The photocatalysts were synthesized by coating of ZnO onto the magnetite by precipitation method and characterized by XRD, SEM and FTIR measurements. Using the Taguchi method, this study analyzes the effect of parameters such as calcinations time, calcinations temperature and molar ratio of Fe3O4:ZnO on the photo activity of Fe3O4/ZnO MNPs. XRD and FTIR analysis confirm that coating process was done successfully. SEM images show that the average particle size of synthesized Fe3O4/ZnO nanoparticles was about 50 nm. The phenol removal efficiency of 88% can be achieved by using a photocatalyst which is synthesized through the optimum conditions: calcinations temperature of 550°C, calcinations time of 2 hours and molar ratio of 1:10 for Fe3O4:ZnO.The photocatalysis of phenol was studied using Fe On the other hand, ZnO which has high photoactivity, chemical stability, low cost and the band gap similar to TiO2, can be a good alternative for it [3O4/ZnO core/shell MNPs have not been sufficiently investigated [3O4/ZnO core/shell composite catalyst was synthesized. This photocatalyst is produced by coating a layer of the zinc oxide onto the surface of magnetite core using precipitation method. The effect of parameters such as calcination temperature, calcination time, and molar ratio of Fe3O4:ZnO on the photocatalytic activity was studied by using Taguchi method. Phenol, which is well known for its biorecalcitrant and acute toxicity, was the organic matter used in this work [Semiconductor photocatalytic materials have been extensively studied in the fields of environmental purification due to their potential to destroy a wide range of pollutants at ambient temperatures and pressures, without producing harmful byproducts ,2. In a n or air . Metal oe for it -6. Many e for it ,8. Howeve for it . Magnetie for it . Althouge for it -14, the stigated . In thisstigated . In thishis work ,12.3.6H2O), ferrous sulfate (FeSO4.7H2O), Zinc acetate (ZnAc2.2H2O), aqueous ammonia (NH3.H2O) and phenol (C6H5OH) were obtained from Merck company and ammonium carbonate ((NH4)2CO3) was purchased from Daejung (South Korea) and used without further purification.Ferric chloride (FeCl3O4 magnetic nanoparticles (MNPs). Co-precipitation is a facile and convenient way to synthesize MNPs from aqueous salt solutions. There are three controllable parameters have been used for the synthetic procedure which each parameter has three levels (Table 3O4 and 200 mL sodium citrate (0.5 M) for 20 min and stirring the mixture for 12 h at 60°C under argon protection. Then the precipitate was collected and rinsed with acetone. The Fe3O4/ZnO core/shell MNPs were prepared by coating the modified Fe3O4 MNPs with direct precipitation using zinc acetate and ammonium carbonate. The modified Fe3O4 was added to 100 mL of deionized water and sonicated for 20 min to make a stable ferrofluid. Then 20, 30 and 50 mL of this ferrofluid were added into a flask to form Fe3O4/ZnO composite with molar ratios of 1:6, 1:10 and 1:15 for Fe3O4:ZnO respectively. Two solutions were made by adding 12.16 g ZnAc2.2H2O and 7.6 g (NH4)2CO3 into 100 mL of deionized water respectively. These two solutions were added dropwise to the flask for each experiment. The collected precipitate was washed with water, aqueous ammonia (pH 9) and ethanol and then it was dried under vacuum for 12 h and calcined according to desired calcination temperature and time. ZnO would be produced when no ferrofluid exists in the flask.A co-precipitation method was used to synthesize the Fels Table . Also, t3O4/ZnO core/shell particles. The X-ray diffraction pattern of the samples was measured in an Equinox 3000 (Inel France). The crystallite dimensions of particles were calculated using Scherrer’s equation and nanoparticles of Fe3O4, modified Fe3O4, ZnO and Fe3O4/ZnO core/shell were examined by a Phillips scanning electron microscopy (SEM). Fourier transform infrared (FTIR) spectra obtained using a KBr method in a Perkin Elmer analyzer, USA.In all analysis, sample number 8 was examined as FePhotocatalytic degradation of phenol was performed in a slurry batch reactor which consisted of cylindrical glass vessel, quartz trap, magnetic stirrer and a Philips 11 W UV-C lamp located at the center of the reactor. The schematic figure of the reactor is shown in Figure COD determination was utilized to analyze the samples. The COD determination tests were performed according to standard dichromate method . The phoi and CODt are photodegradation efficiency, initial chemical oxygen demand, and chemical oxygen demand at time t respectively.where η, COD3O4, Fe3O4, ZnO and Fe3O4/ZnO core/shell presented in Figure 3O4 , the average crystallite sized was calculated about 13.9 nm (a), 11.2 nm (b) for modified Fe3O4 and Fe3O4, respectively. Figure 3O4/ZnO core/shell. Considering this figure, it is shown that after coating, we have enhancement in peak intensity which is caused by overlapping of Fe3O4 peaks. Results were obtained from Hong et al. [The X-ray diffraction patterns of samples modified Fe4 Figure b, which g et al.  confirm 3O4MNPs SEM images with sodium citrate are presented in Figure 3O4/ZnO core/shell particles. The average particle size was obtained about 57 and 48 nm, respectively.Fe3O4/ZnO core/shell MNPs. It can be seen that the characteristic absorption of Fe-O bond is at 582.78/cm and 620.21/cm, while that of -OH bond is at 3449.26/cm. The absorptions at 1395.25/cm and 1591.29/cm are characteristic peaks of the COO-Fe bond, which may be due to the reaction of hydroxide radical groups on the surface of Fe3O4 with carboxylate anion of sodium citrate. These peaks reveal that sodium citrate has been successfully grafted onto the surface of Fe3O4 MNPs. Also, the adsorption at 449.96 refers to Zn-O bond. Combining with XRD results, it is concluded that ZnO had been coated on the Fe3O4, successfully.Figure 3O4: ZnO and calcination time of 2 h. At these conditions, photodegradation of 88% for 100 ppm initial phenol solution (pH = 5) with 1 mg/l catalyst is obtained after 5 hours irradiation which is comparable with results obtained by Pardeshi and Patil [2). These results indicate that this novel catalyst possess high photodegradation ability and could be an effective alternative catalyst in photocatalysis of contaminated water.For each experiment, photo activity was examined according to photo degradation test and results are given in Table nd Patil ,  is achieved after 5 h. Hence, the Fe3O4/ZnO nanopartcles could be an effective recyclable catalyst for photodegradation of phenol.FeThe authors declare that they have no competing interests.MN gave technical support and conceptual advice; MHR and MA designed and performed experiments, analyzed data and wrote the paper; RL performed the experiments and prepared the final manuscript. All authors read and approved the final manuscript.


Saccharomyces cerevisiae Rad52 protein is essential for efficient homologous recombination (HR). An important role of Rad52 in HR is the loading of Rad51 onto replication protein A-coated single-stranded DNA (ssDNA), which is referred to as the recombination mediator activity. In vitro, Rad52 displays additional activities, including self-association, DNA binding and ssDNA annealing. Although Rad52 has been a subject of extensive genetic, biochemical and structural studies, the mechanisms by which these activities are coordinated in the various roles of Rad52 in HR remain largely unknown. In the present study, we found that an isolated C-terminal half of Rad52 disrupted the Rad51 oligomer and formed a heterodimeric complex with Rad51. The Rad52 fragment inhibited the binding of Rad51 to double-stranded DNA, but not to ssDNA. The phenylalanine-349 and tyrosine-409 residues present in the C-terminal half of Rad52 were critical for the interaction with Rad51, the disruption of Rad51 oligomers, the mediator activity of the full-length protein and for DNA repair in vivo in the presence of methyl methanesulfonate. Our studies suggested that phenylalanine-349 and tyrosine-409 are key residues in the C-terminal half of Rad52 and probably play an important role in the mediator activity.The  Homologous recombination (HR) is an important mechanism for the accurate repair of DNA double-strand breaks. Defects in HR lead to mutations in the genome, as well as gross chromosomal rearrangements such as deletions, insertions and loss of heterozygosity, which could lead to genomic instability and cancer . HR consIn yeast, Rad52 is an important factor in HR. Rad52 is a multifunctional protein involved in both the Rad51-dependent and Rad51-independent HR pathways. Increasing evidence suggests that the primary role of Rad52 in the Rad51-dependent HR pathways is to facilitate the loading of the Rad51 recombinase on replication protein A (RPA)-coated single-stranded DNA (ssDNA), which is referred to as the recombination mediator activity ,4. Rad52Previous studies indicated that the N- and C-terminal halves of Rad52 have clearly distinct functions. The N-terminal half is highly conserved among Rad52 orthologs, and contains the self-association region. The isolated N-terminal half of the human Rad52 protein is capable of oligomerizing into a ring structure  and formSaccharomyces cerevisiae Rad52 protein, we prepared and biochemically characterized a C-terminal Rad52 fragment. We found that the Rad52 fragment disrupted Rad51 oligomers and hindered the formation of the Rad51–dsDNA complex. In addition, the alanine substitutions of phenylalanine-349 or tyrosine-409 abolished the observed biochemical activities of the Rad52 fragment. Both of these residues were critical for DNA repair in vivo, and for the mediator activity of Rad52 in vitro.To clarify the molecular roles of the C-terminal half of the S. cerevisiae Rad52, Rad51 and RPA proteins used in all in vitro experiments were expressed and purified as described previously , in which the thrombin protease recognition site was replaced with a PreScission protease recognition site by site-directed mutagenesis. The protease recognition site was changed to minimize non-specific cleavage during hexahistidine tag removal. The Rad52 genes were cloned between the NdeI and BamHI sites of the pET15b-PS vector. These Rad52 derivatives were expressed in the Escherichia coli strain JM109(DE3), bearing a plasmid  to express low-abundance tRNAs. Each protein was purified from a 2-L LB culture that was initially incubated at 30°C. Protein expression was induced at an optical density (A600) of 0.7 with 0.5 mM 1-thio-β-d-galactopyranoside , and the culture was incubated overnight at 18°C. The cells were harvested, resuspended in resuspension buffer  containing 5 mM imidazole and lysed by sonication. The cell lysate was cleared of insoluble material by centrifugation at 30 190 x g for 30 min. The supernatant was mixed with 3 ml of nickel-nitrilotriacetic acid-agarose beads (Qiagen) and was incubated at 4°C with gentle mixing for 1 h. The mixture was poured into an Econo-Column, and the unbound cell lysate was drained out. Rad52 was eluted with 120 ml of resuspension buffer containing 50 mM imidazole. To remove the hexahistidine tag, the eluted protein was mixed with 0.5 U of PreScission protease per milligram of protein, and was immediately dialyzed against Rad52 purification buffer  containing 0.15 M KCl. The dialyzed protein solution was collected, and the removal of the hexahistidine tag was confirmed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The protein solution was then passed through a 6-ml Q Sepharose column , and the flow-through fraction was diluted with water to adjust the KCl concentration to 0.1 M. The diluted fraction was directly applied to a 5-ml Affi-Gel Blue column (Bio-Rad). The column was washed with 100 ml of Rad52 purification buffer containing 0.1 M KCl, and the proteins were eluted with a 100-ml linear gradient of 0.1–1 M KCl in Rad52 purification buffer. The peak fractions were collected, dialyzed against Rad52 purification buffer containing 0.15 M KCl and concentrated to 0.5–2 mg/ml using a Vivaspin ultrafiltration device with a 10 kDa molecular weight cutoff (Sartorius). The concentrations of Rad52233–504 and its point mutants were determined by the absorbance at 280 nm, using an extinction coefficient of 4470 M−1 cm−1. The extinction coefficient was calculated with the ProtParam tool at the ExPASy Web site (http://web.expasy.org/protparam/).The full-length eviously ,24. Rad5233−504 was incubated at room temperature for 1 h. The 250-µl mixture was fractionated through a 24-ml Superdex 200 10/300 GL column  in SE Buffer, containing 20 mM Hepes-KOH, pH 7.5, 0.1 M KCl, 0.5 mM EDTA, 10% glycerol and 2 mM 2-mercaptoethanol. This analysis was performed identically for the Rad52233–504 F349A and Rad52233–504 Y409A mutants.A 250-µl mixture containing 10 µM Rad51 and 20 µM Rad52233–504 complex used for the sedimentation equilibrium analysis was prepared in a 250-µl mixture, containing 25 µM Rad51 and 50 µM Rad52233–504. The mixture was incubated at room temperature for 1 h. The complex was purified by chromatography on a 24-ml Superdex 200 10/300 GL column , using the SE Buffer described earlier. Peak fractions were collected and concentrated using an Amicon Ultra 10 ultrafiltration device (Millipore) until the absorbance at 280 nm reached 0.3. The complex was analyzed using a Beckman Optima XL-I instrument. The concentrated sample was spun in an 8-sector centerpiece, using a Beckman An-50Ti rotor. Equilibrium distributions were analyzed after 20 h of centrifugation, at 16 000 rpm and 20°C. For the molecular mass analysis, an estimated partial specific volume of 0.723 cm3/g and a solution density value of 1.03 g/cm3 were used. These values were calculated from the amino acid composition and the buffer components, respectively, using the SEDNTERP program  was incubated at 37°C for 15 min. To this mixture, 1 µl of 32P-labeled 60-mer ssDNA (5′ GGA ATT CGG TAT TCC CAG GCG GTC TCC CAT CCA AGT ACT AAC CGA GCC CTA TGC TGC TTG 3′) or dsDNA (same length and sequence) was added, and the solution was incubated for 15 min. The complexes were either fixed with 1 µl of 1% glutaraldehyde or treated with 2 µl of deproteinization solution  at 37°C for 15 min. The products were fractionated through a 1% Seakem GTG agarose (FMC BioProducts) gel in 0.5× Tris-borate-EDTA buffer for 2 h at 3.3 V/cm. The gels were dried, exposed to an imaging plate and visualized using an FLA7000 image analyzer (Fuji Film).A 9-µl reaction mixture containing 2 µl of 10× reaction buffer , 1 µl of 20 mM CaClS. cerevisiae XS560-1C-1D2 . The expression vector, pNS31, for the mutant and wild-type RAD52 in S. cerevisiae contains the ADH1 promoter and terminator, ARSH4-CEN6  and the LEU2 marker  of a 10-fold dilution series of each transformant were spotted onto SD plates containing 2 µg/ml uracil and 2 µg/ml histidine with 0.25, 0.59 or 1.18 mM methyl methanesulfonate (MMS). The plates were sealed with Parafilm and incubated at 30°C for 5 days.For the spot test, the synthetic dextrose (SD) liquid medium [2% glucose and 0.67% yeast nitrogen base without amino acids (Difco)] was supplemented with 2 µg/ml uracil and 2 µg/ml histidine. Cells from an overnight culture (10 ml) were concentrated to 1 ml by centrifugation at 200 × g for 3 min. After the dilution of the cell cultures, the cells were spread onto freshly prepared SD plates containing the appropriate supplements, with MMS at 0, 0.13 (0.001%), 0.25 (0.002%), 0.59 (0.005%) or 1.18 mM (0.010%). The plates were sealed with Parafilm and incubated at 30°C for 7 days.For the quantitative test, the SD liquid medium was supplemented with 4 µg/ml adenine sulfate, 2 µg/ml uracil, 2 µg/ml histidine, 4 µg/ml lysine and 4 µg/ml tryptophan (and 6 µg/ml leucine for the YPH499 strain without the plasmid). Cells from an overnight culture (10 ml) were concentrated to 1 ml by centrifugation at 200 × 2, 9 µM Rad51, the indicated concentrations of Rad52, 2 µM RPA and 30 µM ΦX174 circular ssDNA, was prepared. All of the components (9 µl), except for the ssDNA, were mixed and incubated on ice for 45 min. The ssDNA  was then added to the reaction mixture and incubated at 37°C for 10 min. Afterward, 1 µl of 300 µM dsDNA (ΦX174 RFI linearized by PstI digestion) and 1 µl of 50 mM spermidine hydrochloride were added to the reaction mixture and incubated at 37°C for 90 min. The reaction was stopped by the addition of an equal volume of 1% SDS containing 1 mg/ml proteinase K. Deproteinization of the reaction mixtures was performed at 37°C for 20 min. After the addition of 0.2 volume of gel loading dye , the samples were fractionated in 1.0% agarose gels in Tris-acetate-EDTA buffer, stained with ethidium bromide for 60 min and then destained for at least 4 h in a large volume of H2O. Images were recorded with a Gel Doc XR system (Bio-Rad).A ΦX DNA-based DNA strand exchange assay was performed, essentially as described . Briefly32P]ATP (0.1 mM) was incubated with 1.0 µM RPA and 5.0 µM (in nucleotides) pUC119 circular ssDNA in reaction buffer (10 µl), containing 30 mM MOPS-KOH (pH 7.1), 7 mM MgCl2, 50 mM KCl, 1 mM dithiothreitol, 50 µg/ml bovine serum albumin and 0.02% (v/v) IGEPAL CA630 (SIGMA), at 37°C for 10 min. After the incubation, 1.0 µM Rad51 and the indicated concentrations of Rad52 were added to the reaction mixture, which was incubated further for 30 min.  The reaction was terminated by adding 10 µl of stop solution . Aliquots (10 µl) of the sample were spotted on polyethyleneimine plastic film  and developed in a mixture of 0.5 M lithium chloride and 1 M formic acid. The plastic film was exposed to a Phosphor Screen  for 1.5 h, and then the radioactivity was quantitated by a Typhoon 9410 Variable Image Analyzer.[α-233–504) containing only the C-terminal half of the protein, which includes the previously identified Rad51 binding region, but lacks the N-terminal region required for self-association . When Rad52233–504 was mixed with Rad51 and analyzed by size exclusion chromatography, we observed a single peak that clearly indicated a molecular mass larger than that of Rad52233–504, but significantly smaller than that of Rad51 . We found that Rad52233–504 did not appreciably inhibit the formation of the Rad51–ssDNA complex, as judged from the similar amounts of the complex and the absence of the unbound ssDNA , suggesting that divalent ions may inhibit the DNA binding activity of Rad52233–504.To further characterize the function of the C-terminal half of yeast Rad52, we examined the effect of Rad52proteins A. Before233–504 to pre-formed Rad51–ssDNA and Rad51–dsDNA complexes. In this assay, Rad51 was pre-incubated with ssDNA or dsDNA, and then Rad52233–504 was added to the reaction mixture. The amounts of the Rad51–DNA complexes did not change with increasing concentrations of Rad52233–504, suggesting that Rad52233–504 did not destabilize either of the complexes .We next examined the effect of adding Rad52144–147). The crystal structure of the yeast Rad51 filament revealed that the phenylalanine residue interacts with the neighboring Rad51 monomer in a manner resembling a ‘ball and socket’ joint (349–352) resides between the previously identified RPA and Rad51-interacting regions , by size exclusion chromatography, as described earlier. Unlike the Rad52233–504 without the point mutation, the Rad52233–504 F349A mutant did not co-elute with Rad51  and examined its interaction with Rad51 by size exclusion chromatography. Similar to Rad52233–504 F349A, Rad52233–504 Y409A did not co-elute with Rad51 , suggesting that the loss of Rad51 binding was not the result of a disruption of the binding interface. Thus, our results demonstrated that Rad52 has at least two important interaction sites for Rad51 binding.The FVTA sequence of Rad52 is ∼60 amino acid residues apart from the previously identified Rad51 binding region . To deteth Rad51 B, indicain vivo, we replaced the chromosomal RAD52 gene with the rad52 F349A allele, and tested the mutant strain for its MMS sensitivity by a spot assay and a quantitative assay. Increasing the MMS concentration resulted in a significant decrease in the survival of the rad52 F349A mutant (rad52 F349A mutant was similar to that of the rad52 Y409A mutant. When a strain bearing both point mutations (the rad52 F349A/Y409A double mutant) was tested, we observed a higher level of MMS sensitivity. These observations suggested that F349 and Y409 are both important for the function of Rad52 in DNA repair in vivo. Interestingly, the MMS sensitivity of the rad52 F349A/Y409A double mutant was similar to that of a strain carrying a C-terminally truncated RAD52 gene  were completely defective in stimulating the ATPase activity of Rad51 . In vitro studies have failed to detect the mediator activity of human Rad52 . Thus, in these species, Rad52 may not function as a recombination mediator, and BRCA2 fulfills the role instead. Another possibility is that the Rad52 in these species uses a different recombination mediator mechanism, which may involve post-translational modifications or an interaction with another protein  (in part) and the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, ; and the Waseda Research Institute for Science and Engineering supported (to W.K. and H.K.). Funding for open access charge: Waseda University.The Grants-in-Aid from the Conflict of interest statement. None declared.


Protein expression levels were also detected by western blotting. In comparison with the control group exhibiting normal cardiac functions, the mRNA and protein expression levels of Kv4.3α, KvLQT1 and L-Caα1c were significantly reduced in HF patients. By contrast, the mRNA and protein expression levels of NCX were significantly increased in HF patients compared with the control group (P<0.01). The mRNA expression levels of Kv1.5 were not evidently altered. We demonstrated that increased levels of Kv4.3α, KvLQT1 and L-Caα1c and decreased levels of NCX are correlated with the risk of AF in HF patients. Changes in the gene expression of ion channel-related proteins may therefore be used as biological markers of AF occurring in HF patients in future studies.The aim of this study was to investigate the correlation between the altered expression of genes involved in the regulation of ion channels in atrial myocytes and the risk of atrial fibrillation (AF) in patients with heart failure (HF). Right atrial appendages were obtained from 18 HF patients and 18 patients with normal cardiac functions who had undergone surgery. The mRNA expression levels of Kv4.3α, KvLQT1, Kv1.5, L-Caα Congestive heart failure (CHF) is a powerful, independent predictor of atrial fibrillation (AF), with CHF patients exhibiting a 6-fold increase in the relative risk of developing AF . Researcet al (2+ current (ICa2+), the sensitive transient outward K+ current (Ito1) and the slow delayed rectifier K+ current (IKs) in atrial myocytes without altering their voltage dependencies or kinetics. The inward rectifier K+ current (Iki), the ultra rapid rectifier K+ current (Ikur) and the rapid delayed rectifier K+ current (Ikr) were not altered by CHF, while the transient inward Na+/Ca2+ exchanger (INCX) current was increased. In addition, Cha et al  undergoing coronary artery bypass surgery at the Qianfoshan Hospital of Shandong University and the Jinan Central Hospital Affiliated to Shandong University (China) were enrolled. Among these, 18 cases presented with coronary heart disease, 5 with dilated myocardial disease, 6 with rheumatic heart disease and 7 with congenital heart disease. Patients with an eject fraction (EF) <0.4 were defined as the HF group (33.67±3.68%) while those with EF >0.4 were defined as the normal cardiac function group (60.28±4.98%). The study cohort was extremely homogeneous for age, gender and overall etiology constitution. The study was approved by the ethics committee of Shandong Provincial Qianfoshan Hospital, Jinan, China. Written informed patient consent was obtained from the patient’s familyto1 (Kv4.3α), Iks (KvLQT1), Ikur (Kv1.5), ICa2+ (L-Caα1c) and INCX (NCX). Trizol® Reagent was purchased from Gibco BRL . The RNA PCR kit (AMV) Ver. 3.0 was purchased from Takara Bio, Inc. . The primer sequences were provided by Shanghai BioAsia Biotechnology Co., Ltd.  and the target genes included the channel determinant genes of Io., Ltd. .2 (25 mM); 1 μl 10X RT buffer; 3.75 μl RNase free dH2O; 1 μl dNTP mixture (10 mM); 0.25 μl RNase inhibitor; 0.5 μl AMV reverse transcriptase; 0.5 μl oligo dT-adaptor primer; 1 μl RNA. The reverse transcription was then conducted as follows: the reaction mixture was incubated at 30°C for 10 min, annealed at 42°C for 30 min, followed by incubation at 99°C for 5 min and 5°C for 5 min. Following denaturation at 94°C for 2 min, the samples were subjected to 30 cycles of denaturation at 94°C for 30 sec, annealing for 30 sec and extension at 72°C for 50 sec. Then, 35 cycle PCR amplification was used with a 5-min extension time (reaction solution 2). The 10 μl of amplified product was electrophoresed in 1.5% agarose gel containing ethidium bromide, examined and photographed under a UV transilluminator. The intensity of each band was quantified using image analysis software  and the expression levels were calculated by measuring the OD of the target gene and normalized to that of the amplified GAPDH.Open-chest cardiac surgery was performed and 50–100 mg samples of right atrial appendages were rapidly collected with sterilized Eppendorf tubes. The sample was frozen at −80°C immediately following the addition of 0.5 ml TRIzol. The total RNA was extracted using the TRIzol method. The extraction was dissolved by adding 40 μl DEPC and then measured by a spectrophotometer, with the optical density (OD) 260/OD280>1.8 and thereafter stored at −20°C for detection. RT-PCR technology was applied for reverse transcription and cDNA fragment amplification. The first-strand cDNA was synthesized using AMV reverse transcriptase. Total RNA was reverse transcribed in a final volume of 10 μl, containing the following: 2 μl MgCl1c, NCX and actin were purchased from Santa Cruz Biotechnology, Inc. . Secondary antibodies used in this study were donkey anti-goat IgG-HRP , goat anti-rabbit IgG-HRP  and goat anti-mouse IgG-HRP . Bound antibodies were detected using the ECL system . The immunoblot experiments were repeated at least 3 times. The mean normalized OD of detected Kv4.3α, KvLQT1, Kv1.5, L-Caα1c or NCX protein bands relative to the OD of the actin band from the same individual was calculated, respectively.All relevant proteins were harvested from tissue, separated by 10% SDS/PAGE and then subjected to immunoblot analyses. The primary antibodies against Kv4.3α, KvLQT1, Kv1.5, L-CaαConcise Statistics 2000 was used to perform the statistical analyses. All numerical values are expressed as mean ± SD. The t-test was performed for comparison of the experimental group and the control group. P<0.05 was used to indicate a statistically significant result.GAPDH bands. The mRNA expression levels of Kv4.3α, KvLQT1, Kv1.5, L-Caα1c, NCX and GAPDH are shown in GAPDH gene are consistent with theoretically expected sizes.To investigate whether mRNA transcript levels of genes involved in ion channel regulation were changed, right atrial appendages were obtained from 18 HF patients and 18 individuals with normal cardiac functions, who had undergone surgery. The total RNAs were isolated and the mRNA levels were determined by RT-PCR. The PCR products were separated on gels and the OD values of the relevant bands of interest were measured to compare with the OD of Kv1.5 in the HF group (0.30±0.05) was not significantly altered (P>0.05). The mRNA expression levels of Kv4.3α, KvLQT1 and L-Caα1c were significantly reduced in patients with HF (P<0.01) in comparison with those detected in normal individuals . However, the mRNA expression of NCX was significantly increased in HF patients (0.31±0.07) compared with those detected in normal individuals . These results suggest that mRNA levels of some ion channel-related genes may be altered in HF patients.As shown in Kv4.3α, KvLQT1, Kv1.5, L-Caα1c and NCX genes were altered in comparison to those in normal controls, total protein was harvested from tissues, separated by 10% SDS/PAGE and then subjected to immunoblot analyses. The cellular actin protein served as a loading control. Representative blots are shown in To investigate whether levels of proteins encoded by the Kv4.3α, KvLQT1 and L-Caα1c were significantly decreased to 0.41±0.21, 0.23±0.11 and 0.29±0.13, respectively, in the HF groups, when the levels in the normal control group were artificially set as 1. Kv1.5 levels were not altered significantly in the HF group when compared with the normal group with a value of 0.98±0.15. However, expression levels of NCX were significantly increased in HF patients (1.63±0.12) when compared with the normal group  and delayed after depolarization (DAD) as well as the physiological heterogeneity of cardiac myocytes (Iotential . Previou with HF . The dowmyocytes .NCX in the plateau phase is important to the production of EAD. By contrast, the increased activity of NCX during the diastolic spontaneous SR Ca2+ release period may lead to a greater depolarization current and greatly increase DAD and the propensity for triggered arrhythmias in HF patients (Ca2+ and intracellular Ca2+ overload, which provides feedback inhibition of L-Ca2+ channels, a decrease in the inward movement of Ca2+ and a shortened action potential plateau. The overload of extracellular Ca2+ may further influence the K+ channel and promote the electrical instability of cardiac cells, which could induce arrhythmia and trigger activity.In addition, altered activity of NCX may also be strongly correlated with the genesis of AF. The increased inward Ipatients –20. DAD patients . The neuIn conclusion, alterations in the gene expression of ion channels may provide the molecular basis of altered atrial cellular ion currents in patients with HF, and furthermore, may initiate atrial arrhythmia, particularly AF, by either trigger or re-entry activity. HF-induced atrial ionic remodeling may be important in the formation of AF substrate and contribute to the potential mechanisms of AF in HF.


Tityus serrulatus venom (TsV) and its major component, toxin 1 (Ts1), to mediate cytokine and lipid mediator production. Additionally, we demonstrated that TsV induces TLR2- and TLR4/MyD88-dependent NF-κB activation and TLR4-dependent and TLR2/MyD88-independent c-Jun activation. Similar to TsV, Ts1 induces MyD88-dependent NF-κB phosphorylation via TLR2 and TLR4 receptors, while c-Jun activation is dependent on neither TLR2 nor TLR4/MyD88. Therefore, we propose the term venom-associated molecular pattern (VAMP) to refer to molecules that are introduced into the host by stings and are recognized by PRRs, resulting in inflammation.Scorpion sting-induced human envenomation provokes an intense inflammatory reaction. However, the mechanisms behind the recognition of scorpion venom and the induction of mediator release in mammalian cells are unknown. We demonstrated that TLR2, TLR4 and CD14 receptors sense  Human envenomation caused by scorpion stings is a serious public health issue worldwide. The recognition of scorpion venom by mammalian cells triggers a strong inflammatory response that is characterized by increased levels of local and circulating leukocytes Tityus serrulatus venom (TsV) is a complex mixture composed of a wide array of substances. It is composed of insoluble mucus, many neurotoxic proteins that affect Na+ or K+ channels, proteases, hyaluronidase http://web.expasy.org/protparam). Ts1 is a sodium channel β-neurotoxin, which depolarizes the membrane, reduces the amplitude of the action potential and increases its duration 4 (LTB4), and prostaglandin E2 (PGE2) are the primary mediators induced by TsV and its purified toxins, Ts1, Ts2 and Ts6 It has previously been reported that interleukin-1α (IL-1α), tumor necrosis factor-α  interleukin-6 (IL-6), interferon-γ (INF-γ), granulocyte macrophage colony-stimulating factor (GM-CSF), interleukin-10 (IL-10), leukotriene B2 and LTB4 production following VAMP recognition. Specifically, our results demonstrated that TsV interacts with TLR2, TLR4 and CD14. This interaction recruits MyD88 and activates the AP-1 and NF-κB pathways. Similarly, Ts1 induces TNF-α and IL-6 production in a manner that is dependent on TLR2, TLR4 and CD14, although signaling occurs specifically through NF-κB activation.Currently, the mechanisms underlying the innate immune recognition and the cell signaling networks that are involved in the inflammatory response following envenomation by scorpions are unknown. Using knockout mice, we identified, for the first time, the innate immune receptors and cell signaling pathways involved in macrophage activation by TsV and Ts1. In the present manuscript, we describe, for the first time, a role for TLR2, TLR4 and CD14 in the innate immune recognition of TsV and purified Ts1, which we named venom-associated molecular patterns (VAMPs). Additionally, we identified the macrophage signaling pathways involved in TNF-α, IL-6, PGE−/−) mice between the ages of 6 and 8 weeks were donated by S. Akira  and bred in the Animal House of the Faculdade de Medicina de Ribeirão Prêto . Strain-matched WT C57Bl/6  mice of both sexes were bred in the Faculdade de Ciências Farmacêuticas de Ribeirão Prêto . The mice were maintained in a room at 25°C, with a 12 h/12 h light/dark cycle, and provided with free access to food and water. The mice were kept in biohazard facilities. All experiments were approved and conducted in accordance with the guidelines of the Animal Care Committee of Prefeitura of Campus of Ribeirão Preto (PCARP) at the University of São Paulo (Protocol number 11.1.160.53.1).TLR2, TLR4, CD14and MyD88 knockout  test was performed .TsV was extracted from 5 cells/well in RPMI medium supplemented with 10 mM L-glutamine, 100 U/ml penicillin, 100 U/ml streptomycin and 10% fetal bovine serum (FBS). The cells were cultured at 37°C in a humidified 5% CO2 atmosphere for 18 h. After this period, the supernatants were collected and the cells were stimulated either with TsV (50 µg/ml/2×105 cells) for 30 min or 24 h or with Ts1 (50 µg/ml/2×105 cells) for 24 h at 37°C in a 5% CO2 atmosphere. After the stimulation period, the culture supernatants were harvested and stored at −20°C until further use. To verify whether the recognition of TsV or Ts1 by macrophages was dependent on their tertiary structures, TsV and Ts1 were thermally inactivated prior to being added to the cell cultures. For heat inactivation, 50 µg/mL each of TsV and Ts1 were heated for 30 min at 60°C The macrophages were isolated from the peritoneal cavities of mice and plated in 96-well micro culture plates at a density of 2×10To ensure the stimuli were not cytotoxic, lactate dehydrogenase (LDH) levels in the macrophage supernatants were measured after 24 h of incubation with TsV or Ts1. The level of LDH in the cell supernatant was measured using the CytoTox96® non-radioactive assay (Promega) at 490 nm. LDH levels were expressed as percentages of the LDH levels observed in control cultures.The supernatants from the cells stimulated with TsV or Ts1 were used to measure IL-6 and TNF-α levels by enzyme-linked immunosorbent assays (ELISA), with specific purified and biotinylated antibodies and cytokine standards, according to the manufacturer’s instructions (R&D Systems). The optical densities were measured at 450 nm in a micro plate reader . For each sample, the cytokine levels were obtained from a standard curve established with the appropriate recombinant cytokine. The results are expressed as pg/mg of total protein. The sensitivities were >10 pg/ml.4 and PGE2 in the supernatants of cells stimulated with TsV or Ts1, according to the manufacturer’s instructions. The sample absorbance was measured at 420 nm in a micro plate reader , and the concentrations of eicosanoids were calculated based on the standard curve. The detection limit was >13 pg/ml.A specific enzyme immunoassay  was used to quantify LTB2 Profiler PCR Array kit (Qiagen Inc.). Total RNA was isolated using the RNeasy Mini kit (Qiagen Inc.), and the reverse transcription of 500 ng of RNA was performed using the RT2 HT First Strand kit (Qiagen Inc.). Ten nanograms of the cDNA template was used per reaction, and the reactions were performed with specific pre-designed primers for Tlr2 (Toll-like receptor 2), Tlr4 (Toll-like receptor 4), Cd14 (CD 14 molecule), Myd88 (Myeloid differentiation primary response gene 88), Ltb4r1 (leukotriene B4 receptor 1), Alox5 (Arachidonate 5-Lipoxygenase), Alox5ap (Arachidonate 5-Lipoxygenase-Activating Protein), Ptgs2 (Prostaglandin-Endoperoxide Synthase 2 or Cyclooxygenase-2) and Ptges2 (Prostaglandin E synthase 2). The endogenous internal controls used were Tbp (Tubulin), Actb (Beta actin) and Gapdh . Amplification was performed in an Eppendorf Mastercycler® ep realplex 4 (Eppendorf AG) using the SYBR Green Mastermix. Denaturation of the DNA was followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The 2–ΔΔCt method was used for the analysis of the RT-PCR data.mRNA expression was evaluated after 4 h of macrophage stimulation with TsV or Ts1 using a custom RT5 cells/well in DMEM supplemented with Normocin™ (50 mg/mL) and cultured at 37°C in a humidified 5% CO2 atmosphere for 18 h. After this period, the cells were incubated with 10 ng/ml of LPS from Rhodobacter sphaeroides, a TLR4 antagonist (LPS-RS - InvivoGen), with or without LPS from E. coli (0.5 µg/ml), and with TsV (50 µg/ml) or Ts1 (50 µg/ml) for 24 h. In another experiment, the cells were pre-incubated with 100 ng/ml of a purified monoclonal IgG antibody against mouse TLR2 (anti-mTLR2-IgG – InvivoGen) for 30 min and then stimulated with LPS from E. coli (0.5 µg/ml) and TsV or Ts1 (50 µg/ml) for 24 h. After 24 h of stimulation, the medium was harvested, and 50 µl samples were mixed with QUANTI-Blue™ (InvivoGen), which is a SEAP detection medium (150 µl), in 96-well plates at 37°C for 2 h. The optical density was then measured at 650 nm using an ELISA reader.RAW-Blue™ cells, RAW264.7 macrophages that stably express the secreted embryonic alkaline phosphatase (SEAP) gene, which is inducible by the NF-κB/AP-1 transcription factors and is resistant to the selectable marker Zeocin, were donated by Huy Ong . The cells were seeded in 96-well micro culture plates at a density of 2×106 cells/well in RPMI medium and cultured at 37°C in a humidified 5% CO2 atmosphere for 18 h. The cells were then stimulated with TsV or Ts1 (both at 50 µg/ml) for 10 min, 2 h or 24 h at 37°C in a 5% CO2 atmosphere. After treatment, the adherent macrophages were washed with ice-cold PBS. Then, Cell Lysis Buffer  containing protease and phosphatase inhibitors was added to the cells. The cells were scraped and transferred to an Eppendorf tube. The lysates were centrifuged (FANEM) at 10,000 × g for 10 min at 4°C, and the supernatant was stored in a new tube at −80°C. The total protein content of the lysates was determined using the Bradford method (Sigma-Aldrich). The cell lysates were analyzed, and the proteins were semi-quantified using the PathScan® phospho-NFκBp65 (Ser536), phospho-IκBα (Ser32) and phospho-c-Jun sandwich ELISA kits, according to the manufacturer’s instructions . Briefly, the lysates were diluted with sample diluent, and 100 µl of the lysate was added to wells that had been pre-coated with the primary antibody. The plate was incubated overnight at 4°C and then washed 4 times. After washing, the plates were incubated with the detection antibody for 1 h at 37°C. The plates were washed and then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 30 min at 37°C. The plates were then incubated with a 3,3′,5,5′-tetramethylbenzidine (TMB) substrate for 30 min at room temperature. Finally, the reaction was stopped, and the absorbance of the samples was read at 450 nm. The assay was performed in technical duplicates. The results were expressed as percentages of the levels of phosphorylated proteins in the control.To assess the levels of several phosphoproteins that activate inflammation in macrophages, a multi-target ELISA kit was used . Peritoneal macrophages were plated in 12-well microculture plates at a density of 3×102 atmosphere. RPMI medium was used as the negative control. After treatment, the adherent macrophages were washed with ice-cold PBS, and Cell Lysis Buffer containing protease and phosphatase inhibitors was added. The cell lysates were transferred to an Eppendorf tube and immediately placed in a boiling water bath for 5 min. The total protein concentration was adjusted to 1 µg/µl, and the cell lysates were stored at −80°C prior to measurement. The expression levels of p-JNK1/2 (T183/Y185), p-p38 (T180/Y182) and p-ERK1/2 (T202/Y204) were quantitatively measured using antibodies from the multiplex Flex Set Cytometric Bead Array (Becton Dickinson). Serial dilutions (1/2 v/v) of the standards were prepared, and the cell lysates were diluted (1/4 v/v) using the assay diluent. The samples were incubated with 50 µl of the mixed capture beads for 3 h at room temperature, followed by incubation with 50 µl of phycoerythrin (PE) detection reagent for 1 h at room temperature. Then, 300 µl of wash buffer was added, and the samples were centrifuged at 400 × g for 5 min. Flow cytometry was performed using the Becton Dickinson FACSArray Bioanalyzer software. A total of 900 events were acquired, according to the manufacturer’s protocol. The minimum detection levels for each phosphoprotein were 0.38 U/ml for p-JNK and 0.64 U/ml for p-p38 and p-ERK.The sample preparation was performed according to the manufacturer’s protocol for adherent cells (Becton Dickinson). Peritoneal macrophages were plated in 12-well microculture plates as described above. The cells were stimulated with TsV (50 µg/ml) for 15, 30, 60 and 120 min at 37°C in a 5% COp values lower than 0.05 were considered statistically significant. For gene expression, changes in expression levels were considered statistically significant when p<0.05 and a fold-change >2.0 was detected.The data are expressed as the means ± SEM and were analyzed using one-way ANOVA (α = 0.05). Limulus amoebocyte lysate test (data not shown) and TsV polymyxin B treatment , we observed no changes in TNF-α induction  of TsV used in these experiments had no cytotoxic effects . We invenduction .4 and PGE2 are produced in response to Ts2 and Ts6, which are toxins that can be isolated from scorpion venom 2 participates in TsV-induced edematous responses in mice 2 and LTB4 production in peritoneal macrophages, we measured the concentration of these lipid mediators in the cell culture supernatants after 24 h of stimulation. We found that both TsV and Ts1 significantly induced PGE2 and LTB4 release in peritoneal macrophages  analysis of stimulated and non-stimulated macrophages revealed that TsV up-regulated the expression levels of the genes Tlr2, Cd14, Myd88 and Ptgs2 without affecting the expression levels of Tlr4, Alox5ap, Ltb4r1 or Ptges2. Furthermore, Ts1 up-regulated the Cd14 and Ptgs2 genes without affecting the expression levels of Tlr2, Tlr4, Myd88, Alox5ap, Ltb4r1 and Ptges2. The effects of Ts1 on gene expression were less dramatic than those observed with TsV-stimulation , we observed the increased production of both LTB4 and PGE2 following WT macrophage stimulation with either TsV or Ts1. On the contrary, the PGE2 production in response to TsV or Ts1 was almost completely absent in TLR4- and CD14-KO cells. Intriguingly, the amount of PGE2 produced by cells lacking TLR2 was significantly higher than the quantity released by WT cells  and the mitogen-activated protein kinase (MAPK)  120 min . Additio 120 min . LPS, a −/− and TLR4−/− mice were stimulated in vitro with Ts1 for 10 or 120 min. We observed increased levels of p-NF-κB  for 10 min had decreased levels of phosphorylation for NF-κB , mycoplasma diacylated lipoprotein , lipoarabinomannan (AraLAM), zymosan and protozoan GPI anchors Macrophages play a key role in inflammation 2 production following TsV or Ts1 stimulation was dependent on TLR4 and CD14, but independent of TLR2, similar to what has been reported for LPS-stimulated macrophages 4 production following TsV or Ts1 stimulation was dependent on TLR2 and TLR4, but independent of CD14. We have two possible explanations for these results: 1) in the absence of TLR2, the activation of cyclooxygenase-2 is enhanced; or 2) the half-life of cyclooxygenase-2 is enhanced, which is a common and potent negative regulatory mechanism. Regarding the CD14/LTB4 axis, we speculate that, in the absence of CD14, neither TLR4 nor TLR2 act properly. Coffey et al. Moreover, our results demonstrated that PGEAnother objective of the current study was to identify the signaling pathway triggered by TsV downstream of TLR2, TLR4 and CD14. The activation of NF-κB is essential for the coordination of innate and adaptive immune responses, including the production of pro-inflammatory mediators Subsequently, we determined whether the TsV-mediated activation of NF-κB and AP-1 occurred downstream of TLR2 or TLR4 and whether MyD88 played a role in this process. TLR2 often heterodimerizes with TLR1 or TLR6 to respond to many TLR2 ligands −/− and TLR4−/− macrophages upon Ts1 stimulation, our primary findings are that venom recognition occurs through TLR2 and TLR4, resulting in NF-κB activation to generate IL-6 and TNF-α. Our results do not exclude the possibility that c-Jun activation might be due to other membrane receptor activation, which deserves further investigation.To the best of our knowledge, the present study is the first to demonstrate that TsV and Ts1 can induce the production of inflammatory mediators by interacting with TLR2 and CD14/TLR4. We also demonstrated that the phosphorylation of NF-κB is TLR2- and TLR4/MyD88-dependent and is most likely mediated by Ts1, the primary component of TsV. In contrast, TLR2/MyD88-independent phosphorylation of c-Jun may be the result of other components of TsV. Although we observed the increased phosphorylation of c-Jun in TLR2T. serrulatus envenomation. Alternatively, although a number of antivenom treatments that typically attempt to neutralize the harmful venom exist, our results suggest that TLR antagonism may have potential therapeutic benefits following envenomation.These findings indicate that TsV signaling through TLR2 and TLR4/CD14 activates the NF-κB and MAPK signaling pathways, which may serve as important therapeutic targets for the prevention of the deleterious effects that result from the intense systemic inflammatory mediator release following Figure S1TsV and Ts1 do not affect cell viability. (A) Adherent macrophages were stimulated for 24 h with TsV at the indicated concentrations (25–150 µg/ml) in a 5% CO2 atmosphere at 37°C. Cell viability was measured using the MTT assay. Each column represents the mean value of 8 samples from 2 independent experiments. (B) Peritoneal macrophages were stimulated with TsV or Ts1 (50 µg/mL) for 24 h. After this period the disrupt membrane cell was determined by LDH release. *p<0.001 (one-way ANOVA) compared to the medium alone. (C and D) TsV or Ts1 inactivation by heating reduces IL-6 and TNF-α release in peritoneal macrophages. 50 µg/mL of TsV or Ts1 were heated or not at 60°C or 95°C and after 24 hours the cytokines were measured in the supernatant by ELISA. *p<0.001 (one-way ANOVA) compared to the medium alone; #p<0.001 compared to TsV room temperature (RT) and &p<0.001 compared to Ts1 RT.(TIF)Click here for additional data file.Figure S2Peritoneal macrophages from C57Bl/6 mice stimulated with TsV and LPS (0.5 µg/ml) were pre-incubated with or without polymyxin B (poly) to neutralize contaminating LPS. After 24 h, the amount of TNF-α in the supernatant was determined by ELISA. *p<0.001 (one-way ANOVA) compared to the medium alone (dashed line). The values are expressed as the mean ± SD (n = 8). The data are from 2 independent experiments.(TIF)Click here for additional data file.Figure S3p38 and ERK1/2 MAPKs are involved in TsV-induced macrophage activation. p38 (a), ERK1/2 (b) and JNK1/2 (c) expression was determined in peritoneal macrophages from C57Bl/6 mice stimulated with TsV (50 µg/ml) for 10, 30, 60 or 120 min. Medium alone was used as the negative control. The data are expressed as the fold-increase over the control (PBS) ± standard deviation (SD). *p<0.05 (one-way ANOVA) compared to medium alone (dashed line). The values are expressed as the mean ± SD (n = 4), and the data are from 2 independent experiments.(TIF)Click here for additional data file.Figure S4TsV induces the phosphorylation of NF-κB, IκBα and c-Jun in stimulated macrophages. Adherent peritoneal macrophages from WT (C57Bl/6) mice were stimulated with TsV (50 µg/ml) for 10 min, 2 and 24 h in a 5% CO2 atmosphere at 37°C. Medium alone was used as the negative control. p-NF-κB (a), p-IκBα (b) and p-c-Jun (c) protein levels were measured using the PathScan Inflammation Multi-Target Sandwich ELISA kit. The results are presented as the percentage of the relative levels of the phosphoproteins and are normalized to the positive control (100%). *p<0.05 (one-way ANOVA) compared to medium alone. The values are expressed as the mean ± SD (n = 4), and the data are from 2 independent experiments.(TIF)Click here for additional data file.


Organic light-emitting diodes (OLEDs) are attractive for next-generation displays and lighting applications because of their potential for high electroluminescence (EL) efficiency, flexibility and low-cost manufacture. Although phosphorescent emitters containing rare metals such as iridium or platinum produce devices with high EL efficiency, these metals are expensive and their blue emission remains unreliable for practical applications. Recently, a new route to high EL efficiency using materials that emit through thermally activated delayed fluorescence (TADF) was demonstrated. However, it is unclear whether devices that emit through TADF, which originates from the contributions of triplet excitons, are reliable. Here we demonstrate highly efficient, stable OLEDs that emit via TADF by controlling the position of the carrier recombination zone, resulting in projected lifetimes comparable to those of tris(2-phenylpyridinato)iridium(III)-based reference OLEDs. Our results indicate that TADF is intrinsically stable under electrical excitation and optimization of the surrounding materials will enhance device reliability. Usually, a phosphorescent emitter, such as iridium124EQE) of 20% that was comparable to that of phosphorescent OLEDs1 to S1 by thermal activation. To obtain efficient TADF, a small energy gap between the S1 and T1 states (ΔEST) is required, which is realized in molecules with a small overlap between their highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO). Recent designs of molecules that exhibit TADF have been based on a combination of the donor and acceptor units, and have achieved ΔEST comparable to or lower than the thermal energy of 27 meV at 300 K.Recently, we produced high-efficiency OLEDs by using a new route to produce thermally activated delayed fluorescence (TADF)78Phthalonitrile derivatives such as -2,4,5,6-tetra(9H-carbazol-9-yl)isophthalonitrile (4CzIPN) are promising emitters for highly efficient TADF-based OLEDs2 in TADF-based OLEDs while maintaining a high ηEQE that is comparable to those of OLEDs containing iridium complexes.In this work, to investigate the possibility of TADF-based OLEDs with high operational stability, we carefully designed a device architecture that included exciton-blocking layers (EBLs) at the interfaces of the emission layer (EML) and optimized the concentration of the emitter. Here we show that expanding the carrier recombination zone to enhance the electron carrier injection efficiency significantly affects the operational stability of the device. We realize lifetimes of more than 2,500 h at an initial luminance of 1,000 cd/mTo maximize the TADF efficiency, we prepared a guest-host system that escapes concentration quenching, similar to conventional fluorescence and phosphorescence based OLEDs. Also, because TADF is based on an up-conversion process of triplet excitons into a singlet state, improper protection of the triplet excitons induces Dexter energy transfer into the surrounding molecules during the lifetime of the guest's triplet state, which resulted in exciton quenching. It is therefore necessary to introduce host and carrier transport molecules with higher triplet energies than that of a guest TADF molecule.PL) of an EML is one of the main factors in obtaining a high ηint in OLEDs. The use of 3,3-di(9H-carbazol-9-yl)biphenyl (mCBP) as a host matrix with 6 wt.%-4CzIPN showed a slightly higher ΦPL = 85 ± 2% than the ΦPL = 80 ± 2% of 4,4′-bis(carbazol-9-yl)biphenyl (CBP), as shown in 1 = 2.90 eV1 = 2.4 eV) well, and does so better than CBP with T1 = 2.55 eV. Also, in a dilute concentration of 1 wt.%, the maximum ΦPL = 94 ± 2% was obtained, as shown in The high photoluminescence (PL) quantum efficiency , tris(4-carbazoyl-9-ylphenyl)amine  completely quench the excitons of 4CzIPN when they are used as hosts, giving very low ΦPL values of 2%, 5%, 7% and 5%, respectively. This is because of the low triplet energy level of α-NPD, the strong electron donation ability of the triphenylamine units, and the rather shallow HOMO levels of these materials  or an EBL, Φvaluated . We founaterials . TherefoPL for co-deposited films of 6 wt.%-4CzIPN and various EBLs, including bis(2-methyl-8-quinolinolate)-4-(phenylphenolato)aluminum (BAlq2)PL of 22% was obtained in the BAlq2 host, while a high ΦPL of 80% was obtained in the CzTRZ and T2T host matrices. This clearly indicates that the BAlq2 host quenches a triplet exciton of 4CzIPN because of the low triplet energy level of BAlq2.We also determined Φ3)-based OLED to act as a reference device.Based on this preliminary PL experiment, OLEDs were fabricated by the sequential deposition of a 10 nm-thick layer of dipyrazinoquinoxaline-2,3,6,7,10,11-hexacarbonitrile (HAT-CN) as a hole injection layer (HIL), a 30 nm-thick layer of Tris-PCz as an HTL, an EML consisting of a 30 nm-thick layer of mCBP doped with various concentrations of 4CzIPN, a 10 nm-thick layer of T2T as an EBL, a 40 nm-thick layer of 2,7-bistriphenylene (BPy-TP2)J) characteristics of the 3, 6, 10 and 15 wt.% 4CzIPN-doped devices are shown in EQE = 17 ± 0.5%), 49 cd/A (ηEQE = 15.6 ± 0.5%), 48 cd/A (ηEQE = 14.2 ± 0.5%) and 47 cd/A (ηEQE = 14.0 ± 0.5%) at J = 0.01, 0.04, 0.5 and 1.0 mA/cm2, respectively. For the 3 and 6 wt.% 4CzIPN-doped devices, the highest ηEQE was observed at low current density (<0.1 mA/cm2). In contrast, the 10 and 15 wt.% 4CzIPN-doped devices showed a gradual increase in ηEQE with increasing J, indicating that the balance of the hole and electron carrier injection changes with the dopant concentration. At rather high current densities, a high ηEQE of 13.8 ± 0.5% (46 cd/A) was obtained at 1,000 cd/m2 for the 10 wt.%-doped device, which is higher than those of the 3 and 6 wt.%-doped devices.The current efficiency (cd/A) vs. current density  and ηr,T is the proportion of triplet excitons (75%) produced under electrical excitation. From transient PL analysis (F) and the TADF component (ΦTADF) were estimated to be 27% and 51%, respectively, resulting in a reverse ISC (RISC) efficiency (ΦRISC) of 69%, which was estimated from the relation (1 − ΦF) ΦRISC = ΦTADF. The theoretical maximum ηEQE is thus estimated to be 14.2%, assuming a light out-coupling efficiency of 20%. Therefore, near-complete carrier recombination and exciton confinement in the EML were realized in the 10 wt.%-doped device.We derived the theoretical maximum ηanalysis , the con2 to examine the effect of the recombination zone position on the device performance. The OLEDs with CzTRZ and BAlq2 HBLs had inferior characteristics when compared with those of the devices with T2T HBLs  from T2T to CzTRZ and BAlqT2T HBLs . In partT2T HBLs , inset, 0 of 1,000 cd/m2 and 0 = 1,000 cd/m2, we estimated an acceleration factor of 1.92 for each device from the lifetime measurements at L0 = 2,000, 5,000 and 10,000 cd/m2 using the following well-known equation3-doped device , as summarized in To predict LT50 for the devices containing 10 and 15 wt.% 4CzIPN at L1 of 4CzIPN was observed at 1,000 cd/m2 in all devices, another emission peak around the deep blue region was observed in the 3 and 6 wt.%-doped devices at 20,000 cd/m2, suggesting that the carrier recombination zone position changes in these devices, as shown in 2, indicating that the carrier recombination zone moves during constant operation. However, no additional emission signal was observed from the 15 wt.%-doped devices  and hole-only devices (HODs) were fabricated, as shown in the insets of ectively . In cont3. This is an encouraging sign that the emitter itself is quite stable for redox and oxidation processes under electrical excitation. Thus, because Ir(ppy)3 derivative-based OLEDs with optimum materials and device architectures have realized very long lifetimes of over 100,000 hrs.22Finally, we consider the possibility of further enhancement of the device reliability. In this study, we adopted conventional host and carrier transport materials and obtained comparable degradation lifetimes for 4CzIPN and Ir(ppy)EQE of 13.9 ± 0.5% at 1,000 cd/m2) with excellent operational stability, showing LT50 of 2,800 h at 1,000 cd/m2 and of over 10,000 h at 500 cd/m2. We also found that the 4CzIPN molecules act as strong electron trapping sites in the mCBP EML, and that the position of the recombination zone strongly affects the operational lifetime of these devices. Our results confirm that TADF-based OLEDs show great potential for realization of both high efficiency and operational stability.In summary, we clarified that the operational lifetime of 4CzIPN-based OLEDs depends strongly on the emitter concentration in the EML and demonstrated highly efficient TADF-based OLEDs (ηPL) was measured by an absolute PL quantum yield measurement system  under a nitrogen gas flow with excitation wavelengths of 337 or 275 nm. The low-temperature PL intensity and the emission lifetime were measured using a streak camera  and a cryostat  with a nitrogen gas laser  as the excitation light source under a pressure of about 3 Pa.Samples for the optical measurements were fabricated by co-depositing host materials and 6 wt.% 4CzIPN with a thickness of 50 nm on a quartz substrate. The PL quantum efficiency (Φ2 were fabricated on 110 nm-thick indium tin oxide (ITO)-coated glass substrates with a 2 mm stripe pattern. Deposition was performed under vacuum at pressures from 5 × 10−4 to 5 × 10−5 Pa. After fabrication, the devices were immediately encapsulated with glass lids using epoxy glue in nitrogen-filled glove boxes . A commercial calcium oxide desiccant  was included in the encapsulated package. The devices were exposed once to nitrogen gas after the formation of the organic layers because a metal mask was included to define the cathode area. The J-V-luminance characteristics were evaluated using a Keithley 2400 source meter and an absolute ηEQE measurement system . The operational lifetime was measured using a luminance meter  at a constant DC current at room temperature.Green TADF-based OLEDs with an effective area of 1 mmThe experiments were conceived and designed by H.N. and K.M., and were carried out by H.N., K.M. and J.N. H.N. and C.A. wrote the manuscript. The project was supervised by T.S. and C.A. All the authors discussed the results and contributed to the article.Supplementary Info File #1


One of the pathological hallmarks of Alzheimer's disease (AD) is the deposition of the ~4 kDa amyloid β protein (Aβ) within lesions known as senile plaques. Aβ is also deposited in the walls of cerebral blood vessels in many cases of AD. A substantial proportion of the Aβ that accumulates in the AD brain is deposited as Amyloid, which is highly insoluble, proteinaceous material with a β-pleated-sheet conformation and deposited extracellularly in the form of 5-10 nm wide straight fibrils. As γ-secretase catalyzes the final cleavage that releases the Aβ42 or 40 from amyloid β -protein precursor (APP), therefore, it is a potential therapeutic target for the treatment of AD. γ-Secretase cleavage is performed by a high molecular weight protein complex containing presenilins (PSs), nicastrin, Aph-1 and Pen-2. Previous studies have demonstrated that the presenilins (PS1 and PS2) are critical components of a large enzyme complex that performs γ-secretase cleavage.In this study we used RNA interference (RNAi) technology to examine the effects of small-interfering RNA (siRNA) against PS1 on expression levels of PS1 and Aβ42 in IMR-32 Cells using RTPCR, western blotting and immunofluorescence techniques.The results of the present study showed down regulation of PS1 and Aβ42 in IMR32 cells transfected with siRNA against PS1.Our results substantiate the concept that PS1 is involved in γ-secretase activity and provides the rationale for therapeutic strategies aimed at influencing Aβ42 production. A key step in the pathogenesis of AD is proteolysis of APP that results in the formation of the amyloid-β protein (Aβ), the principle protein component of the characteristic cerebral plaques of the disease . Aβ is p2 in Eagle's minimum essential medium supplemented with 2 mM L-glutamine and Earle's BSS, 1.5 g/L bicarbonate, 0.1 mM nonessential amino acids, 1.0 mM Sodium pyruvate and 10% heat-inactivated fetal bovine serum. Media was changed every two days. Passage was done in 1:3 ratios and doubling time was 24 Hours.Human neuroblastoma IMR-32 cells were obtained from NCCS, Pune, India and maintained at 37°C in an atmosphere of 95% air and 5% CO5 cells were seeded per well in 2 ml antibiotic-free normal growth medium supplemented with FBS. The cells were incubated at 37°C in a CO2 incubator until the cells were 60-80% confluent for 18-24 hours. Transfection of PS1 siRNA  and control siRNA duplex was done with the transfection reagent after checking the cell viability with the trypan blue and the cells were incubated for 5-7 hours at 37°C in a CO2 incubator. Then 1 ml of normal growth medium was added, containing 2 times the normal serum and antibiotics concentration  without removing the transfection mixture and the cells were incubated for an additional 18-24 hours. After incubation, media was aspirated and replaced with 1X normal growth medium and incubated for 48 hours. After 48 hours cells were used for RNA (Taurus Scientific) and protein isolation, used for RT-PCR and western blotting analysis respectively.In a six well tissue culture plate, 2 × 10Expression of PS1 after transfecting IMR-32 cells with siRNA against PS1 was evaluated by PCR analysis using sequence specific primers corresponding to the sequence in the open reading frame. 10 μl PCR mixture was prepared in 1× PCR buffer consisting of 1 unit of Taq polymerase, 2 μM of each primer for GAPDH and PS1 along with 200 μM of each dNTP. For amplification of PS 1 and GAPDH, cDNA products (1 μl) were subjected to reverse transcriptase PCR analysis on a gradient thermal cycler instrument . PCR cycle for PS1 and control siRNA comprised of initial denaturation at 94°C for 2 min. The amplification was then carried out for 35 cycles consisting 45 sec each for 94°C (denaturation), 62°C  and 72°C, 1 min (elongation). Final extension was done at 72°C for 10 min. PCR cycle of GAPDH, which was used as internal control, comprised of initial denaturation at 94°C for 2 min. The amplification was then carried out for 35 cycles consisting 45 sec each for 94°C (denaturation), 56.8°C  and 72°C, 1 min (elongation). Final extension was done at 72°C for 10 min. Densitometric analysis of the products was done using SCION IMAGE software  to compare the relative mRNA expression of PS1 gene at various concentrations of PS1 siRNA duplex.Following SDS-PAGE (12%), proteins were transferred to NC membrane with semi dry apparatus (Bio Rad) using transfer buffer . The blots were incubated with 5% non fat dried milk for at least 2 h and then washed three times for 10 min each with PBS plus 0.1% Tween-20. Blots were incubated for 2.5 h at room temperature with anti PS1antibody (diluted 1:250 in PBS) . The primary antibody was removed, and the blots were washed three times for 10 min each with PBS plus 0.1% Tween-20. The blots were then incubated in horseradish peroxidase-labeled goat anti-rabbit IgG  for 45 min at room temperature. After washing the membrane for at least 1 hour, the proteins were visualized by diaminobenzidine (DAB) from Bangalore Genei, India The densitometric analysis of the protein bands was carried out using SCION IMAGE software  to compare the relative expression of proteins.16%), proteins were transferred to NC membrane with semi dry apparatus (Bio Rad) using transfer buffer . The blots were incubated with 5% non fat dried milk for at least 2 h and then washed three times for 10 min each with PBS plus 0.1% Tween-20. Blots were incubated for 2.5 h at room temperature with anti Aβ42 antibody (diluted 1:250 in PBS) . The primary antibody was removed, and the blots were washed three times for 10 min each with PBS plus 0.1% Tween-20. The blots were then incubated in horseradish peroxidase-labeled goat anti-rabbit IgG  for 45 min at room temperature. After washing the membrane for at least 1 hour, the proteins were visualized by diaminobenzidine (DAB), from Bangalore Genei, India. The densitometric analysis of the protein bands was carried out using SCION IMAGE software  to compare the relative expression of proteins.Following SDS-PAGE   for 24 hours at 4°C. After 24 hours incubation, cells were rinsed three times with 1X PBS and incubated with FITC labelled secondary antibody (1:50 dilutions) for 2 hours in dark at 37°C. The cells were visualized under the fluorescence microscope and the images were recorded.pmoles, 75 pmoles, and 100 pmoles) of PS1 siRNA and 100 pmoles of control or scrambled siRNA. Semiquantitative PCR analysis of PS1 showed reduction in PS1mRNA in IMR-32 cells with increasing concentration of PS1 siRNA with maximum reduction at 100 pmoles  using antibodies against PS1 and Aβ42. As is evident from Figure pmole concentration. Therefore, down regulation of PS1 using PS1 siRNA results in down regulation of Aβ42 as well.In order to confirm the results obtained from western blotting further immunoflourescence was carried out after transfecting IMR-32 cells with the control siRNA and PS1 siRNA in the same concentrations (50 γ-Secretase activity is responsible for the cleavage of the transmembrane domain of the amyloid precursor protein (APP), releasing the amyloid peptide Aβ and the APP intracellular domain. Aβ is a major component of the amyloid plaques characteristic of Alzheimer's disease. Presenilins (PS1 and PS2) appear to be essential for γ-secretase cleavage and form an essential part of the γ-secretase complex. PS1 knockout markedly inhibits γ-secretase cleavage [cleavage , whereascleavage , probablcleavage . To datecleavage . On the cleavage ,24. The cleavage . The faccleavage .Based on mutagenesis and inhibitor cross-linking studies, it has been suggested that PS1 constitute the catalytically active subunit of γ-secretase. However, PS1 is not sufficient for γ-secretase activity in vitro and multiple subunits have been identified that are essential for generation of the active enzyme. Several of these subunits are apparently limiting for γ-secretase activity in the cell. Since increase in Aβ42 is consistently linked to AD pathogenesis , the finSo in the present study we have shown that siRNA against PS1 down regulates amyloid beta 42 production in IMR32 cells. Taken together the results of the present study suggest that in pathogenic condition we may target PS1 for anti-amyloidogenic therapy in Alzheimer's disease as IMR32 is an AD featured cell line. These results also reconfirm a role of PS1 in γ-secretase activity.Aβ: Amyloid β; AD: Alzheimer disease; APP: amyloid precursor protein; CTF: C-Terminal fragment; FAD: Familial Alzheimer disease; NTF: N-Terminal fragment; PS: presenilin; siRNA: small-interfering RNA.We don't have any potential conflicts regarding all financial and material support for this research.RJLK planned the experimental design, analyzed and interpreted data, and drafted the manuscript. WYW acquired and interpreted data and drafted the manuscript. BKBK analyzed data. KDG planned the experimental design and analyzed and interpreted data. All authors read and approved the final manuscript.


Objective(s): Different pharmacological effects of carvacrol including relaxant effect, its inhibitory effect on muscarinic and histamine (H1) and stimulatory effect on β-adrenoceptors have been demonstrated on guinea pig tracheal chains in previous studies. In the present study, the effect of carvacrol on blood IL-4, IFN- γ and endothelin levels of sensitized guinea pigs is examined. Materials and Methods: Five groups of guinea pigs sensitized to ovalbumin (OA) were given pure drinking water (group S), drinking water containing three concentrations of carvacrol ) and dexamethasone. The blood IL-4, IFN- γ and endothelin levels of sensitized and control guinea pigs were evaluated . Results: Blood IL-4 and IFN-γ levels (P<0.001 for both cases) as well as endothelin (P<0.01) were increased but IFN-γ/IL-4 ratio decreased (P<0.05) in sensitized animals compared to controls. The treatment of S animals by dexamethasone (P<0.01) and two higher concentrations of carvacrol (P<0.001 for both cases) significantly decreased IL-4 level. The treatment of S animals with dexamethasone did not changed IFN-γ levels but treatment with high concentration of carvacrol significantly increased its level (P<0.001). In addition, IFN-γ/IL-4 ratio was significantly increased in S groups, who were treated with dexamethasone (P<0.05) and two higher concentrations of carvacrol (P<0.001 for both cases). Treatment of S animals by dexamethasone (P<0.01) and all concentrations of carvacrol also significantly decreased endothelin level (P<0.01 to P<0.001).Conclusion: The results show that carvacrol causes the reduction of IL-4 and endothelin, but it increases IFN-γ and IFN-γ/IL-4 ratio in the blood of sensitized guinea pigs. The results also suggest more specific effect of carvacrol compared to dexamethasone due to the absence of the effect of later on IFN-γ. Differe Th1/Th2 . Therefo6H3CH3(OH)(C3H7) is one of the main constituent of everal medicinal plants including Zataria multiflora Boiss 3 dissolved in 1 ml saline i.p. as a booster dose. From day 14 sensitized animals were exposed to an aerosol of 4% OA for 18±1 days, 5 min daily. The aerosol was administered in a closed chamber, dimensions 30 x 20 x 20 cm. Control animals were treated similarly but saline was used instead of OA solution. The study was approved by the ethical committee of the ashhad University of Medical Sciences.Animals were sensitized to OA according the method described previously , 18. BriThe study was performed in control animals  and five different groups of sensitized animals which were pure given drinking water  or drinking water containing treatment agents during sensitization period as follows (n=6 for each group);1. 50 µg/ml dexamethasone (group S+D)2. 40 µg/ml carvacrol (group S+C1)3. 80 µg/ml carvacrol (group S+C2)4. 160 µg/ml carvacrol (group S+C3)Measurement of blood IL-4 and IFN- γlevelsFive ml of peripheral blood was obtained immediately after sacrificing the animals and placed at room temperature for 1 hr. The samples were then centrifuged at 2500 rpm at 4°C for 10 min. The supernatant was collected and immediately stored at 70° C until analyzed. Finally, blood IL-4 and IFN- γ levels were measured using Elisa sandwich (Ab Sandwich) method. The Elisa kits were purchased from Bender Medsystem Sakhte, Austria. The ratio of IFN-γ/IL4 as an index of Th1/Th2 was also calculated.Measurement of blood endothelin levels Five ml peripheral blood was obtained immediately after sacrificing the animals and placed at room temperature for 1 hr. The samples were then centrifuged at 2500 rpm at 4°C for 10 min. The supernatant was collected and immediately stored at 70°C until analyzed. Blood endothelin was measured using the enzyme-linked immunosorbent assay (ELISA) Sandwich method according to the manufacturer’s instructions, .Statistical analysisP<0.05.The data were quoted as mean±SEM. According to the Kolmogorov Smirnov test, the data had normal distribution. The data of sensitized group were compared with the control guinea pigs using unpaired t- test. The data of treated groups was also compared with sensitized guinea pigs using unpaired “t” test. The data of three groups of animals treated with carvacrol were compared with each other using one way ANOVA through Tukey Kermar post hoc test. Significance was accepted at The serum levels of cytokine, endothelin and Th1/Th2 balanceP<0.001 for both cases, In the sensitized group, the serum levels of IL-4 (7.25±0.25) and IFN-γ (2.34±0.04) in group S were significantly higher than that of the group C (2.75±1.17 and 1.26±0.17 for IL-4 and IFN-γ respectively), (P<0.001 for both cases) and dexamethasone (P<0.01) caused significant reduction in IL-4. However, the level IFN-γ was increased in the treated group with the high concentration (160 µg/ml) of carvacrol  and two higher concentration of carvacrol (P<0.001 for both cases) caused significant increase in Th1/Th2 balance  was also significantly decreased in the sensitized group compared to the control group P<0.05, . However balance .P<0.01, P<0.01 to P<0.001, In addition, the serum level of endothelin (2.75±0.10) in group S were significantly higher than group C (1.76±0.14), P<0.01, . The treDifferences in the serum levels of cytokine, endotheline and Th1/Th2 balance between dexamethasone and carvacrol treated groupsP<0.05 to P<0.001, P<0.01 to P<0.001, The effect of the low concentration of carvacrol (40 µg/ml) on IL-4, IFN-γ, IFN-γ/IL4 ratio and endothelin was significantly lower than the effect of dexamethasone  on IL-4, IFN-γ, IFN-γ/IL4 ratio and endotheline was significantly higher than the effect of its low concentration  on IL-4, IFN-γ, endotheline and IFN-γ/IL4 ratio was significantly higher than the effect of dexamethasone.Airway inflammation occurring in asthma is regulated by the balance of Th1 /Th2 cells , 4. Th2 Th1 cells release interleukin 2 (IL-2) and IFN-γ, but Th2 cells produce IL-4 and IL-10 . TherefoTherefore, the results of the present study suggest that carvacrol has inhibitory effect on IL-4 but enhances the production of IFN- γ, indicating the inhibitory effect on Th2 and stimulatory effect on Th1 helper cells. The results of the present study also show raise in the ratio of IFN-γ to IL-4 due to carvacrol treatment in sensitized guinea pigs. However, serum IFN-γ level has also increased in sensitized guinea pigs treated with the last concentration of carvacrol while dexamethasone has not affected it. These results indicate that carvacrol causes inhibitory effect on Th2 similar to dexamethasone but has a stimulatory effect on Th1 helper cells which is different with the effect of dexamethasone. Therefore, the results of the present study show that in inflammatory disorders associated with decreasing the balance of Th1/Th2, carvacrol may show even more therapeutic values compared to dexamethasone.The effect of endothelin on asthmatic inflammation, proliferation of bronchial smooth muscle cells and subepithelial fibrosis has been suggested, that leads to airway remodeling and severe bronchial hyperreactivity . The effThe results also show the concentration-dependent effect of carvacrol on IL-4, IFN-γ and endotheline level as well as IFN-γ/IL-4 ratio. The greater effect of two higher concentration of carvacrol compared to dexamethasone suggests that the effect of carvacrol is more potent than dexamethasone on IL-4, IFN-γ and endotheline level as well as IFN-γ/IL-4 ratio in sensitized guinea pigs at concentrations used.Tymus volgaris) (2-adrnoceptors (1) receptors (Zataria multiflora (Our previous study showed the potent relaxant effect of the extract of other plant containing carvacrol (olgaris)  and carvolgaris)  on trachoceptors  and the eceptors . In addieceptors  and the ltiflora  on muscrThe results of this study showed that carvacrol caused the reduction of IL-4 and endothelin but increased IFN-γ and IFN-γ/IL-4 ratio in the blood of sensitized guinea pigs. The effect of carvacrol was more specific compared to dexamethasone due to the absence of the effect of later on IFN-γ. The results also suggested that the effect of carvacrol was more potent than dexamethasone on IL-4, IFN-γ and endotheline level as well as IFN-γ/IL-4 ratio in sensitized guinea pigs at concentrations used.


They were classified into two types as either the ubiquitous soluble LonA or membrane-inserted archaeal LonB. In addition to the energy-dependent forms, a number of medically and ecologically important groups of bacteria encode a third type of Lon-like proteins in which the conserved proteolytic domain is fused to a large N-terminal fragment lacking canonical AAA+ motifs. Here we showed that these Lon-like proteases formed a clade distinct from LonA and LonB. Characterization of one such Lon-like protease from Meiothermus taiwanensis indicated that it formed a hexameric assembly with a hollow chamber similar to LonA/B. The enzyme was devoid of ATPase activity but retained an ability to bind symmetrically six nucleotides per hexamer; accordingly, structure-based alignment suggested possible existence of a non-functional AAA-like domain. The enzyme degraded unstructured or unfolded protein and peptide substrates, but not well-folded proteins, in ATP-independent manner. These results highlight a new type of Lon proteases that may be involved in breakdown of excessive damage or unfolded proteins during stress conditions without consumption of energy.Lon proteases are a family of ATP-dependent proteases involved in protein quality control, with a unique proteolytic domain and an AAA Most of these specialized proteases are oligomeric and form an enclosed chamber, inside which the proteolytic sites are located and accessible only through a narrow axial pore Escherichia coli (hereafter EcLonA) and was the first known ATP-dependent protease + domain, which contains conserved motifs  for ATP binding and hydrolysis, and the C-terminal protease domain with a conserved serine-lysine catalytic dyad in its proteolytic active site + domains and specific domain organization features in which an N-terminal domain is attached to the AAA+ modules of all LonA but not LonB members, the latters are found only in archaebacteria and possess instead additional membrane-spanning segments integrated into their AAA+ domains that anchors the proteins to the membrane + consensus motifs Thermus thermophilusLon proteases are key components of the protein quality-control systems in bacteria and eukaryotic organelles responsible for degrading abnormal or unfolded proteins; they are also involved in regulation of many cellular processes by selective degradation of regulatory proteins M.taiwanensisFor the present work, we performed multiple sequence comparisons of LonA, LonB, and the Lon-like proteases, which revealed additional sequence features in the protease domain unique only to the latter but not present in the ATP-dependent LonA/B members. Phylogenetic analysis based on sequence alignment results indicated that these Lon-like proteins with unique conserved insertions belong to a clade of Lon proteases distinct from the LonA and LonB groups. This group of Lon-like proteases may be assigned to a new subfamily, LonC, following previous classification scheme Meiothermus taiwanensis WR-220 and identified three Lon homologs in this indigenous thermophilic Gram-negative bacterium. While two were LonA-like, the third gene encoded a novel Lon-like protein of 719 amino acids (hereafter MtaLonC) with a conserved C-terminal Lon protease domain but containing neither LonA-like N-terminal domain, nor transmembrane regions associated only with LonB or the conserved features of AAA+ modules (see below). MtaLonC expressed in M. taiwanensis at 55°C, the organism’s growth temperature, and the expression was induced at 65°C  MtaLonC , during aLonC  + modules were observed from solution studies + ring to generate mechanical power for substrate unfolding and translocation + proteases do. A strong support to this hypothesis comes from sequence alignment of TonLonB and MtaLonC, from which we identified a Walker A-like motif near the N-terminus  and a Walker B-like motif  in the middle portion of the sequence where the conserved Glu (preceding the conserved aspartate) essential for ATP hydrolysis is missing and replaced with a leucine. The conserved AAA+ residues, sensor-1 and sensor-2, are replaced by non-conserved residues; and the arginine finger is disrupted by a short insertion in MtaLonC  was used to analyze the binding isotherms of ATPγS and MtaLonC . The mix MtaLonC . Missing2+  and ATP KM for the ATP-independent reaction by MtaLonC was 14 µM, three-fold higher than EcLonA (4.6 µM), whereas the Vmax was 50-fold lower than EcLonA (0.2 vs. 10 µM/min), the latter however had no cleavage activity at all towards β20 in the absence of ATP , and the nucleotide sequence with similarities to the gene encoding the Lon protease was annotated and cloned into expression vector pET21a. The forward primer contained an ATG initiation codon and an NdeI restriction site. The reverse primer complementarily overlapped the 3′-end of the coding region and contained a HindIII site. After double-digestion with NdeI and HindIII, the product was purified and ligated into the NdeI/HindIII sites of the isopropyl β-d-thiogalactoside (IPTG)-inducible E. coli expression vector pET21a (+) . E.coli strain BL21(DE3)  was transformed with the recombinant plasmids. The nucleotide sequence of the gene encoding the Lon protease homolog MtaLonC was deposited in GenBank (accession number: GQ423488). POP1 with the FAWFP core octapeptide (WRFAWFPA) within β20 appended at the C-terminus (POP1-β20) was created by a PCR-based mutagenesis protocol The thermophilic bacterium M. taiwanensis, together with five and four sequences from the LonA and LonB subfamilies, respectively, were downloaded for phylogenetic analysis. Sequences were aligned using the program MUSCLE Protein sequence of MtaLonC was submitted to the BLAST network service of the Swiss Institute of Bioinformatics (SIB). Selected sequences that are highly similar to Diluted MtaLonC solution (∼ 50 µg/ml) was negatively stained by 2% uranyl acetate (UA). A FEI Tecnai G2 Spirit Twin transmission electron microscope operating at 120 kV was employed to serve the EM data collection. The specimen was imaged on a Gatan 1k×1k CCD at a magnification of 30000x, conferring a pixel size of 5.6 Å. The corresponding defocus values were in the range of −2.0 ∼ −2.3 µm  with an An-60 Ti rotor. The UV absorption (280 nm) of the cells was scanned every 4 min for 250 scans. The data were analysed with the software SEDFIT 2, 1.0 mM ATP, and 5 µg MtaLonC in a total volume of 100 µl. Solutions were incubated at 37 or 50°C for 40 min, and the color of the reaction was developed by adding 800 µl malachite/molybdate solution. The reactions were terminated by adding 100 µl 34% sodium citrate. The absorbance of the final reaction was determined at 660 nm on a Beckman Coulter DU730 UV/Vis spectrophotometer.The ATPase activity of MtaLonC was measured as described previously 2, 10 mM ATP and 30 µM of MtaLonC in buffer containing 20 mM HEPES (pH 7.5), 100 mM NaCl, 10 mM ATP were incubated at 37°C for 5 days. NMR studies were carried out on a Bruker AC 400 spectrometer.30 µM of EcLonA in buffer containing 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2 was analyzed using Origin software .ITC measurements were conducted on an ITC200 Microcalorimeter . The heat produced by the complex formation while 1 mM ATPγS was mixed with 0.1 mM MtaLonC at 25°C in the buffer containing 20 mM sodium citrate pH 5.5, 300 mM NaCl, 10 mM MgCl2)]A}, flanked by a pair of fluorophore (Abz) and quencher (nitrotyrosine) 2, pH 7.5 with 1 mM ATP, ADP, or AMPPNP were used as experimental groups. The reactions were carried out at 55°C. The fluorescence spectra were recorded with a Jobin Yvon Fluorolog-3 fluorescence spectrophotometer. Excitation was at 320 nm, and emission was measured at 420 nm.The fluorescence peptide assays were performed with the F-β20-Q peptide {ortho-aminobenzoic acid (Abz)- QLRSLNGEWRFAWFPAPEAV[Tyr on a MonoS HR5/5 column . Purified αS2-casein (0.5 µM), which also contained a minor amount of isoform S1, was incubated with MtaLonC (1.5 µM) in the buffer containing 0.1 mM NaCl, 20 mM HEPES pH7.5 at 60°C. POP1-β20 (0.25 mg/ml) or E. coli Huβ (0.2 mg/ml) were incubated with EcLonA (0.2 mg/ml) in 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 10 mM ATP, and incubated with MtaLonC (0.2 mg/ml) in 0.1 mM NaCl, 20 mM HEPES pH 7.5. POP1 was incubated with E. coli Lon (0.16 mg/ml) in 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, with or without 10 mM ATP. Samples were analyzed by SDS-PAGE. Hen lysozyme (7 µg) was incubated with or without MtaLonC (5 µg) in the presence or absence of TCEP (1 mM Tris(2-carboxyethyl)phosphine) in 50 mM Tris-HCl (pH 8.0), 10 mM Na2HPO4 (20 µl) at 55°C for 3 hours and analyzed by SDS-PAGE.αS2-Casein was in the buffer containing 0.1 mM NaCl, 20 mM HEPES pH 7.5, whereas MtaLonC was in the buffer containing 10 mM Na2HPO4, 2 mM KH2PO4, 3 mM KF and 70 mM NaF, pH 7.4. The POP1-β20 (0.5 mg/ml) was in 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2 and measured at different temperature. E. coli Huβ (4.0 mg/ml) was in 0.1 mM NaCl, 20 mM HEPES pH 7.5 and measured at different temperature. The far-UV CD spectra were the mean of three accumulations with a 0.1-cm light path.Far-UV CD spectra were recorded with JASCO J-715 spectropolarimeter. αM. taiwanensis was grown in medium containing 0.3% peptone, 0.1% yeast extract, 0.1% glutamic acid, and Castenholz salts, pH 7.8, at 55°C and 65°C. Bacteria cells were lysed in B-PER bacterial protein extraction reagent . Bacteria lysates (100 µg) were separated by SDS-PAGE and electrotransferred to a PVDF membrane. The membranes were incubated at room temperature for 1 h in blocking solution  containing 0.1% Tween 20) and then at room temperature for 2 h with anti-MtaLonC antibody (1∶40000). After three washes in PBS with 0.1% Tween 20, the membrane was incubated with an HRP-conjugated secondary antibody (1∶50000). The membrane was developed using an enhanced chemiluminescence Western blot detection system.2HPO4, pH 8.0 for 0, 15, 30, and 60 min. After incubation, equal volume (60 µl) of 7.4 M guanidine hydrochloride was added to stop enzyme reactions. The samples were analyzed by reverse phase HPLC (C18 column) with line gradient  for 60 min. The sample of each peak was collected and analyzed by Synapt G2 HD mass spectrometer. The MS/MS data was analyzed by Mascot search.Bovine casein (200 µg) was incubated with MtaLonC (15 µg) in the buffer (60 µl) containing 50 mM Tris HCl, 10 mM NaFigure S1Expression of MtaLonC. Western blotting of MtaLonC showed that more MtaLonC was expressed at 65°C. Lane 1: Meiothermus taiwanensis growth at 55°C for one day. Lane 2: Meiothermus taiwanensis growth at 65°C for one day. All samples were loaded at 100 µg of protein per lane. Arrow indicated the signal of MtaLonC.(TIF)Click here for additional data file.Figure S2SDS-PAGE and CD spectra of MtaLonC. (A) Coomassie Brilliant Blue-stained SDS polyacrylamide gel of purified MtaLonC (about 3 µg). The masses of markers are 116.0, 66.2, 45.0, 35.0, 25.0, 18.4, and 14.4 kDa from top to bottom of the gel. (B) Far-UV CD spectrum of MtaLonC. (C) Near-UV CD spectrum of MtaLonC.(TIF)Click here for additional data file.Figure S3Sedimentation velocity analysis of MtaLonC. Sedimentation coefficient distribution of MtaLonC was between 14.1S and 18.3S with a peak at 15.7S.(TIF)Click here for additional data file.Figure S4Temperature- and pH-dependent peptidase activity of MtaLonC. (A) Peptidase activity of MtaLonC at various temperatures showing an optimal temperature between 52 and 65°C. (B) Peptidase activity of MtaLonC under various pH. All fluorescence values were determined after subtracting that of the negative control.(TIF)Click here for additional data file.Figure S5MtaLonC cannot degrade dephosphorylated casein. Protease activity of MtaLonC (50 µg) against dephosphorylated FITC-casein was assayed in the absence or presence of ATP. The fluorescence was measured as described previously (JBiol Chem 279 (2004): 34903–34912; Eur J Biochem 271 (2004): 834–844). Emission was recorded at 525 nm. (A) Cleavage of dephosphorylated FITC-casein at 37°C. EcLonA (4 µg) was used as a positive control. (B) Degradation of the substrate at 55°C. BtLonA (4 µg) was used as a positive control. Blank, no added enzyme.(TIF)Click here for additional data file.Figure S6The cutting site of MtaLonC in E. coli Huβ.E.coli Huβ were degraded by MtaLonC in the buffer containing 50 mM Tris HCl, 10 mM Na2HPO4, pH 8.0 for 17 hours and analyzed by mass spectrometry.(TIF)Click here for additional data file.Table S1The accession codes for the genes in phylogenetic tree.(DOCX)Click here for additional data file.