypes. The genotype of the counted flies is indicated (GEN.). WT: absence of extra-vein. EV: presence of at least one extra-vein. TOT: total number of flies. % EV: percent of extra-veins. A high variability in the penetrance of the paramutants is observed among crosses, independently from the strength of the phenotype of the parents.(PDF)Click here for additional data file.S3 TableDl05151 in heterozygosis with the TM3 or TM2 balancers were outcrossed to ry-/ry- and very few defective wings were found in the progenies containing the balancer chromosomes. WT: wild-type. D: dots. EV: extra-veins. TOT: total number of flies.Females and males carrying the (PDF)Click here for additional data file.


Arthrobotrys oligospora. Two media commonly used in research on this species, cornmeal agar (CMA) and potato dextrose agar (PDA), were chosen for use in this study. The fungus produced a small group of volatile furanone and pyrone metabolites that were associated with the switch from the saprophytic to the pathogenic stage. A. oligospora fungi grown on CMA tended to produce more traps and employ attractive furanones to improve the utilization of traps, while fungi grown on PDA developed fewer traps and used nematode-toxic furanone metabolites to compensate for insufficient traps. Another volatile pyrone metabolite, maltol, was identified as a morphological regulator for enhancing trap formation. Deletion of the gene AOL_s00079g496 in A. oligospora led to increased amounts of the furanone attractant (2-fold) in mutants and enhanced the attractive activity (1.5-fold) of the fungus, while it resulted in decreased trap formation. This investigation provides new insights regarding the comprehensive tactics of fungal adaptation to environmental stress, integrating both morphological and metabolomic mechanisms.The adjustment of metabolic patterns is fundamental to fungal biology and plays vital roles in adaptation to diverse ecological challenges. Nematode-trapping fungi can switch their lifestyle from saprophytic to pathogenic by developing specific trapping devices induced by nematodes to infect their prey as a response to nutrient depletion in nature. However, the chemical identity of the specific fungal metabolites used during the switch remains poorly understood. We hypothesized that these important signal molecules might be volatile in nature. Gas chromatography-mass spectrometry was used to carry out comparative analysis of fungal metabolomics during the saprophytic and pathogenic lifestyles of the model species IMPORTANCE Nematode-trapping fungi are a unique group of soil-living fungi that can switch from the saprophytic to the pathogenic lifestyle once they come into contact with nematodes as a response to nutrient depletion. In this study, we investigated the metabolic response during the switch and the key types of metabolites involved in the interaction between fungi and nematodes. Our findings indicate that A. oligospora develops multiple and flexible metabolic tactics corresponding to different morphological responses to nematodes. A. oligospora can use similar volatile furanone and pyrone metabolites with different ecological functions to help capture nematodes in the fungal switch from the saprophytic to the pathogenic lifestyle. Furthermore, studies with A. oligospora mutants with increased furanone and pyrone metabolites confirmed the results. This investigation reveals the importance of volatile signaling in the comprehensive tactics used by nematode-trapping fungi, integrating both morphological and metabolomic mechanisms. Nematode-trapping fungi (NTF) can detect the presence of nematodes and develop specialized mycelial trap devices to infect and consume prey as a response to nutrient depletion –4. These–Direct physical contact with living nematodes has been assumed to be the crucial biotic factor necessary to induce the trap formation of NTF , 8. Trap9–Interestingly, NTF need an organic energy source other than nematodes in order to remain in an active nematophagous state , 19. Pre16–Arthrobotrys oligospora isolated from The Netherlands  traps in direct contact with nematodes. In order to obtain information on the morphological and metabolic changes in A. oligospora, two common media, CMA and PDA, were used in our analyses to investigate medium-specific metabolic features, as well as to illuminate general aspects of A. oligospora metabolism. There has been no previous report about the response of the fungus just before the fungus starts to form predatory traps via direct physical contact with nematodes, so a bioassay of nondirect contact between the fungus and living/dead nematodes was also performed. Dead nematodes were included in the non-direct-contact bioassay in order to further evaluate if the fungus could make different responses to the approaching living and dead nematodes. The time course designs over short-term intervals have provided successive snapshots of the morphological and metabolic status of A. oligospora (model strain YMF1.01883) in response to a shift from the absence of the nematodes to the presence of nematodes. GC-MS analysis was performed for metabolite profiling to determine the similarities and differences in temporal metabolite responses, and we have identified several volatile compounds that exhibit medium-specific responses during the induction of traps in response to the presence of nematodes.ng fungi , 27. UndA. oligospora grown on CMA and PDA to the presence of nematodes under two modes of contact over 144 h were evaluated and found to be significantly different . In the non-direct-contact bioassay, nematodes were also collected at regular intervals for GC-MS analyses in order to remove the effect of the nematodes on A. oligospora in the direct-contact bioassay. Four replicates for each treatment on one medium at one time point led to, in total, 382 fungal samples and 60 nematode samples for metabolite analysis. At one time point, the metabolite profiles of the fungal strains treated with nematodes were compared with those of the fungal strains treated without nematodes to evaluate the metabolites varying in contents. In order to get more information about the potential metabolites, the peaks were designated metabolites if they were identified with a match of 700 on a scale of from 0 to 1,000 to the data for the metabolites in the inborn library. All the metabolites which showed significant changes in concentrations during the time course metabolite profile analyses were considered (Tables S3 to S4).The time course metabolite profiles of A. oligospora on CMA and PDA under four treatments at 24 h were analyzed . It was obvious that despite four types of treatments, the fungal strains on the same medium shared quite similar metabolite patterns. It seemed that direct contact between nematodes and fungi did not make an obvious difference in the fungal metabolite profiles. It is also clear that the metabolite profiles of the fungi grown on CMA were different from those of the fungi grown on PDA. Comparison with the corresponding control groups without nematodes revealed that 34 out of 70 metabolites from the strains on CMA medium and 16 out of 80 metabolites from those on PDA medium were significantly up- or downregulated during the time course profiles of the fungal contacts with nematodes. Among the 34 various metabolites detected from the fungal strains on CMA, 18 metabolites were found from all the three groups treated with nematodes, 13 metabolites were found from the group treated with nematodes in direct contact, 2 metabolites were found from the group treated with live nematodes in nondirect contact, and 1 metabolite was found from the group treated with dead nematodes in nondirect contact. Among the 16 various metabolites detected from the fungal strains on PDA, 9 metabolites were found from all the three groups treated with nematodes, 5 metabolites were found from the group treated with nematodes in direct contact, 1 metabolite was found from the group treated with live nematodes in nondirect contact, and 1 metabolite was found from the group treated with dead nematodes in nondirect contact. These metabolites included short-chain acids and esters, furanones and pyrones, linoleic acid derivatives, purines, and phenolic compounds (Tables S5 and S6).The metabolite profiles of A. oligospora during the saprophytic and the pathogenic stages of growth on CMA and PDA are depicted . PCA is a useful clustering method for exploratory data analysis and requires no previous knowledge of data structures. The PCA score trajectories of the logarithmically transformed metabolite concentrations from depicted . These ddepicted  and B, iA. oligospora without nematodes (C), (ii) the branch of A. oligospora cohabitating under direct contact with nematodes (DC), (iii) the branch of A. oligospora cohabitating under nondirect contact with live nematodes (NDC-L), and (iv) the branch of A. oligospora cohabitating under nondirect contact with dead nematodes (NDC-D) (The PCA plots of dendrograms from experiments and metabolite data in CMA and in PDA are depicted in  (NDC-D) . The fun (NDC-D) .H-purin-2-amine (compound 3), hexahydro-2,6-epoxyfuro-3-ol (compound 4), 2(5H)-furanone (compound 5), and furan-2-ylmethanol (compound 6) -dione (compound 9), 3-hydroxy-2-methyl-4H-pyran-4-one (compound 10), (R)-1-phenyl-1,2-ethanediol (compound 11), d-(+)-talose (compound 12), n-hexadecanoic acid (compound 13), and -methyl octadeca-9,12-dienoate   . On PDA ic acid) . These cH-purin-2-amine (compound 3), 2(5H)-furanone (compound 5), furan-2-ylmethanol (compound 6), furan-2-carbaldehyde (compound 7), 5-methylfuran-2-carbaldehyde (compound 8), 3-hydroxy-2-methyl-4H-pyran-4-one (compound 10), (R)-1-phenyl-1,2-ethanediol (compound 11), d-(+)-talose (compound 12), n-hexadecanoic acid (compound 13), and -methyl octadeca-9,12-dienoate (compound 14). The use of chemical standards allowed us to test the ability of individual compounds to attract or poison nematodes, as well as to observe their effects on fungal development and morphology.The roles of 12 of the 14 individual metabolites were evaluated using 12 commercially available compounds, including propanoic acid (compound 1), 3-ethoxy-1,2-propanediol (compound 2), 6-methoxy-9C. elegans worms were attracted toward only three metabolites: 2(5H)-furanone (compound 5), furan-2-yl methanol (compound 6), and furan-2-carbaldehyde (compound 7). Interestingly, all three of these metabolites share a furan ring and have similar molecular weights. Compounds 5 and 6 were characterized from the fungus on CMA, while compound 7 was characterized from the fungus on PDA, based on the time course metabolite profiles of the fungal strains (H)-furanone (compound 5) functioned as an attractant through a broad range of concentrations, displaying the strongest nematode-attracting ability at a concentration of 250 μg/ml  showed toxic activity against the nematodes with a 50% lethal concentration (LC50) of 369 μg/ml in 12 h. The other compounds tested did not display obvious toxic effects at the concentrations tested in these experiments (Among the 12 metabolites tested for their toxicity toward eriments .H-pyran-4-one (compound 10), also known as maltol (compound 10), displayed a significant increase in the formation of 3D traps induced by nematodes. Over 12 h, the number of adhesive 3D traps formed by the fungus grown on the media treated with maltol (compound 10) was 189 cm−2 (−2).The same 12 compounds were applied to the fungal cultivation media. In comparison with the findings obtained with the solvent control, fungal strains treated with 2.5 μg/ml of 3-hydroxy-2-methyl-4189 cm−2 , or 30% A. oligospora genome revealed five putative polyketide synthase (PKS) genes, including a type III PKS synthase gene, AOL_s00043g287 (PKS III-1), and four type I PKS synthase genes, AOL_s00043g828 (PKS I-1), AOL_s00079g496 (PKS I-2), AOL_s00215g283 (PKS I-3), and AOL_s00215g926 (PKS I-4) (AOL_s00043g287 (PKS III-1), ΔAOL_s00043g828 (PKS I-1), ΔAOL_s00079g496 (PKS I-2), ΔAOL_s00215g283 (PKS I-3), and ΔAOL_s00215g926 (PKS I-4) mutants, from 20, 7, 8, 22, and 23 transformants, respectively, were screened by genomic DNA isolation and diagnostic PCR . The metabolites from cultures of these mutants and the wild-type strain were extracted and analyzed by high-performance liquid chromatography (HPLC) and GC-MS methods according to standard protocols (AOL_s00215g283 (PKS I-3) and ΔAOL_s00215g926 (PKS I-4) mutants lacked most of the peaks, with retention times ranging from 21 to 40 min (Table S8). The ΔAOL_s00043g287 (PKS III-1) mutant displayed the same HPLC and GC-MS profiles as the wild-type strain. The ΔAOL_s00043g828 (PKS I-1) mutant showed three peaks in the HPLC profile at retention times of 11.68, 17.87, and18.82 min that were not observed in the wild type and that were then characterized as nonfuranone and nonpyrone metabolites by comparison with the standard samples and further GC-MS analysis. Only the HPLC profile of the ΔAOL_s00079g496 (PKS I-2) mutant displayed an obvious difference from the profile of the wild type, which was found in the peak for the attractant compound 2(5H)-furanone (compound 5), while most of the other peaks were similar to those in the wild-type profile (AOL_s00079g496 (PKS I-2) mutant yielded a level of 2(5H)-furanone (compound 5) 200% higher than that yielded by the wild-type strain (AOL_s00079g496 (PKS I-2) mutant than to the wild-type strain (H)-furanone (compound 5).Bioinformatics analysis of the PKS I-4) . Disruptrotocols . In the  profile . It was e strain . Furthere strain , stronglAOL_s00079g496 (PKS I-2) mutant showed the same growth rates and morphological characteristics as the wild-type strain both on CMA and on PDA within 6 days. However, from 9 days on, the ΔAOL_s00079g496 mutant grown on PDA displayed much fluffier aerial mycelia than the wild type   mutant produced more furanone and pyrone metabolites, including furan-2-ylmethanol (compound 6), furan-2-carbaldehyde (compound 7), 1-(furan-2-yl)propan-1-one (an ethyl derivative of compound 7), 5-(hydroxymethyl)furan-2-carbaldehyde , and 3,5-dihydroxy-6-methyl-2H-pyran-4(3H)-one (an hydroxy derivative of compound 10), than the wild-type strain (The Δild type . In addie strain . When nee strain . After 277 cm−2) . Accordie strain . Neverthe strain .Nematode-trapping fungi have fascinated scientists for decades, and many earlier workers have observed the way in which the presence of nematodes alters the morphology and metabolism of trap-forming species. Although earlier studies detected attractant and nematocidal metabolites by their activities, the compounds were never chemically identified –27, 30. –A. oligospora displayed greater morphological transitions in response to the presence of nematodes when it was grown on CMA than when it was grown on PDA.Under direct physical contact with nematodes, fungi grown on CMA produced more 3D traps than those grown on PDA and did so at a higher rate. Similar results were obtained in the non-direct-contact bioassay; however, instead of 3D trap formation, fungal hyphal fusions were observed in the nondirect assay. The fungi grown on CMA developed more hyphal fusions and 3D traps than those grown on PDA. Only at a late stage, after 72 h on CMA and 96 h on PDA, were a few 3D traps observed. Previous studies suggested that trap formation also requires a hyphal fusion event during initial stages , and hypA. oligospora was grown on CMA, it made quicker and stronger responses in both morphology and metabolism even before having direct contact with the nematodes.The time course of metabolite profiling indicated that the growth medium influenced metabolism more profoundly than the mode of contact with nematodes. When grown on CMA, almost half (48%) of the total detected metabolites changed in abundance in response to physical or indirect contact with nematodes; when grown on PDA, only 11% of the metabolites displayed significant changes in abundance in response to direct or indirect contact with nematodes. The fungal strains grown on CMA medium had more extensive metabolic responses to nematodes than those grown on PDA medium. The PCA plot of metabolite data from fungal strains grown on CMA clustered into four main branches, corresponding to the experimental treatments according to the mode of contact (direct or indirect) and the status of the nematodes (living or dead). In other words, the fungal strains grown on CMA had different metabolic patterns in response not only to the approach of nematodes but also to the presence of living or dead nematodes. In summary, our morphological and metabolic analyses indicate complex relationships between media, fungal sensitivity, and morphological transitions. When The metabolomics analyses suggest a role of particular volatile metabolites in initiating the morphological transition of the nematode-trapping fungus. Volatile compounds are emitted by many species of fungi and serve many ecological functions in nature, and they have also been exploited for their role in food flavor and as indirect indicators of the presence of fungal growth –34. Howe–Panagrellus redivivus with nematophagous fungi and nonnematophagous fungi showed that nematophagous fungi preferred to attract nematodes, while nonnematophagous fungi repelled them  emitted by the fungus grown on CMA were found to attract the nematodes to the fungal colony. An early study on comparison of the interaction of the free-living nematode led them . Our datZ,12Z)-methyl octadeca-9,12-dienoate  increased significantly in the fungus grown on PDA under contact with nematodes. Other research had identified linoleic acid to be a nematicidal metabolite in the mycelial extracts of several pathogenic fungi of the genus Arthrobotrys , was found to significantly paralyze and kill the nematodes. There is a long-standing assumption that an unstable nematode-inactivating chemical compound produced by the fungus might be a volatile metabolite , and ourrobotrys . HoweverH-pyran-4-one , was identified to be a morphological regulator. Maltol (compound 10) is found widely in various beans and other plant sources, such as larch tree bark, pine needles, and roasted malt (from which it gets its name)  , but it ts name) , 37. Malts name)  and has ts name) . Maltol ts name) . HoweverH)-furanones associated with fruit aromas act to attract animals to fruits in order to ensure seed dispersal. Furanones may function as interorganism signal molecules in various plant ecosystems -furanone, likely converted by an as yet unknown enzyme in tomato (Solanum lycopersicum) and strawberry (Fragaria ananassa)  , 37. HowA. oligospora. Our previous report revealed that the knockout of the PKS I-3 gene (AOL_s00215g283) led to the abolishment of the morphological regulatory arthrosporols and high levels of trap formation (A. oligospora were constructed. The mutant that lost the PKS I-2 gene (AOL_s00079g496) showed increased production of the furanone and pyrone metabolites on both CMA medium and PDA medium, with no obvious changes in other metabolites or in its morphology. The content of nematode-attracting furanone and pyrone metabolites in the mutant strain was double that in the wild-type strain. The fact that the ΔAOL_s00079g496 (PKS I-2) mutant displayed 150% stronger nematode-attracting activity was in good agreement with the 200% higher levels of attractants in the mutant strain. Although there were fewer traps induced by the strain in which the PKS I-2 gene (AOL_s00079g496) had been knocked out, the overall predatory ability remained the same as that in the wild type. We hypothesize that the extra production of nematode-toxic furanone metabolites compensated for the lower number of traps.Polyketides are the most abundant class of fungal secondary metabolites . Becauseormation . To elucA. oligospora grown on CMA and PDA can sense approaching nematodes and develop hyphal fusions  was used in the fungus-nematode interaction bioassays and cultured on CMA  or PDA . All the bioassays were conducted in 9-cm-diameter glass petri dishes. Inocula of A. oligospora YMF1.01883 were cultured at 28°C on PDA plates for 1 week. Then, one 5-mm-diameter disk of the fungus was cut with a sterile cork borer and was inoculated onto either CMA or PDA. To obtain strong fresh mycelia, the cultures were incubated at 28°C until the fungal lawn occupied half of the petri plate.A. oligospora-nematode interaction: direct physical contact and nondirect contact. Caenorhabditis elegans (strain N2) was cultured in oatmeal medium at 22°C for 6 to 7 days. The fungal strains treated without nematodes on PDA or CMA were used as controls for both treatment bioassays.Two treatment bioassays were performed to evaluate the fungal responses during the 2O was used in place of the nematode suspension. Half of the live nematodes were submerged for 20 min in 45°C water to prepare dead nematodes. In each treatment, the fungus and nematodes were harvested separately at 6-h intervals for the first 48 h and at 24-h intervals for the subsequent 4 days. In total, four biological replicates were performed for metabolomic analysis.For the non-direct-contact bioassay, the bottom of the petri plate containing the fungal lawn was inverted over a second petri plate bottom of identical size containing 1 ml solution of mixed-stage living nematodes or dead nematodes . In this2O was used as a control. The petri dishes were sealed with Parafilm and incubated in a dark chamber at 28°C. The fungal lawn was observed and harvested at 6-h intervals for the first 48 h and at 24-h intervals for the subsequent 4 days. The fungal lawns with nematodes from each time point were extracted with methanol for metabolomic analysis.For the direct-contact bioassay, 1 ml mixed-stage living nematode solution  was directly added in the center of the fungal lawn; 1 ml sterile HFor characterization of fungal growth, development, and morphological transitions, microscopy was performed according to the protocols outlined previously . The morg and 4°C, and the supernatant was concentrated to dryness under vacuum. Each dried extract was resuspended in 1 ml methanol under ultrasonic conditions for 20 min in an ice-cooled bath-type sonicator and then filtered through 0.22-μm-pore-size membranes. The filtrates were stored at −80°C prior to GC-MS analyses.The metabolomic profiling analysis involved sample extraction, metabolite detection, metabolomic data preprocessing , and statistical analysis. The metabolite profiles were obtained from the direct-contact and non-direct-contact bioassays conducted on the two media. Each treatment group consisted of 4 replicates and a corresponding control group with the same number of replicates. The fungal mycelial lawn was harvested and extracted twice with 30 ml methanol under ultrasonic conditions for 30 min in an ice-cooled bath-type sonicator. Each methanol-soluble extract was centrifuged for 3 min at 10,000 × GC-electrospray ionization (EI)-MS analyses were performed as described previously  using a Identification of peaks was performed through determination of the retention time index and mass spectrum. Compounds from the strains were designated metabolites if they were identified with a match of 900 on a scale of from 0 to 1,000 and a retention index (RI) deviation of 3.0 , 46. Thex axis) and the second principal component (y axis) or the second principal component (x axis) and the third principal component (y axis)  . The pri(y axis) . Each poSamples were clustered using unsupervised hierarchical cluster analysis (HCA), which provides an organization of primary data sets without a predefined classification. Data were visualized by the use of dendrograms. Logarithmic values of metabolite relative concentrations were implemented in the J. Craig Venter Institute MeV software in an unsupervised HCA using the Pearson correlation , 49.2, 1 mM MgSO4) according to published protocols (H)-furanone (compound 5), furan-2-ylmethanol (compound 6), furan-2-carbaldehyde (compound 7), 5-methylfuran-2-carbaldehyde (compound 8), n-hexadecanoic acid (compound 13), and -methyl octadeca-9,12-dienoate (compound 14), were obtained from Sigma-Aldrich, USA, and d-(+)-talose (compound 12) was obtained from TCI Tokyo Chemical Industry Co., Ltd. Japan. To evaluate the mutant strains, a 6-mm-diameter disk of mycelium grown for 2 days on CMA medium was used as a test sample. For the negative controls, a 6-mm-diameter disk of the wild-type strain on CMA medium was used. About 100 washed C. elegans adult nematodes in M9 buffer were placed near the center of the plate, equidistant from the two marks. After 1 h, the number of C. elegans nematodes at the putative attractant area and at the control area was counted. A chemotaxis index with the following formula was calculated on the basis of the enrichment of animals at the attractant: chemotaxis index = (number of nematodes at the attractant area − number of nematodes at the control area)/total number of nematodes. The chemotaxis index varied from +1.0 to −1.0. In this assay, a chemotaxis index of 1.0 represents a complete preference for the test sample, and an index of 0 represents an equal distribution.In order to evaluate if these volatile metabolites and mutants have nematode-attracting ability, chemotaxis assays were performed in 9-cm plates containing assay medium  per plate. The same volume solvent of DMSO (0.5% [vol/vol] DMSO) was used as a negative-control group, and 1 μg/ml ivermectin  was used as a positive control. The worms were exposed for 24 h at 20°C, and the number of dead or living worms was determined by the absence/presence of touch-provoked movement when probed with a platinum wire. The median lethal concentration (LC50) value was calculated using the probit method  gene as a selection marker, followed by identification of the desired mutants using diagnostic PCR. The two homologous regions were amplified from A. oligospora genomic DNA using primers containing overlapping regions with the vector pAg1-H3 and the Hyg resistance cassette.The annotation of the genome of 0215g926 . The genn method  for geneA. oligospora was extracted as previously described (r) gene in the pAg1-H3 vector were amplified from the genomic DNA of A. oligospora by PCR  using the primer sets described below. The disruption vector for PKS III-1 gene (AOL_s00043g287) was constructed with primer set 287-5f (TCGAGCTCGGTACCAAGGCCCGGGTAAGACGGTGTAGAGGGCTGC) and 287-5r (GAGGCCTGATCATCGATGGGCCCGGACTTAGACTGGGCACT) and primer set 287-3f (GCGATCGCGGCCGGCCGGCGCGCCGCCGAGGTCTTCTGGAAA) and 287-3r (GAGTCACGAAGCTTGCATGCCTGCAGGTGTGCCGTTGCTTGGTAA). The disruption vector for the PKS I-1 gene (AOL_s00043g828) was constructed with primer set 828-5f (GAGCTCGGTACCAAGGCCCGGGTGCGTCACTTTGTTCATC) and 828-5r (CGAGGCCTGATCATCGATGGGCCCTAAATCTATCGTCGGGTAC) and primer set 828-3f (GCGATCGCGGCCGGCCGGCGCGCCTCACGGAACAGGCACTAC) and 828-3r (TCACGAAGCTTGCATGCCTGCAGGCAGACGATCTATCCCACC). The disruption vector for the PKS I-2 gene (AOL_s00079g496) was constructed with primer set 496-5f (AGCTCGGTACCAAGGCCCGGGTTTGTTATAGAAATGCCTCC) and 496-5r (GAGGCCTGATCATCGATGGGCCCGTCTTACCCAACTTAGCG) and primer set 496-3f (GCGATCGCGGCCGGCCGGCGCGCCAGATAGTAAGGATGGGCAG) and 496-3r (TCACGAAGCTTGCATGCCTGCAGGTGAAACGCAGACGGGTAA). The disruption vector for the PKS I-3 gene (AOL_s00215g283) was constructed with primer set 283-5f and 283-5r and primer set 283-3f and 283-3r (AOL_s00215g926) was constructed with primer set 926-5f (GAGCTCGGTACCAAGGCCCGGGGCCGTAAGTAAATTGTCTG) and 926-5r (AGGCCTGATCATCGATGGGCCCCAAGTGCGTGGTAGGAGC) and primer set 926-3f (TCTAGAGGATCCCCCGACTAGTGTGGCGTTCGTAGTGATG) and 926-3r (CACGAAGCTTGCATGCCTGCAGGTTCCAGTAGGACCGTGTA).Genomic DNA of escribed . Restricd 283-3r . The disA. oligospora (0.5 μl) was added as the template. All PCRs were performed in a Veriti 96-well thermal cycler . The amplification program consisted of predenaturation at 98°C for 4 min, followed by 30 cycles of denaturation at 98°C for 10 s, annealing at 57°C for 15 s, and elongation at 68°C for 2 min, with a final extension step at 68°C for 10 min.The DNA fragments (5′ flanks and 3′ flanks) were purified using a PCR cleanup kit  and NucleoSpin gel and were inserted into the specific sites of the pAg1-H3 vector by the In-Fusion method to generate the completed disruption of the pAg1-H3-5′-3′ vector. Amplifications of the homologous fragments were carried out as follows. An amplification system with a 25-μl PCR mixture with GXL high-fidelity DNA polymerase was applied following the manufacturer's instructions (TaKaRa). Genomic DNA prepared from 4 at pH 6.0. The suspension was incubated for 4 h at 28°C on a rotary shaker at 180 rpm. Protoplasts were collected by filtering through six layers of sterile lens-cleaning tissue and centrifuged at 1,000 × g. The protoplasts were washed twice with KTC  solution and, finally, resuspended in the same solution.PDASS medium  was applied to carry out protoplast regeneration. Four 1- to 1.2-cm diameter mycelial plugs from a 7-day-old fungal strain on YMA medium  were inoculated into 100 ml of TG medium  and cultured at 30°C at 180 rpm for 36 h. The mycelia were harvested and resuspended in 20 ml of a filter-sterilized enzyme solution that contained 120 mg of lysing enzymes , 0.4 ml of cellulase , and 100 mg of snailase  in 0.6 M MgSOA. oligospora was performed as described previously (7/ml) was mixed with 10 μg linear DNA in a 1.5-ml centrifuge tube. After 30 min of incubation on ice, 600 μl of PTC  was added to the mixture and the components were mixed gently. After incubation at 28°C for 1 h and regeneration for 12 h, the putatively transformed protoplasts were plated onto PDAS medium  containing 200 μg/ml of hygromycin B. Transformed colonies were selected after incubation at 28°C for 6 to 8 days, and every single colony was transferred to a new plate containing TYGA medium  with 200 μg/ml of hygromycin B. After incubation for 5 days at 28°C, the genomic DNA of the putative transformants was extracted and was verified by PCR to check for the integration of the genes in the genome. Five mutants deficient in these PKS genes were screened out and confirmed by PCR. Knockout of the PKS I-2 gene (AOL_s00079g496) was further confirmed by Southern blot analysis. Southern analysis was carried out according to the instructions provided by the chemiluminescent nucleic acid detection module . The primer pair KS-5f (TGTATTCCGTTTCGGTCTGC) and KS-3r (TTGAACCAACACGATTCTGC) was used as the Southern hybridization probe, and restriction enzyme AgeI was used to digest the genomic DNA of the wild-type A. oligospora strain and the ΔAOL_s00079g496 mutant for Southern analysis. All the mutants were maintained and cultured on the same media in the same way as the wild-type strain. The metabolites from cultures of these mutants and the wild-type strain were extracted and analyzed by HPLC and GC-MS methods. GC-MS analysis was performed as described above in “Metabolomic profiling.”The protoplast-based protocol for the disruption of the targeted genes in eviously . About 118 column . Mobile phase A was 0.1% formic acid in water, and mobile phase B was 0.1% formic acid in acetonitrile. The LC conditions were described previously  with a Capcell Pak Ceviously  and were


Investigating three-dimensional (3D) structures of proteins in living cells by in-cell nuclear magnetic resonance (NMR) spectroscopy opens an avenue towards understanding the structural basis of their functions and physical properties under physiological conditions inside cells. In-cell NMR provides data at atomic resolution non-invasively, and has been used to detect protein-protein interactions, thermodynamics of protein stability, the behavior of intrinsically disordered proteins, etc. in cells. However, so far only a single de novo 3D protein structure could be determined based on data derived only from in-cell NMR. Here we introduce methods that enable in-cell NMR protein structure determination for a larger number of proteins at concentrations that approach physiological ones. The new methods comprise (1) advances in the processing of non-uniformly sampled NMR data, which reduces the measurement time for the intrinsically short-lived in-cell NMR samples, (2) automatic chemical shift assignment for obtaining an optimal resonance assignment, and (3) structure refinement with Bayesian inference, which makes it possible to calculate accurate 3D protein structures from sparse data sets of conformational restraints. As an example application we determined the structure of the B1 domain of protein G at about 250 μM concentration in living E. coli cells. Escherichia coli (E. coli) cells for the putative heavy-metal binding protein TTHA1718 from Thermus thermophilus HB82in vitro protein structure determination approaches by X-ray crystallography, NMR, or electron microscopy, this approach has unique abilities that permit to investigate the effect of macromolecular crowdingIn-cell NMR spectroscopy was first proposed as a tool for investigating the behavior of bio-macromolecules at high resolution in living cellsE. coli cells at an order of magnitude lower concentration than before. The structure of the Streptococcus protein G B1 domain consisting of 57 amino acids (henceforth referred to as GB1) in living E. coli cells was solved by in-cell NMR at a concentration of approximately 250 μM in the NMR tubes NH, H(CCCO)NH, (H)CC(CO)NH, HCCH-COSY and HCCH-TOCSY spectra were measured. For the collection of NOE-derived distance restraints, three types of 3D NOESY spectra, 15N-separated, 13C-separated and 13C/13C-separated NOESYs, were measured. Methyl-selectively 1H/13C-labeled samples were used for the 3D 13C/13C-separated NOESY experiments. A nonlinear sampling scheme for the indirectly acquired dimensions was employed in order to overcome the problems of low sensitivity and short life times of the in-cell NMR samples CA, HNCO, HN(CA)CO, CBCA(CO)NH, CBCANH, and HCACO, were measured for the backbone  samples .E. coli cells compared to the former caseE. coli cells, in which strong self-correlated diagonal signals and plenty of much weaker correlation cross peaks are observed all together, we examined the reproducibility of the QME processing on reconstructing these spectra. Maximum entropy (MaxEnt) is a widely used method for NMR signal reconstruction that aims at minimizing an objective function, Q(h) = λS(h) – L(h), where h is mock data, S and L are the entropy and residual terms, respectively, and the Lagrange multiplier λ reflects the relative contribution of prior information based on the maximum entropy principle and the error of the experimental data. It is not trivial to manually determine the optimal value of λ based on general criteria. Whereas the conventional MaxEnt approach in the Azara software fixes λ to a used-defined value, QME chooses λ for an entire spectrum by an iterative search procedure for the maximum of an approximated conditional probability distribution for the experimental data given λ. QME improved drastically the quality of in-cell NMR spectra, which suffer from extraordinarily strong background signals from endogenously expressed proteins and a wide dynamic range in peak intensity. The comparison of spectra processed by QME and Azara MaxEnt indicated the superiority of the QME spectra in which the intensities of many cross peaks were clearly enhanced and cross peaks that were undetectable by MaxEnt became visible  by molecular dynamics simulation (MD), which aims at satisfying ranges of distances and dihedral angles derived from experimental data, and subsequent structure optimization in a physical force field. However, this approach did not perform adequately with the in-cell data on account of the sparse experimental structural information and limited conformational search range of SA. A more sophisticated method with a larger radius of convergence is needed in order to accurately evaluate the sparse and ambiguous experimental data derived from proteins in living cells. Thus, we adopted another NMR structure optimization method based on a Bayesian framework, so called Inferential Structure Determination (ISD)2319217 REXMC steps. It converged well  for comparison with the conventional method, the RMSD of these 20 structures is 0.49 Å to its mean and 1.02 Å to the in vitro structure   . The sli13C/13C-separated NOESY were broader than for the 15N-separated and 13C-separated NOESYs, presumably due to the smaller number of peaks, and the concomitant smaller information content.In addition, Bayesian inference provided the distributions of the calibration constants and their standard deviations for the three NOESY spectra. These distributions  reflect in vitro NMR structure determinations, it may obscure deviations from canonical secondary structure manifested in the experimental data because it explicitly fixes standard secondary structure hydrogen bonds for ranges of residues identified by the spectroscopist. It is instructive to improve the TTHA1718 structures by our present approach of data-driven structure determination with prior information. Thus, QME data processing, FLYA automatic resonance assignment, and CYBAY Bayesian structure optimization were applied also to the previously recorded NMR data of TTHA1718 in living E. coli cells. Omitting the hydrogen bond restraints from the conventional in-cell structure determination approach13C-separated, 15N-separated, and 13C/13C-separated NOESY spectra that had previously been processed by Azara MaxEnt were newly reconstructed by QME. As with the in-cell GB1 spectra, the QME reconstruction clearly enhanced the intensities of numerous cross peaks, and additional signals that had previously been obscured by noise could now be observed  gene was used for the protein expression. An additional glycine residue is inserted following the N-terminal methionine. In-cell NMR samples were prepared as follows. JM109 (DE3) E. coli cells harboring the GB1 expression plasmid were first grown in unlabeled M9 minimal medium. The production of uniformly 13C/15N-labeled GB1 was induced by the addition of isopropyl thio-β-D-thiogalactoside (Wako) to a final concentration of 0.5 mM following transfer of the bacteria into M9 minimal medium (100 ml) containing 2 g/l U-13C-glucose (Cambridge Isotope Laboratories) and 1 g/l 15NH4Cl (Cambridge Isotope Laboratories). For uniformly 15N-labeled or 13C-labeled samples, either U-13C-glucose or 15NH4Cl and were replaced with unlabeled compounds, respectively. For the production of GB1 samples with selectively protonated side-chain methyl groups of Ala, Ile, Leu and Val residues in a uniform 2H-background, protein expression was induced in 100% D2O M9 medium containing 2 g/l unlabeled glucose (Wako), 1 g/l 15NH4Cl, 100 mg/l [3-13C] alanine (ISOTEC) and 100 mg/l  α-ketoisovalerate (Cambridge Isotope Laboratories). The incubation was continued for 3 hours at 22 °C. For 13C/15N-labeled and 15N-labeled samples, the cells were harvested by gentle centrifugation and placed as ~60% slurry with M9 medium containing 10% D2O (ISOTEC) into NMR tubes. The 13C-labeled and methyl-selectively 1H/13C-labeled samples were suspended with M9 medium containing ~100% D2O. The concentration of GB1 in E. coli samples was estimated by comparing the density of the Coomassie-stained bands in SDS-PAGE gels with those of the purified GB1. The stability of GB1 E. coli samples was monitored repeatedly by 2D 1H-15N HSQC spectra followed by plating colony testsThe expression plasmid pET47b encoding the in vitro NMR experiments was purified by IgG sepharose 6 Fast Flow affinity column chromatography  following cell lysis by sonication and high temperature (70 °C) treatment for 10 minutes. The final GB1 fractions were concentrated and dissolved in M9 medium containing 10% D2O for NMR experiments.GB1 for z-axis pulsed field gradient coil, using a Bruker AVANCE 600 MHz spectrometer. Backbone 1HN, 15N, 13Cα, 13C’, and side-chain 13Cβ resonance assignments of GB1 in living E. coli cells were performed by analyzing six 3D triple-resonance NMR spectra, HNCA, HN(CO)CA, CBCA(CO)NH, CBCANH, HNCO, and HN(CA)CO measured on 13C/15N-labeled samples, and an HCACO spectrum measured on a 13C-labeled sample. 3D HBHA(CBCACO)NH, H(CCCO)NH, (H)CC(CO)NH experiments on 13C/15N-labeled samples, and 3D HCCH-COSY and HCCH-TOCSY experiments on 13C-labeled samples were performed for side-chain 1H and 13C resonance assignments. A 15 ms 13C isotropic mixing time was employed for the (H)CC(CO)NH, H(CCCO)NH and HCCH-TOCSY experiments. Intraresidue and sequential NOEs involving methyl protons were also utilized for the assignment of Ala/Leu/Val/Ile methyl groups. For the collection of NOE-derived distance restraints, 3D 15N-separated and 3D 13C-separated NOESY-HSQC spectra were measured on uniformly 15N-labeled or 13C-labeled in-cell NMR samples, respectively. In addition, 3D 13C-separated NOESY-HSQC and 3D 13C/13C-separated HMQC-NOESY-HMQC spectra were measured on Ala/Leu/Val/Ile-methyl-selectively protonated samples. A 100 ms NOE mixing period was employed for the 3D NOESY experiments. All 2D and 3D NMR data were recorded using the States-TPPI protocol for quadrature detection in indirectly observed dimensions. Water flip-back 1H pulses and the WATERGATE pulse sequence were used for solvent suppression in the experiments performed on 15N-labeled and 13C/15N-labeled samples, whereas presaturation and gradient-spoil pulses were used for 13C-labeled and methyl-selectively 1H/13C-labeled samples. One protein sample was used to measure one 3D spectrum.NMR experiments were performed at 22 °C probe temperature in a triple-resonance cryoprobe fitted with a t1) x 22 (t2) sampling space. With a ~1 s recycle time (including acquisition) and eight scans par a FID, the duration of each 3D experiment was reduced to ~140 min. Similar approaches were used for the side-chain assignment spectra and the NOESY spectra  and QME, and analyzed using an OpenGL version of the ANSIG 3.3 software and CcpNmr Analysis version 2.4.113C and 15N dimensions. In the analysis of TTHA1718, the identical data as reported previously13C-separated, 15N-separated and 13C/13C-separated NOESY data that had been processed by the Azara Maximum entropy (MaxEnt)NMR spectra were processed using the AZARA 2.7 software  and molecular dynamics simulations (MD) with the Amber physical force field and generalized Born implicit water model. 10 replica Monte Carlo (MC) calculations with different temperatures of 300 to 400 K were performed with 10,000 replica transitions, each consisting of 10,000,000 MC steps for obtaining the conformational prior. The prior of the calibration constants of NOESYs and those standard deviations were described by normal distributions and inverse gamma distribution, respectively, and likelihood was designed by normal distribution. Due to the trace of step evolutions of posterior, ensemble conformations and parameters were sampled from a certain value-equilibrated step. The final ensemble conformations were obtained from trajectories at 300 K. Since the initial steps of the MC samplings were not stationary, the first 1,000,000 and 2,000,000 MC steps of the GB1 and TTHA1718 calculations, respectively, were excluded from the analysis. Selecting a conformer every 5000 MC steps, 1900 GB1 and 1800 TTHA1718 conformers were thus selected as the final structures. For the graphical representation of the structures, we randomly selected 20% of these final conformers.How to cite this article: Ikeya, T. et al. Improved in-cell structure determination of proteins at near-physiological concentration. Sci. Rep.6, 38312; doi: 10.1038/srep38312 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Since Cdc25A degradation is tightly related to its phosphorylation status, we further examined whether CPX alters Cdc25A phosphorylation. The results showed that CPX treatment increased the phosphorylation of Cdc25A (S76 and S82), but only Cdc25A-S82A mutant was resistant to CPX-induced degradation. Furthermore, ectopic expression of Cdc25A-S82A partially conferred resistance to CPX inhibition of cell proliferation. Therefore, our findings indicate that CPX inhibits cell proliferation at least in part by promoting Cdc25A degradation.Ciclopirox olamine (CPX), an off-patent fungicide, has recently been identified as a novel anticancer agent. However, the molecular mechanism underlying its anticancer action remains to be elucidated. Here we show that CPX inhibits cell proliferation in part by downregulating the protein level of Cdc25A in tumor cells. Our studies revealed that CPX did not significantly reduce Cdc25A mRNA level or Cdc25A protein synthesis, but remarkably promoted Cdc25A protein degradation. This resulted in inhibition of G The immunocomplexes were subjected to SDS-PAGE, transferred to a PVDF membrane, and finally autoradiographed at −80°C. NIH Image J was used for semi-quantitative analysis of the intensities of the bands.eviously . BrieflyTo determine the effect of CPX on Cdc25A protein degradation, Rh30 or MDA-MB-231 cells, grown in the growth medium to 70% confluence, were treated with cycloheximide (CHX) , in the presence or absence of CPX (10 μM), for 0-24 h, followed by Western blotting with antibodies to Cdc25A and β-tubulin (loading control), respectively. NIH Image J was used for semi-quantitative analysis of the intensities of the bands.Western blotting was performed as described previously . NIH Imat-test for multiple comparisons. A level of P < 0.05 was considered to be statistically significant.Results were expressed as mean values ± standard error (mean ± SE). The data were analyzed by one-way analysis of variance (ANOVA) followed by post-hoc Dunnett's