Hylaarborea, H. intermedia, and H.molleri) to the upper Miocene, about 5.4–7.1 million yearsago. Sibship analyses of microsatellite polymorphisms revealed that all threespecies have the same pair of sex chromosomes, with complete absence of X-Yrecombination in males. Despite this, sequences of sex-linked loci show nodivergence between the X and Y chromosomes. In the phylogeny, the X and Yalleles cluster according to species, not in groups of gametologs. We concludethat sex-chromosome homomorphy in these tree frogs does not result from a recentturnover but is maintained over evolutionary timescales by occasional X-Yrecombination. Seemingly young sex chromosomes may thus carry old-establishedsex-determining genes, a result at odds with the view that sex chromosomesnecessarily decay until they are replaced. This raises intriguing perspectivesregarding the evolutionary dynamics of sexually antagonistic genes and themechanisms that control X-Y recombination.Non-recombining sex chromosomes are expected to undergo evolutionary decay,ending up genetically degenerated, as has happened in birds and mammals. Why arethen sex chromosomes so often homomorphic in cold-blooded vertebrates? Onepossible explanation is a high rate of turnover events, replacing mastersex-determining genes by new ones on other chromosomes. An alternative is thatX-Y similarity is maintained by occasional recombination events, occurring insex-reversed XY females. Based on mitochondrial and nuclear gene sequences, weestimated the divergence times between European tree frogs ( Non-recombining sex chromosomes, such as the Y chromosome, are expected todegenerate over evolutionary times because they accumulate deleterious mutationsthat cannot be corrected by recombination with a pristine copy. In mostcold-blooded vertebrates, such as frogs, however, sex chromosomes areundifferentiated. Why is that so? On the one hand, the“high-turnover” hypothesis holds that these sex chromosomes areregularly replaced before they had time to decay. On the other hand, the“fountain-of-youth” hypothesis posits that they are regularlyrejuvenated by X-Y recombination in sex-reversed XY females. Here, we show thatthree species of tree frogs that diverged more than 5.4 million years ago sharethe same pair of undifferentiated sex chromosomes. Although male recombinationstopped before species divergence, X and Y alleles show no differentiation, andcluster by species, not gametologs. We conclude that their sex chromosomehomomorphy is not due to a recent turnover but is maintained over longevolutionary times by occasional recombination. Such rare episodes of X-Yrecombination are expected to have long-lasting consequences on the evolution ofsex chromosomes and sex antagonistic genes. SRY) first appeared on an autosome The highly decayed Y chromosome of mammals results from an evolutionary process thatstarted some 170 million years ago (mya), when a new masculinizing gene Is this striking X-Y similarity maintained by occasional recombination, or doesit result from a recent turnover, followed by the rapid loss of malerecombination? To test between these two alternatives, we combinedinvestigations on gene genealogies and recombination patterns in two speciesfrom the sister clade to ealogies  should cealogies . If, howealogies . Furtherealogies , while tealogies . Note thHylaarborea diverged from the sister species (H.intermedia and H. molleri) during the lateMiocene, namely around the Messinian salinity crisis. Estimates point to lowerMessinian  for the mtDNAcytochrome b . We genotyped atotal of 111 families from the three species, each comprising a mating pair andan average of 20 offspring, plus a few additional non-mating adults  may alsogenerate turnover events via sex-ratio selection H. arborea populations were producedin the lab  Ha M2  rangesfrom 100 to 120 bp and has a complex motif including CAG, CAA, and GCA repeats.Ha M3  has a size ranging from 175 to 185 bp and acomplex motif including CAG and CAA repeats. Multiplex PCRs were carried out forthe two loci in a total reaction volume of 10 µl containing 0.2 µMof each primer, 0.6× of Multiplex PCR Master Mix (QIAGEN), and 3 µlof extracted DNA. PCR amplifications were performed on the GeneAmp PCR Systems2700 and 9700  according to the following thermalconditions: initial denaturation at 95°C for 15 min followed by 32 cycles ofdenaturation at 94°C for 30 s, annealing at 58°C for 1 min 30 s,elongation at 72° for 1 min, and then a final elongation step at 60°Cfor 30 min. The same conditions were used to successfully amplify these twomarkers in H. molleri and H. intermedia.We used published primer sequences Ha 1–60 in H. molleri and markersHa 5–22, Ha H-108, andHa D-110 in H. intermedia, which wereamplified individually as follows: 10 µl reaction volume each containing0.25 mM dNTP, 0.5 µM of each primer, 1× QIAGEN PCR Buffer (withMgCl2 15 mM), 0.2 mM MgCl2 (0.5 mM MgCl2 for Ha D-110 and noMgCl2 for Ha 5-22 and Ha H-108), 1×QIAGEN Q-Solution, between 0.03 U and 0.1 U QIAGEN Taq, and between 1 and 3µl of extracted DNA. PCR reactions were performed on GeneAmp PCR Systems2700 and 9700  according to the following thermalprofiles: initial denaturation at 95°C for 15 min  followed by 32–35 cycles at94°C for 45 s (QIAGEN Taq: 40–45 cycles), annealing at 58°C for 45s (60°C for Ha H-108 in H. intermedia andHa 1-60 in H. molleri), elongation at72°C for 1 min, and a final elongation step at 60°C for 30 min (QIAGENTaq: 75°C for 5 min). PCR products were analyzed on an automated sequencer. Allele sizes andgenotypes were determined using GeneMapper 4.0 (Applied Biosystems) followed bymanual proofreading. In order to confirm homology, alleles from eachmicrosatellite locus were cloned and sequenced in all three species.For other primers, PCR reactions were conducted in two independent multiplexreactions (QIAGEN) co-amplifying up to six microsatellites merge.Allele frequencies in males and females were calculated with FSTAT 2.9.3.2 cytochrome b gene was amplified with primersL0 and H1046 The mitochondrial Fibrinogen A,alpha-polypeptide, we used two primers  and aprotocol developed by Jason B. Mackenzie in the Museum of Vertebrate Zoology. PCR were carried out in 12.5 µlreactions containing 7.55 µl H2O, 1.25 µl of PCR bufferincluding 1.5 mM MgCl2, 0.1 µl of dNTPs, 0.1 µl TaqQIAGEN, 0.75 µl of each primer having a concentration of 10 µM, and2 µl of genomic DNA with a concentration of 20 ng/µl. For subsequentcloning, two of such reactions from each individual were pooled to increasevolume. The PCR protocol followed a “touch-up” approach with 10cycles of increasing annealing temperatures (55°C to 60°C) by 0.5degrees each cycle , followed by 25 cycles with 30 s at 94°C, 30 s at 56°C,and 45 s at 72°C, and a final extension of 7 min at 72°C.To amplify ca. 545 bp of intron 1 of HaMed15 (ca. 1 kb fragments including 2exons and 2 introns) was amplified with primers Ha 5-22F (5′-TTACAGCAACAGCAAATGG-3′)and p984R (5′_CGAGTATGCTTAATAGCTAATGCTA_3′). PCRs,72°C 5 min) were carried out in 25 µl reaction volumes containing17.75 µl H2O, 2.5 µl of PCR buffer including 1.5 mMMgCl2, 1.1 µl of a solution containing 2.0 mMMgCl2, 0.25 µl of dNTPs, 0.4 µl Taq QIAGEN, 0.5µl of each primer having a concentration of 10 µM, and 2 µl ofgenomic DNA with a concentration of 10 ng/µl.The sex-linked gene Ha A-103 was amplified (ca. 510bp) with primers Ha A-103F1 (5′_GCCTAGAAATGTGCAGTGATC_3′) and Ha A-103R2(5′_TGGAAAGTTTGCCCATTCAT_3′). PCRs , 72°C 5 min)were carried out in 25 µl reaction volumes containing 19 µlH2O, 2.5 µl of PCR buffer including 1.5 mMMgCl2, 0.25 µl of dNTPs, 0.25 µl Taq QIAGEN, 0.5 µlof each primer having a concentration of 10 µM, and 2 µ µl ofgenomic DNA with a concentration of 10 ng/µl.The sex-linked non-coding marker HaMed15 sequences are JF317989 to JF318012; for themicrosatellite-containing sequence Ha A103: JF318144 toJF318169; for intron 1 of the Fibrinogen A,alpha-polypeptide: JF318013 to JF318047. Those forcytochrome b are provided in For all nuclear markers, PCR products were cloned using the pGEM-easy vectorsystem (Promega). Concentrations were first quantified (NanoDrop ND-1000spectrometer) and adjusted to 25 ng/µl. We mixed 1.5 µl of template,0.075 µl of vector (50 ng/µl), 2.5 µl 2× ligationbuffer, 0.5 µl T4 ligase, and 0.425 µl water and ligated overnight(10°C). Transformations were carried out by incubating a mixture of 2.5µl ligation mix and 12–25 µl JM109 High Efficiency competentcells for 20 min on ice and then heat-shocking them for 45 s at 42°C.Transformed cells were recovered in SOC medium for 1 h 30 min; 80–100µl of cell suspension was applied to LB agar plates supplied withAmpicillin/IPTG/X-Gal. After incubation , templates from anumber of 10–12 white colonies were amplified with forward and reversevector-specific primers M13. Nested vector-specific primers T7 and SP6 (Promega)were used as sequencing primers. All clones were sequenced in both directionsand visualized on an ABI 3730 sequencer and aligned with SEQUENCHER 4.9. For allsex-linked markers we sequenced 10–12 clones from each individual tominimize the risk of allelic dropout; alleles were aligned and screened forsingletons to correct for PCR error. Sequences included in phylogenetic analysesare thus represented by multiple clones each. GenBank accession numbers forPhyML3.0 cytochrome b and HKY model forsex-linked  and autosomal nuclear markers. For each case, we chosea BioNJ tree as a starting tree and used the combined subtree pruning andregrafting (SPR) plus nearest neighbor interchange (NNI) options for treeimprovement. All other parameters were set as default (http://atgc.lirmm.fr/phyml/). Bootstrap values were based on1,000 resampled datasets.Maximum likelihood (ML) phylogenies were generated with cytochrome b gene, weassumed a normal distribution of priors for the substitution rate, with mean0.01 my−1 (±0.007 SD) Fibrinogen alpha gene (intron 1) we followed the sameapproach but used a HKY plus Gamma model of sequence evolution (Modeltestserver1.0) and normal prior distributions for substitution rates with mean valuesranging 0.001 to 0.002 my−1Divergence times were estimated assuming an uncorrelated exponential relaxedmolecular clock. For the mitochondrial Figure S1n = 649 intotal). The distribution  of mean size differences between649 pairs of alleles randomly sampled at the same loci from differentspecies  averages 9.24 bp (±0.35 SD). From thisdistribution, an average difference of 6.26 bp or less has a probabilityp<0.00001 to occur by chance.Distribution of allelic size differences at sex-linked loci. Conspecific Xand Y alleles differ on average by 6.26 bp , obtained asthe size difference between the two alleles expressed in a male Click here for additional data file.Table S1H. arborea, two series of families(respectively 9 and 8) resulted from lab crosses between individuals fromdifferent populations (Cheylas-Laissaud and Lavigny-Flaach).Sampling localities for families. Given are the species, locality withcoordinates, numbers of adult males (Nm), adult females (Nf), families (Na),and offspring (No). In (DOC)Click here for additional data file.Table S2Ha M2and Ha M3 are not included, being strictly linked toHa 5–22 . The consensus matrix was obtained by mergingfamily files from all three species. Corresponding LOD scores are availableupon request.Matrices of recombination rates. Male values (blue) are above diagonal, andfemale values (green) below diagonal. Cells are empty when one of the twomarkers involved could not be amplified for this sex/species, and labeled asNA if the rate could not be assessed, as occurs when markers are notsimultaneously polymorphic within parents. Markers (DOC)Click here for additional data file.Table S3Lavigny population of H. arborea.The four Y haplotypes (marked in color) identified through sibship analysesand sex-specific allelic frequencies differ only at Ha1–60. Null alleles are coded by a star (*). (b) 24 males (top) and24 females (bottom) from the Piazzogna population ofH. intermedia. The five Y haplotypes (marked in color)identified through sibship analyses and sex-specific allelic frequenciesdiffer at three loci . Null alleles are coded by a star(*). (c) 13 males (top) and 2 females (bottom) from theCantera population of H. molleri. Aminimum of two Y haplotypes (marked in color) can be recognized based onsibship analyses and sex-specific allelic frequencies. Presumed null allelesare coded by a star (*). In three males no product could be amplified atHa D-110 (marked as NA).Genotypes of adult tree frogs. (a) 13 males (top) and 13 females (bottom)from the (DOC)Click here for additional data file.Table S4HaD-110 and Ha A-103 in H. arborea). Randompermutations of the matrix  show that such a low number(two cases or less) has a probability p<0.0002 to occurby chance.Matrix of cross-amplification patterns per locus, species, and gametolog. In14 instances no product could be amplified in any individual (entry = 0), but in only two cases did the amplificationpatterns differ between conspecific X and Y (namely for (DOC)Click here for additional data file.Table S5Sample information for sequences used in mtDNA cytb phylogenies. Provided areidentification numbers with species name and voucher information, localitycoordinates, and GenBank accession numbers.(DOC)Click here for additional data file.Text S1Sex differences in allelic frequencies.(DOC)Click here for additional data file.Text S2Non-recombining Y haplotypes in males.(DOC)Click here for additional data file.


SELEX-Seq is now the optimal high-throughput technique for characterizing DNA-binding specificities of transcription factors. In this study, we introduced an improved EMSA-based SELEX-Seq strategy with several advantages. The improvements of this strategy included: (1) using a FAM-labeled probe to track protein-DNA complex in polyacrylamide gel for rapidly recovering the protein-bound dsDNA without relying on traditional radioactive labeling or ethidium bromide staining; (2) monitoring the specificity of SELEX selection by detecting a positive and negative sequence doped into the input DNAs used in each round with PCR amplification; (3) using nested PCR to ensure the specificity of PCR amplification of the selected DNAs after each round; (4) using the nucleotides added at the 5′ end of the nested PCR primers as the split barcode to code DNAs from various rounds for multiplexing sequencing samples. The split barcode minimized selection times and thus greatly simplified the current SELEX-Seq procedure. The reliability of the strategy was demonstrated by performing a successful SELEX-Seq of a well-known transcription factor, NF-κB. Therefore, this study provided a useful SELEX-Seq strategy for characterizing DNA-binding specificities of transcription factors. However, the current techniques based on chromatin immunoprecipitation (ChIP), such as ChIP-Seq in vivo DBSs with the support of DNA-binding specificity characterized in vitro has become more and more important in vitro DNA-binding specificities of many TFs In the human genome, there are about 2000 transcription factors (TFs) et al. determined the in vitro binding sites and relative affinities of TF Zif268 and Aart by using SELEX-Seq for the first time et al. characterized the DNA-binding specificities of 19 TFs belonging to 14 different classes by using SELEX-Seq et al. characterized the DNA-binding specificities of heterodimers formed by eight homoeobox (Hox) TFs with a cofactor extradenticle (Exd) by using SELEX-Seq, and found that Exd binding to the Hox proteins modulated the sequence specificity of these proteins et al. characterized the DNA-binding specificities of four dimers of NF-κB family , and found that NF-κB dimers could specifically bind non-canonical motifs Systematic evolution of ligands by exponential enrichment (SELEX) is an evolutionary process that allows screening the ligands that can be specifically bound by a target of interest from an initially random pool of aptamers The procedure of SELEX-Seq experiment mainly consists of three steps: (i) selecting dsDNAs specifically bound by a TF protein from a random library by SELEX, (ii) sequencing the selected dsDNAs with massive parallel DNA sequencing technique (such as Illumina Solexa), and (iii) bioinformatics analysis of sequence data. In the reported SELEX-Seq studies, the strategies for implementing these steps were not identical, especially those for the first step. This step is critical for the success of SELEX-Seq study. The key to this step is how successfully selecting DNA targets specifically bound by TF protein of interest from the random library. In the traditional SELEX studies, the main strategies for this step were to employ electrophoresis mobility shift assay (EMSA) In this work, we introduced an improved EMSA-based SELEX-Seq strategy for characterizing DNA-binding specificity of TFs in a more rapid, specific and cost-effective manner. This strategy was featured with the following functions: (i) recovering the protein-bound DNA from the native polyacrylamide gels with a support of FAM-labeled probe; (ii) monitoring protein binding specificity by PCR detection of a positive and negative sequence added into the input DNA; (iii) ensuring the specific PCR amplification of the selected DNA by using the nested primers; and (iv) reducing selections by using split barcode added at the 5′ end of PCR primers. The reliability of the strategy was validated by performing a successful SELEX-Seq of a well-known transcription factor, NF-κB.GGGACTTTCC). The negative dsDNA contained no NF-κB binding site. The random dsDNA contained 16-bp random sequences for SELEX selection. At the ends of these dsDNAs, specific primer annealing sequences were designed for their PCR amplification after each round of SELEX selection. The PCR amplifications of positive and negative dsDNAs were used to monitor the specificity of SELEX selection, while the PCR amplification of random dsDNA was used to monitor the selection of random dsDNA and prepare the input DNA for the next round of SELEX selection and the final sequencing  in Klenow reaction. The prepared dsDNAs were checked with polyacrylamide gel electrophoresis (PAGE) and ethidium bromide (EB) staining . The FAMFour rounds of SELEX selections were performed. In each round, six gel shift reactions were set up as described in Materials and Methods. The reaction of positive dsDNA plus p50 protein was used as control binding reaction for tracking protein-bound random dsDNAs in gels. Three reactions of random dsDNA plus p50 protein were performed, in which a reaction without polydI-dC was used to confirm that the gel was correctly cut for recovering the protein-bound random dsDNA, and two reactions with polydI-dC were used to recover the protein-bound random dsDNA. To evaluate the specificity of SELEX selections, trace amounts of positive and negative dsDNAs were added into these two reactions. After each round of SELEX selection, the selected random dsDNA was detected with three parallel PCR reactions amplifying positive, negative and random dsDNAs, respectively. The results revealed that the position of the protein-bound random dsDNA was correctly indicated by the control binding reaction and confirmed by the EB staining after gel cutting . The PCRThe enrichment of random dsDNAs along with SELEX selections were detected with a near infrared fluorescence EMSA (NIRF-EMSA). For this purpose, the selected dsDNAs from each round and the unselected dsDNA were firstly labeled by using PCR amplification with a pair of biotin-labeled universal primers  database under accession number [GSE:48660]. After quality control as described in Materials and Methods, 7347151 clean reads were obtained. These reads were sorted according to their barcode. 444108, 1749128, 2333602, 1779507 and 1040806 reads were obtained for Round 0 to 4, respectively. These reads were used to perform de novo motif analysis. Because most previous studies identified NF-κB binding sites as 10-bp sequence de novo motif analysis.Due to large reads data, motif analysis was performed with small amount of reads that were randomly sampled from reads of each round. To determine the optimal sample size, five samples containing non-overlapping 1000, 5000, 7500, 10000 and 15000 reads from Round 4 were firstly used to perform de novo 10-mer motif analysis was performed with 25 samples of 10000 reads (5 samples for reads of each round). For each sample, five motifs were extracted. The results were shown in To check the enrichment of SELEX selection, new de novo 10-mer motif analysis was performed with 10 samples containing 10000 non-overlapping reads from Round 4. For each sample, ten motifs were extracted. The most enriched motif of each sample was determined according to fold enrichment. As a result, ten motifs with the highest fold enrichment were obtained; these motifs were regarded as intermediate motifs  κ chain enhancer when the transcription factor NF-κB was found for the first time To evaluate the SELEX-Seq data, the reads contained the known κB motifs  between the EMSA values and the SELEX-Seq values of four rounds were calculated. The results revealed that the R2 value increased with the round of SELEX selection, and an excellent correlation was reached at Round 4 (R2>0.9)  . These dIn this study, we introduced an improved SELEX-Seq strategy. The strategy was mainly characterized with the following technical improvements. Firstly, this strategy designed a FAM-labeled probe to track the protein-DNA complex in polyacrylamide gels in SELEX selection. Secondly, this strategy introduced a positive and negative sequence into random dsDNAs. Thirdly, this strategy used the nested PCR to amplify the selected DNAs after each round. Fourthly, this strategy designed split barcode to code dsDNAs from various rounds. These technical improvements made this strategy with several advantages over the current SELEX-Seq procedures.32P-labled dsDNA cannot be directly used to prepare sequencing library by linking sequencing adaptor Firstly, the FAM-labeled positive dsDNA facilitated the recovery of protein-bound dsDNAs from polyacrylamide gel without staining gels with EB and other DNA or protein dyes Finally, the split barcode used in this study greatly simplified the SELEX selection. At present, the reported coding approaches used several consecutive nucleotides  containing 803 11-mer sequences within a generalized NF-κB consensus RGGRNNHHYYB et al. also characterized the DNA-binding specificity of p50 homodimer with two different PBMs, one was a cPBM containing 3285 sequences that represented the top-scoring potential κB site sequences, and the other was an universal PBM (uPBM) containing all possible 10-mer sequences in vivo regulatory elements of NF-κB Until now, there are several SELEX studies on NF-κB p50 homodimers. In 1992, Kunsch GGGACTTTCC in kappa chain of human immunoglobulin Until now, as many as 2000 TFs have been found in the human genome In conclusion, this study provided an improved SELEX-Seq strategy for characterizing the DNA-binding specificities of transcription factors. In comparison with the current SELEX-Seq procedures, this SELEX-Seq strategy had several advantages that significantly facilitated and simplified the SELEX-Seq experiment. This improved SELEX-Seq strategy was fully validated by successfully characterizing the DNA-binding specificity and constructing a high-resolution DNA-binding profile of a well-known transcription factor, NF-κB. Therefore, this study provided a new generalizable EMSA-based SELEX-Seq strategy that would be valuable to laboratories attempting to setup their own SELEX-Seq experiments.2O. The reaction was denatured in boiled water bath for 5 min and then slowly cooled to room temperature. The reaction was added 2 µl Klenow enzymes  and kept at 37°C for 20 min. The Klenow enzyme was inactivated at 75°C for 10 min. The products were purified with Microcon YM-30 (Millipore) and eluted in 25 µl ddH2O. The purified dsDNA was detected with electrophoresis of 2% agarose gel and quantified with NanoDrop 1000 (Thermo Fisher).All oligonucleotides used in this study were listed in Table S1 in 2, 5% glycerol, 2.5 mg/ml BSA, 0.25% NP-40, 0.05 mM DTT), 0.5 µl p50 protein  . The binding reactions were incubated at room temperature for 60 min. The reactions were fractionated by electrophoresis with 6% native PAGE. The gel was detected with UV transilluminator (Bio-Rad). The gel was stained with EB for 30 min and detected again with UV transilluminator.EMSA reactions were set up as follows (10 µl): 2.0 µl 5×DNA-binding buffer (5×DBB) , 0.5 µl diluted positive dsDNA (200 ng), and 7.0 µl ddH2O. Reaction 3 and 4 contained 2.0 µl 5×DBB, 0.5 µl ddH2O or p50 protein (115 ng), 0.5 µl each of diluted positive and negative dsDNA (0.02 ng), 0.5 µl random dsDNA (200 ng), and 6.0 µl ddH2O. Reaction 5 and 6 contained 2.0 µl 5×DBB, 0.5 µl p50 protein, 0.5 µl each of diluted positive and negative dsDNA (0.02 ng), 0.5 µl random dsDNA (200 ng), and 1.0 µl polydI-dC , and 5.0 µl ddH2O. The molar ratio of random dsDNA to protein was maintained at 5∶1 in all TF binding experiments. Before preparing protein-binding reaction, the positive and negative dsDNAs were 1/10000 diluted, so that the ratios of the positive and negative dsDNA to the random dsDNA were 1∶10000. In preparing protein-binding reactions, other components was firstly mixed and incubated at room temperature for 30 min, then dsDNA was added and incubated at room temperature for 60 min.In each round of SELEX selection, six protein-binding reactions  were performed simultaneously. Reaction 1 and 2 contained 2.0 µl 5×DBB, 0.5 µl ddH4Ac, 10 mM Mg(Ac)2, 1 mM EDTA, pH8.0, 0.1% SDS] overnight at 37°C. The diffusion buffer was purified with Microcon YM-30 and DNA was eluted in 25 µl ddH2O.The protein binding reactions were loaded on 6% native polyacrylamide gel and migrated in 0.5×TBE buffer for 1.5 hs. After electrophoresis, the gel was directly visualized on UV transilluminator and the gel slice containing the shifted dsDNA (Reaction 5 and 6) was excised according to the position of the shifted band of Reaction 2. The gel was then stained with EB for 30 min and visualized again on UV transilluminator. The gel slice was socked in 100 µl diffusion buffer [0.5 M NH2O (negative control), and 6.4 µl ddH2O. The PCR reactions were incubated for 3 min at 94°C, and then subjected to 20 cycles of 94°C 1 min, 60°C 1 min and 72°C 2 min, finally incubated for 5 min at 72°C. The PCR products were detected by electrophoresis with 2% agarose gel. The PCR product amplified with nested primers was purified with Microcon YM-30, and the DNA was eluted in 25 µl ddH2O. The purified PCR product was detected by electrophoresis with 2% agarose gel and quantified with NanoDrop 1000 (Thermo Fisher). The purified PCR product was kept at 4°C for final sequencing and also used as input DNA for the next round of SELEX selection.In each round of SELEX selection, 6 PCR reactions were performed: PCR reactions for amplifying positive dsDNA (using primer P-F and P-R), negative dsDNA (using primer N-F and N-R) and random dsDNA , and the corresponding negative controls. PCR reaction (10 µl) consisted of 2 µl 10×Taq buffer, 0.4 µl 2.5 mM dNTP, 0.4 µl 10 µM forward primer, 0.4 µl 10 µM reverse primer, 0.4 µl Taq polymerase , 1.4 µl template dsDNA (10 ng) or ddH2O. The PCR program was as follows: 94°C 3 min, 20 cycles of 95°C 1 min, 60°C 1 min and 72°C 2 min; 72°C 5 min. The PCR product was detected by electrophoresis with 2% agarose gel, and then purified with Microcon YM-30 and dissolved in 25 µl ddH2O. The purified DNA was detected by electrophoresis with 2% agarose gel and quantified with NanoDrop 1000 (Thermo Fisher). The purified DNA was used to prepare the final multiplexed sample for sequencing as DNA of Round 0.To prepare a PCR-amplified product of unselected random dsDNA, the random dsDNA obtained with Klenow reaction and used as input DNA of Round 1 was amplified with a 10-µl PCR reaction including: 2 µl 10×Taq buffer, 0.4 µl 2.5 mM dNTP, 0.4 µl 10 µM forward primer (R-F0), 0.4 µl 10 µM reverse primer (R-R0), 0.4 µl Taq polymerase , 1.4 µl random dsDNA , and 6.4 µl ddH2O. The PCR program was as follows: 94°C 3 min, 20 cycles of 95°C 1 min, 60°C 1 min and 72°C 2 min; 72°C 5 min. The PCR products were purified with Microcon YM-30 and eluted in 25 µl ddH2O. The purified dsDNA was detected by electrophoresis with 2% agarose gel and quantified with NanoDrop 1000 (Thermo Fisher). The EMSA reaction (10 µl) consisted of 2.0 µl 5×DBB, 0.5 µl p50 protein (115 ng), 1.5 µl biotin-labeled dsDNA (200 ng), and 6.0 µl ddH2O. The negative control was performed with no p50 protein (0.5 µl ddH2O in place of 0.5 µl p50 protein). The protein-binding reactions were incubated at room temperature for 60 min. The remained procedures of NIRF-EMSA were performed as previously described The selected DNAs from each round were detected with NIRF-EMSA. The selected dsDNAs were firstly labeled with biotin via PCR amplification with two universal biotin-labeled primers  to generate a multiplexed sample (500 ng) for sequencing. The multiplexed sample was sequenced with Illumina Solexa Hiseq2000 in two directions. The obtained reads were filtered and sorted with Perl scripts. Filters included: definite base calling , valid barcode and constant regions, and unique random sequence at the length of 16 bp. After quality control, the ‘clean’ data of 16-mer reads was obtained and used to perform the following bioinformatics analyses.P-value of 0.001.The motif analysis was performed with MEME4.5.0 in parameters of –dna –revcomp –w 10 -nmotifs 5. Some logos were generated using the enoLOGOS web tool File S1Supporting figures and tables. Figure S1. Contribution of cytosine after the binding site of GGGACTTTCC to the DNA binding affinity of NF-κB. A, all 16-mer sequences containing the site of GGGACTTTCC were collected from reads of Round 4 and classified according to the nucleotides flanking the end of the site. B, EMSA analysis of the relative binding affinities of NF-κB p50 to two dsDNA probes of agttgagGGGACTTTCCTaggc (a) and agttgagGGGACTTTCCCaggc (b). C, the quantified signal intensity of the shifted band. The ends of two dsDNA probes were labeled with biotin. The protein-binding reactions and the EMSA protocol were same as described in Materials and Methods. Figure S2. NF-κB p50 protein was separated by SDS-PAGE and visualized by silver staining. Molecular weight marker (MW marker) was indicated on the left. Table S1. Oligonucleotides designed for SELEX. Table S2. NF-κB motifs reported by other studies. Table S3. Three motifs obtained with reads of Round 3 and 4. Table S4. Enrichments of three motifs obtained with the reads of Round 3 and 4. Table S5. Enrichments of 10 sequences (10-mer) in SELEX-Seq and the relative affinities determined by EMSA.(DOC)Click here for additional data file.


Optimal time of HAART initiation is still unknown. Several studies investigating this issue showed inconsistent results. Our study investigated whether there is a clinical benefit as to treated patients (pts) by an earlier start of therapy at CD4 between 350-449c/µl, compared to the range of 250-349c/µl. An analysis was conducted on basis of the open, retrospective and prospective, multi-center and nationwide cohort of the Competence Network for HIV/AIDS.o), pts had to have 250c/µl <CD4-cells<450c/µl, no prior AIDS defining conditions and CD4 cells never below 200c/µl. Afterwards, pts were stratified in groups by initial CD4 cells between 250-349c/µl (group 1) and 350-449c/µl (group 2). Primary outcomes death, AIDS and first drop of CD4 cell count/µl<200 cells were evaluated as censored event times between to and the date of first event resp. last observation. Time dependent probabilities of event free intervals since start of HAART were estimated by Kaplan-Meier estimation, compared by Log-rank-tests. Cox-regression models were fitted, adjusted for time since infection.For analysis, pts had to be observed at least 3 months before initiation of HAART. Medication had to start later than 1996, with at least three substances. At time of therapy start , in group 2 4.9 years/pt . In group 1, 0.64 deaths occurred per 100 pt years vs. 0.17 events in group 2. 1.38 AIDS events were developed per 100 pt years vs. 0.78 events in group 2. In group 1, 2.64 per 100 pts years dropped <200c/µl vs. 0.77 in group 2. Kaplan-Meier estimations showed borderline significant difference as to developing death , significant difference as to CD4 drop<200c/µl , but no significant differences as to AIDS . Hazard ratio (group 2 vs. 1) was significant for CD4 drop<200c/µl , but not for death  and AIDS .Results showed a tendency for better outcome of pts by start of therapy with CD4 cell count ≥350c/µl regarding death, clear evidence regarding first CD4 drop <200. These results may be a hint to start therapy earlier.


KD and the protein bound fraction of the prototypical uremic toxin indoxyl sulfate (IS) in plasma of healthy and uremic individuals were studied. Binding of IS corresponded to one site binding in normal plasma. DK increased linearly with the NaCl concentration between 0.15 (KD = 13.2 ± 3.7 µM) and 0.75 M (KD = 56.2 ± 2.0 µM). Plasma dilution further reduced the protein bound toxin fraction by lowering the protein binding capacity of the plasma. Higher temperatures also decreased the protein bound fraction of IS in human plasma. Increasing the NaCl concentration was effective to weaken the binding of IS also in uremic plasma: the protein bound fraction decreased from 89% ± 3% to 81% ± 3% at 0.15 and 0.75 M NaCl, respectively. Dilution and increasing the ionic strength and temperature enhance the free fraction of IS allowing better removal of the substance during dialysis. Applied during clinical dialysis, this may have beneficial effects on the long-term outcome of maintenance dialysis patients.Protein binding prevents uremic toxins from removal by conventional extracorporeal therapies leading to accumulation in maintenance dialysis patients. Weakening of the protein binding may enhance the dialytic elimination of these toxins. In ultrafiltration and equilibrium dialysis experiments, different measures to modify the plasma binding affinity and capacity were tested: (i), increasing the sodium chloride (NaCl) concentration to achieve a higher ionic strength; (ii), increasing the temperature; and (iii), dilution. The effects on the dissociation constant  The uremic syndrome is attributed to the accumulation of a large number of compounds, which in healthy individuals are excreted by the kidney. These compounds are called uremic retention solutes or uremic toxins since they have deleterious effects on the human organism. Indoxyl sulfate IS, 213 Da) is a prototypical protein-bound uremic toxin 3 Da is a. KA = 9.1 × 105 to 16.1 × 105 M−1 (corresponding to a dissociation constant KD = 0.6 to 1.1 µM) [Albumin (molecular weight 66.5 kDa) is the most abundant plasma protein with a concentration of about 570 µM 38 g/L) [ g/L [8], 1.1 µM) . In prin 1.1 µM) . Due to  1.1 µM) ,18,19,20Several studies have investigated the protein binding of uremic toxins exclusively in albumin solution as a surrogate for plasma ,11,21,22p < 0.05) and 0.61 M NaCl solution .A rise in temperature from 25 °C to 37 °C significantly reduced the fraction of protein bound IS in both 0.15 M NaCl ) revealed that, independently of the ionic strength, the binding of IS in normal human plasma best fits to a one binding site model. The binding constants of a second binding site in plasma were calculated to be irrelevant. One site-specific binding curve (Equation (2)) at 0.15 M and 0.75 M NaCl are presented in KD = 10.9 ± 2.0, 22.1 ± 6.5, 39.7 ± 11.1 and 54.9 ± 5.9 µM; Bm = 228 ± 52, 251 ± 33, 296 ± 54, and 318 ± 54 µM, at 0.15 M, 0.30 M, 0.50 M, and 0.75 M, respectively) did not significantly differ from the values obtained from non-linear regression using Equation (2). As shown in KD  and the ratio KD/Bm . The maximal binding capacity Bm slightly increased with the ionic strength. However, compared to isotonic NaCl concentration, this finding was statistically significant only at the highest ionic strength of 0.75 M. Increasing the ionic strength resulted in a significant decrease (p < 0.05) of the protein bound fraction from 95% ± 1% at 0.15 M NaCl to 91% ± 2%, 87% ± 3%, and 84% ± 2% at 0.30 M, 0.50 M, and 0.75 M NaCl, respectively, at low toxin-albumin ratio (α = 0.1). The theoretical protein bound fraction for α = 0.1 was 94% ± 2%, 90% ± 2%, 87% ± 3%, and 84% ± 3% at 0.15 M, 0.30 M, 0.50 M, and 0.75 M, respectively. Experimental and theoretical protein bound fractions highly correlated . Mathematical modeling (Equation (2) p < 0.01). There was no significant difference between uremic and IS added healthy plasma regarding the mean total IS concentration . Considering Equation (1) with an experimental albumin concentration of 286 µM after 1:2 dilution, i.e., hypothesizing Bm being constant at 286 µM, a higher NaCl concentration in uremic plasma correlated with KD for IS ).With increasing ionic strength, the protein bound fraction of IS decreased significantly in plasma of uremic and healthy individuals (KD (see Bm was decreased significantly (p < 0.05), being equivalent to the albumin concentration (238 ± 90 µM for 1:2 and 48 ± 18 µM for 1:10 dilution). Fitting models (Equation (2) versus Equation (3)) confirmed a single high affinity binding site in 1:10 diluted human plasma, as also shown by the corresponding Scatchard plot  did not differ from the values obtained by non-linear regression using Equation (2). Experimental and theoretical protein bound fractions highly correlated .For a given NaCl concentration, 1:2 and 1:10 dilution did not affect the binding affinity KD see , while tard plot C. AppropIn vitro experiments have shown its implication in bone and cardiovascular disorders frequently observed in uremic patients. Bone disease is elicited by IS by inhibiting osteoclast differentiation and function, and by inducing osteoblast resistance to parathyroid hormone [IS is a prototypical, highly protein bound uremic toxin associated with the morbidity and mortality of maintenance dialysis patients ,3,4. In  hormone ,25. Card hormone ,27. AlthKD of 0.6 to 1.1 µM [KD of 13.4 ± 3.6 µM. This much lower affinity for IS in plasma may be best explained by the presence of potential competing ligands, such as fatty acids, which may alter the binding properties of albumin by occupying the binding site [KD observed in the present experiments  [In the present experiments a single class of high affinity binding sites were found for IS in plasma. This finding was supported by comparing one site and two site specific binding models (Equations (2) and (3), respectively). Moreover, no difference was found between the Scatchard plot  and non-linear regression (Equation (2)) when calculating ffinity) ,33. The ffinity) ,34. As pffinity) ,11, it mp-cresyl sulfate [p-cresyl sulfate concentration in uremic plasma  and its protein bound fraction  were in the same range. The decrease of the protein bound fraction of IS was associated with a linear increase of KD being 56.2 ± 2.0 µM at 0.75 M NaCl versus 13.4 ± 3.6 µM at 0.15 M. An increase of the ionic strength may result in a modification of electrostatic charges within the binding pocket of albumin and/or a modification of the protein conformation interfering with IS binding. As no decrease of Bm at the given NaCl concentrations was observed, it can be concluded that electrostatic interactions definitely are involved. This result is in agreement with previous findings, which demonstrated that increasing the ionic strength to 0.30 M NaCl decreased the binding affinity of serum albumin for both p-cresol and p-cresyl sulfate [Increasing the NaCl concentration from physiological 0.15 M to 0.75 M effectively decreased the protein bound fraction of IS in both normal plasma (from 93% ± 3% to 83% ± 4%) and plasma from uremic patients (from 89% ± 3% to 81% ± 3%). The lower protein bound fraction in uremic plasma can be explained by competition of other uremic toxins, such as  sulfate , which a sulfate . Further sulfate , a drug  sulfate .KD with rising ionic strength and a decrease of Bm with dilution. Both effects cumulate and lead to an increase in the ratio KD/Bm and, a fortiori, to a reduction of the protein bound fraction  yielded that increasing the NaCl concentration in uremic plasma from 0.15 M to 0.75 M NaCl enhanced DK for IS from 27.3 ± 8.6 µM to 55.9 ± 15.3 µM. This estimated binding affinity reflects the apparent affinity of uremic plasma for IS and is similar to that found in normal plasma. A methodological artifact was excluded by verifying the stability of albumin during freezing of the plasma samples. In three plasma samples, a constant human albumin concentration was confirmed before freezing at −20 °C and after thawing .Finally, it was verified for normal plasma if Equation (1) is suited to allow an accurate prediction of the experimentally bound fraction of IS from the binding constants independently of the plasma dilution. At low toxin concentrations, the protein bound fraction correlated directly with the binding affinity ntration . Advantantration ,19,41, aTo note, performing direct binding studies in native uremic plasma is difficult to interpret because of the competition between the multiple protein bound uremic toxins present in plasma and any newly added ligand ,10,11,30et al. reported interference of heparin with free p-cresol determination [in vitro. With respect to the NaCl concentration (at least 150 mM), the increase in ionic strength resulting from the introduced potassium (the highest concentration being about 0.15 mM) was negligible.Some shortcomings of the present study need to be addressed. The experiments may have been confounded by some differences in anticoagulation, which were due to a matter of blood availability. Despite the fact that De Smet mination , an effeIn summary, uremic and normal plasma do not differ in terms of the binding affinity for protein bound toxins with respect to increased ionic strength as demonstrated using the example of the prototypical IS. Furthermore, Equation (1) is a useful tool to describe binding properties of similar toxins in normal and uremic plasma.KD, and Bm, the maximal binding capacity) and the toxin-protein ratio α = LT/Bm, according to Equation (1), where LT is the total toxin concentration . Samples for the measurement of the total toxin concentration by reversed-phase high performance liquid chromatography (RP-HPLC) were collected from the plasma and the dialysate compartments of the diaylzer when the equilibrium was reached after 1 h. The equilibrium point was determined during a prior pilot experiment (data not shown).Experiments using plasma obtained from four different donors by centrifugation of whole blood were performed at two different ionic strengths. Firstly, 17.5 mL freshly donated heparinized (5 IU/mL) plasma from a single healthy donor was diluted with the same volume of phosphate buffered saline pH 7.4 . Secondly, to obtain euvolemic plasma with higher ionic strength, 17.5 mL heparinized plasma was mixed with 8.75 mL PBS pH 7.4 and 8.75 mL 2.0 M NaCl , resulting in 1:2 diluted plasma of 0.61 M NaCl concentration. Both preparations were dialyzed (flow rate 2.1 mL/min) at 25 °C and 37 °C against a solution of 150 µM IS  of corresponding ionic strength (0.15 M and 0.61 M NaCl) using a mini-dialyzer with a low-flux dialysis membrane , which was incubated for 30 min at room temperature at different NaCl concentrations in PBS pH 7.4 . IS was added to reach a concentration of 8, 15, 23, 30, 90, and 150 µM. After adding IS and NaCl, the volume of PBS was adjusted to finally obtain 1:2 diluted plasma. The toxin/albumin ratio was kept ≤1.0 mol/mol to avoid non-specific binding. In each sample, the free and total IS concentrations were measured by RP-HPLC. Scatchard plots were generated for the free and bound IS concentrations.KD and Bm were determined by nonlinear regression, comparing the one site specific binding model (Equation (2)) with the two sites specific binding model (Equation (3)) by means of the “extra sum-of-squares F test” .RL and L are the bound and free toxin concentration, respectively.The binding constants KD and Bm were also assessed from Scatchard plots, i.e., by calculating the absolute value of the inverse slope and the interception with the X-axis, respectively.The binding constants g) and stored at −30 °C. For the binding experiments, 0.5 mL uremic plasma was incubated for 30 min at different NaCl concentration  in PBS pH 7.4 at room temperature. After adding NaCl, the volume of PBS was adjusted to attain 1:2 dilution of the plasma. For each sample, the free and total IS concentrations were measured by RP-HPLC.After having received approval from the competent institutional review board , written informed consent was obtained from 15 end-stage renal disease patients on maintenance hemodialysis in a single dialysis center . Blood (7.5 mL) was collected in EDTA-tubes from each patient before dialysis. Plasma was obtained by centrifugation  with a final dilution of either 1:2 or 1:10. Plasma (0.5 mL or 0.1 mL) was diluted with PBS, NaCl solution and IS solution to reach a final NaCl concentration of 0.15 M and 0.50 M, respectively, and a final volume of 1.0 mL. To 1:2-diluted samples IS was added to obtain the same concentrations as described above. 1:10-diluted samples contained only one fifth of the IS concentrations targeted in 1:2 diluted plasma. The samples were incubated for 30 min at room temperature, further analyzed by RP-HPLC, and the binding constants g). The clear supernatant was ultrafiltered  to remove remaining larger proteins and the free IS concentration was determined in the resulting filtrate. The obtained clear filtrates were subjected to RP-HPLC . The IS concentration was measured using a C18 column  and fluorescence detection . The samples were eluted at 30 °C using a gradient from 15% to 25% v/v of solvent A  pH 3.4) and solvent B ) at a flow-rate of 1 mL/min.To determine the total IS concentration, all samples were diluted 1:5 with PBS pH 7.4 in glass tubes. Then, protein was precipitated at 95 °C for 30 min and the tubes were centrifuged . Theoretical and experimental protein bound fractions were compared at low toxin-albumin ratio (α = 0.1). The protein bound fractions were determined as follows: for the theoretical approach, α was set at 0.1; for the experimental approach, α was obtained from the total toxin concentration measured by HPLC and the albumin concentration determined using laser nephelometry :The ratio n = 3). The comparative analysis of the binding affinity and capacity in human plasma was performed by using the paired Student’s t-test. Differences between uremic versus healthy plasma were analyzed by applying the Mann-Whitney-U-test, if the results were not normally distributed, and with a one-way ANOVA followed by the Tukey’s post hoc test, if normally distributed. A p-value < 0.05 was considered significant.If not differently indicated, results are presented as mean ± SD of three different donors (Increasing the ionic strength by modification of the NaCl concentration as well as higher temperature are effective to reduce the protein binding affinity and, consequently, the protein bound fraction of the uremic toxin IS in human plasma. Dilution further decreases the protein bound fraction by lowering the binding capacity of the plasma proteins. As only the free toxin fraction is removed during hemodialysis, these measures represent interesting approaches to enhance the removal of protein bound uremic toxins from blood, which may be beneficial for the outcome of patients undergoing dialysis.


Background:Dictyostelium discoideum P2X receptors are found on the contractile vacuole.Results: Four of the five receptors  form ATP-activated channels but differ in their optimal ionic conditions.Conclusion: Properties of five P2X receptors correlate with their rescue of an osmoregulatory phenotype in P2XA-deficient Dictyostelium cells.Significance: A P2X receptor is required for normal contractile vacuole operation. Dictyostelium discoideum genome encodes five proteins that share weak sequence similarity with vertebrate P2X receptors. Unlike vertebrate P2X receptors, these proteins are not expressed on the surface of cells, but populate the tubules and bladders of the contractile vacuole. In this study, we expressed humanized cDNAs of P2XA, P2XB, P2XC, P2XD, and P2XE in human embryonic kidney cells and altered the ionic and proton environment in an attempt to reflect the situation in amoeba. Recording of whole-cell membrane currents showed that four receptors operated as ATP-gated channels . At P2XA receptors, ATP was the only effective agonist of 17 structurally related putative ligands that were tested. Extracellular sodium, compared with potassium, strongly inhibited ATP responses in P2XB, P2XD, and P2XE receptors. Increasing the proton concentration (pH 6.2) accelerated desensitization at P2XA receptors and decreased currents at P2XD receptors, but increased the currents at P2XB and P2XE receptors. Dictyostelium lacking P2XA receptors showed impaired regulatory volume decrease in hypotonic solution. This phenotype was readily rescued by overexpression of P2XA and P2XD receptors, partially rescued by P2XB and P2XE receptors, and not rescued by P2XC receptors. The failure of the nonfunctional receptor P2XC to restore the regulatory volume decrease highlights the importance of ATP activation of P2X receptors for a normal response to hypo-osmotic shock, and the weak rescue by P2XB and P2XE receptors indicates that there is limited functional redundancy among Dictyostelium P2X receptors.The  Dictyostelium discoideum is an intracellular organelle comprising interconnected ducts and small cisternae that periodically coalesce into larger intracellular bladders   or red fluorescent protein (RFP)et al.  . P2X reclucamine , 10. In lucamine , 10. In P)et al.  showed tP)et al.  also pre  at BglII and SpeI sites. Dictyostelium strains were grown and maintained in association with Klebsiella aerogenes or in HL5 axenic medium , 24–48 h after transfection. Cells were voltage-clamped at −60 mV, and membrane currents were amplified with a HEKA amplifier running Pulse and PULSEFIT software . ATP or other potential agonists were applied using an RSC 200 system  for 2-s duration at 2-min intervals. All data were analyzed using PULSEFIT and Prism (GraphPad) software. Numerical data are presented as mean ± S.E. The significance of differences among groups was assessed by nonparametric analysis of variance and Kruskal-Wallis post hoc tests.m NaCl, 10 mm HEPES, and 10 mm EGTA, and the pH was adjusted to 7.3 with NaOH . KCl (pH 7.3) contained 145 mm KCl, 10 mm HEPES, and 10 mm EGTA, and the pH was adjusted to 7.3 with KOH . KCl (pH 6.2) contained 145 mm KCl, 10 mm MES, and 10 mm EGTA, and the pH was adjusted to 6.2 with KOH .Three intracellular (pipette) solutions were used. NaCl (pH 7.3) contained 145 mm NaCl, 2 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 13 mmd-glucose, and 10 mm HEPES, and the pH was adjusted to 7.3 with NaOH. KCl (pH 7.3) contained 149 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 13 mm d-glucose, and 10 mm HEPES, and the pH was adjusted to 7.3 with KOH. KCl (pH 6.2) contained 149 mm KCl, 2 mm CaCl2, 1 mm MgCl2, 13 mmd-glucose, and 10 mm MES, and the pH was adjusted to 6.2 with KOH. All solutions were maintained at 300–320 mosm/liter. Chemicals were purchased from Sigma.Three extracellular solutions were used. NaCl (pH 7.3) contained 147 mI) were fitted to Imax[A]n/(EC50n + [A]n) where n is the Hill coefficient. Adenosine and nucleotides were purchased from Sigma.Concentration-response curves for ATP were generated using either an ascending or a descending order of concentration ([A]). The resulting currents (m (P2XA), 300 μm (P2XE), or 3 mm (P2XB and P2XD) (which correspond to the EC50), and percentage inhibition was calculated from currents in the presence and absence of copper (100 nm).The effect of copper was assessed for each P2X receptor using the ionic conditions found to give the largest currents in response to ATP. ATP concentrations used were 100 μm, rat P2X2; 100 μm, P2XA; 300 μm, P2XB, P2XD, and P2XE) first in 2 mm Ca2+ and then in 10 mm Ca2+. The internal and external solutions used were those found to provide the maximal ATP currents for the particular receptor (rat P2X2 and P2XA: intracellular NaCl (pH 7.3), extracellular NaCl (pH 7.3); P2XB and P2XE: intracellular KCl (pH 6.2), extracellular KCl (pH 6.2); P2XD: intracellular KCl (pH 6.2), extracellular KCl (pH 7.3)). Reversal potentials were measured from ramp voltage commands (−60 to 60 mV in 500 ms) at peak ATP responses. The reversal potential at 2 mm calcium was arbitrarily set to 0 mV to offset junction potentials, and the change in reversal potential (Er) observed at 10 mm calcium ([Ca]o) was then used to compute PCa/PNa from PCa/PNa = (1 + exp(ErF/RT))·([(γK[K]i + γNa[Na]i)(exp(ErF/RT)] − [(γK[K]i + γNa[Na]i)])/4γCa[Ca]o where the activity coefficients γNa, γK, and γCa used were 0.75, 0.75, and 0.3, and where F, R, and T have their usual meanings.For measurements of calcium permeability it was not possible to use simple bionic solutions because increasing the extracellular calcium concentration strongly inhibited the current evoked by ATP . Current2/37 °C incubator. Cells were washed once in Hanks' Balanced Salt solution (Invitrogen) and returned to 5% CO2/37 °C incubator for a further 20 min. The stained cells were then washed twice in phosphate-buffered saline (PBS), fixed in Zamboni  for 10 min at room temperature, washed three times with PBS before permeabilization by incubating in blocking solution (5% goat serum in PBS) containing 0.3% Triton X-100 for 30 min. Cells were then incubated overnight at 4 °C with primary antibody mouse anti-EMPYME (Covance) . Cells were then washed three times in PBS and incubated for 2 h with Cy3-conjugated goat anti-mouse secondary (1:500) (Jackson Laboratories) at room temperature, washed three times, and mounted using ProLong Gold anti-Fade (Invitrogen). Cells were viewed on an inverted microscope  equipped with an oil immersion objective . Image acquisition was controlled with EZ-C1 software, with excitation laser set at either 488 nm (emission of 515 ± 30 nm) for wheat germ agglutinin or 543 nm (emission of 590 ± 50 nm) for P2X receptor staining. Images were processed using Adobe Photoshop and Illustrator SC6 version (Adobe Systems).48 h after transient transfection, serum was removed from the cells by washing with OptiMEM alone, followed by a 10-min incubation in OptiMEM containing a membrane marker, Alexa Fluor 488 wheat germ agglutinin  in 5% COm KH2PO4 and 3.7 mm K2HPO4. Receptors were assayed for their ability to restore the wild type phenotype by placing cells in water for 40 min . Osmotic shock was performed on these clones by placing them in water for 60 min and measuring circularity at 10-min intervals.Dictyostelium P2X receptors (P2XA–P2XE). In each case, antibody staining was evident at the edge of the cell, corresponding to plasma membrane, as well as in large intracellular pools  elicited a large inward current at P2XA receptors that partly desensitized during a 2-s application (m) , 12+ as tion (m) .+ by K+ increased the amplitude of responses to ATP in cells expressing P2XA receptors (50 for ATP from 3.8 ± 0.07 (n = 20 or 21) to 4.0 ± 1.2 (n = 3–7) , and in K+ was 0.35 ± 0.35 (n = 3–7). Currents at P2XA receptors desensitized more rapidly in a K+-based extracellular solution. The amplitude at the end of a 2-s application as a fraction of peak amplitude was 0.71 ± 0.05 in Na+ (n = 11) and 0.28 ± 0.10 in K+ (n = 10). Cells expressing P2XB, P2XC, and P2XE receptors did not respond to ATP with significant currents under these conditions. However, ATP (3 mm) evoked large currents at P2XD receptors (n = 33) of peak amplitude).Substitution of extracellular Naeceptors . This efn = 3–7) . The efflular K+ . The sloeceptors ; these c+-containing extracellular solution had little effect on the peak currents elicited by ATP in cells expressing P2XA receptors (n = 6)). Cells expressing P2XB and P2XC receptors did not respond significantly to ATP. P2XD receptors were strongly inhibited by pH 6.2 (n = 24) of peak amplitude).Acidification (to pH 6.2) of the Keceptors  other thy pH 6.2  whereas y pH 6.2 . These cPCa/PNa) is ideally measured in bi-ionic conditions . However, increasing the extracellular calcium concentration strongly inhibited ATP-evoked currents at all Dictyostelium P2X receptors . Under these conditions, P2XB and P2XE receptors showed no detectable calcium permeability. P2XA and P2XD receptors gave an “apparent” PCa/PNa of 3.2 ± 1.2 (n = 13) and 0.74 ± 0.18 (n = 14), respectively, whereas the value for rat P2X2 receptors under the same conditions was 15.0 ± 2.0 (n = 12). Although these values cannot be compared directly with estimates made under bi-ionic solutions, they indicate that under conditions that provide optimal responses to ATP they are considerably less permeable to calcium than are rat P2X2 receptors.The relative permeability to calcium ions ; P2XB, 84 ± 4.2 (n = 15); P2XD, 30 ± 3.3 (n = 9); and P2XE, 70 ± 9.0 (n = 9).r copper . In the +, and we therefore examined the actions of ATP with recording electrodes containing KCl. At pH 7.3, for all five P2X receptors, ATP-elicited currents were not significantly different whether the recording electrode contained Na+ or K+  evoked currents at P2XB receptors that were not observed under any other conditions. These currents declined to 0.55 ± 0.14 (n = 10) of their peak amplitude during a 2-s application of ATP.The intracellular or pipette solution in the present experiments corresponds to the cytoplasmic or intracellular solution in amoeba. The main cytoplasmic cation would be Ka+ or K+ . Howeverm ATP is used as the agonist. We therefore constructed corresponding concentration-response curves. The results presented above, and summarized in Dictyostelium cells show a prominent osmoregulatory defect in response to hypotonic shock  are functional as ATP-activated ion channels, although they are optimally active under different conditions. However, because these assays were performed in a heterologous system we next sought evidence that they represent functional differences in living amoeba. For this, we tested whether differences in optimal ion channel activity compared with P2XA also reflected differences in the ability of each receptor to complement the function of P2XA. We have previously reported that P2XA-null mutant ic shock . When wiic shock .Dictyostelium cells. The lower left panel of Fig. 6C illustrates that this occurs with very low levels of P2XA expression. Overexpression of P2XB, P2XD, and P2XE receptors also restored the capacity to recover from the hypotonic stimulus, with P2XD being most effective and able to restore RVD at low expression levels. In the case of P2XB the rescue was incomplete even at the highest expression levels, and P2XE also required greater expression levels compared with P2XA. P2XC overexpression failed to rescue the wild type phenotypic response to hypo-osmolarity  ADP, AMP, cAMP, CTP, ITP, GTP, UTP, adenosine, cADPR, GMP, cGMP, UDP-glucose, NAD, reduced NAD (NADH), NADP, reduced NADP (NADPH), and NAADP. No compounds evoked significant currents. The only exception was ADP at P2XA receptors, which gave currents approximately 10% of the amplitude of those evoked by ATP. We also examined a mixture of nucleotides  on each of the five P2X receptors under all the recording conditions described above but in no case observed current greater than 50 picoamperes/picofarad. It is possible that these small currents result from the presence of ATP as an impurity in commercial preparations of other nucleotides.Using HEK cells expressing P2XA, and with NaDictyostelium P2X receptors, and this low level of conservation led us to wonder whether a ligand other than ATP might activate the receptors in the contractile vacuole of Dictyostelium. However, our search of 17 related compounds, many with known intracellular signaling roles, provided no evidence against the view that ATP is the preferred ligand at Dictyostelium receptors.The amino acid residues that make direct contacts with ATP in vertebrate P2X receptors are now known from extensive mutagenesis , 21 and Dictyostelium P2X receptors expressed well in HEK cells, as indicated by immunofluorescence at the plasma membrane  as well as extracellular (vacuolar in Dictyostelium) solutions. We found that P2XA, P2XB, P2XD, and P2XE exhibited robust currents in response to application of ATP in conditions where potassium was the predominant cation on both sides of the membrane and the pH on either side was 6.2    . This re  . Ludlow   inhibits ATP-evoked currents somewhat at P2XA receptors, but profoundly at P2XB, P2XD, and P2XE receptors. This is consistent with observations in mammalian receptors, where external Na+ ions (compared with K+) have been found to inhibit currents at rat P2X2 receptors (+ (cytoplasmic in Dictyostelium) inhibits ATP-evoked currents at P2XB receptors, but has little effect on ATP-evoked currents at P2XA, P2XD, and P2XE receptors.From the perspective of the predominant cations surrounding the P2X receptors, these results can be summarized as follows. External Naeceptors  and rat + concentration has little effect on peak currents at P2XA receptors but accelerates their desensitization. It increases currents at P2XB and P2XE receptors, but inhibits currents at P2XD receptors. In comparison with mammalian P2X receptors, the variability is unsurprising given that currents at some P2X receptors are increased by lower extracellular pH whereas others are decreased  Hecreased , 24.Chaos carolinensis, the vacuole is relatively enriched with sodium compared with the cytoplasm   . These vDictyostelium cells progressively swell over a period of several minutes and then undergo a regulatory volume decrease during which the contractile vacuole can be seen to reach the membrane and discharge its contents. In the AX4 strain, this osmoregulation is markedly impaired in cells in which P2XA gene function has been disrupted , whereas cells became swollen and more rounded when placed in hypotonic solution, with the circularity index reaching 0.84 ± 0.04 (n = 6). Fig. 6B illustrates cells expressing even low levels of P2XA receptors, as judged by the independent measure of RFP fluorescence, showed a restoration of RVD: this essentially confirms our earlier finding  also failed to rescue the osmoregulatory phenotype in intact Fountain  have recproteins , 6 which


Their adipogenic potential decreases with time in the culture. Expressing exogenous genes in 3T3-L1 cells can be challenging. This work tries to establish and characterize an alternative model of cultured adipocytes that is easier to work with than the 3T3-L1 cells.Induced cells were identified as adipocytes based on the following three characteristics: (1) Accumulation of triglyceride droplets as demonstrated by oil red O stain. (2) Transport rate of 2-deoxyglucose increased after insulin stimulation. (3) Expression of fat specific genes such as Fabp4 (aP2), Slc2a4 (Glut4) and Pparg (PPARγ). Among the cell lines induced under different conditions in this study, only NIH/3T3 cells differentiated into adipocytes after prolonged incubation in 3T3-L1 induction medium containing 20% instead of 10% fetal bovine serum. Rosiglitazone added to the induction medium shortened the incubation period from 14 to 7 days. The PI3K/AKT pathway showed similar changes upon insulin stimulation in these two adipocytes. C/EBPα mRNA was barely detectable in NIH/3T3 adipocytes. NIH/3T3 adipocytes induced in the presence of rosiglitazone showed higher 2-deoxyglucose transport rate after insulin stimulation, expressed less Agt (angiotensinogen) and more PPARγ. Knockdown of C/EBPα using shRNA blocked 3T3-L1 but not NIH/3T3 cell differentiation. Mouse adipose tissues from various anatomical locations showed comparable levels of C/EBPα mRNA.NIH/3T3 cells were capable of differentiating into adipocytes without genetic engineering. They were an adipocyte model that did not require the reciprocal activation between C/EBPα and PPARγ to differentiate. Future studies in the C/EBPα independent pathways leading to insulin responsiveness may reveal new targets to diabetes treatment. Much has been learned about the molecular basis of adipocyte formation and insulin action using cell culture models of adipocytes. These models of adipocytes show the accumulation of triglyceride droplets, express adipocyte marker genes and increase glucose uptake in response to insulin stimulation a (C/EBPα) Rosen et al synthesized all available evidence and proposed that the reciprocal activation between CebpThe use of 3T3-L1 cells was sometimes hampered by the gradual loss of their adipogenic potential in the culture over time and their resistance to gene transfer and expression. Adding PPARγ agonists, such as troglitazone or rosiglitazone The 3T3-L1 cells have been used for more than 3 decades. They were always induced after they became confluent so that contact inhibition was considered a prerequisite for adipogenesis The effect of longer induction time to increase adipogenesis was then tested on several murine cell lines, including NIH/3T3, CT26 C/EBPα encoded a member of the bZIP transcription factor family and it could form heterodimers with C/EBPβ and C/EBPδ. During 3T3-L1 adipogenesis, C/EBPβ and C/EBPδ transcription was increased earlier than C/EBPα and activated its transcription. It was possible that C/EBPβ and C/EBPδ might be increased to compensate for the lack of C/EBPα to form NIH/3T3 adipocytes. The mRNA levels of these genes were determined by qPCR before and after differentiation . While tIn addition to the difference in C/EBPα expression, the PI3K/AKT signaling pathway in these two adipocytes were compared by western blotting. NIH/3T3 adipocytes showed AKT phosphorylation upon insulin stimulation as in 3T3-L1 adipocytes . The datDiabetic patients treated with rosiglitazone showed decreased insulin resistance, lower blood pressure, and increased adiposity in utero or soon after birth C/EBPα was generally considered an adipocyte marker and an essential gene for adipogenesis. C/EBPα knockout mice lacked adipose tissue. They were very sick and died either In this work, we tested the hypothesis that increasing the length of induction time during adipogenesis could increase the degree of differentiation to the extent that qualitatively different conclusions would be reached. Our results supported this hypothesis. First we showed that non-confluent 3T3-L1 cells were induced into adipocytes by doubling the induction time or with the help of rosiglitazone. This effect was not limited to 3T3-L1 cells. Multipotential NIH/3T3 is one such cell line that showed increased adipogenicity after prolonged induction. C/EBPα is a member of the bZIP transcription factor family. Theoretically, the lack of C/EBPα during adipogenesis could be compensated by expression of the other members in this gene family, C/EBPβ and C/EBPδ. This possibility could not be easily tested by inactivating C/EBPβ because of its essential role beyond activating C/EBPα. During 3T3-L1 cells differentiation C/EBPα rose and stayed at near peak level in the adipocytes but C/EBPβ and C/EBPδ peaked early then fell in both 3T3-L1 Except for the differences in the expression profiles of the C/EBPs, the insulin signaling patterns of NIH/3T3 and 3T3-L1 adipocytes did not show significant difference. The western blot analysis of the PI3K/Akt pathway in NIH/3T3 shown in in vivo that express as little C/EBPα as the NIH/3T3 cells . Rosiglitazone was purchased from Cayman Chemical . Akt and phosphoAkt(pS473) antibodies were from Epitomics . IRS-1 antibody was from Upstate , and 4G10 was from Millipore . All other antibodies were from Genetex . The RNeasy total RNA kit was from QIAGEN . Reverse transcription reagents were obtained from Promega Corp.  and TaqMan reagents were from PE Applied Biosystems . All other reagents were from Sigma .2-Deoxy-D-[2,6-3T3-L1 fibroblasts  were maintained at no higher than 70% confluence in DMEM (Dulbecco's Modified Eagle Medium)/CS . For differentiation they were grown 2 days post confluence in the same medium and then for 3 days in 3T3-L1 induction cocktail . The medium was then changed to DMEM/FBS  for 6 days (change medium every 48 hours) before use. NIH/3T3 fibroblasts  were maintained at no higher than 70% confluence in DMEM/CS. For differentiation they were grown 2 days post confluence in the same medium and then for 7 days in an adipogenic cocktail containing rosiglitazone  or 14 days in an adipogenic cocktail without rosiglitazone. The medium was then changed to DMEM/FBS for 8 days (change medium on every 72 hours) before use.th day post-induction, in NIH/3T3 adipocytes on 15th day post-induction with rosiglitazone or on 22nd day without rosiglitazone, in 12 (or 24)-well plates.Glucose uptake was measured as described previously 3T3-L1 and NIH/3T3 adipocytes in 12 well plates were washed with PBS 2 times, fixed in 3.7% formaldehyde for 2 minutes, washed with ddH2O, then stained in 1 ml of 0.3% Oil Red O (mixture of 40 ml ddH2O and 60 ml 0.5 g oil red o in isopropanol) for 1 hour, then washed with ddH2O twice. Differentiation efficiency was assessed macroscopically using a Sony cybershot T-900 and microscopically using a Nikon COOLPIX 4500 camera mounted on a ZeissAxio VertA1 microscope.Cells were lysed in a buffer containing 20 mM HEPES, 0.2 M NaCl, 0.5% Triton X-100, 20% glycerol, 1 mM EDTA, 1 mM EGTA, protease and phosphatase inhibitors (Roche Applied Science). After rotating for 30 min at 4°C, cell lysates were centrifuged at 12,000× g for 10 min at 4°C to remove insoluble material. The samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane and probed with the indicated antibodies. The proteins were visualized by enhanced chemiluminescence  or BCIP/NBT 1-Component Phosphatase Substrate . The intensitiy of each band was quantified by densitometry using the ImageJ software GCATGCAGAAGGAGATCACA, TTGTCGATTGTCGTCCTGAG; Gapdh, AATGTGTCCGTCGTGGATCT; CCCTGTTGCTGTAGCCGTAT; Agt, CACCCCTGCTACAGTCCATT, GTCTGTACTGACCCCCTCCA; Scarb1, CAGGCTGTGGGAACTCTAGC, GAAAAAGCGCCAGATACAGC; Cfd, TGCACAGCTCCGTGTACTTC, CACCTGCACAGAGTCGTCAT; Adipoq, GTTGCAAGCTCTCCTGTTCC, TCTCCAGGAGTGCCATCTCT; Lipe, AGACACCAGCCAACGGATAC, ATCACCCTCGAAGAAGAGCA; Pnpla2, TCCGAGAGATGTGCAAACAG, CTCCAGCGGCAGAGTATAGG; Slc2a4 (Glut4), ACTCTTGCCACACAGGCTCT, AATGGAGACTGATGCGCTCT; Cd36, GAGCAACTGGTGGATGGTTT, GCAGAATCAAGGGAGAGCAC; Cebpa (C/EBPα), TTACAACAGGCCAGGTTTCC, CTCTGGGATGGATCGATTGT; Pparg (PPARγ), TTTTCAAGGGTGCCAGTTTC, AATCCTTGGCCCTCTGAGAT; Fabp4, TCACCTGGAAGACAGCTCCT, AATCCCCATTTACGCTGATG. Cebpb (C/EBPβ), CAAGCTGAGCGACGAGTACA, AGCTGCTCCACCTTCTTCTG; Cebpd (C/EBPδ), ATCGCTGCAGCTTCCTATGT, AGTCATGCTTTCCCGTGTTC.RNA was extracted from the cells following the manufacturer's instructions. Real time quantitative PCR was performed using an ABI-PRISM 7700 Sequence Detection System instrument and software  as described previously. The primers from 5′-3′ for each gene were as follows : Actin, Data are presented as mean ± SE. Statistical significance of treatments was determined by the t test using the Stata 8 software . P <0.05 was considered statistically significant. The results in http://rnai.genmed.sinica.edu.tw/en/Protocols.asp.The preparation of transfection quality DNA for the TRC library and production of viruses was done following the protocols of TRC at http://rnai.genmed.sinica.edu.tw/en/Protocols.asp.Lentivirus infection was done following the protocols of TRC at The lentiviruses expressing shRNA targeting C/EBPα were RNAi consortium clone ID TRCN0000009502 (C1) and TRCN0000009504 (C3). Those targeting PPARγ were RNAi consortium clone ID TRCN0000001658 (P3), TRCN0000001660 (P5). The construct targeting luciferase was used as the control, Luc(TRCN0000072266).To avoid unnecessary sacrifice of animals, murine adipose tissues were obtained from the control mice of a related project, which had been approved by the Institutional Animal Care and Use Committee, Tzu Chi University. . C57BL/6 mice were purchased from the National Laboratory Animal Center of Taiwan. Tissues were isolated from CO2 euthanized animals by the veterinarian of the animal facility.


The thermal stability of this compound was studied in detail by thermogravimetric/differential thermal analysis and temperature-dependent X-ray diffraction. Moreover, the electrochemical uptake and release by the galvanostatic cycling method of pure and carbon-coated NbO1.3(1)N0.7(1) versus lithium was investigated as an example of an Li-free transition-metal oxynitride. The results showed that reversible capacities as high as 250 and 80 A h kg−1 can be reached in voltage ranges of 0.05–3 and 1–3 V, respectively. Furthermore, a plausible mechanism for the charge–discharge reaction is proposed.The decomposition reaction of niobium(V) oxytrichloride ammoniate to the oxynitride of niobium in the 5+ oxidation state was developed in a methodological way. By combining elemental analysis, Rietveld refinements of X-ray and neutron diffraction data, SEM and TEM, the sample compound was identified as approximately 5 nm-diameter particles of NbO For example, Nb2O5 and various niobates like AlNbO4, KNb5O13, and K6Nb10.8O30 exhibit outstanding electrochemical properties as anodes of Li-ion batteries.Nowadays, it is a consensus that the rechargeable Li-ion battery is one of the best solutions for energy storage. Even though intensive research on Li-ion batteries has been carried out in recent decades, the development of electrode materials with high energy densities still remains a big challenge.7.9MnN3.2O1.6, which exhibited a similar electrochemical behavior to Li7MnN4 but showed improved chemical stability.We believe that transition-metal oxynitrides are promising candidates for both anode and cathode applications, too. Recent studies started to investigate transition-metal oxynitrides in various applications, such as ionic conductivity, catalysis, pigments, and thermoelectrics.3 with an excess of NH4Cl at 900–1000 °C and used to determine its crystal structure.2) structure with monoclinic symmetry (space group P21/c). As shown in Figure 3N4], which are connected by edge-sharing N and corner-sharing O atoms. NbV oxynitride is a semiconductor due to the empty d band of niobium. NbON has a calculated band gap of 1.7 eV, which results in the intrinsic blue-colored appearance of the compound.4 and most of the other commonly used electrode oxides. In the present work, we investigated reaction conditions, thermal analyses, crystal structures, and micro-characterization of the products. Pure and carbon-coated compounds were prepared to study their electrochemical performance in Li-ion batteries, and a cycling mechanism was proposed as well.TaON was the first ternary oxynitride with a full oxidized state obtained by the ammonolysis method. It was first reported by Brauer et al. in 1966 and recently studied as a new pigment candidate.3Reaction of NbOCl3with NH: Niobium(V) oxytrichloride ammoniate NbOCl3(NH3)x was prepared by treating NbOCl3 with ammonia gas at room temperature. Figure 3(NH3)x in argon atmosphere. It indicates that decomposition of NbOCl3(NH3)x starts at 200 °C and is complete at 400 °C. Considerable heat of reaction is released. According to the weight loss during the decomposition, the molecular formula of the starting yellow niobium(V) oxytrichloride ammoniate was determined to be NbOCl3(NH3)4. A constant product weight is reached above 500 °C.xNy , which was reported previously.As plotted in Figure Reaction (1) is active at room temperature. When the temperature is increased to 500 °C, reaction (2) takes place.3(NH3)4 can be formulated as Equation .On the basis of the TG and DTA results discussed above, the decomposition process of NbOCl4Cl decomposes into NH3 and HCl above 380 °C. In a first attempt of this synthesis, dynamic vacuum and inert gas (Ar) were employed to remove NH3 and HCl. Surprisingly, a black-colored Nb2O5 phase, indicating oxygen deficiency and/or N doping, was obtained instead of NbON. We then changed the procedure to heating the starting material in a closed Pyrex tube and successfully produced NbON. However, overpressure results in the closed tubes from the formation of NH3 and HCl. To eliminate the overpressure two modifications were employed; either a long Pyrex tube in a vertical tube furnace was used so that the NH3 and HCl gases formed from the decomposition of NH4Cl at the hot bottom of tube the gases could recombine into solid NH4Cl on the cold top of the tube outside the furnace, or the NH4Cl was treated before it started to decompose. This process could be formulated as Equation .NHThe reaction conditions used to prepare five different samples are presented in Table Crystallographic characterization: Figure 3 impurities. This could be explained in the following way: excess LiI can form eutectic mixtures with LiCl/NH4I and acts as a proper flux to provide homogeneous conditions for the reaction. In this case, unlike in the direct decomposition reaction of NbOCl3(NH3)4, little gas is released and overpressure in the reactor can be avoided. Figure 0 to 4 have similar N/O content, and to elucidate the crystal structure in detail, sample 3 was chosen for further investigation. The composition of final product 3 was determined as NbO1.3(1)N0.7(1) by hot-gas N/O elemental analysis, which means that 1 of 1.3 O atoms occupy the oxygen positions in the structural model of NbON from ref. Uiso had negative values that are physically meaningless. In contrast, refinement with unrestrained N/O content gave reasonable results: instead of full occupancy of nitrogen in NbON, the nitrogen position was occupied by 0.64(5) O and 0.36(5) N atoms, which also agreed with the results of elemental analysis.Considering the various possible oxidation states of niobium, the oxygen and nitrogen ratio in the NbON phase obtained here is likely to be greater than 1:1 with Nb valence between +4 and +5. Elemental analysis showed samples 1.3(1)N0.7(1) and lactose-treated samples. The SEM images . In fact, this phenomenon usually occurs when interparticle magnetic interactions are sufficiently weak in an assembly of nanoparticles.Owing to the oxidation valence +4.7 of niobium in NbOThermal behavior: The thermal stability of NbO1.3(1)N0.7(1) was investigated by temperature-dependent XRD under vacuum. According to the XRD patterns in Figure 2N  exhibit even higher coefficients of thermal expansion than the corresponding isostructural oxides.1.3N0.7 investigated here, niobium is connected with seven O/N ions to form irregular octahedra. By comparison with symmetric and regular octahedra in perovskites, it becomes evident that tilting of [NbO3N4] polyhedra hardly affects the thermal expansion of compound, whereas extension of Nb—O/N bonds by heating apparently plays a more important role. Shilling et al. reported that baddeleyite- and fluorite-type oxynitrides always have lower volume thermal expansion coefficients than isostructural oxides.1.3(1)N0.7(1) would exhibit significantly low positive coefficients of thermal expansion. Shifting of the Bragg reflections which represent the [010] and the [001] zones after heating hints to a slight conversion of the compound. Compared with the initial compound, the sample after cooling to 40 °C has slightly shifted on reflections, which could originate from thermally induced changes in O or N content.Galvanostatic cycling: The electrochemical performance of pure and lactose-treated NbO1.3(1)N0.7(1) against Li was tested and compared under galvanostatic cycling conditions. During the annealing process the lactose is decomposed and gives a carbon coating of the oxynitride particles. Considering different lithiation mechanisms in different potential ranges, measurements were carried out at two potential cutoffs, 0.05 and 1 V. The first discharge was measured from open-circuit voltage (OCV) to final potentials and the subsequent charge cycled up to 3 V.1.3(1)N0.7(1) exhibits an extraordinarily high capacity of 500 to 700 A h kg−1, which afterwards drops rapidly to 200–300 A h kg−1 during subsequent cycles. Apparently, carbon-coated samples show better cycling performance than uncoated ones: the first discharge is followed by cycling with a quite stable capacity around 250 A h kg−1. The first voltage profiles of pure NbO1.3(1)N0.7(1) show sloping plateaus at 0.9 V and below 0.5 V. In contrast, an extra 1.5 V plateau is observed for the carbon-coated sample. To investigate these potential peaks in detail, their differential capacity curves are plotted in Figures 4 and CaMoO4. The observed plateaus around 0.9 V are ascribed to breakdown of the metal oxide framework, catalytically enhanced by carbon.When the potential ends at 0.05 V Fig>ures  and 11 a5+/Nb4+ redox potential versus lithium. This behavior is similar to that of other niobates like Nb2O5, AlNbO4, and KNb5O13.4+/Nb3+ occurs at a potential plateau of 1.2 V. However, in our case, NbO1.3(1)N0.7(1) only has an Nb4+/Nb3+ redox couple in the first cycle, which then disappears gradually. When the potential cutoff is set to 1.0 V, as shown in Figures −1 in stable running. The oxidation peaks shift to smaller potential as charging continues, and simultaneously the peaks corresponding to the Nb4+/Nb3+ reduction start to fade. This may be explained by lithium insertion into various environments of the crystal structure. First, lithium is inserted into different voids formed by N/O anions during discharge, and then as more and more lithium remains in these specific positions, a different potential is needed to remove the rest of the lithium ions during charging.As the insets in Figures 1.3(1)N0.7(1) is proposed here. Basically, this process can be discussed in two potential regions (0–1 and 1–3 V), as indicated in Figure + into interstitial sites of the crystal structure of NbO1.3N0.7. A capacity as high as 110 A h kg−1 corresponds to intercalation of about 0.5 Li into the host [Eq. ].On the basis of this discussion and previous studies on other similar niobates and molybdates, a charge–discharge reaction mechanism of NbO1.3(1)N0.7(1) and Nb2O5 have similar oxidation–reduction peaks, but there is a large difference in ability to insert lithium. No matter which structure Nb2O5 has, as much as 1.6–1.8 mol of Li+ can be inserted into 1 mol of Nb2O5. This can be ascribed to the existence of a number of easily traversed tunnels between edge- and corner-sharing [NbO6] octahedra. However, the space between [NbO3N4] units is irregular, small, and hardly fitting for lithium ions.This reaction is reversible when cycling is continued in the range from 1 to 3 V. NbO2O and Li3N, respectively. In the oxynitride studied here, we suppose that lithium preferably forms Li2O, and in parallel, Nb4.7+O1.3N0.7 is reduced to Nb2.7+O0.3N0.7. Obviously at room temperature, amorphous products appear after the conversion reaction. Consequently, after the conversion reaction powder XRD only shows broad angular distributions. Further lithium ions might be inserted into the reduced oxynitride NbO0.3N0.7 or another conversion reaction takes place.In the second voltage range, 1–0.05 V, the decreasing potential forces more lithium ions to diffuse into the compound, and breakdown of the host structure results. Such a conversion below 1.0 V occurs very frequently in oxide and nitride anodes.+ enter into each mole of the oxynitride and Nb changes its valence from 4.7+ to 2.2+. However, the proposed reaction mechanism needs to be confirmed by further investigations.In total, during the first discharge, 2.3 moles of Li1.3(1)N0.7(1) with Ni4.7+ oxidation state was synthesized by decomposition of niobium (V) oxytrichloride ammoniate and chemically and electrochemically analyzed. Two methods were developed for preparation of samples: 1) direct decomposition of NbOCl3(NH3)4 and 2) an LiI-assisted method. In both cases, pure compounds were obtained and used to characterize the crystal structures, morphologies, and electrochemical performances against lithium. From elemental analysis and neutron diffraction, the compound was determined to be NbO1.3(1)N0.7(1) instead of the expected NbON. The reaction products consist of 3–5 nm particles, but a few micrometric single crystals were found as well. NbO1.3(1)N0.7(1) loses nitrogen on increasing temperature and transforms in air into Nb2O5 above 400 °C.The oxynitride NbO1.3(1)N0.7(1) was investigated with regard to its electrochemical performance in Li-ion batteries as an example of a lithium-free transition-metal oxynitride. Our study indicates that NbO1.3(1)N0.7(1) coated with 4.6 wt % C is much more stable under reversible cycling than uncoated samples. When the cutoff potential is set to 0.05 and 1 V, respectively, the measured capacities reach 500 and 100 A⋅h kg−1 during the first discharge and then stabilize at 250 and 80 A⋅h kg−1 in subsequent cycling, respectively. A plausible mechanism has been proposed for the discharge–charge process of this oxynitride. This shows the potential of transition-metal oxynitrides as electrode materials of Li-ion batteries. Since the average potential of NbO1.3(1)N0.7(1) is practically the same as that of Nb2O5, replacement of oxygen by nitrogen, which formally enhances the Li uptake capacity by 1 Li, does not necessarily lower the average electrochemical potential of the material. We believe that the increased charge of nitride anions compared to oxide anions is responsible for this observation. Clearly oxynitrides provide new opportunities as electrode materials in place of oxides.NbO3 was prepared by gas-transport reaction. Nb2O5  and NbCl5  were mixed in a molar ratio of 1:3, sealed in Pyrex tubes, heated to 400 °C over 4 h, and kept at this temperature for 40 h. This reaction yielded deep green needle-shaped crystals of NbOCl3. The compound was ground into a fine white powder and treated with ammonia at RT until the color changed into the bright yellowish of niobium(V) oxytrichloride ammoniate NbOCl3(NH3)x. To make sure the reaction was complete, a second grinding was applied. Because NbCl5 and NbOCl3 are both water-sensitive, all handling was carried out in a glove box under a dry Ar atmosphere.The precursor compound NbOCl3(NH3)x (0.8 g) was sealed in a Pyrex tube (8 mm inner diameter and 1 m length), which was placed in a vertical tube furnace so that about 20 cm of the tube was outside of furnace. The temperature was set to 500 °C for 10–30 h. Black NbON powder was obtained at the bottom of the container and white NH4Cl condensed at the top of the Pyrex tube. If a flux salt was used, typically NbOCl3(NH3)x (0.8 g) and LiI  were sealed in a Pyrex tube (8 mm inner diameter and 12 cm length), heated up 500 °C (100 K h−1) and kept at this temperature for 5–40 h in a muffle furnace. The black product was washed twice with deionized water, centrifuged, and dried at 100 °C for 2–3 h to give a final black powder of pure NbON.NbON was synthesized by decomposition of niobium(V) oxytrichloride ammoniate. In a typical reaction, NbOCl2 flow furnace. The temperature was increased to 300 °C (10 K h−1) and then to 500 °C over 1 h, kept at 500 °C for 5 h, and cooled down as the furnace was switched off. 4.6 wt % of carbon was detected in the final sample.Carbon-coated samples were prepared by mixing pure NbON and 15 wt % of lactose in water followed by subsequent drying at 100 °C. This product was transferred into a N3(NH3)x (48.1 mg) was loaded in an alumina crucible The temperature was increased and decreased by 10 K min−1 from 20 to 1400 °C.Thermogravimetric (TG) and differential thermal analysis (DTA) were carried out on a Netzsch STA 409 C/CD instrument in argon atmosphere. For the study of the reaction, NbOClλCuKα1θ range from 15 to 120°. Temperature-dependent XRD was carried out at steps of 0.015°, each for 0.2 s over the 2θ range from 5 to 90° while heating at a rate of 0.5 K s−1.Powder X-ray diffraction (XRD) data were collected on a Bruker diffractometer (AXS mod. D8 Advance) with Bragg–Brentano geometry (θ range from 10 to 135°. The sample was contained in a vanadium can. Rietveld refinements for XRD and NPD were both performed with the GSAS software.Neutron powder diffraction (NPD) data were collected with the PUS two-axis diffractometer at the JEEP-II reactor at Kjeller, Norway. A neutron wavelength of 1.5561 Å was used. The step size was 0.05 in the 2The carbon content of the lactose-treated sample was analyzed in Laboratorium für Organische Chemie (ETH Zürich). The O/N content of the samples was measured by the hot-gas extraction method by using a LECO TC500 analyzer at EMPA .6 cathode) and a Tecnai F30 microscope  the material was deposited onto a holey carbon foil supported on a copper grid.Scanning electron microscopy (SEM) analysis was carried out on a Zeiss Gemini 1530 operated at 1 kV. For transmission electron microscopy on a CM30ST  as electrolyte. Electrodes of NbON/Super P carbon/PVDF (80:10:10 wt %) were ground and then ultrasonically dispersed in N-methyl-2-pyrrolidone (NMP) for 30 min at 40 °C. The obtained slurry was printed on titanium current collectors from a dropper, and then the solvent was evaporated at 150 °C for 1 h and 100 °C overnight. The typical weight of such electrodes is about 5 mg. Galvanostatic discharge/charge curves were measured with an applied current of 10 A h kg−1.A lithium metal  was taken as anode and Merck Selectipur LP30, which consists of 1