Among advanced non-small cell lung cancer (NSCLC) patients with an acquired resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI), about 50% carry the T790M mutation, but this frequency in EGFR-TKI-naïve patients and dynamic change during therapy remains unclear. This study investigated the quantification and dynamic change of T790M mutation in plasma cell-free DNA (cf-DNA) of advanced NSCLC patients to assess the clinical outcomes of EGFR-TKI therapy.We retrospectively investigated 135 patients with advanced NSCLC who obtained progression-free survival (PFS) after EGFR-TKI for >6 months for their EGFR sensitive mutations and T790M mutation in matched pre- and post-TKI plasma samples, using denaturing high-performance liquid chromatography (DHPLC), amplification refractory mutation system (ARMS), and digital-PCR (D-PCR). Real-time PCR was performed to measure c-MET amplification.Detection limit of D-PCR in assessing the T790M mutation was approximately 0.03%. D-PCR identified higher frequency of T790M than ARMS in pre-TKI (31.3% vs. 5.5%) and post-TKI (43.0% vs. 25.2%) plasma samples. Patients with pre-TKI T790M showed inferior PFS  and overall survival  compared with those without T790M. In patients harboring EGFR sensitive mutation, high quantities of pre-TKI T790M predicted poorer PFS (p = 0.001) on EGFR-TKI than low ones. Moreover, patients who experienced increased quantity of T790M during EGFR-TKI treatment showed superior PFS and OS compared with those with decreased changes .Qualitative and quantitative T790M in plasma cf-DNA by D-PCR provided a non-invasive and sensitive assay to predict EGFR-TKI prognosis. Inhibition of epidermal growth factor receptor (EGFR) kinase activity by EGFR tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib and erlotinib, can result in improved response and prolonged progression-free survival (PFS) in selected non-small cell lung cancer (NSCLC) patients harboring sensitizing EGFR mutations, especially the exon 19del and exon 21 L858R mutations T790M mutation was previously regarded as a secondary mutation that was acquired following EGFR-TKI therapy of tumors harboring sensitizing EGFR mutations. Recently, increasing evidences suggested that T790M might co-exist at a low frequency before EGFR-TKI therapy Cell-free DNA (cf-DNA) in plasma is a kind of fresh and real-time sample, and has been shown to be promising for the detection of sensitizing EGFR mutations In this study, we used qualitative and quantitative methods, including highly-sensitive D-PCR, to assess the EGFR T790M mutation in plasma DNA samples from patients with advanced NSCLC before and after EGFR-TKI therapy. We then correlated our findings with clinical outcomes.We retrospectively analyzed 135 advanced NSCLC (stage IIIb or IV) patients who received EGFR-TKI treatment (gefitinib or erlotinib) at the Peking University Cancer Hospital between April 1st, 2005 and July 31st, 2012. Inclusion criteria were: 1) PFS after EGFR-TKI >6 months; and 2) sufficient plasma samples for analyses of EGFR mutations before and after EGFR-TKI treatment. EGFR-sensitive mutations (19del and 21L858R) were analyzed in tumor tissues of 130 patients before EGFR-TKI treatment. We collected the plasma samples when PD after EGFR-TKI was observed but a subsequent treatment did not begin. The interval time between PD after EGFR-TKI and plasma extract was less than 21 days.PFS after EGFR-TKI was defined as the time interval between beginning EGFR-TKI and disease progression or death. The overall survival (OS) was defined as the time interval between disease diagnosis and death. Clinical data, including age, gender, histological type of cancer, smoking status, imagery and clinical outcomes after EGFR-TKI were reviewed. Light smokers were defined as patients who had smoked less than 100 cigarettes in their lifetime.The study was approved by the Institutional Review Boards of the Peking University Cancer Hospital. All patients provided written informed consent before the collection and use of their tumor specimens and blood samples.Specimen collection, DNA extraction, and denaturing high-performance liquid chromatography (DHPLC) were performed according to methods described in our previous publication 2O, which was used as a negative control. We pipetted 9 µl of the reaction mixture into the chip inlets, and the nanoscale valves and channels in the chip delivered the reaction mixture to the 765 individual reaction chambers. This partitioning of the mixture allowed the target sequences to be amplified separately. We analyzed all matched samples in the same chip in order to standardize the detection conditions. The digital array underwent thermocycling on a BioMark real-time PCR system (Fluidigm). The thermocycling conditions were: 30 s at 50°C, hot start at 95°C for 5 min, and 15 cycles of 95°C for 25 s, 64°C for 20 s, 72°C for 20 s, followed by 31 cycles of 95°C for 25 s, 60°C for 35 s with fluorescence reading (FAM and HEX), and 72°C for 20 s (as suggested by Amoy). The internal reference gene (HEX) is located at the conserved region of exon 2 of EGFR gene.D-PCR analyses were conducted using the EGFR T790M mutation detection kit  and the nanofluidic 12.765 digital array chip . Each chip contains 12 individual panels, and each panel contains 765 independent 6-nl reaction chambers. We prepared 10 µl of reaction mixture that contained 8.2 µl of T790M reaction mixture (Amoy), 0.1 µl of Taq polymerase, 0.5 µl of 20×GE sample loading reagent (Fluidigm) and 0.2 µl of 50×6-carboxy-X-rhodamine (ROX) stock solution . We then added 1 µl of DNA sample or ddHThe D-PCR analysis software was used to process the data and count the numbers of both FAM-positive chambers (mutated T790M) and HEX-positive chambers  in each panel. For the T790M mutation assay, a chamber was considered to contain a mutant allele if the mutant signals were positive; a chamber was considered to contain a wild-type sequence if it was positive for the reference signal and negative for the mutated signal. The total numbers of positive chambers for the two signals were then used to estimate the true numbers of mutated T790M alleles in each panel, and to calculate the ratio of mutated T790M/.−ΔΔCT >1.5  The c-MET copy number was determined by quantitative real-time PCR using a Stratagene Mx3000P Real-Time PCR System  with TaqMan Universal PCR Master Mix . The reference used was TaqMan Copy Number Reference Assay RNase P(4403326), and the MET primer and probe were designed by Applied Biosystems (Hs01432482_cn). Normal human genomic DNA was used as a control . We defined c-MET gene amplification as: 22 tests. The Kaplan-Meier method was used to estimate survival curves for PFS and OS. Log-rank tests were used to compare the survival curves between different subgroups. A two-sided P<0.025 was considered statistically significant.All analyses were performed using SPSS 17.0 . Comparisons of proportions were performed by χA total of 135 EGFR-TKI-treated patients with advanced NSCLC were selected for the present study , includiTo assess the detection limit of D-PCR in detecting T790M mutation, a pilot test was performed using a plasmid carrying T790M or genomic DNA from normal human peripheral-blood mononuclear cells (PBMCs). When the probe specific for the T790M mutation or the human genomic DNA was used separately, the dots were distinctly visualized as red  or blue , but not vice versa.Sensitivity assays were performed using the serially diluted T790M mutation in genomic DNA solutions . ARMS were performed for DNAs isolated from individualized dots to confirm their positivity. The positive signal ratios  for T790M in each DNA dilution were: 1∶100 (115/659), 1∶300 (20/686), 1∶1000 (15/692), 1∶3000 (5/702) , and 1∶1The T790M mutation was detected by ARMS and D-PCR, respectively, in pre-TKI and post-TKI plasma . In pre-Noticeably, among 32 patients identified with pre-TKI T790M mutation by D-PCR, 6 cases presented this gene aberration alone, accompanying with neither common sensitizing EGFR mutation (19del or 21L858R) nor uncommon mutation  and the rest possessed concurrently EGFR mutations.Among 103 pre-TKI plasma samples, 6 patients were T790M-positive by ARMS. The median PFS after EGFR-TKI among these patients was not significantly different from the median PFS of patients without T790M by ARMS (p = 0.308). However, the p-value reached statistical significance when D-PCR was used for T790M detection. T790M-positive patients showed an inferior median PFS  and median OS  compared with T790M-negative patients by D-PCR. In contrast, when post-TKI samples were analyzed, no correlations between T790M positivity and survival (PFS and OS) after EGFR-TKI were observed by either ARMS or D-PCR.Given that sensitizing EGFR mutations (19del and L858R) are the most important predictor of the response to EGFR-TKI, de novo T790M mutation quantitative analysis was performed in patients with sensitizing EGFR mutations (n = 83). In the cohort of 83 cases, patients were subdivided into three groups based on the quantity of T790M in pre-TKI plasma samples by D-PCR . The median PFS was 7.1 vs. 9.5 vs. 12.8 months . The other 44 patients showed different T790M status before and after EGFR-TKI  . In the To identifying the effect of other resistant gene variation on the change of T790M quantity, we then detected c-MET amplification in post-EGFR-TKI plasma samples and found that 32% of patients harbored this gene variation in group 2 (6/19), and that only 12% of patients harbored a c-MET amplification in group 1 (4/34).With the development of T790M-specific drugs such as AZD9291 In the present study, we explored the availability of a noninvasive assay for T790M mutation in plasma cf-DNA, and reported the largest series on quantitative and dynamic monitoring T790M mutation in matched plasma samples before and after EGFR-TKI therapy. Our results indicated that D-PCR was a highly sensitive and useful method for detecting the T790M mutation. Qualitative and quantitative T790M in plasma cf-DNA can predict survival on EGFR-TKI. Moreover, dynamically monitoring T790M change might help determining EGFR-TKI prognosis.As a highly sensitive and quantitative method, D-PCR had been used in the assessment of sensitizing EGFR mutations An Increasing of studies including our previous’ have explored the availability of plasma cf-DNA for the assessment of gene variations such as sensitizing EGFR mutation By utilizing D-PCR to analyze the T790M mutation status in plasma cf-DNA, we demonstrated that patients with de novo T790M showed inferior PFS compared with those without T790M, whether stratified by sensitizing EGFR mutations or not. Several studies by Maheswaran et al de novo T790M mutation, which leaded to a long EGFR-TKI sensitive phase. To understand why patients with a decreasing trend in T790M quantity from pre- to post-TKI displayed inferior survival with EGFR-TKI treatment, we analyzed c-MET amplification in these patients and found that a higher rate of c-MET amplification in T790M-decreased patients than T790M-increased ones. These results suggested that cancer cells harboring other driver genes, such as c-MET, may rapidly proliferate, inducing resistance to EGFR-TKI, and diluting the concentration of the T790M mutation. However, these speculations need to be confirmed in basic researches.Noninvasive genotyping in plasma cf-DNA facilitates the dynamic monitoring of gene mutation during treatment, which was important in optimizing personalized therapy Although the EGFR T790M mutation was considered to occur in patients carrying an sensitizing EGFR mutation de novo T790M in pre-TKI plasma samples predicted poor survival on EGFR-TKI. Moreover, dynamic monitoring the change of T790M quantity might help determining EGFR-TKI prognosis.In summary, utilizing D-PCR in plasma cf-DNA provided a noninvasive and promising assay to qualitative and quantitative T790M mutation. By this approach, relatively high frequency of T790M could also be detected in plasma samples, thereby facilitating the dynamic detection of the T790M mutation. T790M in EGFR-TKI naïve samples was a negative predictive factor for survival on EGFR-TKI. Through quantitative assays, high quantity of


Sipunculus nudus L. polysaccharide (SNP) in combination with WR-2721, rhIL-11 and rhG-CSF on irradiated mice. A total of 70 Imprinting Control Region (ICR) mice were divided into seven groups: the control group, the model group and five administration groups. All groups, except the control group, were exposed to a 5 Gy 60Co γ-ray beam. Blood parameters  were assessed three days before irradiation, and the on the 3rd, 7th and 14th days after irradiation. Spleen, thymus and testicular indices, DNA contents of bone marrow cells, bone marrow nucleated cells, sperm counts, superoxide dismutase (SOD), malondialdehyde (MDA), testosterone and estradiol levels in the serum were assessed on the 14th day after irradiation. The combined administration of SNP, WR-2721, rhIL-11 and rhG-CSF exerted synergistic recovery effects on peripheral blood WBC, RBC and platelet counts and hemoglobin levels in irradiated mice, and synergistic promotion effects on spleen, thymus, testicle, bone marrow nucleated cells and sperm counts in irradiated mice. The synergistic administration increased the serum SOD activities and serum testosterone content of irradiated mice, but synergy decreased the content of serum MDA and estradiol in irradiated mice. These results suggest that the combined administration of SNP, WR-2721, rhIL-11 and rhG-CSF should increase the efficacy of these drugs for acute radiation sickness, protect immunity, hematopoiesis and the reproductive organs of irradiated-damaged mice, and improve oxidation resistance in the body.This study investigated the radioprotective effect of  Radiation affects people's lives. People who work in nuclear plants, nuclear reactors, nuclear medicine, nuclear science and nuclear weapons may receive low-dose irradiation. Nuclear accidents may expose people to high-dose irradiation. Radiotherapy is an important modality for cancer therapy, but there are major drawbacks, primarily because of the severe side effects that are generated as a result of tissue damage. Some attention has been paid to the treatment of acute radiation sickness. Several types of antiradiation drugs have been developed, such as WR-2721 (amifostine), estrogen, cytokines, natural antioxidants and immune stimulators. However, the inherent side effects of these antiradiation drugs and their limited pharmacodynamic action create the need for further research for new drugs and new therapy strategies, including combination administration, which can be advantageous.2 and 250 mg/m2 for 15–42 days could increase significantly during radiotherapy and/or chemotherapy [in vivo thrombopoietic effects in normal and myelosuppressed non-human primates, and rhIL-11 may be an important therapy for reducing the severity and duration of thrombocytopenia resulting from chemotherapy [Sipunculus nudus L. polysaccharide (SNP) is a bioactive polysaccharide that is extracted from Sipunculus nudus L. We reported previously that oral SNP administration in beagle dogs facilitated the recovery of hematopoietic bone marrow damage induced by γ-radiation [Sipunculus nudus L. exhibit some physiological functions, such as antiradiation, anti-oxidation, antisenescence and antifatigue, and improve immunity [Previous studies have demonstrated that WR-2721 administration for head and neck cancer is associated with a high rate of serious adverse effects, including hypotension, vomiting and allergic reactions [otherapy . rhIL-11otherapy . The comotherapy . Sipuncuadiation . Extractimmunity –15. ReceSipunculus nudus L. was purchased from Beihai ; it was identified by Professor Jia Fuxing from the Naval Medical Research Institute of the PLA. The SNP was prepared in our laboratory as described in a previous study [us study , and theThree-week-old male imprinting control region (ICR) mice, weighing 17–19 g, were purchased from the Sino–British SIPPR/BK Laboratory. Animal Co. Ltd mice were maintained under conditions of standard lighting (12:12 h light:dark cycle), temperature (23–27°C) and humidity (50–60%) with freely available food and water. The Institutional Animal Care and Use Committees of the Naval Medical Research Institute approved all procedures for animal care and treatment.The animals were randomly divided into seven groups of 10 mice each. The administration techniques and doses for each group are shown in Table 60Co γ- radiation  in a specially designed, well-ventilated acrylic box at a dose rate of 0.8 Gy/min 30 min after WR-2721 administration on the 8th day. Animals were sacrificed via cervical dislocation on the 14th day after irradiation, according to the experimental schedule.Animals in all groups, except the control group, were whole-body exposed to 5.0 Gy of Whole blood was collected from the mouse tail into EDTA-coated tubes three days before irradiation and on the 3rd, 7th and 14th days after irradiation. Peripheral blood cell counts were obtained using a veterinary hematology analyser  according to the operator's manual.Animals were sacrificed on the 14th day after irradiation. The spleen, thymus and testis were separated, and organ indices were determined using the following equation:2 solution and incubated at 4°C for 30 min. Cell suspensions were centrifuged at 2500 rpm for 15 min, and the pellet was resuspended in 5 ml of a 0.2-mol/l HClO4 solution. The suspension was incubated at 90°C for 15 min and filtered through a 0.22-μm membrane. The absorbance at 260 nm was detected using a 759-UV spectrophotometer (Shanghai APL instrument Co. Ltd).Animals in all groups were sacrificed 14 days after irradiation. The right femur of each animal was removed and cleaned of adherent tissues. The bone marrow was flushed into 5 ml of a 5-mmol/l CaClThe left femur of each animal was removed 14 days after irradiation, and the bone marrow was flushed into 2 ml of physiological saline containing 20-U/ml heparin. A total of 0.39 ml of 2% acetic acid was added to 10 μl of the cell suspension, and the number of nucleated cells was counted using a cell counter.The left parorchis of each animal was removed 14 days after irradiation, and sperm was flushed into 3 ml of physiological saline. Sperm was quantified under a microscope after a 15-min incubation at 37°C.g for 15 min. SOD and MDA were determined using kits from Nanjing Jiancheng Bioengineering Institute according to the manufacturer's instructions, and testosterone and estradiol were determined using ELISA kits from R&D Systems China Co. Ltd.Blood was collected from an eye socket vein into clean centrifuge tubes. Serum was prepared by centrifugation at 3000t test was performed for statistical analyses. The analysis was performed using the SAS software package.Data are presented as means ± standard deviation (SD). The unpaired Student's P > 0.05). However, WBCs in all irradiated groups decreased significantly from the 3rd to the 14th day after irradiation compared with the control group (P < 0.01). WBCs in the W, S + W, W + I + G and S + W + I + G groups increased significantly on the 3rd and 7th days after irradiation compared with the model group (P < 0.05 or P < 0.01). WBCs in the S + W + I + G group increased significantly on the 14th day after irradiation compared with the model group (P < 0.01). The S + W + I + G group had the highest elevation of WBCs in the irradiated mice. These results suggest that the combination of SNP, WR-2721, rhIL-11 and rhG-CSF produced a synergistic promotion effect on peripheral blood WBCs in irradiated mice.The results are presented in Table P > 0.05). Platelets in all irradiated groups decreased significantly on the 7th day after irradiation compared with the control group (P < 0.01). Platelets in the W + I + G and S + W + I + G groups increased significantly on the 7th day after irradiation compared with the model group (P < 0.05 or P < 0.01). Platelets in the model, W, S + W and W + I + G groups decreased significantly on the 14th day after irradiation compared with the control group (P < 0.05 or P < 0.01). Platelets in the S + W + I + G group returned to normal levels on the 14th day after irradiation. These results suggest that irradiation decreases the level of peripheral blood platelets 7 days after irradiation, and that the combined administration of SNP, WR-2721, rhIL-11 and rhG-CSF effectively restores platelet levels.Table P > 0.05). RBC counts in the model, W, S + I + G and W + I + G groups decreased significantly on the 7th day after irradiation compared with the control group (P < 0.05 or P < 0.01). RBCs in the S + W + I + G group returned to normal levels. RBCs in the model, W, S + I + G and S + W groups decreased significantly on the 14th day after irradiation compared with the control group (P < 0.05 or P < 0.01). RBCs in the S + W + I + G and W + I + G groups returned to normal levels. These results suggest that irradiation decreases the level of peripheral blood RBCs 7 days after irradiation, and that the combined administrations of SNP, WR-2721, rhIL-11, rhG-CSF and the combination of WR-2721, rhIL-11, rhG-CSF exhibit a synergistic promotion effect on peripheral blood RBCs in irradiated mice.Table P < 0.05 or P < 0.01). The hemoglobin levels in the W, S + W, W + I + G and S + W + I + G groups increased significantly on the 14th day after irradiation compared with the model group (P < 0.01). These results suggest that the combined administrations of SNP, WR-2721, rhIL-11 and rhG-CSF exhibit a synergistic promotion effect on peripheral blood hemoglobin in irradiated mice.Table P > 0.05). The thymus indices of the model, W, S + I + G and S + W groups decreased significantly compared with the control group (P < 0.05 or P < 0.01). The thymus indices of the S + W, W + I + G and S + W + I + G groups increased significantly compared with the model group (P < 0.05 or P < 0.01). The testicle indices of the model, W and S + I + G groups decreased significantly compared with the control group (P < 0.01), and the testicle indices of the S + I + G, S + W, W + I + G and S + W + I + G groups increased significantly compared with the model group (P < 0.05 or P < 0.01). These results suggest that irradiation has no effect on spleen, but that it damages the thymus and testicles, and that the combined administration of SNP, WR-2721, rhIL-11 and rhG-CSF alleviates thymus and testicular injury in irradiated mice.Table P < 0.01). The DNA contents of the five administered groups increased significantly compared with the model group (P < 0.05 or P < 0.01), and the DNA contents of the S + W + I + G group returned to control levels (P > 0.05). These results suggest bone marrow cells were damaged during irradiation, and that all administration combinations ameliorated the damage. The combined administration of SNP, WR-2721, rhIL-11 and rhG-CSF produced the most improvement in the DNA contents of the bone marrow cells in the irradiated mice.Figure P < 0.05 or P < 0.01). BMNCs in the S + W + I + G group increased significantly compared with the model group (P < 0.01). These results suggest that the combined administration of SNP, WR-2721, rhIL-11 and rhG-CSF produced a synergistic protection effect on BMNCs in irradiated mice.Figure P < 0.05). Sperm counts in the W + I + G and S + W + I + G groups increased significantly compared with the model group (P < 0.05 or P < 0.01) and returned to normal levels. These results suggest that the combination of SNP, WR-2721, rhIL-11 and rhG-CSF produced a synergistic restoration effect on sperm counts in irradiated mice.Figure P < 0.01). The serum testosterone content of all treated groups, except the W + I + G group, increased significantly compared with the model group (P < 0.05 or P < 0.01). The testosterone level in the S + W + I + G group was significantly higher than in the control group (P < 0.01). These results suggest that the combinations of SNP, WR-2721, rhIL-11 and rh-CSF synergistically improved serum testosterone content in irradiated mice.The results are shown in Table P < 0.01). Serum estradiol contents of all treated groups decreased significantly compared with that of the model group (P < 0.01), and the estradiol level in the S + W + I + G group was lower than in the control group (P < 0.01). These results suggest that the various combinations of SNP, WR-2721, rhIL-11 and rh-CSF exerted a synergistic regulation effect on serum estradiol contents in irradiated mice.Serum estradiol content in the model group was significantly higher than in the control group (P < 0.01). SOD activities of all treated groups, except for the S + I + G group, increased significantly compared with that of the model group (P < 0.01). SOD activity in the S + W + I + G group was higher than in the control group (P < 0.01). These results suggest that the combination of SNP, WR-2721, rhIL-11 and rh-CSF synergistically improved serum SOD activity in irradiated mice.The results are shown in Table P < 0.05 or P < 0.01). Serum MDA contents of the W + I + G and S + W + I + G groups decreased significantly compared with the model group (P < 0.05 or P < 0.01), and MDA content in the S + W + I + G group returned to normal levels (P > 0.05 compared with the control group). These results suggest that the combination of SNP, WR-2721, rhIL-11 and rhG-CSF synergistically reduced serum MDA in irradiated mice.Serum MDA contents in all irradiated groups, except for the S + W + I + G group, increased significantly compared with the control group  and higher than in the other irradiated groups. Serum estradiol levels in the S + W + I + G group were significantly lower than in the model group (P < 0.01) and lower than in the other irradiated groups.Previous studies showed that irradiation reduced the testicular index in rats, decreased sperm counts, changed sex hormone levels in serum, and caused sexual dysfunction . The preP < 0.01). The effects of the combination of SNP, WR-2721, rhIL-11 and rhG-CSF on serum SOD and MDA were better than other administrations. These results suggest that the combined administration of SNP, WR-2721, rhIL-11 and rhG-CSF exerts a significant synergistic promotion effect on the peripheral hemogram, and a significant synergistic protective effect against thymus, testicle and bone marrow damage in irradiated mice. The combined administration of SNP, WR-2721, rhIL-11 and rhG-CSF protected against immune, hematopoietic, and reproductive organ damage in irradiated mice and improved antioxidation in the body. This combination treatment may be feasible as a new strategy for the clinical treatment of acute radiation sickness.Irradiation produces oxygen-free radicals, which cause lipid peroxidation and large amounts of abnormal metabolic material. Serum SOD and MDA in the model group differed significantly from those in the normal group . Funding to pay the Open Access publication charges for this article was provided by National Science and Technology Major Project of China (No. 2008ZXJ09004-027).


Some R-M systems rely on specialized transcription factors called C (controller) proteins. These proteins play a vital role in the temporal regulation of R-M gene expression, and function to indirectly modulate the horizontal transfer of their genes across the species. We report novel regulation of a C-responsive R-M system that involves a C protein of a poorly-studied structural class - C.Csp231I. Here, the C and REase genes share a bicistronic transcript, and some of the transcriptional auto-control features seen in other C-regulated R-M systems are conserved. However, separate tandem promoters drive most transcription of the REase gene, a distinctive property not seen in other tested C-linked R-M systems. Further, C protein only partially controls REase expression, yet plays a role in system stability and propagation. Consequently, high REase activity was observed after deletion of the entire C gene, and cells bearing the ΔC R-M system were outcompeted in mixed culture assays by those with the WT R-M system. Overall, our data reveal unexpected regulatory variation among R-M systems.Restriction–modification (R-M) systems are highly prevalent among bacteria and archaea, and appear to play crucial roles in modulating horizontal gene transfer and protection against phage. There is much to learn about these diverse enzymes systems, especially their regulation. Type II R-M systems specify two independent enzymes: a restriction endonuclease (REase) and protective DNA methyltransferase (MTase). Their activities need to be finely balanced  Many species of bacteria and archaea possess restriction-modification (R-M) systems ,2 that, Three control mechanisms for Type II R-M system expression have been outlined: MTase, antisense RNAs, and C proteins, but none of these mechanisms is understood in great detail. In the first case, a MTase represses its own transcription via cognate operator binding within the promoter –17. Addiin trans alleviates REase toxicity after loss of the R-M system  being a member of the understudied C.EcoO109I family. A crystal structure for C.Csp231I is available ,46, but EcoO109I . We findCitrobacter sp. RFL231, and was kindly supplied by Dr. A. Janulaitis, MBI Fermentas, Lithuania. Despite the fact the E. coli and Citrobacter are both members of the Enterobacteriaceae, expression of the Csp231I R-M system in E. coli cells seemed to be toxic. Accordingly, to clone the WT Csp231I R-M system the competent cells were prepared from E. coli MM294 strain, which expresses the MTase gene from pEcoVIIIM to ensure the protection of host genome. M.Csp231I and M.EcoVIII both recognize the same nucleotide sequence 7697, ΔlacX74, galU, galK, hsdR, strA] was used in all lacZ reporter assays . The transcription start points of the genes encoding the Csp231I R-M system were determined by the primer extension method. The 5′ ends of appropriate primers were labeled with 5 pmol of [γ-32P]ATP . Twenty-μl primer extension reactions containing 10 μg RNA, 0.6 pmol of labeled primer, buffer , 1 mM of each dNTPs and 10 u RiboLock RNase Inhibitor were denatured at 95°C for 3 minutes, and then incubated at 50°C for 1 hour. Next, 200u RevertAid H Minus Reverse Transcriptase (Fermentas) was added and samples were incubated at 42°C for 30 min. Finally, 4 μl of loading dye  was added, samples were denatured at 75°C and then loaded onto 6% acrylamide:bis (19:1)–7 M urea in 1× TBE gel ( TBE gel . SequencFor reverse transcription PCR (RT-PCR), 5 μg of total RNA was DNase I treated in solution at 37°C for 1 h using the RNAse-free DNase I ). After a 20 min inactivation at 65°C, the cDNA synthesis was performed using RevertAid H Minus Reverse Transcriptase (Fermentas) kit with random hexamers according to the manufacturer's protocol. The resultant cDNA was then used as template for PCR with indicated primer pairs. The resultant PCR products were separated on 2% TBE agarose gels.C promoter/operator region. The non-specific DNA substrate (515 bp) containing the PM promoter region was amplified with C2-C30 primers. Reactions containing 100 nM DNA and the indicated protein concentrations were prepared in binding buffer  to a final volume of 20 μl, and incubated for 20 min at 22°C. Samples were electrophoresed on 6% native polyacrylamide gels in 0.5× TBE buffer at 22°C. The location of dsDNA in the gels was determined by ethidium bromide staining.DNA specific substrate was double-stranded PCR-amplified  fragments that were 576 bp in length and included the entire PCitrobacter sp. RFL231 genomic DNA using primers CNco and CRev. NcoI and EcoRI treated PCR fragments were cloned into pET28(+) linearized with the same restriction enzymes to generate pET-CWT, which produces a C protein with a C-terminal His5-tag. The other C gene variants were generated using Quick-Change mutagenesis on the pET-CWT template. Resultant plasmids were: pET-Csqe (primers sqe1 and sqe2) producing a C protein variant with the following triple amino acid residue substitution S16A; Q17A; E18A, and pET-Carq (primers arq1 and arq2) producing the A33G; R34E; Q37A C protein variant. The vectors were sequenced confirmed and are called C-SQEmut and C-ARQmut respectively throughout the text.The coding sequence of C gene was PCR-amplified from E. coli Rosetta strain was used to overexpress all three His5-tagged C protein variants  and sonicated (40 × 10 s). The lysates were cleared by centrifugation and applied to a column packed with TALON cobalt resin (Clontech), and washed with buffer C. The bound proteins were eluted with 150 mM imidazole in C buffer. Protein-containing fractions were pooled, and dialysis was performed over night in buffer containing 50 mM MgCl2, 5% glycerol, 50 mM Tris–HCl pH 8.0 to remove excess of imidazole. Finally, purified protein samples were concentrated in 50% glycerol and stored at –20°C. The protein concentration was determined by densitometry in Tricine–SDS-PAGE  is defined in this study as the relative number of transformants obtained from a given preparation of competent cells using a non-saturating amount of plasmid DNA. EOT is calculated from the ratio of transformants with a test plasmid relative to those with the control vector. This term is equivalent to the term ‘relative transformation efficiency’. In this particular case due to toxicity of the investigated R-M system, was used .E. coli cells carrying Csp231I R-M system and its variants was measured by determining plating efficiency of bacteriophage λvir. The efficiency of plaquing (plaque-formation) (EOP) of λvir was calculated as the ratio of plaques formed on E. coli MM294 [pEcoVIIIM] containing plasmid pBR322 (restriction negative) to those formed on the same strain containing a plasmid with the Csp231I R-M system or its variants. Relative restriction ( = 1) refers to the WT R-M system.The restriction activity of E. coli MG1655 obtained from a fresh transformation grown on LB-agar plates with appropriate selective antibiotic. One colony carried the p18amp plasmid bearing the WT R-M system, and was selective on ampicillin. The second colony, selected on tetracycline, contained its variant - the p30tet plasmid wherein the C gene and its C-box sequence were deleted. The bla gene of p30tet was disrupted to change the plasmid selection marker to tetracycline resistance. In parallel control cultures, plasmids with restriction-negative and modification-positive variants (p17amp versus p31tet) or empty vectors (pBRamp versus pBRtet) were used. Time zero of the competition experiment marks the point at which 1:1 mixed cultures of competing cells were inoculated. Every 15–18 hrs of incubation at 37°C with shaking, the co-cultures were diluted 106 into fresh minimal media without antibiotics. A sample of each mixed competition culture was immediately taken, appropriately diluted and spread quantitatively either onto LB-agar containing the appropriate selective antibiotic or on antibiotic free LB agar. The colonies were counted and the ratio of colony-forming units (CFU) of the two competing cell populations was calculated using T = (CFUamp/CFUtet); their generation number was also determined. Data were normalized using the results from the vector control (T = (CFUamp/CFUtet)). The relative competitive fitness (W) was then calculated as W = log[T/V] for each tested generation time-point , from which cells would be protected if they had a MTase with corresponding specificity  and MTase (csp231IM) genes, the regulatory C gene (csp231IC) is present. The csp231IC gene is upstream of csp231IR in a typical colinear location. The genes do not overlap, unlike other C and REase genes (csp231IR and csp231IM genes (www.softberry.com).The genetic organization of Csp231I R-M system is shown in Figure se genes , which rlacZ reporter gene. LacZ enzyme activity was measured via ONPG hydrolysis assays. Promoter activity was detected for all three DNA fragments in: pM4 (PM of MTase gene), p20 (PC of C gene) and pR3 (PR of REase gene)  Figure ,36,40.in trans  generating plasmid p12. The promoter activity this variant remained unchanged regardless of the C protein presence or absence. These results indicate there is at least one promoter that can be stimulated for higher activity (directly or indirectly) by C protein. In the putative promoter for the MTase gene (PM), no inverted repeats have been detected. A similar observation of the C protein effect on MTase expression of unclear origin was reported previously  locatedeviously .E. coli cells carrying the complete, functional Csp231I R-M system. For the MTase gene, a single primer extension product was produced   probably indicating weak activity from the second PR2 promoter . For th) Figure , and thir Figure .C promoter .Mapping the start of mRNA for the C gene showed a single spot of multiple products within a stretch of five ‘T's located on the DNA template just upstream of the translational start Figure . This in5-CTTAG separated by an extended AT-rich 18-nt spacer, that showed no obvious similarity to the binding site of its homolog C.EcoO109I (70 of RNA polymerase to achieve transcription activation (14LSQE18) in the C.Csp231I amino-acid sequence . We also constructed a C protein variant  that is predicted to impair DNA binding (We next tested if the observed stimulation of transcription in presence of the C gene is due to C protein binding the inverted repeats (C-boxes) located upstream of the C gene. To date, most of the tested C proteins (C.PvuII-like) recognize two operators comprising nearly palindromic sequences of GACT-tat-AGTC, separated by a highly conserved central spacer with GT conserved at the center ,68. C.CsEcoO109I ,45. MoreEcoO109I  that mayEcoO109I  shows thtivation . We alsoe Figure , and gen binding . We desi binding . The pre binding . The idein vitro  fused to the C.Csp231I operator/promoter sequence (p20), which is the target for C protein action. C expression is controlled by the araBAD promoter, which is repressed by glucose and induced by arabinose over a wide range  upstream of the PC promoter containing the two inverted repeats CTAAG-n5-CTTAG is associated with activation or repression , a bicistronic C-REase mRNA initiating from the C gene promoter has been found. While our data for Csp231I indicate that the REase gene has its own promoter, it did not rule out possible readthrough transcription of the REase gene from the C promoter. To test whether this occurred we used reverse transcription PCR (RT-PCR) with primers encompassing the C/R region. PCR products were generated for each primer set located within the C or REase genes clearly indicating that a bicistronic transcript is made . PR1 knock out showed a drastic effect on REase expression (WT/C+ versus PRmut/C+) making it clear that the major effect on REase gene expression is via PR1 and not the C gene  did not fully restore the WT REase expression. Instead the REase expression was significantly increased at lower C concentration (Cmut/C+ versus Cmut/C-)  Figure . Overallvir bacteriophage, which carries six recognition sites for R.Csp231I. Changes to amino acid residues within the C protein that prevent binding to its C-box sequence  also showed about 4-fold less restriction activity. The knockout of the major REase promoter (p32) had the most pronounced effect in reducing restriction activity at about 100 times less than WT, which is also in accord with our reporter assay  resulted in the highest phage restriction, that is, about three times that of the WT Csp231I R-M system  and in parallel controlled flasks restriction-negative R-M systems: p17amp (C+R-M+) versus p31tet (C-R-M+) were tested in identical conditions. Vector control (pBRamp versus pBRtet) served to normalize the experiments in case the antibiotics-resistance expression conferred some fitness advantage. We started the cultures and let them grow for about 130 generations in antibiotic-free minimal medium. Sub-cultures were created every 24 generations, and the number of colony-forming units were counted  is a small fraction of the entire C protein number ,44. C.Cs5-CTTAG separated by 18nt with A/T rich spacers  via a short highly conserved amino acid sequence (SQE) found in most C proteins  to interact with the DNA phosphate backbone , which are overlapped by the -35 hexamer of PC  is linked to the highly cooperative binding of two C proteins dimers, and was identified for C.AhdI, C.PvuII, C.Esp1396I . In add residue . Overall residue ,78. In a binding , and it o Figure , inabilio Figure , decreaso Figure  and dimio Figure . We alsoC Figure . Saturattetramer  leads toEsp1396I ,38,79. BEsp1396I ,46. InteR1 and PR2, for the REase gene in the region between the C and REase genes  . Deletion of the entire C gene resulted in a 3-fold increase in phage restriction (Figure The Csp231I R-M system contains collinearly oriented C and REase genes that are separated by 152 nt. We found that there are two functional promoters, Ps Figure . This ins Figure . The tans Figure . Our in activity . In thise Figure . The rese Figure , and wasn Figure  and seven Figure . The redIn this work, we report the first case of a C protein-associated R-M system in which the C gene positively affects REase production, but is not required for its expression. In general, the C protein control operates at two important stages: R-M system maintenance and establishment in a new cell . The maiSupplementary Data are available at NAR Online.


Neuroguidin (NGDN) is a eukaryotic translation initiation factor 4E binding protein. The purpose of this study was to clarify the function of NGDN and its possible mechanism of action in human myeloid leukemia cells. Proliferation inhibition and apoptosis in NGDN over-expressing myeloid multidrug-resistant leukemia cells (K562/A02-NGDN) was significantly higher than in control K562/A02 cells following treatment with vincristine, etoposide, and epirubicin, indicating that NGDN over-expression can increase the sensitivity of multidrug-resistant leukemia cells to chemotherapeutic drugs. Furthermore, NGDN knock-down in K562/A02 cells resulted in the activation of multiple tumor-related signaling pathways, especially the mammalian target of rapamycin (mTOR) pathway.The online version of this article (doi:10.1186/s13045-015-0108-6) contains supplementary material, which is available to authorized users. We previously reported the expression of some genes with unknown functions in myeloid leukemia cell lines and primary leukemia cells from clinical patients -3, one oP < 0.05)     by lentiviral transduction [see Additional files ) Figure A,B,C. Fo) Figure C. These ) Figure D,E,F. Fo) Figure F. These P < 0.05). A number of genes were up-regulated in K562/A02-KD cells compared to K562/A02-NC cells  was generated from K562/A02 cells by lentiviral transduction , and a negative control was also generated with unrelated RNA (K562/A02-NC) [see Additional files ls Table . For exa62/A02-KDin vitro confirmed that NGDN over-expression can increase the sensitivity of human myeloid multidrug-resistant leukemia cells to chemotherapeutic drugs, indicating that the high expression of NGDN may be a favorable prognostic factor for patients with acute myeloid leukemia [see Additional file Overall, the results of this study


Here, we show that in vitro binding of synthetic RNA mimicking that of Mononegavirales  leader sequences to purified RIG-I does not induce RIG-I oligomerization. Furthermore, in cells devoid of endogenous functional RIG-I-like receptors, after activation of exogenous Flag-RIG-I by a 62-mer-5′ppp-dsRNA or by polyinosinic:polycytidylic acid, a dsRNA analogue, or by measles virus infection, anti-Flag immunoprecipitation and specific elution with Flag peptide indicated a monomeric form of RIG-I. Accordingly, when using the Gaussia Luciferase-Based Protein Complementation Assay (PCA), a more sensitive in cellula assay, no RIG-I oligomerization could be detected upon RNA stimulation. Altogether our data indicate that the need for self-oligomerization of RIG-I for signal transduction is either dispensable or very transient.Effective host defence against viruses depends on the rapid triggering of innate immunity through the induction of a type I interferon (IFN) response. To this end, microbe-associated molecular patterns are detected by dedicated receptors. Among them, the RIG-I-like receptors RIG-I and MDA5 activate IFN gene expression upon sensing viral RNA in the cytoplasm. While MDA5 forms long filaments  In vertebrates, the first step of innate immunity is the detection of microbe-associated molecular patterns (MAMPs) by specific pattern-recognition receptors (PRRs) The precise mechanisms of RIG-I activation are still not fully understood. It has been proposed that RIG-I-mediated activation relies on RIG-I oligomerization via dimerization of RIG-I C-terminal domain (CTD), multiple oligomerization sites within RIG-I, and/or RNA-mediated oligomerization 2.Huh7.5 Moraten-eGFP measles virus was recovered by reverse genetics as described by Radecke et al. ko  cDNA were subcloned into pEF-BOS expression vector using PCR amplification of cDNA fragments and in vitro recombination . HA, Cl25  and Flag tag coding sequences were fused to RIG-I cDNA during the PCR amplification step. RIG-I insert constructs were entirely verified by sequencing (Eurofins).Wild-type human RIG-I and RIG-IGaussia Luciferase-Based Protein Complementation Assay (PCA) , were modified into pCI-glu1 and pCI-glu2 to eliminate the Gateway insert without changing the flanking vector sequence in order to preserve the linker bridging glu domains and inserts. HA-RIG-I and Cl25-RIG-I fragments were subcloned upstream or downstream of gaussia glu1 and/or glu2 domains by InFusion recombination of PCR-amplified fragments. Gcn4 sequence The two original expression vectors used for For immunoblotting the following primary antibodies were used: anti-Flag , Cl25 anti-MeV N  For DNA plasmid transfection, JetPRIME reagent (Polyplus transfection) was used in 293T cells and Transit-LT1 reagent (Mirus) was used in Huh7.5 cells. RNA transfection was performed with Oligofectamine reagent (Invitrogen). Poly(I:C) was purchased from Amersham Biosciences.in vitro T7 transcribed and purified by excising the band after denaturing urea-PAGE Rabies leader 5′ppp-RNA (GGACGCUUAACAACAAAACCAGAGAAGAAAAAGACAGCGUCAAUUGCAAACGAAAAAUGUGC), measles leader 5′ppp-RNA (GGACCAAACAAAGUUGGGUAAGGAUAGAUCAAUCAAUGAUCAUAUUCUAGUACACUUGAAUUC) and Ebola leader 5′ppp-RNA (GGACACACAAAAAGAAAGAAAAGUUUUUUATACUUUUUGUGUGCGAAUAACUAUG) were GGCACCGGGGAACCAAGGCGAACACGGACACGCAACAAACGAGACCGACAACAGACAGGACC). Both 62-mer-5′ppp-ssRNA were made from only three nucleotides to avoid secondary structure and preclude T7 polymerase re-initialization on and copy of the nascent RNA The 62-mer-5′ppp-dsRNA was obtained by annealing two T7 transcribed and purified complementary 62-mer-5′ppp-ssRNA (GGUCCUGUCUGUUGUCGGUCUCGUUUGUUGCGUGUCCGUGUUCGCCUUGGUUCCCCGGUGCC) and . Scattering data was collected for different protein concentrations and from the merged curves the radius of gyration (Rg) was determined from the Guinier plot. Next, the maximal distance in the size distribution function was adjusted, so the calculated Rg from the fit would be in agreement with the experimental value.Cells were seeded into 96-well plates and, 18 h later, transfected with 50 ng DNA of IFN-β luciferase, 17 ng DNA of renilla luciferase and 33 ng DNA of RIG-I plasmid. One day after DNA transfection, cells were transfected with Poly(I:C), synthetic RNA or infected with Moraten-gfp virus at MOI 1. The following day, the luciferase assay was performed using the Dual-Glo system from Promega. Firefly luciferase values were normalized to renilla luciferase to measure transfection efficiency.Gaussia Luciferase-Based Complementation Assay (PCA) For 2, 0.05% NP-40, 25 mM EDTA) or NP-40 buffer  both complemented with Complete (Roche) protease inhibitor cocktail for 20 minutes on ice. The proteins were then separated from cell debris by centrifugation at 7,000×g for 10 minutes. Proteins were denatured by addition of Laemmli 1X buffer and heating at 100°C for 3 minutes before analysis by SDS-PAGE and immunoblotting.For immunoblot analysis, transfected or infected cells were suspended in lysis buffer, either PLB buffer  beads (Sigma). Beads were washed four times with lysis buffer and proteins were eluted by addition of 22.5 µg 3xFlag Peptides (Sigma). Lysates were then analysed by SDS-PAGE and immunoblotting.GCTGCTACTCGGCTGATCTCAC.RNA immunoprecipitated with RIG-I was purified by Trizol/chloroform extraction, then amplified by stem-loop RT-PCR as set up for miRNA detection in vitro was tested by Multi-Angle Laser Light Scattering coupled to Size Exclusion Column (SEC-MALLS) analysis. Purified recombinant hRIG-I was able to bind to synthetic copies of the leader RNAs from three different Mononegavirales families: Ebola (Filoviridae), rabies (Rhabdoviridae) and measles (Paramyxoviridae) viruses (g for RIG-I without RNA appears to be 38.5±0.27 Å and with a short panhandle RNA of influenza virus to be slightly larger 42.2±0.21 Å. This represents an elongation of the molecule but no dimerization. P(R) functions of the scattering curves  protein to bind different 5′ppp RNA  viruses . The incg curves  that werSince RNA sensing by RIG-I results in IFN-β gene expression, the ability of the synthetic RNAs to induce the expression of a luciferase reporter gene under the control of the IFN-β promoter was tested. To avoid any interference of the endogenous innate immune response of the host cell, we selected Huh7.5 cells since they lack TLR3 and MDA5 expression, express a defective T55I RIG-I mutant and exhibit a poor feedback upregulation of RLR genes due to an IFNAR signalling defect in cellula complexes with RIG-I that are stable enough to be detected by immunoprecipitation of RIG-I. This synthetic RNA was transfected in Huh7.5 cells expressing wild type RIG-I or a RIG-Iko construct associating a T55I mutation in the first CARD domain (inhibition of TRIM25 recruitment) with Q229A, T697A, E702A and K888/907A mutations in the helicase and CTD that prevent RNA binding to the corresponding domain ko counterpart  or RNA transfection and finally harvested 18 hours after stimulation for an analysis in a co-immunoprecipitation assay. Cl25-RIG-I could not be co-immunoprecipitated with Flag-RIG-I, since similar trace amounts were detected in the absence or presence of RNA stimulation in Flag-RIG-I eluates  or synthetic dsRNA, we tried to stimulate RIG-I by infecting cells with measles virus Since we did not observe any RIG-I oligomerization Since La Crosse virus (LACV) nucleocapsids that exhibited a triphosphorylated 5′ (5′ppp) terminus as does MeV can be co-immunoprecipitated with RIG-I from infected cells th amount of RNA, the amount of Cl25-RIG-I found in the anti-Flag immunoprecipitate decreased with the 62-mer-5′ppp-dsRNA but increased with the 62-mer-5′ppp-ssRNA. Reducing the amounts of transfected RNA and thus the number of RNA molecules accessible for one RIG-I would increase the chance for RIG-I to oligomerize. Since this assessment was only verified for the non-stimulatory ssRNA, we interpret the co-immunoprecipitation of Cl25-RIG-I with Flag-RIG-I as an experimental artefact likely due to over expression of RIG-I in 293T cells. All of these results were repeatedly observed using various experimental conditions, including the use of different lysis buffers.RIG-I oligomerization was also tested in 293T cells stimulated by transfection of Poly(I:C), synthetic dsRNA or ssRNA, or Moraten-eGFP infection and 18 hours later submitted to the immunoprecipitation assay. Both Flag-RIG-I and Cl25-RIG-I were strongly expressed , inputs.in cellula, Huh7.5 cells were co-transfected with Flag-RIG-I and Cl25-RIG-I constructs, stimulated by either Poly(I:C) transfection or Moraten-eGFP infection and harvested 4 hours after stimulation for a co-immunoprecipitation assay. Both Flag-RIG-I and Cl25-RIG-I were expressed in Huh7.5 cells . PCA has been described to be highly sensitive and have allowed us to detect interactions between monomers in the 0.2–1 µM range Gaussia glu1 and glu2 split domains. All chimeric proteins were strongly expressed in Huh7.5 cells  (We reasoned that the co-immunoprecipitation assay might not be sensitive enough to detect RIG-I oligomerization induced by a cognate RNA. We therefore switched to the .5 cells . However-I-glu1) . It shou-I-glu1) . The add-I-glu1) . Similar-I-glu1) . Moreove-I-glu1) . Correla-I-glu1) .RIG-I oligomerization was proposed to occur during activation by a RNA ligand by two groups in 2007–2008 in vitro analysis by gel filtration of a mixture of pure RIG-I protein and short (from 19 bp to 135 bp) 5′ppp-RNA Mononegavirales leader 5′ppp-RNA (this work) was compatible only with RNA/RIG-I 1∶1 monomer complexes. In agreement with our observations, RIG-I and hairpin duplexes of 10, 20 or 30 base pairs with a single 5′ppp end form 1∶1 complexes as analysed by analytical ultracentrifugation-sedimentation velocity in cellula. This could be explained by an unbalanced molar ratio of RIG-I protein to 5′ppp-dsRNA in the intracellular milieu, a competition with other 5′ppp-RNA binding proteins and/or the highly dynamic interaction of RIG-I with 5′ppp-dsRNA despite a Kd in the 160 pM range The RIG-I oligomerization concept originated from The incubation of very stable 5′ppp- panhandle RNA with dsRNA of variable length with cellular extracts from RIG-I transfected cells allows the observation of RIG-I oligomerization, at least if the dsRNA exceeds 46 bp in length In vivo, RIG-I oligomerization was reported once by pull down assay of Flag- and Myc-tagged RIG-I  and % of GFP expressing cells (right). Figure S2, The anti-Flag immunoprecipitation procedure can detect complex formation between MeV N and FLAG-P proteins. Vero cells were infected with two measles viruses expressing a wt P protein or a Flag-tagged P protein at MOI 0.1. The cell extracts, collected 20 h after infection, were immunoprecipitated with Flag antibody coupled to beads. Proteins eluted with Flag peptide were analyzed by western blot using anti-P 49.21 and anti-N Cl25 monoclonal antibodies. Note the exclusive pull-down of N from cells infected with the Flag-P virus. Figure S3, Ability of RIG-I/glu/gcn4 constructs for self-binding in absence or presence of Poly(I:C) determined by PCA. Luciferase activity was measured 4 hours after transfection or not with Poly(I:C) in 293T cells expressing RIG-I/glu1/2/gcn4 constructs. Figure S4, Accessibility of gcn4 sequence in RIG-I/glu/gcn4 constructs for determination of RIG-I oligomerization by PCA. Luciferase activity was measured 24 hours after transfection of RIG-I/glu1/2/gcn4, gcn4/glu1/2 and MeV Ntail/XD/glu1/2/gcn4 constructs in 293T cells.(PDF)Click here for additional data file.


Bacillus anthracis, loss of hmgA, encoding homogentisate 1,2-dioxygenase, results in accumulation of a melanin-like pigment called pyomelanin. Pyomelanin is produced in the mutant as a byproduct of disrupted catabolism of L-tyrosine and L-phenylalanine. Accumulation of pyomelanin protects B. anthracis cells from UV damage but not from oxidative damage. Neither loss of hmgA nor accumulation of pyomelanin alter virulence gene expression, sporulation or germination. This is the first investigation of homogentisate 1,2-dioxygenase activity in the Gram-positive bacteria, and these results provide insight into a conserved aspect of bacterial physiology.Melanin production is important to the pathogenicity and survival of some bacterial pathogens. In  Bacillus anthracis is a Gram-positive, endospore-forming bacterium that is the etiological agent of anthrax. Virulence in the mammalian host depends on production of anthrax toxin and capsule, carried on virulence plasmids pXO1 and pXO2, respectively [ectively .Production of melanin pigments contributes to microbial pathogenesis, as it is associated with virulence in many microorganisms (reviewed in ). MelaniPyomelanin is a melanin-like pigment produced by the spontaneous oxidation of homogentisic acid . HomogenhmgA) of B. anthracis. Loss of hmgA results in accumulation of pyomelanin in the presence of L-tyrosine and L-phenylalanine. Pyomelanin protects B. anthracis cells from UV damage but loss of hmgA does not alter other metabolic and virulence characteristics.In this report, we have identified and characterized the gene encoding homogentisate 1,2-dioxygenase (B. anthracis strain 34F2 (pXO1+ pXO2-) and its derivatives were routinely grown in LB or brain heart infusion (BHI) broth supplemented with the appropriate antibiotics at the following concentrations: chloramphenicol (7.5μg/ml), spectinomycin (100μg/ml), and kanamycin (7.5μg/ml). As indicated, B. anthracis was grown in a modified formulation of defined R-Media [3 under 5% CO2. 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside  (40μg/ml) was added to LB agar to monitor β-galactosidase activity as necessary. Competent cells of B. anthracis were prepared following the method of Koehler et al [ R-Media  buffereder et al , and eleE. coli TG1, C600 and DH5α competent cells were used for the propagation and isolation of all plasmid constructs. E. coli transformation was performed in chemically competent cells as previously described [escribed . TransfoTransposition was performed in the 34F2 strain using the Mariner-based transposon delivery system pAW068  and the E. coli. The presence of the pUC origin of replication allows re-ligated DNA containing the transposed sequence to replicate in E. coli as a spectinomycin-resistant plasmid. Following isolation of plasmid from E. coli, sequencing of transposon-flanking DNA was performed using transposon-specific primers oMarSeq1 (5’-GCTTGTCATCGTCATCCTTGT) and oMarSeq2 (5’-GGCCGCGAAGTTCCTATT).To identify the site of insertions, mutant strains were grown overnight in BHI containing 0.5% glycerol and spectinomycin at 28°C. Genomic DNA was extracted from the overnight cultures using UltraClean Microbial DNA Isolation Kit . The site of insertion was identified by restriction digestion of genomic DNA using NsiI. Digested genomic DNA was then re-ligated and used to transform GGATCC GCGTTTGAGCAGCATGTAAA) and BAS0228U3’Sal (5’-TGCGTCGACGGAATTGTACATGTCGTTTATG) while a 638 bp region downstream of BAS0228 was amplified using primers BAS0228D5’Sal (5’-CACCGTCGACAAAGAGGCATGCTCAATAACG) and BAS0228D3’Pst (5’- TGCCTGCAGCGTTACATCCGCAACCATC) using OneTaq DNA polymerase (NEB). The upstream PCR fragment was cloned into the BamHI and SalI restriction sites of pORI-I-SceI [B. anthracis were generated through a modification of the technique of Janes and Stibitz [Strains and plasmids are listed in I-I-SceI  to gener Stibitz , as prevCultures of parental and mutant strains were grown for 48 hours at 37°C. Cell-free supernatants were prepared by centrifugation of cultures at 10,000xg followed by transfer of supernatant to new tubes. The optical spectra of the supernatants were recorded on a Cary 5000 UV-Vis-NIR spectrophotometer at room temperature in 50 mM potassium phosphate buffer pH 7.5 as previously described .B. anthracis strains grown in LB were diluted to OD600 of 0.01 in fresh LB and grown for 6 hours at 37°C. Cells were pelleted by centrifugation and resuspended in phosphate-buffered saline. Cells were then disrupted by sonication, cell debris removed by filtration, and cell-free extracts transferred to new tubes. Amino acids were added as indicated to cell-free extracts at the following concentrations based on composition of R-medium: L-phenylalanine 125mg/L; L-tryptophan 35mg/L; L-tyrosine 144mg/L. After overnight incubation at 37°C, samples were assayed visually for color change and spectroscopically at OD400.Overnight cultures of B. anthracis strains were grown in LB at 37°C for 28 hours. The cultures were transferred into a petri dish and irradiated with UV at 302 nm as indicated in figure. Serial dilutions were performed and plated on LB-agar. After 18 hours at 37°C, colony forming units (CFU) were counted.For UV protection assays, 2O2 sensitivity by disk diffusion analysis, B. anthracis strains were grown in LB at 37°C to an OD600 of 0.6. Aliquots of bacterial cultures were then added to LB-agar cooled to 50°C and poured into petri dishes. Cooled and solidified plates were overlaid with H2O2-saturated paper disks at concentrations indicated. Plates were incubated overnight at 37°C and the diameters of the zones of clearance were measured.For H2O2 protection was also assayed in liquid cultures similar to assays described previously [B. anthracis strains were grown in LB at 37°C for 24 or 48 hours. Bacteria were then pelleted by centrifugation and resuspended in cell-free supernatants. H2O2 was added at concentrations indicated and cultures incubated for 15 minutes at 37°C. Serial dilutions were performed and plated on LB-agar. After 18 hours at 37°C, CFU of pre- and post-treatment cultures were counted.Heviously . B. anthB. anthracis [Sporulation assays were performed using strains grown in SM (Schaeffer’s sporulation medium)  at 37°C nthracis . Sporula3 spores were resuspended in 1 ml LB broth, incubated for 60 minutes at 37°C, serially diluted, and plated on LB-agar. After 18 hours at 37°C, CFU were counted.For germination experiments, strains were grown in SM media for 72 hours at 37°C. Spores were harvested by heating samples to 80°C for 30 minutes followed by 3 washes in sterile water. Spores were enumerated by microscopy using a hemocytometer. 5x10B. anthracis strains harboring gene promoter fusions on the replicative vector pTCV-lac were grown at 37°C in LB supplemented with kanamycin. β-galactosidase activity was assayed as described previously and specific activity was expressed in Miller units [er units ,27.+ pXO2-) strain of B. anthracis was randomly mutagenized by Mariner transposition using plasmid pAW068. pAW068 carries a Mariner-based transposon system which delivers a transposon containing a spectinomycin-resistance cassette for selection of transposon mutants and a Gram-negative origin of replication for plasmid rescue and identification of mutants [hmgA-like genes are found among members of the Bacillus cereus group, such as B. cereus, B. anthracis, and B. thuringiensis, but are not found in the related model organism Bacillus subtilis or in other pathogenic firmicutes. Genetic organization of the BAS0228 region is shown in hmgA and the 3’ end of BAS0227. BAS0227 is predicted to encode a fumarylacetoacetate hydrolase (HmgB), the final enzyme in a tyrosine and phenylalanine catabolism pathway that converts fumarylacetoacetate to fumarate and acetoacetate in many species.The 34F2 . The ΔhmgB strain was not further analyzed.As the genes predicted to encode B. anthracis strains were grown in a derivative of a commonly used defined medium, R-medium, without added bicarbonate (R-bic). Using a defined medium allows removal and addition of specific amino acids to determine contributions to pyomelanin production. Growth of 34F2 and the ΔhmgA strain were similar when grown in R-bic or R-bic without L-phenylalanine, L-tyrosine, and L-tryptophan . The protective effects of pyomelanin-containing supernatants on the 34F2 strains were assayed similar previous studies [hmgA supernatants to protect 34F2 against H2O2 damage, suggesting that pyomelanin does not protect B. anthracis against H2O2-induced oxidative damage.Initially, H studies ,24. As sB. anthracis, the effect of loss of hmgA on virulence gene expression was assayed by β-galactosidase analysis. Transcriptional fusions of the promoters of a toxin subunit, pagA, and the master virulence regulator, atxA, to a lacZ reporter were introduced into the 34F2 and ΔhmgA strains. No difference in β-galactosidase activity was observed with either reporter between the 34F2 and ΔhmgA strains in exponential or stationary phase growth  which could function to convert L-phenylalanine to L-tyrosine, allowing L-phenylalanine to be degraded in the homogentisate pathway. A number of genes are predicted to encode aminotransferases  potentially capable of converting L-tyrosine to 4-hydroxyphenylpyruvate. A predicted 4-hydroxyphenylpyruvate dioxygenase, which converts 4-hydroxyphenylpyruvate to homogentisate, is encoded by BAS0226. HmgA (BAS0228) then converts homogentisate to maleylacetoacetate. The next steps in maleylacetoacetate degradation are unclear. BAS0227 encodes a predicted fumarylacetoacetate hydrolase that converts fumarylacetoacetate to fumarate and acetoacetate, but a gene encoding maleylacetoacetate isomerase, converting maleylacetoacetate to fumarylacetoacetate, cannot be identified in the B. anthracis genome. A previous report indicates that in some Bacillus species, maleylacetoacetate can be degraded by a maleylacetoacetate hydrolase to acetoacetate and maleic acid [B. anthracis genome. Total transcriptome analysis indicates that BAS0226, BAS0227 (hmgB), and BAS0228 (hmgA) are coordinately transcribed as part of an operon [Loss of eic acid . A gene n operon , suggesthmgA strain are grown without agitation, the development of pigment is delayed compared to a culture grown with agitation controlled for differences in cell density (data not shown), likely due to decreased oxidation of homogentisic acid. The rate of homogentisic acid production and the oxidation of homogentisic acid to pyomelanin cannot be established from these experiments. Further analysis of homogentisic acid production and oxidation under specific conditions would be needed to optimize pyomelanin production.The production of pigment in cell free extracts of cells in the early stationary phase of growth indicates that the enzymes required to convert L-phenylalanine and L-tyrosine to homogentisic acid are produced at this time; however, pigmentation in batch culture is just becoming detectable in early stationary phase. The appearance of pigment relies on conversion of 4-hydroxyphenylpyruvate to homogentisic acid followed by spontaneous oxidation of homogentisic acid to pyomelanin . When bahmgA-like genes are not found widely in Gram-positive bacteria. In fact, hmgA-like genes in the firmicutes are largely restricted to the Bacillus cereus group, which includes B. anthracis, B. cereus, and B. thuringiensis. The genome of Bacillus subtilis, the model organism for Bacillus physiology and genetics, does not encode a predicted homogentisate 1,2-dioxygenase. Bacillus subtilis does produce a protein coat that contains a brown pigment around the endospore. This melanin-like pigment is produced by the copper-dependent laccase, CotA [B. anthracis does possess a CotA-like protein [se, CotA . CotA cose, CotA ,32. B. a protein , but thiB. anthracis is unclear. Neither loss of hmgA nor pyomelanin accumulation affect expression of key virulence factors. Pyomelanin production does not appear to protect B. anthracis cells from oxidative damage, a feature important to anthrax pathogenesis [B. anthracis would mostly likely be exposed to UV radiation outside the host when in the form of UV-resistant spores. It may be possible that pyomelanin protects cells from other stresses found during the course of infection that were not assayed. Further analysis of pyomelanin-producing mutants in cellular and animal models of infection may be of value.The relevance of pyomelanin accumulation to pathogenesis of ogenesis . While p


Dodonaea viscosa led to the isolation of six new compounds including four isoprenylated flavonoids, dodovisones A-D (1–4), and two clerodane diterpenoids, dodovislactones A and B (5 and 6). Their structures were established by extensive spectroscopic analysis. Phytochemical investigation of the aerial parts of Supplementary material is available for this article at 10.1007/s13659-013-0053-4 and is accessible for authorized users. Supplementary material, approximately 1.79 MB.


Streptococcus agalactiae (group B Streptococcus) is a common commensal strain in the human gastrointestinal tract that can also cause invasive disease in humans and other animals. We report here the complete genome sequence of S. agalactiae SG-M1, a serotype III, multilocus sequence type 283 strain, isolated from a Singaporean patient suffering from meningitis. Streptococcus agalactiae  is a Gram-positive bacterium commonly found as a commensal in the gastrointestinal and genitourinary tract of up to 40% of humans  after delivery; invasive disease  in all age groups; and urinary tract infections , SG-M1 was determined to be a multilocus sequence type 283 strain (ST283); previously, ST283 strains were isolated from a meningitis patient in Hong Kong  in the SMRT Analysis suite version 2.3 using default parameters (SG-M1 was grown overnight in brain heart infusion broth at 37°C with shaking. The bacterium was collected by centrifugation and lysed in a buffer containing 20 mM Tris-Cl (pH 8.0), 2-mM sodium EDTA, 1.2% Triton X-100, and 20 mg/mL lysozyme from chicken egg white  at 37°C for 45 min. Genomic DNA was then extracted using a Qiagen QIAamp DNA minikit and sheared to a size of approximately 10 kbp using g-Tube (Covaris). A 10-kb SMRTBell library was prepared for sequencing according to the protocols recommended by Pacific Biosciences, loaded with a MagBead-bound library protocol, and sequenced using P5-C3 chemistry on the PacBio RS II instrument (Pacific Biosciences) with a 240-min movie time on two SMRTCells. rameters . In totaS. agalactiae SG-M1 harbors a single chromosome of 2,116,810 bp with a G+C content of 35.5%; no plasmids were found. Annotations of the CI5 genome and plasmid were performed using the NCBI Prokaryotic Genome Annotation Pipeline (PGAAP) (S. agalactiae SG-M1 will aid in further understanding the causative basis for invasive disease caused by GBS.S. agalactiae strain SG-M1 chromosome has been submitted to GenBank under the accession number CP012419.The complete sequence of the


The aim of this study was to investigate PD-1/PD-L1 involvement in the hyporesponsiveness of rheumatoid arthritis (RA) synovial fluid (SF) CD4 T cells upon stimulation by thymic stromal lymphopoietin (TSLP)–primed CD1c myeloid dendritic cells (mDCs).Expression of PD-1 on naïve (Tn), central memory (Tcm) and effector memory (Tem) CD4 T cell subsets was assessed by flow cytometry. PD-L1 expression and its regulation upon TSLP stimulation of mDCs from peripheral blood (PB) and SF of RA patients were investigated by quantitative RT-PCR and flow cytometry. The involvement of PD-1/PD-L1 interactions in SF T cell hyporesponsiveness upon (TSLP-primed) mDC activation was determined by cell culture in the presence of PD-1 blocking antibodies, with or without interleukin 7 (IL-7) as a recognized suppressor of PD-1 expression.PD-1 expression was increased on CD4 T cells derived from SF compared with PB of RA patients. TSLP increased PD-L1 mRNA expression in both PB and SF mDCs. PD-L1 protein expression was increased on SF mDCs compared with PB mDCs and was associated with T cell hyporesponsiveness. Blockade of PD-1, as well as IL-7 stimulation, during cocultures of memory T cells and (TSLP-primed) mDCs from RA patients significantly recovered T cell proliferation.SF T cell hyporesponsiveness upon (TSLP-primed) mDC stimulation in RA joints is partially dependent on PD-1/PD-L1 interactions, as PD-1 and PD-L1 are both highly expressed on SF T cells and mDCs, respectively, and inhibiting PD-1 availability restores T cell proliferation. The potential of IL-7 to robustly reverse this hyporesponsiveness suggests that such proinflammatory cytokines in RA joints strongly contribute to memory T cell activation. Rheumatoid arthritis (RA) is characterised by progressive joint inflammation that results in tissue damage . This isThymic stromal lymphopoietin (TSLP) has recently been considered as a potential trigger to activate CD1c mDCs in the joints of RA patients. TSLP cytokine levels are significantly increased in the SF of RA patients compared with SF of osteoarthritis patients ,13. TSLPBecause of the prominent role of CD4 T cells in arthritic processes and the potential of SF-derived mDCs and TSLP-primed mDCs to activate autologous PB-derived CD4 T cells, in this study we investigated the potential of these mDCs to activate autologous SF-derived CD4 T cells. An evident hyporesponsiveness of SF-derived CD4 T cells upon mDC or TSLP-primed mDC activation was observed. Several observations led us to investigate the role of programmed death 1 (PD-1) and its ligand interactions in this hyporesponsiveness, because ligation of PD-1 by PD-L1 or PD-L2 leads to inhibition of T cell proliferation ,16. FirsSF was obtained from 13 RA patients during effusion of the knee and 70 ml of heparinized PB was collected from 16 RA patients. RA was classified according to the American College of Rheumatology criteria . Ethical+ T cell isolation kits . PB memory T cells were isolated from the CD4 T cell fraction by their lack of CD45RA expression using the CD45RA+ isolation kit (Miltenyi Biotec). Isolations were performed according to the manufacturer’s instructions.Mononuclear cells (MNCs) were isolated from PB and SF by density centrifugation using Ficoll-Paque Plus . Prior to MNC isolation, PB or SF was diluted 1:1 with RPMI 1640 medium  containing penicillin (100 U/ml), streptomycin (100 μg/ml) and glutamine (2 mM) . PB- and SF-derived CD1c mDCs and CD4 T cells were isolated from the MNC fraction by magnetic-activated cell sorting (MACS) using CD1c (BDCA-1)-positive dendritic cell and CD46 cells/ml with or without recombinant TSLP at 20 ng/ml  for 20 hours in Sarstedt tubes  at 37°C.All cells were cultured in RPMI GlutaMAX medium (Gibco/Life Technologies) supplemented with penicillin, streptomycin and 10% (vol/vol) GemCell human serum AB . The mDCs were cultured at a cell concentration of 0.5 × 106 cells/ml and kept at 37°C in full culture medium before coculturing with the (TSLP-primed) mDCs. Washed TSLP-activated mDCs and unstimulated mDCs were added to the autologous T cells (mDC:T cell ratio 1:5 or 1:10) in triplicate in the absence of additional stimuli and cocultured for 6 days. To test the effects of PD-1 blockade or IL-7 effects in cocultures of mDCs and memory T cells from PB and SF, anti-PD-1 mAB  or IL-7  was added at the start of the cocultures. Proliferation was measured by [3H]thymidine incorporation .The functional capacities of PB- and SF-derived mDCs were assessed by measuring activation of autologous CD4 T cells derived from PB and SF. For this purpose, isolated T cells were seeded in round-bottomed 96-well plates at a concentration of 0.25 × 10Ex vivo or cultured mDCs were stained with CD1c phycoerythrin , CD19 peridinin chlorophyll  and CD274 (PD-L1)-APC (BioLegend). mDCs were gated as CD1c-positive and CD19-negative. Ex vivo CD4 T cells were stained with CD45RO fluorescein isothiocyanate , CD27-APC (Invitrogen), CD279 (PD-1)-PE and CD4-PerCP (BioLegend) using isotype antibodies or autofluorescence as controls. All samples were analysed using FlowJo software . To compare mean fluorescence intensity (MFI) values, the autofluorescence intensity was subtracted from the MFI of the stains to reveal true expression values.PD-1 expression on CD4 T cells and PD-L1 expression on CD1c mDCs of RA patients were analysed by flow cytometry using a FACSCanto II flow cytometer . 6 cells/ml) were stimulated with 20 ng/ml recombinant TSLP (R&D Systems) or left untreated for 20 hours in Sarstedt tubes at 37°C. mDCs were collected and lysed in Buffer RLT  prior to RNA isolation. Total RNA was isolated using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. RNA quantity was measured by using a NanoDrop spectrophotometer , and RNA quality was assessed on a 2100 Bioanalyzer . cDNA synthesis was performed with an automated system , starting with 70 ng of total RNA from each sample, as previously described in detail [To examine TSLP-mediated PD-L1 gene regulation in isolated CD1c mDCs from PB and SF of RA patients, CD1c mDCs . Data were considered statistically significant at P < 0.05.Differences between conditions were assessed by paired-samples evaluation using the nonparametric Wilcoxon signed-rank test unless indicated otherwise. Unpaired data analysis was performed using the nonparametric Mann–Whitney in vivo and in vitro activated mDCs /PD ligand 1 (PD-L1) interactions could play a role in the regulation of synovial T cell hyporesponsiveness to (TSLP-primed) mDCs from RA patients. First, microarray analysis had shown that PD-L1 mRNA was highly expressed compared with PD-L2 mRNA expression and significantly upregulated upon TSLP activation in addition to upregulated costimulatory and antigen-presenting molecules such as CD80, CD86, HLA class II and CD1c (data not shown). In addition, preliminary data had shown us that PD-L1 was upregulated on SF mDCs of RA patients. Therefore, we set out to investigate the expression levels of PD-1 on T cells, PD-L1 on mDCs from PB and SF of RA patients and the capacity that this interaction has to regulate T cell responsiveness.CD4 T cell subsets can be divided into naïve, central memory and effector memory T cells , based on the expression of CD27 and CD45RO. In RA patients, PB-derived T cells consist mainly of naïve and central memory T cells, whereas SF-derived T cells hardly contain any naïve T cells and consist mainly of central memory and effector memory T cells . Blocking PD-1 interactions in cocultures of memory T cells activated by mDCs resulted in a significant increase of the T cell proliferation  CD1c mDCs. SF-derived CD4 T cells consist mainly of memory T cells, which express elevated levels of PD-1, whereas SF-derived CD1c mDCs abundantly express its ligand PD-L1, which is upregulated by TSLP. Memory T cell hyporesponsiveness is partially dependent on PD-1/PD-L1 interactions because blockade of these interactions by anti-PD-1 antibodies partially restores the T cell proliferation. Importantly, IL-7, an inhibitor of PD-1 expression, also overcomes hyporesponsiveness and robustly activates synovial memory T cells in the context of activated (TSLP-primed) mDCs, which could be further enhanced by blocking PD-1.Similarly to TSLP-primed mDCs from PB, SF-derived mDCs have a strongly enhanced capacity to activate T cells derived from PB that is even upregulated upon TSLP stimulation . Associain vivo activated synovial mDCs and in vitro activated TSLP-mDCs, which, in contrast, expressed high levels of PD-L1. PD-1-blocking antibodies, blocking interactions with PD-L1, resulted in an increase in the T cell proliferation, indicating that SF-derived T cell hyporesponsiveness is at least partially dependent on PD-1/PD-L interactions. However, based on the strong expression of both PD-1 on T cells and PD-L1 on activated mDCs, more robust reversal of T cell hyporesponsiveness was expected. The lack of response might be due to the fact that PD-1 is also expressed on regulatory T cells, preventing immunosuppression by these cells [In line with our results showing increased PD-1 expression on SF-derived memory T cells compared with PB-derived T cells, PD-1 expression was previously described to be increased on SF-derived CD4 T cells . These Sse cells . Becausese cells , blockadse cells ,25. Addise cells . AlthougHyporesponsiveness of SF-derived CD4 T cells upon mDC or TSLP-mDC stimulation was robustly reversed by IL-7, which is in line with previous data demonstrating vigorous activation and even hyperresponsiveness of synovial CD4 T cells in the context of monocytic cells . Recentlin vivo activated SF mDCs or in vitro TSLP-primed mDCs contribute to T cell hyporesponsiveness. The potential of IL-7 to robustly counteract this hyporesponsiveness suggests that such cytokines in joints of RA patients might strongly contribute to the activation of memory T cells, in addition to the potential of SF-derived mDCs to activate autologous peripheral (naïve) CD4 T cells attracted to the joint.The present study demonstrates that PD-1/PD-L1 interactions between SF-derived T cells and


Next, we identified a molecular pathway by which the increased expression of miR-200c induced by natural substances led to ZEB1 inhibition and E-cadherin induction. These results indicate that our method is a valuable tool for a fast identification of natural molecules that exhibit tumour-suppressor activity in human cancer through miRNA activation.Certain dietary agents, such as natural products, have been reported to show anti-cancer effects. However, the underlying mechanisms of these substances in human cancer remain unclear. We recently found that resveratrol exerts an anti-cancer effect by upregulating tumour-suppressor microRNAs (miRNAs). In the current study, we aimed to identify new dietary products that have the ability to activate tumour-suppressor miRNAs and that therefore may serve as novel tools for the prevention and treatment of human cancers. We describe the generation and use of an original screening system based on a luciferase-based reporter vector for monitoring miR-200c tumour-suppressor activity. By screening a library containing 139 natural substances, three natural compounds — enoxolone, magnolol and palmatine chloride — were identified as being capable of inducing miR-200c expression in breast cancer cells at 10 μM. Moreover, these molecules suppressed the invasiveness of breast cancer cells  In vitro screening using human cancer cell lines enabled the classification of these products according to their cytotoxicityDiet is one of the most important environmental factors that can modulate gene expression and affect animal physiology23MicroRNAs (miRNAs) are small, non-coding RNAs that repress the expression of a wide variety of genes at the post-transcriptional level via sequence-specific base pairing to the 3′ UTR region of multiple target mRNAs9in vitro and in vivo through the transcriptional activation of tumour-suppressor miRNAsNumerous studies have demonstrated that the knockdown of oncogenic miRNAs or the reexpression of tumour-suppressor miRNAs can significantly promote drug sensitivity, inhibit cancer cell proliferation and suppress invasion and metastasis1112To identify natural compounds that activate tumour-suppressor miRNAs, we designed a reporter system to monitor miR-200c activity. We selected miR-200c to serve as a reporter in this study because we previously demonstrated that resveratrol suppresses breast cancer cell malignancy by increasing the expression of miR-200c, and we provided evidence of the tumour-suppressor activity of this specific miRNARenilla luciferase as a control reporter for normalisation. In this system, firefly luciferase activity decreases when miR-200c activity is increased and vice versa  . We idenreatment . Moreovereatment .Then, from our screening data, we selected three substances — enoxolone, magnolol and palmatine chloride — that strongly induced miR-200c expression , Fig. 2CA recent study demonstrated that miR-200c strongly inhibits the invasion ability of breast cancer cellsTo confirm that the invasiveness inhibition induced by these three substances is regulated by miR-200c, MDA-MB-231-luc-D3H2LN cells were transfected with an antisense oligonucleotide targeting miR-200c (miR-200c inhibitor) in the presence of these molecules. As shown in ZEB1 (zinc-finger E-box-binding homeobox 1) is an important EMT activator in human breast cancer. It has been reported that the overexpression of ZEB1 promoted the metastasis of colorectal cancer in a mouse xenograft modelRenilla luciferase activity after cells were transfected with a plasmid containing the ZEB1 3′ UTR , the trypsin-EDTA mixture (containing 0.05% trypsin and EDTA), FBS  and donkey anti-goat Alexa 594 were obtained from Invitrogen . Goat polyclonal anti-E-cadherin  was obtained from Santa Cruz Biotechnology . Rabbit monoclonal anti-ZEB1 (D80D3) was purchased from Cell Signaling Technology . Hoechst 33258 was obtained from Dojindo . Enoxolone (G10105), magnolol (M3445) and palmatine chloride (361615) were purchased from Sigma-Aldrich .The Natural Product Library was purchased from Selleck Chemicals . This library contains a collection of 139 natural compounds supplied as solutions dissolved in DMSO.The pmiRGLO Dual-Luciferase miRNA Target Expression Vector was purchased from Promega . For the miR-200c reporter assay, pmiRGLO-200c was constructed by introducing tandem-binding sites with a sequence that is perfectly complementary to miR-200c into the multiple cloning site of the pmirGLO vector at the XhoI and SalI sites. The sequences of the binding sites are as follows: 5′- AAACCTAGACTCGAGCCACATTACCCGGCAGTATTAAAGAATTCTTTCCATCATTACCCGGCAGTATTAGTCGACTGGCCGCAA -3′ (sense) and 5′- TTGCGGCCAGTCGACTAATACTGCCGGGTAATGATGGAAAGAATTCTTTAATACTGCCGGGTAATGATGGACTCGAGTCTAGGTTT -3′ (antisense).2.MDA-MB-231 cells (American Type Culture Collection), MCF7 cells (American Type Culture Collection) and MDA-MB-231-luc-D3H2LN cells  were cultured in RPMI 1640  containing 10% heat-inactivated FBS and the antibiotic solution at 37 °C in 5% COStable MCF7 cell lines used to monitor miR-200c activity were generated by selection with geneticin (500 μg/mL). MCF7 cells were transfected with 0.5 μg of the pmirGLO vector at 90% of confluency in 24-well dishes using Lipofectamine LTX reagent in accordance with the manufacturer’s instructions. Twelve hours after transfection, the cells were replated in a 15-cm dish, followed by a 3-week selection with the antibiotic. Ten surviving single colonies were selected from each transfection and were then cultured for 2 additional weeks.A total of 5,000 cells per well were seeded in 96-well plates. The following day, the cells were treated with natural products. Three days after culturing, cell viability was measured using the Cell Counting Kit-8  in accordance with the manufacturer’s instructions. Absorbance was measured at 450 nm using the EnVision system .Renilla luciferase reporter vector (pmiR-200c-MCF7) were seeded onto 96-well plates at a density of 10,000 cells per well. The following day, the cells were treated with 139 natural substances at 10 μM. After 2 days, the cells were harvested and the firefly luciferase activity was measured and normalised to the Renilla luciferase activity. All assays were performed in triplicate and repeated at least three times. Representative data are shown in the manuscript.MCF7 cells stably expressing our firefly luciferase and Breast cancer cell invasion was assayed in 24-well BioCoat Matrigel Invasion Chambers  in accordance with the manufacturer’s protocol. Briefly, cells were treated with natural products. The following day, 20,000 cells were plated in the upper chamber, which contained RPMI 1640 without FBS. The lower chambers were filled with RPMI 1640 containing 10% FBS as a chemoattractant. Twenty-two hours later, the non-invasive (upper chamber) cells were removed with a cotton swab. Cells that migrated through the membrane to the lower surface of the membrane were fixed with methanol and stained with Diff Quick staining . For quantification, the cells were observed microscopically and counted in four random fields. All assays were performed in triplicate. Invasive values were normalised to the values obtained from cells treated with DMSO.Total RNAs were extracted from cultured cells using the QIAzol and miRNeasy Mini Kit  in accordance with the manufacturer’s protocol. PCR was performed in 96-well plates using the 7300 Real-Time PCR System . All reactions were performed in triplicate. TaqMan qRT-PCR kits and human E-cadherin and human β-actin TaqMan Expression Assays were purchased from Applied Biosystems . Reverse transcription  and TaqMan quantitative PCR  were performed in accordance with the manufacturer’s instructions. SYBR Green I qRT-PCR was performed, and the β-actin housekeeping gene used to normalise the variation in the cDNA levels. The following pairs of primers were used for gene amplification: for vimentin, 5′-TCTGGATTCACTCCCTCTGG-3′ (forward) and 5′-GGTCATCGTGATGCTGAGAA-3′ (reverse); for c-Met, 5′-CAGGCAGTGCAGCATGTAGT-3′ (forward) and 5′-GATGATTCCCTCGGTCAGAA-3′ (reverse); and for β-actin, 5′-ACTCTTCCAGCCTTCCTTCC-3′ (forward) and 5′-AGCACTGTGTTGGCGTACAG-3′ (reverse).After washing three times with PBS (–), the cells were fixed in cold methanol . Then, the cells were incubated in RPMI 1640 containing E-cadherin primary antibodies at a dilution of 1:100 for 60 min and subsequently incubated in RPMI 1640 containing Alexa Fluor fluorescent secondary antibodies. Nuclei were visualised using Hoechst 33258  staining for observation under a confocal microscope .t-test.The data are presented as the mean ± S.E.M. Each experimental point is the average of at least triplicates. All experiments were repeated at least 3 times. Statistical analyses were performed using Student’s How to cite this article: Hagiwara, K. et al. A robust screening method for dietary agents that activate tumour-suppressor microRNAs. Sci. Rep.5, 14697; doi: 10.1038/srep14697 (2015).