Inhibition of the TLR3, TLR/TIR-domain-containing adaptor-inducing interferon β (TRIF), NF-κB, and IRF3 pathways decreased the polyI:C- and IL-17A/polyI:C-induced G-CSF and IL-8 mRNA expression. Comparing the levels of mRNA induction between co-treatment with IL-17A/polyI:C and treatment with polyI:C alone, blocking the of NF-κB pathway significantly attenuated the observed synergism. In western blotting analysis, activation of both NF-κB and IRF3 was observed in treatment with polyI:C and co-treatment with IL-17A/polyI:C; moreover, co-treatment with IL-17A/polyI:C augmented IκB-α phosphorylation as compared to polyI:C treatment alone. Collectively, these findings indicate that IL-17A and TLR3 activation cooperate to induce proinflammatory responses in the airway epithelium via TLR3/TRIF-mediated NF-κB/IRF3 activation, and that enhanced activation of the NF-κB pathway plays an essential role in synergistic induction after co-treatment with IL-17A and polyI:C in vitro.Viral respiratory infections activate the innate immune response in the airway epithelium through Toll-like receptors (TLRs) and induce airway inflammation, which causes acute exacerbation of asthma. Although increases in IL-17A expression were observed in the airway of severe asthma patients, the interaction between IL-17A and TLR activation in airway epithelium remains poorly understood. In this study, we demonstrated that IL-17A and polyI:C, the ligand of TLR3, synergistically induced the expression of proinflammatory cytokines and chemokines , but not type I interferon  in primary culture of normal human bronchial epithelial cells. Synergistic induction after co-stimulation with IL-17A and polyI:C was observed from 2 to 24 hours after stimulation. Treatment with cycloheximide or actinomycin D had no effect, suggesting that the synergistic induction occurred without  Bronchial asthma is a chronic inflammatory disease of the airway involving many cells and cellular elements –3. AirwaViral respiratory infections have been demonstrated to be one of the most common causes of acute exacerbation in asthma patients , 4. AirwThe proinflammatory cytokine IL-17A is mainly produced by Th17 and γδT cells and is a part of the “IL-17 family” with other five members (IL-17B-F). IL-17A is well known for its protective properties during bacterial infections and its involvement in autoimmune diseases . IL-17A In this study, we examined the molecular basis of the interaction between TLR3-mediated innate immune responses and intracellular signaling of IL-17A in airway epithelial cells. We showed that IL-17A synergistically enhances polyI:C-induced expression of proinflammatory cytokines and chemokines  but not type I interferon , in NHBE cells and BEAS-2B, which is a bronchial epithelial cell line. The subsequent mechanistic study reveals a crucial role for the transcriptional factors NF-κB and interferon regulatory factor 3 (IRF3) in IL-17A/polyI:C-provoked synergistic induction in airway epithelial cells.4 cells/cm2 in commercially available bronchial epithelial growth medium . The BEAS-2B (ATCC® CRL-9609™) cell line was obtained from American Type Culture Collection (ATCC) through Summit Pharmaceuticals International Corporation . BEAS-2B cells were plated in 6-well plates at 0.3–1.0 × 104 cells/cm2 in LHC-9 serum-free medium . Both types of cells were incubated at 37°C in a humidified atmosphere with 5% CO2. Submerged cells were stimulated with polyI:C and/or IL-17A. Conditioned media were collected from the cultured NHBE cells and stored at -80°C for immunoassays.Primary NHBE cells were purchased from Lonza . NHBE cells were seeded in 6-well plates at 1.8 × 10PolyI:C (high molecular weight) was purchased from Imgenex . Recombinant human IL-17A was from R&D Systems . Concentrations of polyI:C and IL-17A used in this study were 50 μg/ml and 10 ng/ml, respectively. In our preliminary data and previous report . Thus, w® ND-1000; Thermo Scientific, Chicago, IL) before reverse transcription PCR. Preparation of first strand cDNA was performed using the ReverTra Ace® qPCR RT Master Mix  from 2 μg of total RNA. The PCR mixture consisted of 10 μl of the THUNDERBIRD® SYBR® qPCR Mix (TOYOBO), 0.3 μM of forward and reverse primers, and the cDNA samples . Real-time PCR analysis was performed using the 7500 FAST Detection System  according to the manufacturer’s instructions as described previously [-(Ct of gene of interest–Ct of β-actin or GAPDH) in arbitrary units. The list of primers used in real-time RT-qPCR analysis is described in Total RNA was extracted using RNA the TRIzol reagent , and stored at -80°C. Total RNA was quantified using a spectrophotometer . Absorbance was read at 450 nm with wavelength correction at 540 nm using a microplate reader . All the measurements were performed in duplicate.To determine the concentration of the granulocyte-colony stimulating factor (G-CSF) and the IL-8 protein in the conditioned media, double-sandwich ELISAs for human G-CSF and IL-8 were performed using a Quantikinede novo protein synthesis, 5 μg/ml of cycloheximide  was administrated together with IL-17A and/or polyI:C treatment. To explore the stability of the mRNA, the cells were stimulated with polyI:C overnight (approximately 15 hours) to induce the expression of cytokines. Then, actinomycin D  was added together with IL-17A and/or polyI:C to block further mRNA synthesis, and mRNA was harvested at different time points  after actinomycin D treatment. BAY11-7082 , an IκB-α phosphorylation inhibitor, was added 1 hour before stimulation with IL-17A, polyI:C, and co-treatment of IL-17A/polyI:C to inhibit IκB-α phosphorylation. Cycloheximide, actinomycin D, and BAY11-7082 were dissolved in dimethyl sulfoxide before use.To investigate the influence of The siRNA for TLR3, Toll-like receptor adaptor molecule 1 , IRF3, and tumor necrosis factor receptor 1 (TNFR1) were purchased from Santa Cruz Biotechnology . NF-κB p65 siRNA and random oligomer for negative control were obtained from Ambion Biotech . BEAS-2B cells were transiently transfected with siRNAs using a DharmaFECT-based transfection kit (Thermo Scientific), as described previously , 18, 19.® TGX gel (Bio-Rad) and transferred electronically to PVDF membranes. The membranes were blocked with 3% bovine serum albumin (BSA) in 50mM Tris-buffered saline (TBS) or 5% nonfat milk for 30 minutes at room temperature before incubation with each primary antibody overnight at 4°C or 2 hours at room temperature. Then, the membranes were incubated with HRP conjugated secondary antibodies for 30 minutes at room temperature. The ECL chemiluminescence reagent was used to detect the signal bands as described previously [Total protein lysates from different treatments were harvested using RIPA lysis buffer  and quantified with a DC protein assay . Before loading, 20 μg of the cell lysate and 4× reducing sample buffer were mixed and heated at 95°C for 8 minutes. The proteins were separated on a Mini-PROTEANeviously  and semiPhospho-IκBα mouse monoclonal antibody (mAb) , phospho-IRF3 rabbit mAb (#4947), and IRF3 rabbit mAb (#4392) were purchased from Cell Signaling Technology  and diluted 1:1000 in 3% BSA/TBS or 5% nonfat milk. The monoclonal anti-β-actin antibody was produced in mice  and used at a 1:3000 dilution.HRP conjugated goat anti-mouse IgG (W492B) was purchased from Promega  and HRP-linked anti-rabbit goat IgG (#7074) was purchased from Cell Signaling Technology. The secondary antibodies were used at 1:16700 and 1:3000 dilutions, respectively.p values that were less than or equal to 0.05 were considered statistically significant.Measurements are described as mean ± S.E. Experiments were carried out in duplicate and repeated in at least three independent cultures. Differences among the groups were compared using one-way or two-way analysis of variance (ANOVA) and the Tukey-Kramer multiple comparison test. The To investigate the proinflammatory responses induced by viral respiratory infection in airway epithelial cells, primary NHBE cells in submerged cultures were challenged with polyI:C, which is a synthetic double-stranded RNA analogue and a ligand of TLR3, for 24 hours. The mRNA levels of cytokines and chemokines were measured by real-time RT-qPCR. The mRNA expression of proinflammatory mediators such as G-CSF, IL-8, CXCL1, CXCL5, and IL-1F9 was increased after 24 hours of exposure to polyI:C (50 μg/ml) as compared to untreated NHBE cells . NotablyTime course analyses indicated that the mRNA expression of G-CSF and IL-8 increased after stimulation with polyI:C . Notablyde novo protein synthesis was involved in the IL-17A/polyI:C-provoked synergistic induction of proinflammatory cytokines, BEAS-2B cells were treated with the protein synthesis inhibitor cycloheximide in the presence of IL-17A and polyI:C. Cycloheximide had no effect on the IL-17A/polyI:C-provoked synergistic induction of G-CSF and IL-8 mRNA levels  axis using TLR3 and TICAM-1 siRNAs. TRIF/TICAM-1 is an adapter in responding to activation of TLR3. BEAS-2B cells were transiently transfected with siRNAs for TLR3 or TICAM-1.The TLR3 siRNA suppressed 90% of its own message . At 72 hNext, we examined the role of the NF-κB pathway, which is related to signaling pathways downstream of both TLR3 and IL-17A, using a p65 siRNA. The p65 siRNA attenuated 90% of its own message . The IL-To further confirm the involvement of IRF3 in the IL-17A/polyI:C-induced synergistic proinflammatory response, we used an siRNA approach to knock down IRF3. As shown in To date, several groups have demonstrated the synergistic induction of proinflammatory cytokines by IL-17A and tumor necrosis factor (TNF) α treatment , 26. We To evaluate the activation of NF-κB signaling, we performed western blotting analysis to detect phosphorylation of IκB-α. BEAS-2B cells were cultured in submerged conditions until the cells were 90% confluent, and then stimulated with IL-17A and/or polyI:C. PolyI:C treatment or co-treatment with IL-17A and polyI:C induced IκB-α phosphorylation from 30 to 240 minutes after treatment  Files. IPhosphorylation of IRF3 was also analyzed to assess the activation of IRF3. PolyI:C treatment or co-treatment conditions with IL-17A and polyI:C induced IRF3 phosphorylation at 60 to 120 minutes after treatment  Files. Nde novo protein synthesis and mRNA stabilization. Attenuation of TLR3, TICAM-1 , NF-κB, and IRF3 using specific inhibitors or siRNAs decreased the synergistic effects on G-CSF and IL-8 mRNA expression. Comparing the ratio of mRNA induction between co-treatment with IL-17A/polyI:C and treatment with polyI:C alone, blocking the NF-κB pathway significantly attenuated the synergism. Western blotting analysis revealed that both NF-κB and IRF3 activation were observed in polyI:C single treatment and co-treatment with IL-17A and polyI:C. In addition, NF-κB activation was potentiated by co-stimulation with IL-17A and polyI:C as compared with the single treatment. These findings provide evidence that IL-17A and TLR3 signaling cooperate to enhance the expression of proinflammatory cytokines in the airway epithelium via TLR3/TRIF-mediated NF-κB/IRF3 activation; moreover, enhanced NF-κB dependent pathway activation may play a key role in the synergism.In this study, we investigated the interaction between IL-17A and TLR3 signaling in airway epithelial cells, and the regulatory mechanism of the proinflammatory response mediated by IL-17A and polyI:C, a ligand of TLR3. We found that IL-17A and polyI:C synergistically induced proinflammatory cytokines and chemokines expression , but not antiviral gene expression (IFN-α1 and –β) in primary cultures of NHBE cells. The IL-17A/polyI:C-induced synergistic proinflammatory response occurred in the absence of Our data demonstrates that IL-17A and TLR3-signaling synergistically enhanced proinflammatory cytokine and chemokine expression in airway epithelial cells. Viral respiratory infections activate innate immune responses through pattern recognition receptors, particularly TLRs and RLRs, in airway epithelial cells , which cet al. [To date, few studies describe the interaction between IL-17A and TLR signaling in airway epithelial cells, and the molecular mechanism governing the synergism elicited by IL-17A and TLRs has not been fully elucidated. Wiehler et al.  has showet al.  reportedSynergism between IL-17A and other cytokines, including TNF-α, IL-1β, IL-4, and IL-13, in inflammatory gene expression has been appreciated in various cell types , 32–36. de novo protein synthesis is dispensable for the synergism. Moreover, blocking TNF receptor signaling did not attenuate the IL-17A/polyI:C-induced synergistic expression. Based on these findings, IL-17A/polyI:C-induced synergistic G-CSF and IL-8 expression in airway epithelial cells is mediated by transcriptional activation, possibly through NF-κB and IRF3 activation. The different mechanism by which IL-17A augments proinflammatory gene expression may be partly explained by the different cell types and stimulants used in each study.Several groups have shown the synergistic proinflammatory cytokines expression induced by IL-17A and TNF-α treatment , 26. Theet al. [The detailed molecular mechanisms of synergistic inflammatory cytokine induction may be considerably complicated. Recently, Qiao et al.  demonstrIn summary, our data provide an explanation for the IL-17A/polyI:C-induced synergistic proinflammatory cytokine and chemokine expression in airway epithelial cells. IL-17A and polyI:C synergistically induced proinflammatory  but not antiviral (IFN-α1 and -β) gene expression in primary cultured NHBE cells, which promotes the attraction of other immune cell types  in the airway and excessive airway inflammation. Analysis of regulatory mechanisms for IL-17A/polyI:C-induced synergistic proinflammatory responses revealed the importance of TLR3/TRIF-mediated NF-κB/IRF3 activation; moreover, enhanced activation of the NF-κB dependent pathway may play an essential role in the observed synergism. Patients with severe asthma are highly susceptible to viral respiratory infection, which results in severe airway inflammation and acute exacerbation of asthma. This study highlights an important interaction between IL-17A and TLR3 signaling in excessive airway inflammation, and control of IL-17A/TLR3-mediated NF-κB/IRF3 activation may be a novel therapeutic target to prevent exacerbation of asthma.S1 File(TIF)Click here for additional data file.S2 File(TIF)Click here for additional data file.S3 File(TIF)Click here for additional data file.S4 File(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Claviceps purpurea is an important food contaminant and well known for the production of the toxic ergot alkaloids. Apart from that, little is known about its secondary metabolism and not all toxic substances going along with the food contamination with Claviceps are known yet. We explored the metabolite profile of a gene cluster in C. purpurea with a high homology to gene clusters, which are responsible for the formation of epipolythiodiketopiperazine (ETP) toxins in other fungi. By overexpressing the transcription factor, we were able to activate the cluster in the standard C. purpurea strain 20.1. Although all necessary genes for the formation of the characteristic disulfide bridge were expressed in the overexpression mutants, the fungus did not produce any ETPs. Isolation of pathway intermediates showed that the common biosynthetic pathway stops after the first steps. Our results demonstrate that hydroxylation of the diketopiperazine backbone is the critical step during the ETP biosynthesis. Due to a dysfunctional enzyme, the fungus is not able to produce toxic ETPs. Instead, the pathway end-products are new unusual metabolites with a unique nitrogen-sulfur bond. By heterologous expression of the Leptosphaeria maculans cytochrome P450 encoding gene sirC, we were able to identify the end-products of the ETP cluster in C. purpurea. The thioclapurines are so far unknown ETPs, which might contribute to the toxicity of other C. purpurea strains with a potentially intact ETP cluster. Claviceps purpurea infects a broad range of grasses including economically important cereal crop plants  um/z 100–800 with a resolution of 60 000 in the positive and negative ion mode was used. All observed ions showed errors less than 5 ppm. The HRMS data were analyzed by Metworks  in order to screen for the characteristic sulfur isotopic pattern. To get information on the structure of the novel metabolites, the new substances were analyzed by stepwise mass spectrometric fragmentation experiments in the positive mode. The experiments included collision-induced dissociation (CID) with a relative energy of 20% to 45%, depending on the ionization and an isolation width of m/z 1.5. The fragments were analyzed with the Orbitrap detector at a resolution of 60 000.In order to identify biosynthetic pathway intermediates, the culture filtrates were measured by RP-HPLC-DAD-HRMS. For the HRMS measurement, an Accela LC 60057-60010 system  was linked to a LTQ Orbitrap XL mass spectrometer (Thermo Fisher Scientific). A SPD-M20A Shimadzu PDA Detector  was coupled to the MS spectrometer. Data acquisition was performed with Xcalibur 2.07 SP1 (Thermo Scientific). Separation was carried out on a 150 x 2.1, 3 μm, Nucleodur phenyl-hexyl column  using a binary gradient at a column temperature of 40°C. The injection volume was 15 μL and the autosampler was cooled to 7°C. The flow rate was set to 250 μL/min. Solvent A was MeOH with 1% of formic acid (v/v) and solvent B was water with 1% of formic acid (v/v). The HPLC was programmed as follows: in the first 5 minutes isocratic 10% of A, afterwards a binary gradient to 100% in 35 min. Then the column was washed with 100% A and equilibrated at starting conditions. For the detection of intermediates, a total ion scan of a mass range from n data.After three weeks the sclerotia were ground and extracted with a mixture of ethyl acetate and water as well as acidified ethyl acetate. 500 μL water and 500 μL of organic solvent were added to the ground sclerotia in a 1.5 mL E-cup and shaken for 15 min. In the next step, phases were separated and the solvent evaporated. Afterwards resolved in 500 μL methanol/water  and 10 μL used for injection. The three extracts (ethyl acetate/ acidified ethyl acetate/ water) were analyzed in the same way as in the axenic cultures by RP-HPLC-DAD-HRMS. The metabolites were identified by their retention time, exact mass and HRMSThe liquid fungal culture was filtrated through Miracloth  to remove the mycelium. 1 L of this filtrate was fractionated on a Strata C18-E 55 μm, 70 Å, 10 g/60 mL solid phase extraction cartridge . First, the cartridge was activated by use of 150 mL methanol and 200 mL water. All steps were performed under vacuum at the column outlet. The culture filtrate was applied to the cartridge and to prevent plugging of the column 100 mL methanol/water  were applied in between to wash off salts and sugars and other polar compounds. After application of the whole amount of filtrate it was again washed with 200 mL of water. Afterwards five fractions with different amounts of methanol/water were collected (each 200 mL). The fractions were as follows: 20%, 40%, 60%, 80% and 100% methanol/water (v/v). An aliquot of the five fractions were diluted 1:10 with water and used for fraction control via HPLC-HRMS. Therefore, the same MS-parameters were used as described above. For further steps only the 60% and 80% fractions containing the relevant metabolites were used. The solvent of these fractions was removed using a rotary evaporator  at a temperature of 40°C under vacuum. Then the samples were dried using a freeze-drier LyoVac GT2  and resolved in 15 mL methanol/water . In the next step the fractions were further purified using preparative HPLC-UV. All laboratory work was carried out under reduced light conditions and every flask or tube was wrapped with aluminum foil. All samples or fractions were stored at -25°C.The biomass was suspended in 200 ml of water and cell disruption was carried out with an Ultra-Turrax  under cooling with ice. The rotor speed was 12 000 rpm. Afterwards the mycelium was extracted several times with a mixture of n-pentan and methyl-t-butyl ether. The organic solvents were evaporated, and the extract directly purified by preparative HPLC-UV.For the isolation and purification a Jasco HPLC-UV system  was used. The system consists of PU-2087 pumps (Jasco), a dynamic mixing chamber  and a degasys populaire DP 4010 degasser  and system controller LC-NetII/ADC (Jasco). Injection was performed manually via rheodyne model 7125 with a 2 mL sample loop. The detector was an UV-2075 UV/Vis detector (Jasco), the installed software was Jasco-Borwin, Version 1.5 (Jasco). The column used was a 250 mm x 10 mm, 5 μm Nucleodur phenyl-hexyl column  with a 4.0 mm x 2.0 mm phenyl-hexyl guarding column . Solvent A was methanol with 1% of formic acid (v/v) and solvent B was water with 1% of formic acid (v/v). The gradient was as follows: 10 min at 40% of A and afterwards a binary gradient to 80% of A during 40 min. Afterwards the column was washed with 100% A and equilibrated at starting conditions. The injection volume was 500 μL and the detection wavelength was set to 260 nm.m/z 1.5. The fragments were analyzed with the Orbitrap detector at a resolution of 60 000. The molecular composition was assigned on base of the accurate mass of the [M+H]+- and [M+Na]+ ions with a mass tolerance of 5.0 ppm or less.An aliquot of 20 μL of each isolate obtained after preparative HPLC was 1:10 diluted with methanol/water  and analyzed via RP-HPLC-HRMS. The column and solvent were the same as described above. The HPLC-parameters were as follows: 2 min with 20% of A isocratically. Then a binary gradient for 8 min to 100% of A. Afterwards the column was washed with 100% A and then equilibrated. The flow rate was set to 250 μL/min. To get information on the structure of the novel metabolites, the new substances were analyzed by stepwise mass spectrometric fragmentation experiments in the positive mode. The experiments included collision-induced dissociation (CID) with a relative energy of 20% to 48%, depending on the ionization of the ions, and an isolation width of The LTQ Orbitrap XL was used with a heated electrospray ionization technique. The sheat gas flow was 40 arbitrary units, the aux gas flow 20 arbitrary units and the sweep gas flow 5 arbitrary units. In the positive mode, vaporizer temperature was set to 350°C and capillary temperature to 285°C. The source voltage was 3.5 kV, capillary voltage 43 V and Tube Lens 145 V. In the negative mode, vaporizer temperature was set to 300°C and capillary temperature to 285°C. The source voltage was 3 kV, the capillary voltage -35 V and the Tube Lens -110 V.The Shimadzu PDA-Detector had the following parameters: starting wavelength 200 nm, ending wavelength 600 nm, with a wavelength step of 4 nm. The sampling frequency was consequently 4.16 Hz.The mass spectrometer used was an AB SCIEX QTRAP 5500  with ESI. The curtain gas was set to 40 psi, the collision activated dissociation gas to medium, GS1 to 30 psi, and GS2 to 45 psi. The source temperature was 450°C. The ion spray voltage was 5500 V in positive mode and 4500 V in negative mode. Entrance potentials of 10 V and -10 V were used in positive and negative modes, respectively. Unit resolution was applied and a neutral loss of 64 Da measured. The scan rate was set to 200 Da/s. The same chromatography as for the HRMS screenings was used.S1 FigTransformants with ectopic integration of the overexpression vector (A) were identified via PCR (B) using primer pair PoliC_F and Tgluc_R.(PDF)Click here for additional data file.S2 FigtcpZ and tcpN, the tcpN overexpression vector (A) was transformed into the OE::tcpZ transformant T2. Transformants with ectopic integration of the tcpN overexpression vector were identified via PCR (B) using primer pair PoliC_F and cp2672_R1 (5´), and Tgluc_R and cp2672_F1 (3´). Overexpression of tcpC as well as tcpN was verified by northern analysis (C).To generate double overexpression transformants of (PDF)Click here for additional data file.S3 FigtcpZ and sirC, the sirC overexpression vector (A) was transformed into the OE::tcpZ transformant T2. Overexpression of sirC was verified by northern analysis (B).To generate double overexpression transformants of (PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S5 Fig1H-NMR, (B) H,H-COSY, (C) HSQC.(A) (PDF)Click here for additional data file.S6 Fig1H-NMR, (B) H,H-COSY, (C) HSQC.(A) (PDF)Click here for additional data file.S7 Fig1H-NMR, (B) 13C-NMR, (C) H,H-COSY, (D) HSQC, (E) HMBC.(A) (PDF)Click here for additional data file.S8 Fig1H-NMR, (B) 13C-NMR, (C) H,H-COSY, (D) HSQC, (E) HMBC.(A) (PDF)Click here for additional data file.S9 Fig1H-NMR, (B) 13C-NMR, (C) H,H-COSY, (D) HSQC, (E) HMBC.(A) (PDF)Click here for additional data file.S10 Fig1H-NMR, (B) 13C-NMR, (C) H,H-COSY, (D) HSQC, (E) HMBC.(A) (PDF)Click here for additional data file.S11 Fig1H-NMR, (B) 13C-NMR, (C) H,H-COSY, (D) HSQC, (E) HMBC.(A) (PDF)Click here for additional data file.S12 Fig1H-NMR, (B) 13C-NMR, (C) H,H-COSY, (D) HSQC, (E) HMBC.(A) (PDF)Click here for additional data file.S13 Fig1H-NMR, (B) HMBC.(A) (PDF)Click here for additional data file.S14 Fig1H-NMR, (B), H-H COESY, (C) HSQC, (D) HMBC.(A) (PDF)Click here for additional data file.S15 Fig(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 TableThe structures are depicted in (PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.


Pest insects develop resistance to this class of insecticide by acquisition of one or multiple amino acid substitution(s) in this channel. In Southeast Asia, two major Vssc types confer pyrethroid resistance in the dengue mosquito vector Aedes aegypti, namely, S989P+V1016G and F1534C. We expressed several types of Vssc in Xenopus oocytes and examined the effect of amino acid substitutions in Vssc on pyrethroid susceptibilities. S989P+V1016G and F1534C haplotypes reduced the channel sensitivity to permethrin by 100- and 25-fold, respectively, while S989P+V1016G+F1534C triple mutations reduced the channel sensitivity to permethrin by 1100-fold. S989P+V1016G and F1534C haplotypes reduced the channel sensitivity to deltamethrin by 10- and 1-fold (no reduction), respectively, but S989P+V1016G+F1534C triple mutations reduced the channel sensitivity to deltamethrin by 90-fold. These results imply that pyrethroid insecticides are highly likely to lose their effectiveness against A. aegypti if such a Vssc haplotype emerges as the result of a single crossing-over event; thus, this may cause failure to control this key mosquito vector. Here, we strongly emphasize the importance of monitoring the occurrence of triple mutations in Vssc in the field population of A. aegypti.The voltage-sensitive sodium (Na + channel (Vssc) consisting of about 2100 amino acid residues. In this study we generated several types of Vssc with a single or multiple mutations, expressed in Xenopus oocytes, and examined their electrophysiological properties using two-electrode voltage clamp method. We confirmed that Aedes aegypti Vssc harboring a triple mutations exhibited extremely high levels of resistance to pyrethroid insecticides. This Vssc type can be generated by a single crossing-over event of two resistant Vssc genes that are widely distributed in Southeast Asia, one of the greatest dengue endemic areas. Our results highlight the importance of intensive monitoring for the triple mutations in Vssc in the A. aegypti mosquito.Pyrethroids are one of the major classes of insecticides that is widely used to control mosquito vectors. The target site of pyrethroids is found in the voltage-sensitive Na Aedes aegypti is the major mosquito vector of dengue fever (DF), yellow fever, and chikungunya fever. DF causes more illness and death in humans than any other arboviral disease A. aegypti+ channel (Vssc) Pyrethroid insecticides target the voltage-sensitive NaVssc haplotypes confer pyrethroid resistance to A. aegypti, i.e., S989P+V1016G and F1534C. A. aegypti with these two haplotypes are widely and sympatrically distributed in this area Aedes albopictusA. aegypti although heterozygous form of these two haplotypes were confirmed Vssc haplotypes. Moreover, the sensitivity of such a triple-mutated Vssc to pyrethroids has not yet been investigated.In Southeast Asia, one of the largest DF endemic areas, two major In this study, we analyzed the pyrethroid sensitivity of Vssc harboring triple substitutions, using a two-electrode voltage-clamp method, and we discussed the effect thereof on the control of the dengue mosquito vector by pyrethroids. We also investigated the synergistic effect of S989P on the sensitivity of Vssc harboring the V1016G mutation to pyrethroid insecticides.Xenopus frogs was conducted under benzocaine anesthesia and all efforts were made to minimize suffering.This study complied with the guidelines for animal experiments performed at the National Institute of Infectious Diseases, Japan. The protocols for the use of mice and frogs were approved by the Animal Use Committee of the National Institute of Infectious Diseases (registration number: 211031) and Nippon-soda Co. Ltd. (registration number: 046), respectively. Animal experiments were carried out in adherence to the Guidelines for Proper Conduct of Animal Experiments established by Science Council of Japan in 2006 and the Animals (Scientific Procedures) Act 1986 by the Parliament of the United Kingdom. All surgery on A. aegypti were used: SMK, a laboratory reference strain susceptible to pyrethroid insecticides, and SP, originally collected from Singapore in 2009, which has developed a high level of resistance to permethrin  after permethrin selection for 10 generations Two strains of In vitro metabolism and synergistic studies revealed that both reduced permethrin susceptibility and enhanced metabolic enzyme activity, via cytochrome P450 monooxygenases, confer the high level of resistance in strain SP Vssc homozygously Vssc cDNA in the SP strain, however, has not yet been sequenced.2, 1 mM MgCl2, 5 mM HEPES, pH 7.6) immediately prior to experiments. The DMSO concentration in the final solution was <0.1% and it did not affect the function of Na+ channels in the experiments (data not shown).Permethrin (purity>99.1%) and deltamethrin (purity>99.0%) were purchased from LGC Standards  and Wako Pure Chemical Industries, Ltd. , respectively. The stock solutions of insecticides (100 mM) were prepared in dimethyl sulfoxide (DMSO). Working solutions were prepared in standard oocyte saline  and reverse transcriptase ReverTra Ace  according to the manufacturer's instructions. The full-length cDNAs of A. aegypti Vssc were amplified by PCR using PrimeStar GXL DNA polymerase  and the primers aegSCF1 (5′-ATGACCGAAGACTCCGATTCGA-3′) and aegSCR2 (5′-TCAGACATCCGCCGATCGT-3′). Cycling conditions for PCR were as follows: 95°C for 2 min, followed by 35 cycles of each consisting of 98°C for 10 s, 60°C for 15 s, and 68°C for 6 min. Nested PCR was performed under the same conditions, but using the primers aegSCF3 (5′-ATGACCGAAGACTCCGATTCGATAT-3′) and aegSCR4 (5′-TCAGACATCCGCCGATCGTGAA-3′). PCR products were purified by agarose gel electrophoresis and inserted into the pTA2 plasmid using the TArget Clone -Plus system . The insert sequence was confirmed using the following primers: T7b (5′-CGTAATACGACTCACTATAGGGC-3′), aegSCF5 (5′-GTCGAATCTACCGAGGTGAT), aegSCF9 (5′-TGGTGTTACGATCAGCTGGA-3′), aegSCR8 (5′-CAGCCCTCTTGGAAGTAGTA-3′), AaSCF3 (5′-GTGGAACTTCACCGACTTCA-3′), aegSCF7 (5′-GAGGCGTTCAATCGGATATC-3′), AaSCR21 (5′-GCAATCTGGCTTGTTAACTTG-3′), aegSCF10 (5′-CGTTTCAGCTCATCGAGAAC-3′), AaSCR8 (5′-TAGCTTTCAGCGGCTTCTTC-3′), AlSCR8 (5′-AACAGCAGGATCATGCTCTG-3′), aegSCR6 (5′-TGCCTGAACTTCACCCAGTT-3′), and T3b (5′-CGCAATTAACCCTCACTAAAGGGA-3′). Eight and six clones of full-length Vssc cDNAs from the SMK and the SP strains, respectively, were sequenced. Nucleotide polymorphisms that were confirmed only in a single clone were considered to reflect polymerase errors.Total RNA was extracted from 10 three-day-old females using ISOGEN . Genomic DNA was removed by digesting the total RNA samples with DNase I using TURBO DNase . cDNA was synthesized using RNA and QT' primer , which was isolated from insecticide susceptible SMK strain as described below. The forward primer aegSCF13 (CGGAATTCATGACCGAAGACTCCGATTCGATAT-3′5′-) and reverse primer aegSCR14 (GAAGGCCTTCAGACATCCGCCGATCGTGAA-3′5′-) included EcoRI and StuI restriction sites (underlined), respectively, to facilitate cloning. Full-length cDNAs encoding the substitutions V1016G (vR6AaNa), F1534C (vR7AaNa), S989P (vR8AaNa), S989P+V1016G (vR9AaNa), and S989P+V1016G+F1534C (vR10AaNa) in Vssc were amplified, as shown in vS2AaNa (wild-type), each fragment containing the smallest number of polymorphisms were subjected to oligonucleotide-mediated mutagenesis following a standard protocol 5′-GGAAATCTAGTAGGACTTAACCTTTTCTTAGC-3′), aegSCR16 (5′-GCTAAGAAAAGGTTAAGTCCTACTAGATTTCC-3′), aegSCF17 (5′-CTTCATCATCTGCGGGTCGTTCTT-3′), aegSCR18 (5′-AAGAACGACCCGCAGATGATGAAG-3′), aegSCF19 (5′-AGTGGATCGAACCCATGTGGGATT-3′), and aegSCR20 (5′-AATCCCACATGGGTTCGATCCACT-3′). Amplified PCR products were digested with restriction enzymes and cloned into the pTS1 vector . Clones were fully sequenced and each amino acid substitution was confirmed. Drosophila melanogaster temperature-induced paralytic E (tipE) cDNA was also amplified and cloned into pcDNA3.1(+)  using published primer sequences Drosophila tipE with Vssc of other insects has been well documented Site-directed mutagenesis was performed by PCR using PrimeStar GXL DNA polymerase and primers that were designed based on the 5′ and 3′ end sequences of in vitro transcription were PCR-amplified from plasmids using KOD plus Neo (Toyobo) and the primers pTS1_cRNA_F (5′-GCGCATAATACGACTCACTATAGGGAG-3′) and pTS1_cRNA_R (5′-AGCTGTGCGCGCAAATTAACCCTC-3′). Capped RNA transcripts were synthesized using the mMessage mMachine T7 Ultra Kit . The quantity and quality of cRNAs were verified by agarose gel electrophoresis and absorption spectroscopy, and cRNAs were stored at −80°C until required for use.Templates for Xenopus laevis were purchased from Hamamatsu Seibutsukyozai  and maintained in dechlorinated water at 18°C. Oocytes were surgically obtained following anesthesia of a Xenopus frog using 0.03% benzocaine , and enzymatically defolliculated by incubating them for 1–1.5 h in a Ca2+-free SOS containing 1 mg/ml collagenase . Vssc cRNA and tipE cRNA (0.9 ng each) were co-injected into healthy stage V–VI oocytes, which were incubated at 18°C in SOS medium supplemented with 50 µg/ml gentamycin, 100 U/ml penicillin, 100 µg/ml streptomycin, 2.5 mM Na pyruvate, and 4% horse serum, for 2–4 days after injection. The medium was replaced daily and unhealthy oocytes were discarded. It is generally recognized that the knockdown resistance is a recessive trait Xenopus oocytes, theoretically we reproduced the environment of the homozygous Vssc form in the cell.Female + currents were recorded using a TEV-200A  amplifier and digitized at 40 kHz with PowerLab 8/30 . Signals were filtered at 2 kHz using a Bessel four-pole filter . The P/N method was used to subtract capacitive transient currents Na+-current recordings were performed using standard two-electrode voltage clamping, as described by Du et al. + currents were elicited by a 20-ms test pulse from a holding potential of −120 mV to −20 mV. The peak current ensuing from 20-ms step depolarization, in response to a test potential (V) in the range of −80 mV to +40 mV, in 5-mV increments, from a holding potential, at 10-s intervals was measured. The voltage dependence of activation was then calculated by dividing by (V – Vrev), where Vrev is the reversal potential as determined from the current-voltage curves. Peak conductance values were plotted against V and fitted with the Boltzmann equation to obtain the half-maximal activation voltage.Methods for NaThe voltage dependence of Vssc inactivation was determined using a 200-ms pre-pulse in the range of −100 mV to 0 mV, in 5 mV increments, followed by a 20-ms test pulse at −20 mV. Data were plotted as normalized current vs. inactivation pre-pulse voltage, and fitted with the Boltzmann equation to obtain the half-maximal inactivation voltage.25).Application of permethrin and deltamethrin were conducted using the disposable perfusion system 1/2) were compared statistically between wild-type and mutant channels using one-way ANOVA followed by Dunnett's multiple comparison test. The decays of tail currents were fitted with single functions to determine time constants . Statistical comparisons (one-way ANOVA followed by Dunnett's multiple comparison test) were made for time constants of the decays between the wild-type and the mutant channels at P<0.05. Tukey-Kramer HSD (Honestly significant difference) procedure was used to test for the differences of % modified channels among Vssc haplotypes after the treatment of insecticides.The recorded data were analyzed using GraphPad Prism 5.04 software . Voltage dependences (both activation and inactivation of VVssc cDNA obtained from a pyrethroid-susceptible SMK strain were sequenced. The Vssc cDNA (six clones) of SP was also sequenced to determine if S989P and V1016G were the only substitutions present in Vssc in this strain. The SMK strain harbored two polymorphisms that both generated a substitution at amino acid 427 (R or K). K427 was not confirmed in the Vssc of strain SP. It is known that Vsscs have two mutually exclusive alternative exons (c and d) vS2 , which contains R427 and exon c, as does the wild-type Vssc. We also confirmed that S989P and V1016G are the only amino acid substitutions present in the full-length Vssc cDNA in the SP strain.Eight clones of full-length A. aegypti Vssc (AaNavS2) and Drosophila tipE, which is required for robust expression of insect Vssc, were co-expressed in Xenopus oocytes. No peak current was detected in oocytes that had not been injected with cRNA (data not shown). A peak current of approximately 0.5–3 µA was detected on days 2–4 in most oocytes that had been co-injected with wild-type vS2AaNa and tipE cRNA. A representative trace is shown in + currents.The wild-type + currents through AaNavS2 were detected at approximately −40 mV, and peaked at −15 mV , alone or in combination, on the voltage dependence of activation, steady-state inactivation, and reversal potential were evaluated. Na+ currents recorded in oocytes expressing mutant channels exhibited voltage-dependent activation and inactivation, similar to those expressing the wild-type AaNavS2 (vR9 (S989P+V1016G) and AaNavR10 (S989P+V1016G+F1534C) mutants were slightly shifted, from +35 mV in the wild-type (vR6 (V1016G), AaNavR7 (F1534C), and AaNavR8 (S989P) significantly (P<0.05 by one-way ANOVA followed by Dunnett's multiple comparison test) shifted the voltage-dependence of activation in the depolarizing direction , or in combination with V1016G (AaNavR9) (vR10), exhibited 1100-fold more resistance, which represents an 11-fold reduction (1100/100) and a 44-fold (1100/25) reduction in Vssc sensitivity to permethrin as compared to Vssc harboring S989P+V1016G or F1534C, respectively. This suggested that these substitutions acted synergistically (The S989P mutation had no effect on permethrin sensitivity on its own (AaNaAaNavR9) . The V10AaNavR9) . The comstically .vR10 (S989P+V1016G+F1534C) had a lower amplitude and recovered to baseline more rapidly than did the wild-type  decayed very quickly (vR10 (S989P+V1016G+F1534C) because the tail current tended to keep recovering to baseline. The deltamethrin-induced tail current of AaNavR10, however, clearly decayed faster than that of the wild-type.Deltamethrin-induced tail currents initially increased, and then gradually recovered in the wild-type channel . Howeverild-type . As comp quickly . We coulvR8) had no effect on deltamethrin sensitivity, but the combination of S989P and V1016G (AaNavR9) reduced deltamethrin-sensitivity of Vssc by 10-fold (vR6 and AaNavR9 at deltamethrin concentration of 1.0 µM (P = 0.0091 by Tukey-Kramer test). Furthermore, S989P+V1016G+F1534C (AaNavR10) exhibited a 90-fold greater resistance than wild-type. This represents a 9-fold (90/10) and 90-fold (90/1) greater reduction in sensitivity than that induced by S989P+V1016G and F1534C, respectively, indicating a strong synergistic effect of the triple substitutions (vR9 and AaNavR10 at deltamethrin concentration of 10 µM (P = 0.034).V1016G reduced the sensitivity of Vssc to deltamethrin by 2-fold, while F1534C alone exerted no effect on Vssc sensitivity . S989P a 10-fold , which witutions . The perA. aegypti distributed in Southeast Asia, including Thailand A. aegypti if such a new type of Vssc emerges in the field population of this mosquito species. Therefore, we strongly emphasize the importance of surveying of the triple mutations in Vssc in the field population of this key mosquito vector. Early detection of this unique haplotype may assist in vector control strategies by providing early warning of insecticide insensitivity.To date, the V1016G mutation  of Vssc has been confirmed only in vR10 (S989P+V1016G+F1534C) shifted slightly, although there were no statistically significant differences in the voltage dependence of activation or in the voltage dependence of steady-state inactivation between AaNavR10 and the wild-type AaNavS2. In North and South America, where no V1016G mutation has been confirmed in the Vssc of A. aegypti to date, V1016I is widely distributed Vssc gene with this mutation Vssc haplotypes, but the pyrethroid-susceptibility of Vssc harboring V1016G+F1534C or S989P+F1534C double mutations may also elucidate the synergistic effects of these mutations.The likelihood of occurrence of such a Vssc with triple mutations may depend on its fitness cost. In this study, we observed that the reversal potential of AaNa+ currents of each Vssc at five concentrations of deltamethrin, with at least 4 independent replications, and used EC25 values as a measure of sensitivity. The percentages of modified channels were significantly different between AaNavR6 (V1016G) and AaNavR9 (S989P+V1016G) at deltamethrin concentration of 1.0 µM (P = 0.0091 by Tukey-Kramer test; A. aegypti collected especially in Southeast Asia.Our results exhibited that S989P has a synergistic effect on the sensitivity of V1016G-harboring Vssc to deltamethrin, but not to permethrin . This re


Kr) current in the heart. The N-terminal ‘eag’ domain, which is composed of a Per-Arnt-Sim (PAS) domain and a short PAS-cap region, is a critical regulator of hERG channel function. In previous studies, we showed that isolated eag (i-eag) domains rescued the dysfunction of long QT type-2 associated mutant hERG R56Q channels, by substituting for defective eag domains, when the channels were expressed in Xenopus oocytes or HEK 293 cells.Here, our goal was to determine whether the rescue of hERG R56Q channels by i-eag domains could be translated into the environment of cardiac myocytes. We expressed hERG R56Q channels in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and measured electrical properties of the cells with whole-cell patch-clamp recordings. We found that, like in non-myocyte cells, hERG R56Q had defective, fast closing (deactivation) kinetics when expressed in hiPSC-CMs. We report here that i-eag domains slowed the deactivation kinetics of hERG R56Q channels in hiPSC-CMs. hERG R56Q channels prolonged the AP of hiPSCs, and the AP was shortened by co-expression of i-eag domains and hERG R56Q channels. We measured robust Förster Resonance Energy Transfer (FRET) between i-eag domains tagged with Cyan fluorescent protein (CFP) and hERG R56Q channels tagged with Citrine fluorescent proteins (Citrine), indicating their close proximity at the cell membrane in live iPSC-CMs. Together, functional regulation and FRET spectroscopy measurements indicated that i-eag domains interacted directly with hERG R56Q channels in hiPSC-CMs. These results mean that the regulatory role of i-eag domains is conserved in the cellular environment of human cardiomyocytes, indicating that i-eag domains may be useful as a biological therapeutic.Human Ether á go-go Related Gene potassium channels form the rapid component of the delayed-rectifier (I Like other Kv channels, hERG channels have six transmembrane domains and intracellular N- and C-terminal regions}, where V1/2 is the half-maximal activation potential and k is the slope factor. Steady-state inactivation was measured using a three-pulse protocol, as previously described[Icorrected = (gdifference)(20-Erev); gdiference = (Ipeak—Iend)/(Emem-Erev), where Ipeak is the peak current during the 15 ms conditioning pulse, and Iend is the current at 15 ms. The resulting values were normalized, plotted versus voltage, and fit with a Boltzmann function. Deactivation current was fit with a double exponential function (y = A1et/τ1- + A2et/τ2-), where t is time and τ is the time constant of deactivation. Current deactivation was fit with a double exponential function (y = A1et/τ1- + A2et/τ2-), where t is time and τ is the time constant of deactivation.HEK293 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% penicillin and 1% streptomycin at 37°C and 5% COdescribed. Errors 2. After 48 hours, iCPM was replaced with cell culture media , which was exchanged every 48 hrs. Cardiomyocytes were maintained on coverslips for 4 to 21 days prior to use[Human iPSC-CMs (iCell Cardiomyocytes) were purchased from Cellular Dynamics International . For single cell patch-clamp recordings, glass coverslips were coated with 0.1% gelatin, placed into each well of a 12-well plates, and 2 mL of iCell Cardiomoycytes Plating Medium (iCPM) containing 40,000~60,000 cardiomyocytes were added to each coverslip. Cardiomyocytes at a low density were plated to permit culture of single cells and stored in an incubator maintained at 37°C and 5% COor to use.2, 1 MgCl2, 10 Glucose, and 10 HEPES (pH to 7.4 with NaOH), and the internal solution contained (in mM): 120 KCl, 1 MgCl2, 10 EGTA, 3 MgATP, and 10 HEPES (pH to 7.2 with KOH).hERG currents and action potentials (APs) were recorded from single cardiomyocytes with perforated whole-cell patch-clamp techniques (using 240 μg/mL amphotericin B) at room temperature. For AP recordings with heterologous expression of WT hERG1a and hERG1a R56Q, cells were infected with the identical amount of adenovirus, cultured for the same period of time (24hrs) and AP recordings were performed on the following day and interleaved, as a control. For iPSC-CMs, APs were evoked by a current injection. The external bath solution contained (in mM): 140 NaCl, 5.4 KCl, 1.8 CaCltotal) free of donor contamination. F436total contained two components—one due to direct excitation of Citrine at 436nm (F436direct) and one due to FRET (F436FRET). The F436total spectrum was normalized to the Citrine emission spectrum with excitation at 500nm (F500) and was termed Ratio A: Ratio A = F436total/F500 = (F436direct/F500) + (F436FRET/F500). To solve for the F436FRET, the ratio of F436direct/F500, termed Ratio A0, was calculated from cells expressing acceptor (hERG-Citrine) only. We calculated the difference between Ratio A and Ratio A0 (from 530 to 560 nm) as Ratio A—Ratio A0 = F436FRET/F500, which is directly proportional to FRET efficiency.To explore a direct interaction between adenoviral hERG R56Q and i-eag domains, we used Förster resonance energy transfer (FRET) spectroscopy. FRET spectroscopy directly tests protein-protein proximity when the distance between proteins is less than approximately 80Å. The FRET donor was i-eag fused to monomeric, enhanced CFP, and the FRET acceptor was hERG R56Q fused to Citrine. We measured FRET as stimulated emission of the acceptor by the donor (CFP). CFP and Citrine are FRET pairs and following excitation of the donor (CFP), energy is transferred from the donor to the acceptor. For both cell and spectroscopic imaging, two filter cubes (Chroma) we used a YFP cube and a custom CFP cube with a long-pass emission filter, as previously described. Two spet-test and one-way ANOVA. p<0.05 was considered as significant.The average data were expressed as mean ± S.E.M.. Statistical analysis was performed using unpaired Student’s In this study, our goal was to test whether the genetically encoded, isolated eag (i-eag) domains could rescue the function of a hERG LQTS mutant channel in the environment of cardiac myocytes. We chose to use hiPSC-CMs because they are human cells, can be cultured for several weeks  and have some properties of cardiomyocytes, such as APs. We found that transfection of hERG channels in mammalian expression vectors (e.g. pcDNA3) was not effective for hiPSC-CMs, so to carry out these experiments, we first performed a positive control by testing the expression of adenovirally delivered wild-type hERG, hERG(R56Q) and hERG(R56Q) + i-eag domains  in HEK291/2 of -13.6 mV compared with -32.2 mV for WT hERG (p<0.05). Coexpression with adenoviral i-eag domains restored the steady-state inactivation curve .Ad and hERG(R56Q).Ad + i-eag.Ad in hiPSC-CMs and measured robust currents using the perforated whole-cell patch-clamp method Fig  and 4C. We characterized the altered deactivation of hERG R56Q channels and examined the rescue of adenoviral i-eag domains in more detail, using a voltage command protocol to elicit a series of deactivating traces. Compared with that of hERG.Ad, hERG(R56Q).Ad channels exhibited accelerated deactivation kinetics Fig  and 5E. + current were from heterologously expressed hERG channels. The I-V relationship shows less rectification for hERG R56Q channels than for WT hERG channels and rectification was restored when hERG R56Q was co-expressed with i-eag domains  proximity to one another. We expressed hERG(R56Q).Citrine alone Fig  and 7C aWe next examined the effects of hERG channels on action potentials of hiPSC-CMs . CompareXenopus oocytes or HEK293 cells. The faster deactivation kinetics and I-V relationship and the peak current amplitude and timing of hERG R56Q could be restored to levels similar to that of WT hERG by co-expresssion with i-eag domains.The primary goal of these studies was to determine whether i-eag domains could associate with and regulate hERG R56Q channels in the cellular environment of cardiomyocytes. We report here that hERG R56Q channels heterologously expressed in hiPSC-CMs had faster deactivation kinetics and a less-rectifying I-V relationship and a larger and earlier peak current in response to an AP waveform than that of WT hERG channels, similar to the properties of hERG R56Q expressed in Xenopus oocytes and HEK293 cells[We detected FRET between hERG R56Q channels which were tagged with Citrine and i-eag domains which were tagged with CFP, which suggests a direct interaction between these two proteins. Based on the functional restoration of deactivation gating and FRET between i-eag domains and hERG R56Q channels, we propose that the regulatory effect of i-eag domains is due to a direct association with hERG R56Q channels. In a model we previously proposed to explain the rescue of gating properties by i-eag domains, hERG R5293 cells,22, i-eaWe assessed the role of eag domains on the excitable properties of hERG R56Q in hiPSC-CMs. We first asked whether cells expressing hERG R56Q channels had a change in the APD. Our result (a prolonged AP in cells with hERG R56Q) is opposite to what might be expected due to the similar peak tail current and larger peak current measured in the I-V relationship for hERG R56Q compared to that of WT hERG . RecordiWe found that heterologous expression of i-eag domains with hERG1a R56Q increased the outward current relative to the tail current, decreased the decay of the repolarizing current either with a square Figs , 5 and 6S1 FigA, Tail currents at -50 mV from versus command voltage. B, Currents at the end of each depolarizing pulse were normalized to cell capacitance (pF) and plotted versus command voltage.(TIF)Click here for additional data file.


RET proto-oncogene, and a phenotypic variability among carriers of the same mutation has been reported. We recently observed this phenomenon in a large familial MTC (FMTC) family carrying the RET-S891A mutation. Among genetic modifiers affecting RET-driven MTC, a role has been hypothesized for RET-G691S non-synonymous polymorphism, though the issue remains controversial. Aim of this study was to define the in vitro contribution of RET-G691S to the oncogenic potential of the RET-S891A, previously shown to harbour low transforming activity.Hereditary medullary thyroid carcinoma (MTC) is caused by germ-line gain of function mutations in the RET-S891A and RET-G691S/S891A mutants were generated by site-directed mutagenesis, transiently transfected in HEK293T cells and stably expressed in NIH3T3 cells. Their oncogenic potential was defined by assessing the migration ability by wound healing assay and the anchorage-independent growth by soft agar assay in NIH3T3 cells stably expressing either the single or the double mutants. Two RET-S891A families were characterised for the presence of RET-G691S.The RET-G691S/S891A double mutant displays a higher oncogenic potential than RET-S891A single mutant, assessed by focus formation and migration ability. Moreover, among the 25 RET-S891A carriers, a trend towards an earlier age of diagnosis was found in the MTC patients harboring RET-S891A in association with RET-G691S.The functional studies demonstrated that RET-G691S non-synonymous polymorphism enhances in vitro the oncogenic activity of RET-S891A. Moreover, an effect on the phenotype was observed in the RET-G691S/S891A patients, thus suggesting that the analysis of this polymorphism could contribute to the decision on the more appropriate clinical and follow-up management.We demonstrate that the The online version of this article (doi:10.1186/s13023-015-0231-z) contains supplementary material, which is available to authorized users. RET) proto-oncogene. The RET gene, which encodes a tyrosine kinase receptor with a crucial role in development, comprises 21 exons and generates a transcript subjected to alternative splicing leading to two main isoforms: a protein of 1114 residues displaying 51 C-terminal-specific amino acids (RET51) and a shorter protein of 1072 residues displaying nine unrelated C-terminal-specific amino acids (RET9) )/Start wound × 100. Data and graphs were analyzed using GraphPad Prism 5.02.NIH3T3 cells stably expressing RET mutants were suspended in DMEM containing 0.33% agar, 10% serum and GDFN 10 ng/ml (50000 cells/1.5 ml medium) and added into a layer of medium containing 0.5% agar and 10% serum in 60 mm dishes. After 3 weeks incubation, plates were analyzed for colonies number and size. Colony number was determined in plates stained with p-iodonitrotetrazolium chloride violet (Sigma Aldrich).For anchorage-independent growth assay, NIH3T3 cells stably expressing Agar colonies size, calculated as mean diameter, was determined by the count/size tool in Image-Pro Plus 7.0.1 software analyzing the agar colonies images the day before staining. A cutoff area of 250 pixel was manually set on NIH3T3 control cells. Only foci surpassing the cut-off area were automatically selected and scored by the software. Diameter data relative to the total foci analyzed per each field were exported and the mean diameter for each sample was calculated.RET-G691S polymorphism expression/penetrance was assessed in two FMTC families carrying the germline activating mutation RET-S891A. Family 1 is an extremely large family with members distributed in several cities of the North of Italy, with a common ancestor [RET gene carriers were screened by means of yearly basal and stimulated CT measurement and neck ultrasound. Prophylactic surgery was performed at the first evidence of a positive provocative test.ancestor . Data reancestor . Family ancestor  and the ancestor ,28; in pRET-G691S polymorphism was assessed by PCR and direct sequencing as previously described [Genomic DNA was extracted from peripheral blood leucocytes of FMTC families members and the presence of escribed . All patStatistical analysis and graphs were generated using GraphPad Prism version 5.02. Comparisons between two groups were performed by two-tailed Student’s t-test with unequal variance. A value of P < 0.05 was considered statistically significant.RET-G691S polymorphism to the in vitro oncogenic potential of RET-S891A mutant, both localized in the intracellular domains of RET protein  and the empty vector were transiently transfected into HEK293T cells and the RET proteins and the associated downstream signaling on ERK1/2 were analyzed  and RET-M918T mutant (positive control), were stably transfected in NIH3T3 cells and the transforming activity was assessed , suggesting for this polymorphism a role of transforming potential enhancer. This is also suggested by the cells morphology  and low levels of RET (691/891 clone 1 vs. 891 clone 4).To better characterize the tumorigenic properties of y Figure . The wouy Figure A. Data iRET-G691S/S891A double mutants give rise to a statistically significant higher number of agar colonies than RET-S891A single mutants . Among the 39 RET-S891A carriers, 14 also harbored the RET-G691S polymorphism either in heterozygosis (n = 13) or in homozygosis (n = 1), showing an overall allele frequency of 19% . Thus, in the present series, the prevalence of the co-segregation RET891/RET691 was similar to that reported in general population by the publicly available ARUP database [RET-G691S appears to display a trans presentation with RET-S891A in Family 1, whereas a cis presentation is observed in Family 2 , though no lesion suspicious for pheochromocytoma (PHEO) have been yet identified using specific imaging screening . Finally, no significant differences were noted in the outcome of patients with or without RET-G691S, likely as a consequence of the bias introduced by prophylactic thyroidectomy in gene carriers.Interestingly, even though not statistically significant, a trend towards an earlier age at diagnosis was found in MTC patients harboring the RET-G691S, not oncogenic per se, enhances the in vitro oncogenic potential of the RET-S891A, a mutant causing hereditary MTC. Furthermore, we showed in two FMTC families that carriers of RET-S891A mutation associated with the RET-G691S polymorphic variant display a trend towards an earlier age at diagnosis of MTC and the adrenal medulla involvement.In this study we demonstrated for the first time that the functional polymorphic variant RET mutations in hereditary MTC are a paradigmatic example of clinical decision based on molecular diagnosis. Nevertheless, even within families and patients with the same RET mutation, the age of disease onset and the phenotypes are unpredictable, suggesting the existence of modifier loci influencing the expression and severity of the disease whose identification could drive the diagnostic and therapeutic management. Accordingly, though RET-S891A mutation has been associated to isolated FMTC for several years after its first identification in 1997 [RET-S891A mutation [ in 1997 , a MEN2 mutation .RET gene polymorphisms have been associated with susceptibility and/or disease progression [RET-G691S. Although debated, RET-G691S has been suggested to have a role in MTC susceptibility and in the modulation of the age of onset [RET-G691S, not oncogenic per se, enhances in vitro the oncogenic potential of the rare germline mutation RET-K666E [RET 666 codon [Among the potential genetic modifiers, gression -5, and tof onset -9,13,31.ET-K666E , accordi66 codon .RET-G691S in the modulation of the oncogenic capacity and phenotype expression of RET mutants, we focused on the RET-S891A. This intermediated risk mutation displays in vitro low transforming activity [RET mutations localized in the tyrosine kinase domain, such as RET-M918T causally related to MEN2B [RET-S891A mutation the association with RET-G691S polymorphism correlates in vitro with enhanced tumorigenic properties such as transforming activity and migratory and clonogenic ability. These findings, indicating a possible role of genetic modifier for this polymorphism, are concordant with the expression/penetrance pattern of the two analyzed RET-S891A FMTC families, that include members harboring the RET-S891A mutation associated or not with the RET-G691S polymorphism. Though not statistically significant, an earlier age at diagnosis of MTC was observed in RET-G691S/S891A carriers compared with RET-S891A carriers, suggesting a possible role in the modulation of the age at disease onset.In the present work, to get more insights and to confirm the possible role of activity  that mayto MEN2B . In agrein vitro studies, mimicking the cis presentation of RET-S891A mutation and RET-G691S polymorphism observed in the Family 2, demonstrate the oncogenic enhancer role of the polymorphism in the case of cis presentation. Moreover, the clinical data from both families suggest a trend towards an earlier age at diagnosis of MTC in the RET-G691S/S891A carriers irrespectively of the cis or trans presentation of the RET-S891A mutation and the RET-G691S polymorphism (Additional file RET-G691S polymorphism may act as genetic modifier also in the case of trans presentation with RET-S891A mutation. However, further studies are necessary to demonstrate that RET-G691S polymorphism may enhance the oncogenic activity of RET-S891A, or of other RET mutants, also in trans.Our RET-G691/S891A patients had elevated normetanephrine levels, suggesting that the full expression of MEN2A phenotype could be related to the association with this non synonymous polymporphism, especially in the case of cis presentation with RET-S891A mutation.Moreover, clinical data from Family 2 indicate that two out of three RET-G691S polymorphism has been shown in vitro to enhance the oncogenic potential of the RET-S891A mutation and in vivo to modify the clinical expression of the disease.In conclusion, RET-G691S in other series with RET-S891A or other RET mutations is desirable to confirm the role of this polymorphism in the modulation of the phenotype, particularly in intermediate risk forms. If confirmed, present data suggest that the evaluation of RET-G691S in RET carriers should be always recommended in order to select the more appropriate clinical and follow-up management.The genetic analysis of


While naive CD8+ T-cell numbers hardly changed during follow-up, naive CD4+ T-cell counts continually declined. We show that a fine balance between increased T-cell division and loss in the peripheral naive T-cell pool can explain the observed short- and long-term changes in TRECs and naive T-cell numbers, especially if T-cell turnover during the acute phase is more increased than during the chronic phase of infection. Loss of thymic output, on the other hand, does not help to explain the biphasic loss of TRECs in HIV infection. The observed longitudinal changes in TRECs and naive T-cell numbers in HIV-infected individuals are most likely explained by a tight balance between increased T-cell division and death, suggesting that these changes are intrinsically linked in HIV infection.Naive T cells in untreated HIV-1 infected individuals have a reduced T-cell receptor excision circle (TREC) content. Previous mathematical models have suggested that this is due to increased naive T-cell division. It remains unclear, however, how reduced naive TREC contents can be reconciled with a gradual loss of naive T cells in HIV-1 infection. We performed longitudinal analyses in humans before and after HIV-1 seroconversion, and used a mathematical model to investigate which processes could explain the observed changes in naive T-cell numbers and TRECs during untreated HIV-1 disease progression. Both CD4 The CD8+ T-cell pool expands during the acute stage of infection and starts to decline at the AIDS stage [+ T-cell pool is severely reduced throughout HIV infection [+ and CD8+ T-cell pools are still debated.Both CD4nfection : in the are lost ,3 while nfection –7. The cHIV infection of the thymus, and a resulting decline in thymic output, has been suggested to contribute to the gradual loss of naive T cells in HIV infection –10. In t+ and CD8+ T-cell pools during HIV infection are thus not trivially explained. Similarly, it remains unclear to what extent loss of thymic output can explain the changes in the T-cell pool during HIV infection, because naive T cells are very long-lived, with an average lifespan of 6–9 years in healthy individuals [in vivo deuterium labeling study among treatment-naive HIV-1 infected individuals revealed that during chronic HIV-1 infection, naive T-cell production and loss rates are at least 3-fold increased, yielding life-expectancies of 1.7 and 0.7 years for CD4+ and CD8+ naive T cells, respectively [Although increased naive T-cell division is indeed expected to lead to a reduction in the average TREC content, it is not clear how it can be reconciled with declining naive T-cell numbers. The increased naive T-cell loss that probably counteracts the effect of increased T-cell division on the size of the naive T-cell pool in HIV infection, is in fact expected to increase the average TREC content through "rejuvenation" of the T-cell pool ,14, therividuals , and thyividuals . Our recectively . With supercentage of naive cells in the CD4+ and CD8+ T-cell pools during HIV infection [+ and CD8+ T-cell counts during disease progression and over seroconversion. By studying TRECs and naive T-cell numbers longitudinally we circumvented the problems intrinsic to cross-sectional studies that are hampered by the large inter-individual differences in TREC levels and naive T-cell numbers. Indeed, a considerable overlap between healthy and HIV-infected individuals has repeatedly been reported [+ and CD8+ TREC contents decrease biphasically, with a rapid decline during the first year and a much slower decline during the chronic phase of infection. Naive CD4+ T-cell numbers declined throughout infection, with no sign of a biphasic pattern, while naive CD8+ T-cell numbers remained stable during the 5-year follow-up. Using a mathematical model, we show that a combination of increased naive T-cell division and loss suffices to explain the biphasic TREC loss and the observed naive T-cell changes during HIV-1 disease progression, but only if the changes in T-cell division and loss are tightly linked. Thymic impairment, on the other hand, does not help to explain the observed biphasic TREC loss.Here, we collected longitudinal data on naive T-cell numbers and TRECs over HIV-1 seroconversion and during the first five years of untreated HIV-1 infection, and used a mathematical model to study which factor(s) can explain their changes. In contrast to most previous studies, which followed the nfection –7, we alreported ,13,18,19Samples from HIV-infected individuals for the longitudinal study (n = 18) were derived from the Amsterdam Cohort Studies (ACS) on HIV infection and AIDS. Cross-sectional data were derived from the ACS and from previous studies ,20,21. N+ and CD8+ T-cell counts were determined by dual-platform flow cytometry. Effector/memory  and naive (CD27+CD45RA+) CD4+ and CD8+ T-cell fractions were assessed by flow cytometry as described previously and analyzed on a FACSCalibur with CellQuest software (Becton Dickinson (BD), San Jose, California) [+CD4+ T cells were positively selected by magnetic beads. Because CD45RA+CD27- effector CD4+ T cells are virtually absent in healthy individuals (≤1.5% in 10 controls in whom we used magnetic beads and were able to measure the effector subset), this fraction represents CD45RA+CD27+ naive CD4+ T cells. To measure the TREC content within CD4+ and CD8+ T cells, these subsets were purified from thawed PBMC by positive selection using magnetic beads according to manufacturer’s instructions .Peripheral blood mononuclear cells (PBMC) were obtained by Ficoll-Paque density gradient centrifugation from heparinized blood and viably frozen until further processed. Absolute CD4ifornia) . In 14 oDNA was isolated using the QIAamp Blood Kit according to manufacturer’s instructions . Signal joint (Sj) T-cell receptor excision circle (TREC) numbers were quantified using a real-time PCR method as previously described .+ T cells N and the total amount of TRECs T in the naive T-cell population, as a function of the time-dependent source of naive T cells from the thymus σ(t), the average number of TRECs per recent thymic emigrant c, the rate d of naive T-cell loss through cell death and priming of naive into memory cells, and the rate of peripheral naive T-cell division p. More details about the model and the parameters are given in Supplemental Materials  before seroconversion. Since the expected TREC content decline in the period until seroconversion is negligible  and fractions of naive and effector/memory CD4+ and CD8+ T cells were determined.We performed longitudinal analyses of the average TREC content of CD4ividuals ), we use+ and CD8+ T-cell TREC contents revealed a biphasic decline during HIV-disease progression , differed significantly from those of 38 healthy age-matched controls (−4), underlining the importance of longitudinal follow-up in TREC analyses. In contrast, TREC changes in the CD8+ T-cell pool could also be observed cross-sectionally . For CD8+ T cells, we found that naive percentages decreased significantly during phase I  HIV-1 [+ T cells and stable naive CD8+ T-cell numbers during HIV-1 infection.Our recent deuterium labeling studies have pointed out that naive and memory CD4d) HIV-1 . Here, wσ(t) and by peripheral T-cell division at a rate p and are lost at a rate d  increasing the naive T-cell loss rates d in accordance with the turnover estimates of our deuterium labeling studies among chronic HIV-1 infected individuals [+ and CD8+ T cells from the combination of d and the observed net loss of these cells in HIV infection + T-cell numbers during the first 5 years of HIV infection, the observed increase in the naive CD8+ T-cell loss rate [+ T-cell division rate . Assuming an increased value of c in HIV-infected individuals would yield even more restrictive constraints on the parameters  and it has been suggested that this population may be the one that is quickly lost after HIV infection, thereby explaining both changes in the TREC content and naive T-cell declines. However, our ividuals ,17 have ividuals  and therS1 Fig(PDF)Click here for additional data file.S2 Fig(PDF)Click here for additional data file.S3 Fig(PDF)Click here for additional data file.S4 Fig(PDF)Click here for additional data file.S1 File(PDF)Click here for additional data file.


Taken together, our study provides a guideline for rational design the promoter-targeting siRNA to sequence-specifically enhance gene expression.Small interfering RNAs (siRNAs) are widely used to repress gene expression by targeting mRNAs. Some reports reveal that siRNAs can also activate or inhibit gene expression through targeting the gene promoters. Our group has found that microRNAs (miRNAs) could activate gene transcription via interaction with the TATA-box motif in gene promoters. To investigate whether siRNA targeting the same region could upregulate the promoter activity, we test the activating efficiency of siRNAs targeting the TATA-box motif of 16 genes and perform a systematic analysis to identify the common features of the functional siRNAs for effective activation of gene promoters. Further, we try various modifications to improve the activating efficiency of siRNAs and find that it is quite useful to design the promoter-targeting activating siRNA by following several rules such as (a) complementary to the TATA-box-centered region; (b) UA usage at the first two bases of the antisense strand; (c) twenty-three nucleotides (nts) in length; (d) 2′-O-Methyl (2′-OMe) modification at the 3′ terminus of the antisense strand; (e) avoiding mismatches at the 3′ end of the antisense strand. The optimized activating siRNAs potently enhance the expression of  WAF1/CIP1 (p21) or VEGF showed long-lasting and sequence-specific induction of targeted genes SiRNAs are widely used to repress genes expression by targeting mRNAs + T cells isolated from HIV-1-infected patients receiving suppressive highly active antiretroviral therapy (HAART) Recently, our group revealed that an HIV-1-encoded miRNA, miR-H3, could target the TATA-box region in HIV-1 5′ LTR and enhance viral replication. Moreover, the chemically-synthesized siRNAs targeting the same site could activate HIV-1 viral production from the CD4in vitroin vivoCaenorhabditis elegansFor rational design of the conventional siRNAs to degrade target mRNAs, many guidelines have been developed. For instance, the G/C content of the siRNA sense strand should be 30–50%; bases at positions 1–3 should be A/U; the 10th and 19th bases of the sense strand should be A or U; the 1st base of the sense strand should be G/C A large amount of chemical modifications have been developed to improve the properties of siRNAs Although several criteria have been suggested for efficient design of repressive siRNAs that target mRNAs, there is no suggestion about the effective design of activating siRNAs that target gene promoters. In this study, we attempted to investigate the role of TATA-box-targeting siRNAs in the regulation of gene expression and increase their efficiency by testing various modifications including the target site, sequence characters, length and chemical modifications. We identified several helpful characteristics and have combined them together to obtain a reasonable strategy for rational designing siRNAs to activate the gene promoters specifically.This research was approved by the Ethics Review Board of Sun Yat-Sen University. Healthy donors were comprised of a group of local volunteers, who were seronegative and had no reported history of chronic illness or intravenous drug use. All mouse experiments were approved by the Sun Yat-Sen University Institutional Animal Care and Use Committee. BALB/c mice were obtained from the Animal Experimental Center of Sun Yat-Sen University. All mice were maintained under specific pathogen-free conditions and used at 8–10 weeks of age.http://epd.vital-it.ch/) which is a collection of experimentally defined eukaryotic POL II promoters http://www.bioinformatics.org/yapp/cgi-bin/yapp.cgi). The sequences of the two mutated IL-2 promoter (−50 to +1) were as follows: IL-2mt-27/−25, CACAAATTGCATCTCTTGTTCAAGAGTTCTAATATTTTTCCAGAATTAACAG; IL-2mt-17/−16, TACTCTTGTTCAAGAGTTCTAATATTTTTCCAGAATTAACAGTATAAATTGC. Mutations were italicized and underlined.The sequences of gene promoters were downloaded from the Eukaryotic Promoter Database  with the sequence −400 to +1 bp relative to the TSS of the human IL-2 promoter. The vectors containing HIV-1, apolipoprotein E (APOE), insulin (INS), c-Myc, or other gene promoters were generated in the same way. Hsa-let-7i mimics, ssRNAs and the corresponding negative control RNAs were purchased from Genepharma . SiRNAs targeting the TATA-box motif of each gene were synthesized and chemically modified by Ribobio . The sequences of the small RNAs used in this study are listed in The + T lymphocytes were then isolated from PBMCs with human CD4+ T Lymphocyte Enrichment kit (BD). The mouse primary CD4+ T cells were isolated from spleens of 8- to 10-week-old female BALB/c mice with mouse CD4+ T Lymphocyte Enrichment kit (BD). The isolated human or mouse primary CD4+ T cells were grown in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS), 50 U/ml penicillin and 50 µg/ml streptomycin in a humidified incubator with 5% CO2.Jurkat and HEK293T cells were obtained from ATCC  and cultured according to the recommendations. Human peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood of healthy donors with Ficoll-Hypaque Solution . The human primary CD4+ T cells with Lipofectamine RNAiMAX (Invitrogen) at final concentrations of 120 nM. At 12 hrs post transfection, Jurkat cells were treated with 50 ng/ml phorbol 12-myristate 13-acetate  and 1 µM ionomycin (Sigma) for 36 or 84 hrs to activate the cells. Human CD4+ T cells were activated with 1 µg/ml anti-CD3 (R&D Systems) and 5 µg/ml anti-CD28 (R&D Systems) for 84 hrs. Mouse CD4+ T cells were stimulated with 2 µg/ml anti-mouse CD3 (R&D Systems) and 1 µg/ml anti-mouse CD28 (R&D Systems) for 84 hrs.Small RNAs and plasmids were transfected into HEK293T cells with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. SiRNAs were transfected into Jurkat and human or mouse primary CD44 cells per well and grown to 40–60% confluence overnight. Five to ten ng of gene promoter-driven firefly luciferase (FL) reporter and 2 ng renilla luciferase (RL) vector were co-transfected with activating siRNAs or negative control siRNA at final concentrations of 25 nM into HEK293T cells using Lipofectamine 2000 (Invitrogen) by following the manufacturer’s instructions. At 36 hrs post transfection, FL and RL activities were measured with the Dual-Glo Luciferase assay system (Promega) according to the instructions of the manufacturer. FL signals (sample) were normalized to RL signals (transfection control).HEK293T cells were seeded in 48-well plates (Corning) at a density of 2.5×10+ T cells were isolated with TRIzol reagent (Invitrogen) and then subjected to cDNA synthesis using PrimeScript RT reagent Kit (Takara). Quantitative PCR was performed with SYBR Premix ExTaq II Kit (Takara) by using the CFX96 Real-Time System (Bio-Rad). The instructions of the manufacturer were followed. Quantification was normalized to mRNA levels of the housekeeping gene GAPDH or β-actin. The sequences of the primers used in this study are listed in the supplementary data.Total RNAs from Jurkat or primary CD4After transfection, cells were collected and lysed with lysis buffer . The lysates were then separated by 15% SDS-PAGE and analyzed by immunoblotting using primary antibodies specific for human IL-2  or β-actin . The LI-COR Odyssey scanner was used to detect and quantify fluorescent signals as previously described IL-2 promoter activity. We wondered whether siRNAs targeting the TATA-box region in gene promoters could also activate their promoter activities. For this purpose, we randomly designed three siRNAs targeting a ∼30 bp range around the individual TATA-box of sixteen genes: siRNAs targeting the TATA-box-centered position (CEN), siRNAs targeting the positions in upstream (UP) or downstream (DN) of the TATA-box in each gene promoter  stands for the enhancement of siRNA on promoter activity in percent compared with NC) (Table S1 in The most effective siRNA for each gene was picked up and the promoter activities relative to NC were listed in IL-2 core promoter (IL-2 promoter (IL-2-pro) or the mutated IL-2 promoter (IL-2mt-27/−25 or IL-2mt-17/−16) were co-transfected with siRNA IL2-CEN into HEK293T cells. The dual-luciferase assay indicated that the siRNA IL2-CEN had no enhancement effect on the mutated promoters any longer, especially the mutation which impaired the binding in seed sequence (IL-2mt-17/−16)  . Further-17/−16) .IL-2 promoter by let-7i for its regulation function (see the related manuscript). Therefore, it is intriguing to examine whether the regulation of the activating siRNAs is also through an AGO-dependent way. When AGO1 or AGO2 was knocked down by siRNAs, the activation of IL-2 promoter transcription by the activating siRNA was significantly impaired . To confirm this result, we chemically synthesized various siRNAs against different sites of human IL-2, INS or APOE promoter  and 1–3-A/U in the antisense strand  effects .IL-2 promoter activity more potently than siRNAs with other lengths  at their 3′ termini could accumulate in the nucleus et al. reported that the 3′ end of the antisense strand of siRNA hTF167i had a general tolerance to mutations (G/C transversions), while the 5′ end (within the seed region) exhibited low tolerance to mutations IL-2 or INS promoter and named them according to the mutated position relative to the 5′ end of the antisense strand  and ionomycin (1 µM). All the chemical modified siRNAs enhanced IL-2 mRNA expression in Jurkat cells at two days post transfection  and normalized to human β-actin and mouse GAPDH respectively. Human IL-2 protein levels were analyzed by Western blot and normalized to human β-actin. Compared to normal siRNAs, the optimized siRNA 5ua23Me3 enhanced IL-2 mRNA and protein expression potently in human CD4+ T cells  (see the related manuscript). The specific target sites of the functional activating siRNAs in our study may enable the TATA-box-targeting siRNAs to facilitate the recruitment of PICs members onto the TATA-box in gene promoters and subsequently promote the transcription.TATA-box represents the most conserved and a wide-spread core promoter In consistence with previous studies, the optimized siRNAs with U/A in the 5′ termini of antisense strands exerted enhanced activities , becauseThe results above may be due to the different thermodynamic properties of the binding of RNA-RNA and RNA-DNA hybrids. RNA/RNA duplex is more stable than DNA/DNA double helix and RNA/DNA hybrid duplex with the same nearest-neighbor sequence et al. showed that 2′-F sugar was generally well-tolerated on the antisense strand, whereas siRNAs with three 2′-OMe residues at the 3′ ends of the antisense strands were more active than the 5′-modified ones et al. found that siRNAs with 2′-OMe modification at position 2 in the antisense strands can reduce off-target effects et al., except that our functional activating siRNAs contained four 2′-OMe modified bases at the 3′ ends of the antisense strands  or DNA repair genes (BRCA2), may provide a new safe treatment strategy for many diseases such as cancer and diabetes. Further studies should be conducted to find the target position(s) and the sequence characteristics of activating siRNAs that could enhance the promoter activities of the genes without the TATA-box motif.In conclusion, we have demonstrated that the siRNAs targeting the TATA-box-centered region effectively activated 14 out of 16 genes promoter activities, and the siRNA targeting TATA-box-centered region, with UA at the first two bases of the antisense strand, 23 nts in length, and with 2′-OMe modification at the 3′ terminus of the antisense strand, is most effective in activating gene transcription. Taken together with other reports, these results imply the potential of using appropriate modified siRNAs for technical or therapeutic applications. For instance, it is well known that some primary cells  are very difficult to allow the entrance of transfected plasmids File S1Supporting Figures and Tables. Table S1. Feature analysis of siRNAs. For each siRNA, only antisense strand is shown. Efficiency was the FL/RL  ratio relative to the negative control siRNA (NC) for each siRNA. SiRNAs were classified according to their individual fold upregulation of the efficiency in percent (E (%)) compared with NC. Relative position was the distance of 3′ end of siRNA antisense strand to 5′ end of TATA box. Nuclear import sequences in the 3′ region of antisense strand were in bold. S-G/C, N-A/G/C/U. Table S2. Sequences of single-stranded oligonucleotides targeting human IL-2 promoter and negative control. Nucleotides in bold were corresponding to the TATA-box motif. Modified bases were in bold lowercase. ss, single strand; as, antisense; s, sense. Table S3. Sequences of siRNAs targeting the TATA-box of c-Myc, NPPA, or IL-6 with or without modified nuclear import sequence (Mip). Modified bases were in lowercase. Nuclear import sequences in the 3′ region of the antisense strand of modified siRNA were in bold. Figure S1. Effects of inhibiting AGO1, AGO2 or both genes on the activation of IL-2 promoter by siRNA. After transfection of 50 nM siRNAs for AGO1or AGO2 or the negative control siRNA (NC) into HEK293T cells for 24 h (1st transfection), 25 nM siRNA IL-2-CEN or NC were co-transfected with IL-2 promoter-driven firefly luciferase (FL) and renilla luciferase (RL) constructs (2nd transfection). Thirty-six hrs later, promoter activities were determined by dual-luciferase assay. P-values were calculated using the two tailed unpaired Student’s t-test with equal variances. n = 3. *, p<0.05. **, p<0.01. ***, p<0.001. Figure S2. Distribution of functional activating siRNAs with low G/C content. The frequency of siRNAs with low G/C content (30%<G/C<50%) in the antisense strand in each functional activating class . Figure S3.Impacts of ssRNAs (single strand RNAs) on IL-2 promoter activity. SiRNAs were co-transfected into HEK293T cells with the target promoter-driven firefly luciferase (FL) and renilla luciferase (RL) constructs. Thirty-six hrs later, promoter activities were determined by dual-luciferase assay. ss-asRNA, single strand antisense RNA; ss-sRNA, single strand sense RNA. P-values were calculated using the two tailed unpaired Student’s t-test with equal variances. n = 3. *, p<0.05. **, p<0.01. ***, p<0.001. Figure S4. Nuclear import sequence modified siRNAs showed no significant improvement of neither the enrichment in the nucleus nor the activation of gene promoters. (A) Analysis of reported nuclear import sequence in 3′ ends of siRNAs antisense strands. S-G/C, N-A/G/C/U. ND, not detected. Subcellular distributions of antisense strands of siRNAs with (middle) or without (left) modified nuclear import sequence (Mip) targeting (B) c-Myc, (C) NPPA or (D) IL-6 promoter. Thirty-six hrs after siRNAs were transfected, nuclear and cytoplasmic fractions were separated. The expression levels of siRNAs, tRNA-Lys and U78 were then tested with qRT-PCR. Effects of siRNAs on gene promoter activities were determined with dual-luciferase assay as described above (right). P-values were calculated using the two tailed unpaired Student’s t-test with equal variances. n = 3. *, p<0.05. **, p<0.01. Figure S5. The mRNA levels of IL-4 and IL-5 were not affected. “On target” and “Off target” activities. At 12 hrs post transfection of 120 pmol siRNAs into Jurkat cells, cells were subsequently treated with PMA (50 ng/ml) and ionomycin (1 µM) for (A–B) 2 days or (C–D) 4 days. The mRNA levels of IL-4 and IL-5 were determined by qRT-PCR as described above. P-values were calculated using the two tailed unpaired Student’s t-test with equal variances. n = 3. *, p<0.05.(DOCX)Click here for additional data file.