Invitro formation of diverse QDH by selective SLQS hits were successfully verified by nuclear magnetic resonance spectroscopy. Folding topologies of two SLQS were elucidated in detail. We also demonstrated that sequence changes at mutation/single-nucleotide polymorphism loci could affect the structural conformations adopted by SLQS. Thus, our predicted SLQS offer novel insights into the potential involvement of QDH in diverse (patho)biological processes and could represent novel regulatory signals.Duplex stem-loops and four-stranded G-quadruplexes have been implicated in (patho)biological processes. Overlap of stem-loop- and quadruplex-forming sequences could give rise to quadruplex–duplex hybrids (QDH), which combine features of both structural forms and could exhibit unique properties. Here, we present a combined genomic and structural study of stem-loop-containing quadruplex sequences (SLQS) in the human genome. Based on a maximum loop length of 20 nt, our survey identified 80 307 SLQS, embedded within 60 172 unique clusters. Our analysis suggested that these should cover close to half of total SLQS in the entire genome. Among these, 48 508 SLQS were strand-specifically located in genic/promoter regions, with the majority of genes displaying a low number of SLQS. Notably, genes containing abundant SLQS clusters were strongly associated with brain tissues. Enrichment analysis of SLQS-positive genes and mapping of SLQS onto transcriptional/mutagenesis hotspots and cancer-associated genes, provided a statistical framework supporting the biological involvements of SLQS.  For instance, the quadparser algorithm  identified >350 000 PQS in the human genome (L > 7) have also been investigated . A typical intramolecular G4-forming sequence would consist of four G-tracts interspersed with three loops Figure , while en genome . On the stigated –43 and iL > 7) for PQS, could reveal as yet unknown biology of these motifs.Another non-canonical DNA structural conformation that has been extensively studied is the duplex stem-loop (SL) (or hairpin) motif Figure . These mHere we developed a prediction model for the family of QDH-forming sequences, provided mapping of such QDH sequences onto the human genome and verified this model using nuclear magnetic resonance (NMR) spectroscopy. We performed a bioinformatics search to identify SL-containing quadruplex sequences (SLQS) in the human genome, a substantial number of which were found to reside within regulatory meaningful loci. Enrichment analysis revealed that SLQS display strand specificity and are preferentially distributed within specific genic and gene promoter regions, RNA polymerase II (Pol2) binding sites and other transcriptional regulatory DNA sites. In addition, these SLQS are enriched within specific functional classes of genes, and they occur at especially high frequencies in many hundreds of brain tissue-related and cancer-associated genes. NMR characterization on a selected list of human genome SLQS hits validated their adoption of diverse QDH-forming structures, with the folding topology of two particular sequences being elucidated in detail. We also explored the effects of sequence changes, which could arise at mutation/single-nucleotide polymorphism (SNP) loci, on the structural conformations adopted by these SLQS.hg18 of the human genome were downloaded from the UCSC Genome Browser . PQSL20 for which all three loops are shorter or equal to 7 nt were sub-classified as PQSL7. For the remaining PQSL20, the long loops (L > 7) were extracted and screened for the existence of duplex stem-loop elements. During the extraction step, the loop sequences were extended both ways to include guanines in excess of three from the two flanking G-tracts (e.g. 2 G's will be added to the loop sequence from a flanking G-tract of G5). As such, the extracted loop sequences would range from 8 to 26 nt. Only for cases in which all of these long loops could form stable stem-loops  probability function was used to characterize and fit the distribution of the number of SLQS in a given gene of human genome ,52. The escribed ,52. In t\documentX is restricted to sample size and the rarest events are non-observed. Thus, random variable X is doubly truncated, i.e. the range 1, 2, …, J (In order to apply the probability function Equations  or 2) t t2) to tJ, where the occurrence values 0 and J + 1, J + 2, … are not detected. Details of the curve-fitting computational algorithm have been previously published  and CGAP_SAGE_QUARTILE were utilized. The false discovery rate (FDR) method is the default P-value adjustment method in this study. In this method, the P-values are first sorted and ranked. The smallest value gets rank 1, the second rank 2 and the largest gets rank N. Then, each P-value is multiplied by N and divided by its assigned rank to give the adjusted P-values.Gene ontology (GO) analysis was carried out separately using two systems. First, the database for annotation, visualization and integrated discovery (DAVID) bioinformatics resources   cut-off of 1 × 10−5 was exercised.Second, the MetaCore™  software was used to calculate the biological process enrichment statistics for genes encompassing SLQS in different gene segments, namely transcriptional hotspots and 5′-UTR. The list of genes (in terms of RefSeq IDs) was supplied as the input, and the output consists of a Unlabelled and site-specific labelled DNA oligonucleotides were chemically synthesized on an ABI 394 DNA/RNA synthesizer. The oligonucleotides were de-protected, purified, dialyzed successively against ∼20 mM KCl and against water, and prepared in a buffer containing 20 mM KCl and 20 mM potassium phosphate (pH 7.0).15N-labelling  spectra were recorded on a JASCO-815 spectropolarimeter over the range of 220–320 nm using a 1-cm path length quartz cuvette with a reaction volume of 500 μl. For CD-melting experiments, cooling and heating were successively performed across the temperature range of 15–95°C over a total of 14 h. At intervals of 1°C, the full spectrum was recorded as an average of three scans, the spectrum of the buffer was subtracted and the data were zero-corrected at 320 nm. The molar ellipticity at 295 nm was extracted for melting analysis. Two baselines corresponding to the completely folded (low temperatures) and completely unfolded (high temperatures) states were manually drawn in order to determine the fractions of folded and unfolded species during the melting process. CD-melting experiments for G4ST02001786748 were carried out at strand concentrations of 2, 4, 20 and 100 μM.X ≥ 3NL = 1 – 7GX ≥ 3NL = 1 – 7GX ≥ 3NL = 1 – 7GX ≥ 3) in the human genome revealed more than 350 000 candidates (A previous survey of PQS (based on the definition Gndidates . It has ndidates  (TSS) anndidates . These oX = 3 – 6, L = 1 – 20; denoted as PQSL20) to accommodate longer loops that might harbour duplex SL elements. Based on this model, a total of 2 933 131 PQSL20 were identified  and genlgorithm . For all package  (see ‘MaL7 (out of 360 438 unique clusters) and 80 307 SLQS (out of 60 172 unique clusters) were identified in the human genome (build hg18). The complete list of SLQS hits (each tagged with a unique SLQS ID), accompanied with gene annotations, is presented in Supplementary Dataset 1. Among these, 48 508 SLQS (out of 35 469 unique clusters) are spread across 12 315 RefSeq genes and/or their gene promoter regions . Thus, these 33 148 SLQS would represent a novel set of potential regulatory genomic signals, distinct from the canonical PQS that have previously been reported in the literature (L7 were otherwise not found (gene list presented in Supplementary Dataset 2). On the other hand, SLQS that coincide with PQSL7 would extend the range of the putative quadruplex-forming regions, as in the case of the RET gene promoter , which has been previously characterized , we examined the distribution of SLQS with SL length ranging from 8–100 nt across Chromosome 1 (which covers ∼15% of the entire human genome) as a representative sample Figure . In thisL7 is presented in Table L7  in comp7 Figure , suggestP = 0.00021), ‘alternative splicing’ (P = 0.00018), ‘phosphoproteins’ (P = 3.7 × 10−16), ‘mutagenesis sites’ (P = 0.00016), ‘repressor’ (P = 0.00005), ‘activator’ (P = 0.000028), ‘proto-oncogene’ (P = 0.000054), ‘regulation of transcription from RNA polymerase II promoter’ (P = 1.85 × 10−7) and ‘transcription regulation’ (p < 2.86 × 10−7). In these cases, the adjusted P-values after Benjamini-Hochberg correction were used . Genes with the highest number of SLQS (14–32 per gene) in Pol2 binding sites include RNF213, BCOR, RNPC3, FAM38A, C14orf43, PIK3CD, KLF13, AHRR, ZMIZ1, UBE2E1, TP73, PREX1, MKNK2.Genes for which Pol2 binding regions are highly enriched with SLQS could exert a regulatory role. We found 13 914 SLQS located in the vicinity of Pol2 binding sites (at least 1-nt overlap between SLQS and Pol2 binding sites) from 6367 genes. Most of these genes were associated with one or two SLQS. A total of 297 genes were associated with more than 6 SLQS across their genic and/or gene promoter regions (Supplementary Dataset 3). Gene enrichment analysis revealed specific functional enrichment of these genes (Supplementary Dataset 4), e.g. ‘splice variants’  systems wherein the species birth-death processes are occurring and driving a system by evolution towards the complexity and self-organization function (θ = 1) may help us better understand the roles of common and rare SLQS in gene functions and the origin of G-quadruplexes, stem-loop-forming sequences, and QDH, and their natural co-evolution in the genome.A log–log plot of the distribution of SLQS (with respect to the count of SLQS per gene) for a protein-coding gene is presented in Figure 2 Figure , SBNO2, function ,52 with L7- and SLQS-containing genes as the background set (Supplementary Table S5), showing that P-values (with Benjamini correction) of GO terms using all human genes as the background set were more significant.To investigate if there is any evolutionary selection pressure for SLQS across specific biological classes of genes, GO analysis was carried out for the group of protein-coding genes encompassing at least one SLQS motif, using the database for annotation, visualization and integrated discovery (DAVID) bioinformatics resources  with allP-value < 1 × 10−5, after Bonferroni correction; Supplementary Table S6), ranging from ‘regulation of transcription’, ‘negative regulation of cell proliferation’ and ‘positive regulation of apoptotic process’, to ‘axon guidance’, ‘protein phosphorylation’ and ‘heart development’. These process categories overlap considerably with the GO categories previously reported for promoter PQS  with regulatory PQSL7, with ‘learning or memory’ being the sole exception (Supplementary Table S6). Interestingly, we note that the latter constitutes part of a larger group of genes related to brain tissue that corresponds to the long right tail in Figure inutero embryonic development’ and ‘apoptotic process’ are enriched with regulatory SLQS located on the non-template strand, whereas the GeneGo process categories ‘regulation of transcription’, ‘nervous system development’ and ‘protein phosphorylation’ are enriched with regulatory SLQS located on the template strand.Next, to explore the transcriptional regulatory potential of SLQS across different functional classes of genes, GO analysis was performed on 2866 gene symbols (represented by 4366 RefSeq IDs) for which SLQS located in promoter regions have been found to coincide with gene regulatory loci . Compared against the control , 10 of these GeneGo process categories were enriched with 5′-UTR SLQS but not 5′-UTR PQSL7 (Supplementary Table S8), including ‘response to drug’, ‘activation of adenylate cyclase’, ‘cell cycle’ and ‘angiogenesis’. Different preferences in strand placement were also observed for 5′-UTR SLQS from distinct functional classes of genes  for which SLQS have been localized to 5′-untranslated region (5′-UTR) , neurite growth-promoting factor midkine (MDK) and cell cycle regulator cyclin Y (CCNY), are summarized in Table L7 motifs . These observations lent support to the formation of QDH in the context of double-stranded genomic DNA. The sequence hits were also cross-checked against mutation ) me Atlas , NCBI) dRICTOR promoter), G4ST08067688172 (MYBL1 promoter) and G4ST17001906030 (HIC1 promoter); Figure MYT1L Intron 22) and G4ST07001717478 (ELFN1 Intron 1) displayed distinct sharp peaks amenable for detailed structural characterization ; Figure CCNY promoter); Figure To investigate the potential formation of QDH ,46 by SLMYO9B gene sequence G4ST19017166594 and its mutated counterpart G4ST19017166594del with a GAGAGT deletion (Table BMP8A gene segment G4ST01039729840A and G4ST01039729840G (Table Effects of nucleotide changes (arising from mutation or SNP) on the formation of QDH by SLQS were also examined. The on Table ), showedon Table  and a’, on Table ,62. Sing0G Table  and b’.MYT1L gene sequence G4ST02001786748  was performed. One-dimensional imino proton NMR spectrum of G4ST02001786748 indicated the adoption of a single predominant QDH structure; five major peaks at 12.5–13.8 ppm corresponded to the formation of five base pairs while twelve major peaks at 10.9–12.0 ppm corresponded to the establishment of three G-tetrads , as monitored at 295 nm, indicated the formation of an intramolecular structure by G4ST02001786748. Selective guanine imino  facilitated the determination of the H8/H6-H1′ sequential connectivity of the oligonucleotide , whereas the other tetrad guanine residues adopt anti glycosidic conformation. Cyclic imino-H8 NOE connectivity patterns within the tetrads .The ide loop ,41,65,66ide loop , charactide loop ,68. NMR ide loop –c. In thX1NL1GX2NL2GX3NL3GX4) exercising an arbitrary loop-length cut-off of 7-nt . Based on a loop length of 8–20 nt, our prediction model identified 80 307 SLQS, 48 508 of which were located across genic and gene promoter regions. More than 68% (33 148 out of 48 508) of these SLQS do not overlap with PQSL7, representing a pool of genomic regions that could exert novel (previously uncharacterized) gene regulatory signals. Note that for practical considerations, here we limited the G-tract and initial loop length of SLQS to 3–6 nt and 20 nt, respectively, while stable QDH structures comprising only two G-tetrad layers (X = 2), or containing longer stem-loops (L > 20), may well exist. Nevertheless, we expect that our model would cover close to half of total SLQS population in the entire human genome.In this study, we explored the existence of SLQS in the human genome, which would otherwise have been overlooked by conventional PQS algorithms . For instance, single-stranded segments of genomic DNA may persist up to hundreds of nucleotides behind a DNA helicase, which could reasonably encompass the SLQS motifs examined in this study.Bioinformatics analyses were performed on the distribution of SLQS across genic and gene promoter regions of the human genome. The 48 508 SLQS motifs were found to spread across a total of 12 315 genes. Distribution of these loci across protein-coding genes, according to the count of SLQS per gene, assumes the pattern of a skewed function exhibiting a high level of inequality, which can be approximated based on the K–W model ,52. By tPTPRN2 gene locus and its products. PTPRN2 is the most SLQS-populated human gene, containing 129 SLQS motifs. PTPRN2 protein encoded by this gene is a member of the protein tyrosine phosphatase (PTP) family. PTPs are known signalling molecules that regulate a variety of cellular processes including cell growth, differentiation, mitotic cycle and oncogenic transformation. PTPRN2 protein was identified as a major autoantigen associated with insulin-dependent diabetes mellitus. Three alternatively spliced transcript variants of this gene, which encode distinct proteins, have been reported. PTPRN2 is also one of the most R-loop-forming sequences (RLFS)-populated genes in the human genome, containing 140 RLFS  and in 73% of central neural system disorder tissues. It cannot be ruled out that SLQS motifs localized in RLFS loci are cooperative with RLFS in promoting R-loop formation and/or its stabilization, maintaining open chromatin and stalling RNA polymerase.Strong tissue specificity with regards to the brain was observed for the group of genes encompassing SLQS motifs. Many of these brain tissue related genes exhibit high frequencies of SLQS motifs. Specifically, our study predicts important role of SLQS motifs in the biological function of the 140 RLFS . During 140 RLFS . High frThe SLQS motifs were found across numerous genomic loci of biological and medical interest. Out of 8598 SLQS identified in the gene promoter regions, more than 56% were mapped onto chromatin accessibility regions and transcription factor binding sites. Moreover, distributions of SLQS in TSS-proximal regions indicated their high enrichment, similar to PQS. Some of these could conceivably exert a regulatory function in transcription as that posited for promoter-associated PQS ,18. Our Recently, it has been shown that non-coding RNAs are produced in a pervasive manner  and theiSeveral SLQS were identified at mutation and SNP loci. These observations suggested the possible associations of SLQS with genome instability, genetic diseases and specific biological processes. Some of the SLQS were found to reside within experimentally defined G-quadruplex-forming regions , corroboRAB3D, RAB12, HIC1, RET and CCNY. Most of these SLQS displayed NMR signatures of both duplex and quadruplex elements, supporting their adoption of QDH structures. Structural polymorphism was observed in numerous cases, whereas a single predominant conformation was observed in a handful of other cases. These examples showcase the structural diversity that can arise from SLQS motifs. We determined the folding topology of the QDH adopted by two particular SLQS hits (G4ST02001786748 from Intron 22 of the MYT1L gene and G4ST07001717478 from Intron 1 of the ELFN1 gene). Detailed understanding on the structures that can be adopted by various SLQS will aid in future drug design efforts targeting these non-canonical nucleic acid structures.Accurate identification and prediction of QDH-forming genomic sequences will be helpful towards the understanding of their potential biological functions. Towards this end, we carried out structural characterization on a selected list of SLQS using NMR. Among the sequence hits investigated include promoter-associated SLQS from cancer-associated genes such as invitro. In addition, G4 structures with more complex scaffold have been previously observed (We have shown previously that QDH with three SL in three separate loops  and two observed ,29,75–81Supplementary Data are available at NAR Online.


Insulin induces the activation of Na,K-ATPase while translationally controlled tumor protein (TCTP) inhibits this enzyme and the associated pump activity. Because binding of insulin with its membrane receptor is known to mediate the phosphorylation of multiple intracellular proteins, phosphorylation of TCTP by insulin might be related to the sodium pump regulation. We therefore examined whether insulin induces TCTP phosphorylation in embryonic kidney 293T cells. Using immunoprecipitation and Western blotting, we found that insulin phosphorylates serine (Ser) residues of TCTP. Following fractionation of the insulin-treated cells into cytosol and membrane fractions, phosphorylated TCTP at its Ser residue (p-Ser-TCTP) was detected exclusively in the cytosolic part and not in the membrane fraction. Phosphorylation of TCTP reached maximum in about 10 min after insulin treatment in 293T cells. In studies of cell-type specificity of insulin-mediated phosphorylation of TCTP, insulin did not phosphorylate TCTP in HeLa cells. Computational prediction and immunoprecipitation using several constructs having Ser to Ala mutation at potential p-Ser sites of TCTP revealed that insulin phosphorylated the serine-9 and -15 residues of TCTP. Elucidations of how insulin-mediated TCTP phosphorylation promotes Na,K-ATPase activation, may offer potential therapeutic approaches to diseases associated with vascular activity and sodium pump dysregulation. This efInsulin is known to activate a number of kinases that activate signaling pathways. TCTP contains potential phosphorylation sites at its serine , threonine , and tyrosine  residues. To test whether insulin promotes phosphorylation of TCTP, lysates of 293T cells transfected with pEGFP N1-TCTP-GFP construct, were immunoprecipitated with anti-phospho-Ser-, -phospho-Thr-, and -phospho-Tyr-specific antibodies. Phosphorylated TCTP was detected through immunoblotting with anti-GFP antibody. As shown in Phosphorylation of proteins induces intracellular translocation of specific proteins, between membrane and cytosol. We examined whether insulin treatment affects the localization of TCTP by specifically detecting the p-Ser-TCTP in cytosol and plasma membrane of the transfected 293T cells treated with insulin. We obtained the membrane and cytosolic fractions of the insulin treated cells; obtained immunoprecipitates with anti-p-Ser-antibodies and subjected them to immunoblotting to detect p-Ser-TCTP using anti-GFP antibodies. i.e., exogenous vs. endogenous origins) and the cell type . Exogenous TCTP was introduced by transfection. Cells were incubated with insulin and the cytosolic fraction was subjected to immunoprecipitation using anti-p-Ser-specific antibodies; and immunoblotting performed with anti-GFP or -TCTP-specific antibodies. We found that both exogenous , transfected 293T cells with them and treated the transfected cells with insulin to induce TCTP phosphorylation. This study showed that both S9A and S15A mutant constructs exhibited reduced phosphorylation by insulin compared to that of wild-type TCTP (WT) A. InsuliWe also confirmed that Ser-9 and -15 are the sites of insulin-promoted TCTP phosphorylation in studies on the phosphorylation of S9A and S15A double mutant (pEGFP-N1-S9AS15A). In cells overexpressing S9AS15A TCTP, there was a total absence of phosphorylation of TCTP by insulin B. This cTCTP has been shown to be phosphorylated by polo-like kinase (Plk) at its Ser-46 and -64 residues in a hierarchical manner . The preIt has been suggested that an unknown cytoplasmic factor regulated by insulin, may regulate the pump activity of Na,K-ATPase . The preInsulin-induced TCTP phosphorylation appears to be cell-type-specific because in our hands insulin did not induce TCTP phosphorylation in HeLa cells as it did in 293T cells C. PrevioRegulation of TCTP can occur at transcriptional, translational, and/or post-translational levels. It is well established that TCTP functionally responds to various physiological and pathological stimuli at transcriptional and translational levels ,21. But Conceivably other posttranslational modifications of TCTP, such as oligomerization and truncation, may also contribute to the regulation of TCTP. Additionally, specific pathological conditions may provoke some of the posttranslational modifications and contribute to TCTP-related diseases such as diabetes, and allergy , and sta2. After serum starvation, the 293T cells were incubated in media containing 100 nM insulin  for indicated times. For transient expression of TCTP, pEGFP-N1 constructs containing the TCTP gene were transfected in 293T cells using WelFect EX Plus reagent  according to the manufacturer’s guidelines.Cells from a human embryonic kidney cell line, 293T, obtained from American Tissue Culture Collection (ATCC), were cultured in cell culture dishes containing Dulbecco’s Modified Eagle’s Medium , supplemented with 10% fetal bovine serum , and penicillin/streptomycin (Gibco) at 37 °C under humidification with 5% COFull-length rat TCTP was subcloned into pEGFP-N1 vector using the following primers (forward: 5'-CCC AAG CTT (Hind III) ATC ATC ATC TAC CGG GAC-3'; reverse: 5'-ACG CGT CGA C  GA CAT TTT TCC ATC TCT AA-3'). Serine to alanine (Ser-Ala) point mutations at specific serine residues , were accomplished employing polymerase chain reaction (PCR)-based site-directed mutagenesis employing Quickchange II kit . All point mutation constructs including pEGFP-N1-S9A, -S15A, -S37A, S53A, -S9AS15A, and -S46A64A98A  were prepared using primers that were specifically designed to generate Ser to Ala substitution . Additional point mutations at indicated residues were accomplished as described. The point mutations at specific residues were verified by nucleotide sequencing (data not shown).Cells were washed with phosphate-buffered saline (PBS), and harvested by scraping from the culture dish. They were lysed with a cold lysis buffer , 1% Triton X-100), briefly sonicated and centrifuged at 12,000 rpm for 10 min at 4 °C. The protein content of the lysates was assessed by Bradford assay . The lysate samples were diluted with sample buffer and boiled. Aliquots of the lysates containing equivalent quantity of proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins on gel were transferred to nitrocellulose  or polyvinylidene difluoride  membrane and blocked. The proteins were then probed with primary antibodies against anti-β-actin , -GFP , and -TCTP  and then incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Bio-Rad). Protein bands were analyzed using an enhanced chemiluminescence  and UV Products Image Reader .After transfection of pEGFP N1-TCTP-GFP or its Ser to Ala point mutants, the cells were serum starved. The quiescent 293T cells were then treated with insulin (Sigma) at a concentration of 100 nM for indicated times, washed, and lysed in a buffer devoid of detergent (200 mM Tris (pH 7.5) 135 mM NaCl, phosphatase inhibitor I and II, and protease inhibitor (Sigma)) on ice. The lysates were subjected to brief sonication and centrifuged at 3120 rpm for 15 min at 4 °C. The supernatants were collected and centrifuged again at 14,000 rpm for 90 min at 4 °C. The resulting pellet gave crude membrane and the supernatant, cytosolic fractions. The pellet was then diluted in the lysis buffer now containing a detergent, 1% Triton X-100. The cytosolic and membrane fractions were subjected to SDS-PAGE and the protein bands were analyzed by Western blotting.After transfection with pEGFP N1-TCTP-GFP or Ser to Ala mutant constructs, the 293 T cells were serum starved, treated with insulin (100 nM) for indicated times and their cytosolic and membrane fractions were prepared as previously described. Phosphorylated TCTP was detected by immunoprecipitation with anti-phospho-serine (p-Ser), -phospho-tyrosine (p-Tyr), and -phospho-threonine (p-Thr) antibodies (Sigma) for 1 h. The reaction mixtures were then reacted with protein A-agarose overnight in a rocking platform at 4 °C. In addition, the fractions were treated with anti-p-Ser agarose (Sigma) to immunoprecipitate the TCTP phosphorylated at its Ser residue(s). The immune complexes were washed, diluted with sample buffer containing SDS and separated by SDS-PAGE. The resolved proteins were electro-transferred to NC membrane (Whatman) and immunobloted using anti-GFP antibody (SantaCruz).To predict the phosphorylation residues of TCTP, several database servers providing phosphorylation prediction functions including NetPhos  and PHOSIn the present study, we found that insulin induces phosphorylation of TCTP in 293T cells at Ser residues. Phosphorylation of TCTP by insulin was independent on the origin of substrate, endogenous or exogenous origins of TCTP, but dependent on the types of cells. Insulin specifically phosphorylates the Ser-9 and -15 residues of TCTP, possibly through the stimulation of the still unidentified insulin-dependent kinase. Phosphorylated TCTP was not found in the plasma membrane but detected in the cytosolic fraction of cells. Study on the responsible kinase and the possible involvement of TCTP phosphorylation in sodium pump regulation is needed for further verification. Use of insulin can be considered in the treatment of TCTP-induced hyperreactive vascular disease such as hypertension.


The solubility of the heavy metals in 1 mol dm−3 NH4NO3 was 1–49 % for zinc, 5–45 % for cadmium, and <1–10 % for lead. In 1 mol HCl dm−3, the solubility of the studied metals was much higher and obtained values depending on the collection site, from 45 to 92 % for zinc, from 74 to 99 %, and from 79 to 99 % for lead. The lower solubility of the heavy metals in 1 mol dm−3 NH4NO3 than 1 mol HCl dm−3 is connected with that, the ammonium nitrate has low extraction power, and it is used in determining the bioavailable (active) form of heavy metals. Toxicity assessment of the soil samples was performed using two tests, Phytotoxkit and Microtox®. Germination index values were between 22 and 75 % for Sinapis alba, between 28 and 100 % for Lepidium sativum, and between 10 and 28 % for Sorghum saccharatum. Depending on the studied soil sample, Vibrio fischeri luminescence inhibition was 20–96 %. The sensitivity of the test organisms formed the following series: S. saccharatum > S. alba = V. fischeri > L. sativum. Significant positive correlations (p ≤ 0.05) of the total and soluble contents of the metals with luminescence inhibition in V. fischeri and root growth inhibition in S. saccharatum were found. The general trend observed was an increase in metal toxicity measured by the biotest with increasing available metal contents in soils. All the soil samples were classified into toxicity class III, which means that they are toxic and present severe danger. Biotest are a good complement to chemical analyses in the assessment of quality of soils as well as in properly managing them.This research is aimed at assessing the total content and soluble forms of metals  and toxicity of soils subjected to strong human pressure associated with mining of zinc and lead ores. The research area lay in the neighbourhood of the Bolesław Mine and Metallurgical Plant in Bukowno (Poland). The study obtained total cadmium concentration between 0.29 and 51.91 mg, zinc between 7.90 and 3,614 mg, and that of lead between 28.4 and 6844 mg kg The obtained information may provide a better understand the limitations and benefits of chemical and ecotoxicological methods for conducting the environmental risk assessment of heavy metal polluted soils.The aims of this study were as follows: (1) to investigated total content and soluble forms of metals  in soil collected in the zinc and lead ore mining and metallurgy area; (2) to use two biotests  and another one further from the plant , at a distance of 3,000 and 4,000 m . Cadmium, lead, and zinc were determined with the use of a Jobin–Yvon Ultrace 238 JY Inductively Coupled Plasma Atomic Emission Spectrometer (ICP-AES). An internal standard and a certified reference material, CRM023-050-Trace Metals-Sandy Loam 7 (RT Corporation), were added to each series of determinations in order to verify their precision. Each sample of the soil material was analysed in two replications. If the replications analysis results differed from one another by more than 5 %, another two analyses have to be conducted for that same sample. A Microsoft Excel 2007 spreadsheet and Statistica 10 package were used for analysis and presentation of the obtained results.The collected soil samples were dried and then sifted through a sieve with a 2 mm mesh. In soil prepared this way, basic soil properties were determined: pH in 1 mol KCl by the potentiometric method; granulometric composition (using the Casagrande method modified by Prószyński); the content of organic carbon using the Tiurin method; and the sorption capacity of soil  , Sinapis alba (L), and Lepidium sativum (L). The measurement parameters were the inhibition of seed germination (IG) and root length inhibition (IR) in the tested soil in comparison with the control soil. The test was conducted in accordance with the procedure recommended by the manufacturer  appears to be a good method for assessing the toxicity of bottom sediments, composts, and soils , and the highest from sampling site 4 (32.3 g kg−1 d.m)  to 563.5 (sampling site 2) mmol (+) kg–1 d.m (Table –1 d.m.) and the lowest from sampling site 7 (65.8 mmol (+) kg–1 d.m.).Basic physico-chemical properties of the soils are presented in Tables −1 of soil d.m., total lead concentration between 7.90 and 3,614 mg, and total zinc concentration between 28.4 and 6,844 mg  to 49 % (sampling site 6) for zinc, from 5 % (sampling site 3) to 45 % (sampling site 6) for cadmium, and from <1 % (sampling sites 2 and 4) to 10 % (sampling site 6) for lead  to 92 % (sampling site 2) for zinc, from 74 % (sampling site 5) to 99 % (sampling sites 1 and 2) for cadmium, and from 79 % (sampling site 4) to 99 % (sampling sites 2 and 6) for lead in comparison with their total concentrations in the soils  fraction of heavy metals  and in 1 mol NH4NO3 dm3 . The concentration of metals in the studied soils was generally positively correlated with pH, organic carbon content, sorptive capacity, and percentage content of the finest fraction <0.02 mm , and the lowest in S. alba (between 0 and 20 %). Among the studied soil samples, the strongest inhibition of S. saccharatum seed germination was observed in the case of soil from sampling site 2, while for L. sativum the strongest inhibition was observed in soil from sampling site 4, and for S. alba, sampling site 5. These soils are located very close to the Bolesław Mine and Metallurgical Plant in Bukowno. The plant root growth inhibition was between −11 and 82 % (Table S. saccharatum (60–82 %). L. sativum was the plant most resistant to the phytotoxic influence of the studied soils. Root growth inhibition of this plant was between −11 and 70 %. The root growth inhibition of S. alba varied from 22 to 74 %. Soils collected from sampling sites 2, 5, and 6 caused the highest root growth inhibition of the test plants  and 75 % (sampling site 2)  to 100 % (sampling site 7). Only soil samples collected from sampling sites 2 and 7 were not toxic for L. sativum (GI of 90–110 % indicates no effect/non-toxicity), and the remaining ones caused growth inhibition of the test plant. The lowest GI values were found for S. saccharatum (between 10 % and 28 %)  and in 1 mol NH4NO3 dm−3 (cadmium and zinc) were significantly positively correlated (p ≤ 0.05) with the luminescence inhibition of V. fischeri and with the root growth inhibition of S. saccharatum   Table .S. saccharatum and showed 29 % root growth inhibition , which evaluate habitat function, as well as the aquatic test (V. fischeri), which evaluate soil retention function, proved to be adequate to assess the quality of these mine-contaminated soils. Due to their specificity, ecotoxicological tests constitute a good complement to chemical analyses in the assessment of quality of soils as well as in properly managing them.To sum up, biotests carried out on soils collected in the area of zinc and lead ore mining and metallurgy showed that they are toxic for the test organisms. The general trend observed was an increase in metal toxicity measured by the biotest with increasing available metal contents in soils. These soils were classified into toxicity class III. At the same time, their toxicity was higher in soil samples collected at the shortest distance from the Mine and Metallurgical Plant in Bukowno. The direct biotest (


Yet antibodies have multiple copies of these residues throughout their macromolecular structure, making control over the location of the conjugation reaction impossible. This lack of site specificity can lead to the formation of poorly defined, heterogeneous immunoconjugates with suboptimal in vivo behavior. Over the past decade, interest in the synthesis and development of site-specifically labeled immunoconjugates—both antibody-drug conjugates as well as constructs for in vivo imaging—has increased dramatically, and a number of reports have suggested that these better defined, more homogeneous constructs exhibit improved performance in vivo compared to their randomly modified cousins. In this two-part review, we seek to provide an overview of the various methods that have been developed to create site-specifically modified immunoconjugates for positron emission tomography, single photon emission computed tomography, and fluorescence imaging. We will begin with an introduction to the structure of antibodies and antibody fragments. This is followed by the core of the work: sections detailing the four different approaches to site-specific modification strategies based on cysteine residues, glycans, peptide tags, and unnatural amino acids. These discussions will be divided into two installments: cysteine residues and glycans will be detailed in Part 1 of the review, while peptide tags and unnatural amino acids will be addressed in Part 2. Ultimately, we sincerely hope that this review fosters interest and enthusiasm for site-specific immunoconjugates within the nuclear medicine and molecular imaging communities.Due to their remarkable selectivity and specificity for cancer biomarkers, immunoconjugates have emerged as extremely promising vectors for the delivery of diagnostic radioisotopes and fluorophores to malignant tissues. Paradoxically, however, these tools for precision medicine are synthesized in a remarkably imprecise way. Indeed, the vast majority of immunoconjugates are created  Over the last three decades, medical imaging has revolutionized cancer care, providing clinicians with the means to noninvasively acquire anatomical, functional, and biological information about tumors. Due to their remarkable affinity and specificity for cancer biomarkers, antibodies—as well as an ever-growing array of antibody fragments—have played an increasingly important role in this field Fig.  1, 2]. , 2. 1, 2Yet paradoxically, these agents designed to enable “precision medicine” are synthesized in a rather imprecise manner. At present, the vast majority of bioconjugation techniques rely on reactions between bifunctional probes and amino acids, typically lysines Fig.  5–7]. F–7. F5–7]These random bioconjugation approaches produce immunoconjugates that are poorly defined and heterogeneous on three different levels –10. FirsH3 region, for example, will likely exhibit in vivo pharmacokinetics different from that of an antibody bearing five fluorophores attached to lysines in the VH and CH1 domains. Furthermore, without the ability to control the precise location of the conjugation reactions, cargoes may become appended to the antigen-binding domains of the antibody, thus impairing the immunoreactivity of the conjugate TETA-Fab′ construct could be found in the initial report or any follow-up publications. In a third example, over 20 years ago, Slinkin et al. created an anti-carcinoembryonic antigen (CEA) Fab′ via the digestion of a full-length IgG with pepsin and the reduction of the hinge region disulfides of the F(ab′)2 intermediate with mercaptoethylamine (MEA) . In. In54]. ted Fig.  56]..et al. het al. developed the phage ELISA for selection of reactive thiols (PHESELECTOR) biochemical assay, a procedure that provides information on the influence that the site of the introduced cysteine has on antigen-binding affinity as well as the ability to covalently modify the thiol in question DOTA-cysDb exhibited higher tumoral uptake and more favorable tumor-to-background activity concentration ratios than its randomly labeled cousin. In addition, the site-specific labeling of the cysDb also seemed to exert a significant influence on the pharmacokinetic profile of the radioimmunoconjugate, increasing kidney uptake and decreasing liver retention compared to the randomly labeled construct.In the last few years, a number of laboratories have employed genetic engineering to create site-specifically modified immunoconjugates based on antibody fragments. In 2014, for example, Anna Wu’s laboratory reported the development of anti-activated leukocyte cell adhesion molecule (ALCAM) cysDb . After rvia reactions with C-terminal cysteines, including an anti-TNC [111In]DTPA-scFv, an anti-human epidermal growth factor receptor 2 (HER2) [111In]DTPA-sdAb, an anti-MUC1 [111In]DOTA-di-scFv, and an anti-CEA [64Cu]DO3A-GLGK-cysDb  bearing a vinyl sulfone moiety to site-specifically modify an anti-TAG-72 diabody with a C-terminal cysteine  and therapy (using I-131) DTPA-satumomab has been used in the clinic in patients with colorectal cancer DOTA-trastuzumab specifically targeted the HER2-expressing xenografts but did not offer a significant improvement over an [111In]DOTA-trastuzumab construct synthesized using a traditional, nonsite-specific conjugation method . Ku. Ku88]. n method .Over the last 15 years, a number of alternative chemoenzymatic methods for the specific functionalization of glycoproteins have emerged. Using both natural and engineered enzymes, it is now possible to introduce carefully tailored sugars into the glycans to enable chemoselective modifications. Not surprisingly, this work has been enthusiastically applied to the creation of site-specifically labeled radioimmunoconjugates.N-acetylglucosamine (GlcNAc) residues in the glycans , a mutant β-1,4-galactosyltransferase developed by Qasba and coworkers. This substrate-permissive galactosyltransferase facilitates the attachment of modified galactose monomers to ans Fig.  91]. Tw. TwN-aceications , 93.et al. employed a three-step modification procedure: (1) the removal of terminal galactose residues of the glycans using β-1,4-galactosidase, (2) the attachment of Gal-NAz to the sugar chains using Gal-T1(Y289L), and (3) the conjugation of chelator-modified dibenzocyclooctynes to the glycans via the strain-promoted azide-alkyne click reaction  than an analogous agent produced using a traditional, nonsite-specific modification strategy (57.5 ± 5.3 %ID/g at 96 h postinjection). In a subsequent study, the same group used an improved, one-pot modification strategy to create a series of DFO- and AlexaFluor® 680-bearing immunoconjugates based on the huA33 antibody for the multimodal PET/near-infrared fluorescence imaging of colorectal cancer -based methodology for the construction to nuclear imaging agents was published in 2013 , 97et alAs the recency of these citations illustrates, the use of metabolic glycans engineering to create site-specifically modified antibodies—let alone site-specifically modified antibodies for imaging applications—remains a very young field. Thus, in order to maximize the benefits of this technology, it is crucial that imaging-focused laboratories continue to investigate the use of existing metabolic engineering technologies and prove quick to leverage any new advances in the years to come , 97.engineered cysteine residues provides an exquisite level of stoichiometric and regiochemical control but requires genetic engineering, which limits its modularity and broad applicability. Likewise, while the modification of glycans via bioorthogonal click chemistry is modular, facile, and straightforward, the usefulness of this approach is necessarily limited to immunoglobulins with pendant sugar chains. Setting specifics aside, however, each of these strategies offers a route to immunoconjugates that are more homogenous and better defined than constructs created using traditional bioconjugation techniques. Furthermore, preclinical studies have shown that these site-specifically labeled immunoconjugates often boast superior in vivo behavior compared to their randomly constructed cousins. Somewhat curiously, while the clinical validation of site-specifically labeled immunoconjugates is of the utmost importance, work in this area seems to have stalled just short of the clinic. In our humble opinion, the move from bench to bedside is the most pressing imperative for the field.In the preceding pages, we have discussed an array of site-specific bioconjugation strategies that are predicated on two simple functionalities: cysteine residues and glycans. Of course, each approach has its own intrinsic advantages and disadvantages. For example, while the modification of natural cysteine residues is both modular and straightforward, it does not offer the same degree of homogeneity and stoichiometric control as other approaches. Conversely, conjugation to two of the four families of site-specific modification strategies. In Part 2 of this work, which will appear in the next issue of the journal, we will shift gears and discuss bioconjugation approaches based on peptide tags and unnatural amino acids, methods which elegantly harness chemoselective ligations and enzymatic transformations to create site-specifically modified immunoconjugates.In closing, it is important to note that in this installment of the review, we have only covered


Results indicated that (Z)-1-benzyl-4-(hydroxyimino)-1H-benzo[f]indol-9(4H)-one (10) showed a potent dual inhibitory effect on NE release and superoxide anion generation with IC50 value of 2.78 and 2.74 μM respectively. The action mechanisms of 10 in human neutrophils were further investigated. Our results showed that compound 10 did not alter fMLF-induced phosphorylation of Src (Src family Y416). Notably, phosphorylation of Akt (S473) and mobilization of [Ca2+]i caused by fMLF was inhibited by compound 10. Further structural optimization of 10 is ongoing.Certain benzo[ Human neutrophils play an important role in the defense system against invasion by microorganisms and in the pathogenesis of various diseases such as rheumatoid arthritis, ischemia-reperfusion injury, chronic obstructive pulmonary disease, and asthma ,2,3,4,5.Tabebuia sp.) and evaluated for their biological activities. Among them, β-LAPA was found to be able to inhibit the expression of nitric oxide (NO) and PGE2 in alveolar macrophages [d]imidazole derivatives and evaluated for their anti-inflammatory activities. Our results indicated that (E)-2-[2-(5-nitrofuran-2-yl)vinyl)]-furonaphthoimidazole (1) naphthoimidazole (2) furophenazinecarboxylic acid (3) [4) [f]indole-4,9-dione derivatives which belong to a new structural type possessing versatile iminoquinone moiety. Their cytotoxicities were also evaluated due to the structural similarity of these tricyclic compounds to the cytotoxic (Z)-4-(hydroxyimino)naphthofuran-9(4H)-one (5) indole-4,9-dione (8) and 1-benzyl-2-methyl-1H-benzo[f]indole-4,9-dione (9) respectively in a moderate yield. Treatment of 8 with NH2OH proceeds in the regiospecific and the stereospecific manners to give (Z)-1-benzyl-4-(hydroxyimino)-1H-benzo[f]indol-9(4H)-one (10) as a sole product. The regiospecific oximination occurred at C-4 rather than C-9 carbonyl was established based on the 13C-NMR in which the more downfield C-4 carbonyl shifted from 180.98 to 141.42 ppm while the more upfield C-9 carbonyl shifted from 176.23 to 174.12 ppm indole-4,9-dione (12) [7 with acetylacetone. Treatment of 12 with NH2OH or NH2OMe gave 1-benzyl-3-[1-(hydroxyimino)ethyl]-2-methyl-1H-benzo[f]indole-4,9-dione (13) and its methoxyimino analog 14 respectively. Reaction of 8 and 9 with procainamide proceeds in the regiospecific and the stereospecific manners to afford (Z)-4-(1-benzyl-9-oxo-1H-benzo[f]indol-4(9H)-ylideneamino)-N-[2-(diethylamino)ethyl]benzamide (15) and (Z)-4-(1-benzyl-2-methyl-9-oxo-1H-benzo[f]indol-4(9H)-ylideneamino)-N-[2-(diethylamino)ethyl]benzamide (16) respectively in a fairly good overall yield. The formation of Z-form product rather than the E-isomer can be realized in which the 4-substituted phenyl group proximate the pyrrole ring is less sterically hindered indole-4,9-dione derivatives were synthesized and evaluated for their inhibitory effects on superoxide anion generation and neutrophil elastase (NE) release in formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLF)-activated human neutrophils and results are shown in H-benzo[f]indole-4,9-dione (8) and its 2-methyl derivative 9 exhibited weak inhibitory effects on superoxide anion generation and were inactive on the inhibition of NE release. (Z)-1-Benzyl-4-(hydroxyimino)-1H-benzo[f]indol-9(4H)-one (10) showed a potent dual inhibitory effect on NE release and superoxide anion generation with IC50 value of 2.78 and 2.74 μM respectively. In contrast, its 2-methyl derivative 11 exhibited only marginal activity on superoxide anion generation and was inactive on the inhibition of NE release. These results indicated that the oxime moiety enhanced anti-inflammatory activities while methyl substituent at C-2 position was unfavorable especially in the inhibition of NE release. The same structure-activity relationships were observed in which 3-acetyl-1-benzyl-2-methyl-1H-benzo[f]indole-4,9-dione (12) was inactive while its oxime derivative 13 exhibited a weak dual inhibitory effect on NE release and superoxide anion generation. Compound 14, the methyl derivative of 13, was inactive. Among these benzo[f]indole-4,9-dione derivatives, (Z)-4-(1-benzyl-9-oxo-1H-benzo[f]indol-4(9H)-ylideneamino)-N-[2-(diethylamino)ethyl]benzamide (15) was the most potent dual inhibitor on NE release and superoxide anion generation with IC50 value of 0.51 and 2.05 μM respectively. Although (Z)-4-(1-benzyl-2-methyl-9-oxo-1H-benzo[f]indol-4(9H)-ylideneamino)-N-[2-(diethylamino)ethyl]benzamide (16) exhibited a strong inhibitory effect on superoxide anion generation with an IC50 value of 0.52 μM, it induced NE release of human neutrophils.Certain benzo[f]indole-4,9-dione derivatives were evaluated in vitro against a panel of cell lines consisting of MCF7 (breast), NCI-H460 (lung), and SF-268 (CNS) as described previously indol-9(4H)-one (10) as a novel structural type of potential anti-inflammatory agent. Compound 10 exhibited dual inhibitory activities on NE release and superoxide anion generation in formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLF)-activated human neutrophils with IC50 value of 2.78 and 2.74 μM respectively. Mechanism studies indicated that compound 10 did not alter fMLF-induced phosphorylation of Src (Src family Y416). Notably, phosphorylation of Akt (S473) and mobilization of [Ca2+]i caused by fMLF was inhibited by compound 10. Further structural optimization of 10 is ongoing.We have identified (


The direct binding of Fe65 to APP not only regulates target gene expression, but also contributes to secretase-mediated processing of APP, producing cytoactive proteolytic fragments including the APP intracellular domain (AICD) and cytotoxic amyloid β (Aβ) peptides. Given that the accumulation of Aβ peptides in neural plaques is a pathological feature of Alzheimer’s disease (AD), it is essential to understand the mechanisms controlling Aβ production. This will aid in the development of potential therapeutic agents that act to limit the deleterious production of Aβ peptides. The Fe65-APP complex has transcriptional activity and the complex is regulated by multiple post-translational modifications and other protein binding partners. In the present study, we have identified Ser289 as a novel site of UV-induced phosphorylation. Interestingly, this phosphorylation was mediated by ATM, rather than ATR, and occurred independently of APP. Neither phosphorylation nor mutation of Ser289 affected the Fe65-APP interaction, though this was markedly decreased after UV treatment, with a concomitant decrease in the protein levels of APP in cells. Using mutagenesis, we demonstrated that Fe65 Ser289 phosphorylation did not affect the transcriptional activity of the Fe65-APP complex, in contrast to the previously described Ser228 site.Fe65 undergoes a phosphatase-sensitive gel mobility shift after DNA damage, consistent with protein phosphorylation. A recent study identified Ser Fe65 is an adaptor protein consisting of an N-terminal WW domain and two C-terminal phosphotyrosine binding domains PTB1 and PTB2)  and PTB2. AICD is668 , pFR-Luc (Agilent) and pRL-TK (Promega) were used.228 site. Characterisation of this antibody revealed that mutation of Fe65 Ser228 reduced, but did not abolish, the DNA damage-induced Western blot signal after p-Fe65x detection. We hypothesised that p-Fe65x was thus recognising additional Fe65 phosphorylation sites and used this antibody and mutagenesis in the present study to identify a new Fe65 phosphorylation site. After incubation with the relevant HRP-conjugated secondary antibody, western blots were visualised using ECL Prime  and images captured using a Syngene G:Box gel documentation system. When images were quantified, ImageJ software was used, with levels of p-Fe65x normalised to Fe65 levels in the corresponding immunoprecipitate. For statistical analysis, a paired t-test (two-tailed) was used, with level of significance defined as: p < 0.05 (*), p < 0.01 (**) and p < 0.001 (***). All Western blot images are representative of at least three independent experiments.After the indicated treatments, cells were washed in cold PBS before lysis in NETN buffer . After incubation at 4C for 15 min, lysates were cleared by centrifugation and supernatants isolated. For immunoprecipitation, cell lysates were incubated with either GFP-Trap  or FLAG-M2 agarose beads  for 2 hours at 4C before washing three times in TBST . For immunoprecipitation of endogenous Fe65, cell lysates were incubated overnight with 4μl of Fe65 antibody . For lambda phosphatase treatment, beads were resuspended in 50μl of reaction buffer and 200U of lambda protein phosphatase  for 15 min at 30C before washing again in TBST. Beads were resuspended in an equal volume of 2X LDS sample buffer (Life Technologies) containing 2.5% 2-mercaptoethanol before heating at 95C for 5 min. Samples were analysed by Western blotting using 4–12% bis-tris gels (Life Technologies) before transferring to nitrocellulose using an iBlot machine (Life Technologies). Membranes were blocked in 2.5% BSA/TBST for 1 hour prior to overnight incubation (4C) in blocking buffer containing the following primary antibodies: GFP (2956), APP (2452), phospho-S/T-Q , CHK1 phospho-Ser317 (12302), GAPDH (2118), all from Cell Signalling Technology and FLAG . The primary antibody referred to as p-Fe65x in this study was raised against a phosphopeptide encompassing the previously described SerFor analysis of GFP-Fe65 and GFP-Fe65 S289A localisation, HEK293 cells were transfected with the relevant plasmid using calcium phosphate. After 24h, cells were fixed on glass coverslips with 4% formaldehyde and stained with DAPI. Fluorescence images were captured using a Zeiss Axioscope fluorescence microscope. Data are representative of at least three independent experiments.The APP-dependent luciferase assay has been described previously and was performed according to our earlier study ,17.228 as a DNA damage-induced site of phosphorylation using an anti-ATM/ATR motif antibody [raised against a library of peptides phosphorylated on Ser/Thr followed by Gln (SQ/TQ)–the ATM/ATR ‘consensus sequence’]. We attempted to generate a phosphospecific antibody to Ser228 and tested the reactivity/specificity of this antibody in immunoprecipitations. Throughout the manuscript, this antibody is referred to as p-Fe65x. Cells were transfected with GFP-Fe65 or GFP-Fe65 S228A and Fe65 immunoprecipitated from untreated cells or UV-treated cells (to induce DNA damage). As shown in 228, consistent with our previously published data. The same samples were analysed using the p-Fe65x antibody. Again, GFP-Fe65 was detected by this antibody and the signal was increased after DNA damage. However, when Ser228 was mutated, the p-Fe65x signal was reduced but not completely abolished, and was still increased after UV. This strongly suggests that p-Fe65x recognises Ser228, as well as additional DNA damage-induced phosphorylation site(s). To confirm that p-Fe65x was binding other phosphorylation sites in GFP-Fe65 S228A, immunoprecipitates were analysed in the presence and absence of λ-phosphatase. As shown in As part of an earlier study, we identified Fe65 Ser228 mutated to Ala, to prevent p-Fe65x recognising this site. As shown in 289. Therefore, Ser289 was mutated in GFP-Fe65 S228A to generate the S228A/S289A double phosphomutant. These Fe65 variants were immunoprecipitated from UV-treated cells and tested for reactivity with p-Fe65x. As shown in 289 was additionally mutated. This confirms that Ser289 is a novel site of phosphorylation (likely to be targeted by ATM/ATR due to SQ sequence) and that the p-Fe65x antibody is able to detect both Ser228 and Ser289. Analysis of the sequence context of these two phosphorylation sites reveals significant similarity, with an identical SQGS sequence present in both . Using fluorescence microscopy, it was demonstrated that mutation of Ser289 in GFP-Fe65 did not affect cellular localisation, with Fe65 and S289A being present in both the cytoplasm and nucleus  being recognised by p-Fe65x, a series of Fe65 deletion mutants  were imm in both . This is nucleus . The locafter UV . This sh289 phosphorylation after UV, GFP-Fe65 S228A was transfected into cells. By using GFP-Fe65 S228A for these studies, it was ensured that p-Fe65x was specifically binding only to Ser289 and thus data was specific for this site. Cells were exposed to the indicated doses of UV for the indicated times and GFP-Fe65 S228A immunoprecipitated before Western blotting with p-Fe65x. As shown in 289 phosphorylation was increased as early as 15min after UV treatment (~2-fold) and continued to increase up to 240min (~3-fold). In 289 phosphorylation was increased after UV doses as low as 4J/m2 (1.5-fold), and continued to increase up to 40J/m2 (~4.5-fold). These data confirm that Fe65 Ser289 is phosphorylated after UV in a dose- and time-dependent manner.To investigate the kinetics and dose response of Fe65 Ser289, specific inhibitors were used. Ser289 conforms to the SQ/TQ target site of the ATM/ATR protein kinases. Generally, ATR is the major kinase activated after UV, though ATM can also contribute to UV-induced DNA damage signalling. Cells were transfected with GFP-Fe65 S228A before treatment with either DMSO, AZ20 (ATR inhibitor) or KU55933 (ATM inhibitor) followed by UV and analysis of Fe65 Ser289 phosphorylation using p-Fe65x. As shown in 289 phopshorylation was increased after UV. Interestingly, inhibition of ATR had no effect on this UV-induced phosphorylation, whilst ATM inhibition significantly reduced Ser289 phosphorylation (317) was investigated in the cell extracts. As shown in 317 phosphorylation was markedly increased after UV and completely abolished by ATR inhibition, confirming that ATR was inhibited by AZ20. Together, these data suggest that Fe65 Ser289 is phosphorylated after UV in an ATM-dependent manner.To identify the kinase that is phosphorylating Fe65 Serrylation . To conf289. An Fe65 mutant that was unable to interact with APP was generated by mutating Cys652 to phenylalanine in the PTB2 domain of Fe65 (C652F). Cells were then transfected with FLAG-Fe65 S228A or FLAG-Fe65 S228A/C652F before treatment with UV, Fe65 purification and analysis of Ser289 phosphorylation using p-Fe65x. Again, performing all studies in a S228A mutant background ensured that results were specific to Ser289. As shown in 289 phosphorylation in FLAG-Fe65 S228A or FLAG-Fe65 S228A/C652F .The Fe65-APP interaction can be disrupted by APP Thr289 phosphorylation in Fe65-APP transcriptional activity, a luciferase-based reporter gene assay was used in HEK293 cells. This system has been used in multiple studies and has revealed a complex regulatory network controlling Fe65-APP mediated transcription [289 in Fe65 was investigated. As shown in 289 phosphorylation is not involved in regulating the transcriptional activity of the Fe65-APP complex.Previous studies have demonstrated that Fe65 markedly enhanced APP-mediated gene transcription. To investigate the potential role of Fe65 Sercription ,17,19,30To confirm the reproducibility of data, repeats of relevant studies are included in the supplementary material –S4 Figs.289 as a novel site of UV-induced phosphorylation. Phosphorylation increased in a dose- and time-dependent manner and was found to be targeted by ATM, rather than ATR, after UV in an APP-independent manner. Mutation of Ser289 to Ala did not affect the Fe65-APP interaction nor the Fe65-APP transcriptional activity. These findings are in contrast to the Fe65 Ser228 site that we previously identified, which we found to be targeted by ATR after UV and contributed to the regulation of Fe65-APP transcriptional activity.In the present study, we identified Fe65 Ser228 described recently and Ser289 identified in the present study [228 being ATR-dependent and Ser289 being ATM-dependent. To gain further insight into this differential regulation, it will be important to compare the phosphorylation kinetics of each site after different forms of DNA damage and investigate the kinase involved.Previous studies demonstrated that Fe65 displayed a phosphatase-sensitive gel mobility shift after DNA damage . Our recnt study . These snt study . Given ont study ,31,32. I228 and Ser289 occurs independently of the APP interaction. Assuming that the above model is correct, this suggests that the ‘closed’ conformation of Fe65 can still be targeted by the ATM/ATR kinases and that the phosphorylation occurs independently of chromatin interaction. Interestingly, as well as multiple studies demonstrating that Fe65 contributes to the cellular DNA damage response, recent publications have implicated Fe65 in breast cancer biology. More specifically, Sun et al (2014) provided some evidence that Fe65 expression is increased in breast cancer cell lines and that Fe65 was able to act as an activator of estrogen receptor-α (ERα), promoting cell growth [228 and Ser289) in these particular research areas. Studies are in progress to functionally characterise these new phosphorylation sites in response to DNA damage by expressing phosphomutant versions of each in Fe65-deficient cells.Fe65 knockout (KO) mouse embryonic fibroblasts (MEFs) showed elevated levels of DNA damage after exposure to etoposide or ionising radiation, caused (at least in part) by inefficient recruitment of the histone acetyltransferase TIP60 to sites of DNA double strand breaks (DSBs), inefficient histone H4 acetylation and defective chromatin relaxation  ,22. Whill growth . Interesl growth . Given t289 to Ala did not affect the Fe65-APP interaction or the transcriptional activity of this complex. On the contrary, we previously demonstrated using both phosphoblocking and phopshomimetic mutations, that Fe65 Ser228 phosphorylation inhibited Fe65-APP transcriptional activity [Mutation of Seractivity . In addiactivity ,23,35. O289 as a novel site of ATM-dependent phosphorylation after UV-induced DNA damage. Whilst this site appears to have no role in mediating the Fe65-APP interaction or transcriptional activity of this complex, it will be important to investigate how this site contributes to the DNA damage response roles of Fe65.Given the roles of Fe65 in Alzheimer’s biology and DNA damage response pathways, it is important to fully elucidate the mechanisms regulating Fe65 and how these impact on the function of Fe65 and Fe65-binding partners within the cell. This study has identified SerS1 Fig2 UV for 2h. Cell extracts were subjected to GFP immunoprecipitation and samples analysed by Western blotting using the indicated antibodies. Please note, we have previously published the data in S1A . (B) HEK293 cells were transfected with GFP-Fe65 S228A 1–349 for 24h before treatment with 40J/m2 UV for 2h. After GFP immunoprecipitation, each sample was split and half treated with lambda phosphatase for 15min at 30C, before western blot analysis with the indicated antibodies.(A) HEK293 cells were transfected with GFP-Fe65 or GFP-Fe65 S228A for 24h before treatment with 40J/m(PDF)Click here for additional data file.S2 Fig2 UV for 2h and GFP immunoprecipitation. Samples were analysed by Western blotting with the indicated antibodies.HEK293 cells were transfected with FLAG-Fe65 S228A for 24h before treatment with DMSO, 5μM AZ20 or 10μM KU55933 for 45min, followed by 40J/m(PDF)Click here for additional data file.S3 Fig2 UV for 2h. Cell extracts were subjected to FLAG immunoprecipitation before Western blot analysis with the indicated antibodies.HEK293 cells were transfected with FLAG-Fe65 S228A or FLAG-Fe65 S228A/C652F for 24h before treatment with 40J/m(PDF)Click here for additional data file.S4 Fig2 UV for 2h. Cell extracts were subjected to FLAG immunoprecipitation before western blot analysis with the indicated antibodies.HEK293 cells were transfected with FLAG-Fe65 or FLAG-Fe65 S289A for 24h before treatment with 40J/m(PDF)Click here for additional data file.


Oncorhynchus keta), thyrotropin-releasing hormone gene expression increased immediately after release from a hatchery into the natal stream, and the expression of the essential NR1 subunit of the N-methyl-D-aspartate receptor increased during downstream migration. Gene expression of salmon gonadotropin-releasing hormone (sGnRH) and NR1 increased in the adult chum salmon brain during homing from the Bering Sea to the natal hatchery. Thyroid hormone treatment in juveniles enhanced NR1 gene activation, and GnRHa treatment in adults improved stream odour discrimination. Olfactory memory formation during juvenile downstream migration and retrieval during adult homing migration of chum salmon might be controlled by endocrine hormones and could be clarified using NR1 as a molecular marker.The olfactory hypothesis for salmon imprinting and homing to their natal stream is well known, but the endocrine hormonal control mechanisms of olfactory memory formation in juveniles and retrieval in adults remain unclear. In brains of hatchery-reared underyearling juvenile chum salmon ( Salmon migration is iconic in ecology and is also culturally and economically important to many individuals; however, migratory salmon are under threat globally from overharvesting, anthropogenic pollution, climate change, and environmental degradation123The olfactory hypothesis for salmon homing to their natal stream was proposed in the 1950s57810Oncorhynchus keta) has recently been cloned and characterizedO. kisutch) during a sensitive period for imprintingO. nerka) in Lake Shikotsu 25The N-methyl-D-aspartate receptor (NMDAR) is a glutamate receptor channel subtype and mediates most of the fast excitatory synaptic transmission in the central nervous system. It plays important roles in memory formation and retrieval in higher vertebrates1315161718Here, we show that the gene expression of NR1 and thyrotropin-releasing hormone (TRH) increased during downstream migration to the sea in the juvenile chum salmon brain and that NR1 and salmon gonadotropin-releasing hormone (sGnRH) gene expression increased in the brain during adult homing from the Bering Sea to the natal hatchery. Juvenile treatment with thyroid hormone enhanced NR1 gene activation, and adult treatment with GnRHa increased stream odour discrimination ability. This work demonstrates that NR1 expression in the brain plays crucial roles in olfactory memory formation and retrieval that might be controlled by endocrine hormones in juvenile and adult chum salmon, respectively. Investigation of this mechanism will allow researchers to predict responses to altered biochemical environments and provides tools to explain migratory salmon behaviour, including straying rates.Using underyearling juvenile chum salmon reared in a hatchery and then released into the Chitose River  to allow migration to the sea , the relB  when the fish were in the hatchery. Expression was also prominent in the ventral TE, which is connected with the olfactory bulb (OB)C C .B and D compared to the fish at the hatchery  water in juvenile chum salmon collected at Sites hatchery . These dG) in July to the Chitose Hatchery (Site A) in October, the mRNA expression levels of salmon gonadotropin-releasing hormone (sGnRH-I and II) and NR1 in the OB and TE appeared to increase, with some differences between males and females that might be related to sex-specific gonadal maturationH was significantly higher for both the Ishikari River and Chitose River, their home stream waters, than for the Toyohira River, a nearby tributary not on their migratory path Prior work has shown that treatment with thyroid hormone can prompt olfactory cellular proliferation in coho salmonF) prior to entering the Ishikari River. GnRHa implantation increased the serum levels of 17α, 20β-dihydroxy-4-pregnen-3-one (DHP) after 48 h , which can accelerate homing and gonadal maturation in sockeye salmon25ter 48 h , and tenter 48 h . GnRHa iter 48 h . The relter 48 h , revealiThe endocrine control of osmoregulation in relation to seawater adaptability has been intensively studied in teleost fish, including salmon10C, and a second significant peak at the mouth of their natal river (Site E). The results of in situ hybridization confirmed that NR1 mRNA localization increased prominently in the ventral TE at Site C compared to the hatchery immediately before release into the river. Moreover, the oral administration of T4 significantly increased NR1 expression levels for 4 days.Although the effects of salinity change on NR1 gene expression have been reported in juvenile chum salmon, demonstrating that NR1 expression increases with increased salinityB and D during downstream migration compared to those at the hatchery. The olfactory memory formation capacity for stream odours likely develop when juvenile chum salmon encounter different streams during their downstream migration, and these capabilities are clearly linked to NR1 and BPT hormones. Cumulatively, our data suggest that during the initiation of the downstream migration of juvenile chum salmon, the environmental changes involved in the release into the river may induce the expression of the BPT hormones, which then stimulate the upregulation of NR1, enhancing the olfactory memory formation capability.Natal stream odour discrimination abilities were examined using the EOG response to stream water odours, revealing significantly greater discrimination ability for the Ishikari River (home stream) water in juvenile chum salmon collected at Sites F) to the natal hatchery (Site A), the gene expressions levels of sGnRH-I/II and NR1 in the OB and TE showed some sexually differentiated patterns, likely due to sex-specific gonadal maturationThe homing migration of adult chum salmon is closely related to gonadal maturation, which is regulated mainly by the brain-pituitary-gonad hormones. In particular, sGnRH in the olfactory system, terminal nerve, and preoptic area is believed to play brain region-dependent leading roles in salmon homing migration9H showed significantly higher EOG responses to their natal stream water than to non-natal stream water. GnRHa implantation in adult male chum salmon caught in Site F prior to entering their natal stream produced different EOG responses whether including straying fish from another hatchery or only originating from the natal hatchery. The relative EOG response of adult male chum salmon originating from the Chitose Hatchery showed significant discrimination of the Chitose River water, but that including straying fish only showed significant discrimination of the Ishikari River water. These data suggest that straying fish and accurately homing fish show different olfactory discrimination ability for their natal stream waters.Adult male chum salmon collected at Site Our data on adult homing migration suggest that the activation of sGnRHI/II may cause both the onset of homing migration and the upregulation of NR1, facilitating olfactory memory retrieval as well as natal stream odour discrimination during upstream migration. This study demonstrates that the gene expression levels of NR1 in the brain are a useful molecular marker to clarify the olfactory imprinting and homing abilities of both juvenile and adult chum salmon. While measurement of NR1 transcription is a good first step, it is the protein that will be directly involved in memory formation, and this has not been measured in the data presented. It is unclear whether the relatively small changes in transcription  result in increased protein levels. Further biochemical and molecular biological studies investigating the protein levels and the gene expression profiles of NR1 and NR2A-D with treatments of NMDAR antagonist will be able to reveal olfactory long-term potentiation in salmon brain.These results have important ramifications for salmon conservation and production. Straying, or lack of appropriate homing, is present in all migratory populations to varying degrees, and its expression may be either positive  or negative . Adult chum salmon were captured with fish traps in the Chitose River at the Chitose Hatchery (Site A) and the Indian Waterwheel (Site H), with set nets in Ishikari Bay (Site F), and with longlines as part of the R.V. Hokko-Maru 2013 summer cruise in the Bering Sea (Site G). All fish were collected in accordance with the animal care guidelines of Hokkaido University.Juvenile chum salmon were captured in the river using cast nets (Sites A), Hokkaido National Fisheries Research Institute, Chitose, Hokkaido, Japan were sampled monthly from January (A1) to April (A4) of 2013. After release into the Chitose River, they were sampled at the second bridge of the Chitose (Site B), the Chitose River in the Kamaka Ward (Site C), the confluence point of the Chitose and Yubari Rivers (Site D), the mouth of the Ishikari River (Site E), and Ishikari Bay (Site F) in April 2013. The fork length (FL) and body weight (BW) of the fish are provided in Underyearling juvenile chum salmon reared at the Chitose Hatchery (Site A), the second bridge of the Chitose (Site B), the Chitose River in the Kamaka Ward (Site C), the confluence point of the Chitose and Yubari Rivers (Site D), the mouth of the Ishikari River (Site E), and Ishikari Bay (Site F). The FL and BW of the fish are provided in For the EOG experiment on juvenile chum salmon during downstream migration in April 2015, three fish were used at each sampling point: the Chitose Hatchery  containing T4  for 14 days. T4 (100 mg) was dissolved into 220 μl of 4 M NaOH, added to 3.5 ml of ethanol, sprayed over 50 g of pellets, and dried, for a T4 final concentration of 2 mg/g pellet. Fish were sampled at day 0 , 1 (Control and Experiment 1d), 4 (Control and Experiment 4d), 7 (Control and Experiment 7d), and 14 (Control and Experiment 14 d). The FL and BW of the fish are provided in G) in late July 2013, and in Ishikari Bay (Site F), the Indian Waterwheel (Site H), and the Chitose Hatchery (Site A) in October 2013. The FL, BW and GSI (gonad weight/body weight × 100) of the fish are provided in Adult chum salmon (average of 4 years old) of both sexes were sampled in the Bering Sea  (Site H) in October 2014. We only use adult male chum salmon due to limited experimental time, but we have previously reported no significant differences in EOG response between male and female salmonFor the EOG experiment on adult male chum salmon during homing, seven fish were collected at the Indian Waterwheel (Site F) prior to entering the Ishikari River were transferred to the Sapporo Salmon Museum and reared in seawater from Ishikari Bay maintained at 13 °C using a refrigerator truck in October 2014. They received a 2 mm intramuscular capsule implant via a 17 gauge needle; the capsule contained 300 μg of GnRHa . To measure TRHa/b and NR1 gene expression, fish were decapitated, and the whole brain was removed. To measure TSHβ gene expression, fish were decapitated and the upper head, which contains the pituitary gland, was removed. To measure T4 and T3 levels, we used the lower jaw, containing the thyroid gland, for some fish and the entire body without the head for other fish. The samples were frozen in liquid nitrogen and stored at −80 °C.For sampling adult chum salmon, fish were quickly immobilized by a gentle hit on the snout and then immediately decapitated, after which the brain was removed. Three regions of the brains were collected: the olfactory bulb (OB), telencephalon (TE), and hypothalamus (HT). To measure serum DHP levels, blood was collected from the caudal vasculature and centrifuged at 1,200 g for 15 min at 4 °C. The resulting brain and serum samples were frozen in liquid nitrogen and stored at −80 °C.E), the Toyohira River water (Site I), or the Chitose River water (Site B). The amplitude of the EOG response was quantified by the displacement of the odour-evoked peak relative to the pre-stimulus electrical baseline. The magnitude of the response is expressed as the mean percentage of the response to 0.1 mM Ser ± SEM.The EOG response35Total RNA was extracted from each sample using TRIzol  according to the manufacturer’s protocol. The quality of RNA was measured at A260 nm and the purity was assessed using the A260/A280 nm ratio . Only RNAs with A260/A280 nm ratios of 1.6–2.0 were used for cDNA synthesis. Reverse transcription was performed on 0.5 μg of total RNA using a ReverTra Ace® qPCR RT Master Mix with gDNA Remover  according to the manufacturer’s protocol.Real-time PCR was used to detect the expression levels of TRHa/b, TSHβ, sGnRH-I/II, and NR1 mRNA in all samples. The β-actin mRNA expression levels were also measured in all samples as a housekeeping gene. The following primer sets were designed: TRHa qPCRF/TRHa qPCRR, TRHb qPCRF/ TRHb qPCRR, TSHβ qPCRF/ TSHβ qPCRR, sGnRH-I qPCRF/sGnRH-I qPCRR, sGnRH-II qPCRF/sGnRH-II qPCRR, NR1 qPCRF/NR1 qPCRR, and β-actin qPCRF/β-actin qPCRR . QuantitA) and after release into the Chitose River at the Kamaka site downstream on the Chitose River (Site C) in April 2013. The FL and BW of the fish are provided in ®-T Easy Vector . Using the linearized plasmid, in vitro transcription was performed using T7 or SP6 RNA polymerase. Sections were treated with the following incubation steps: fixation with 4% paraformaldehyde-PB (pH 7.2) for 10 min, washing in PBS (pH 7.2) for 10 min, acetylation with 0.25% acetic anhydride in 0.1 M triethanolamine-HCl (pH 8.0) for 10 min, and pre-hybridization for 30 min in a hybridization buffer , 0.02% Ficoll, 0.02% polyvinylpyrrolidine, 0.02% bovine serum albumin, 0.6 M NaCl, 200 μg/ml of tRNA, 1 mM EDTA, and 10% dextran sulfate). Hybridization was performed at 63.5 °C for 12 h in the hybridization buffer supplemented with sense or antisense cRNA probes at a dilution of 1:1,000. Post-hybridization washing was performed successively at 61 °C with 5 × SSC (1 × SSC: 150 mM NaCl and 15 mM sodium citrate) for 30 min, 4 × SSC containing 50% formamide for 40 min, 2 × SSC containing 50% formamide for 40 min, and 0.1 × SSC for 30 min. Sections were dipped at room temperature in 0.1 × SSC for 15 min, NTE buffer  for 20 min, 20 mM iodoacetamide in NTE buffer for 20 min, and TNT buffer  for 20 min. For immunofluorescent detection, sections were blocked in TNT buffer for 10 min, DIG blocking solution : normal sheep serum: TNT buffer = 1:1:8) for 30 min, and 0.5% TSA blocking reagent  in TNT buffer for 30 min. They were subjected to detection by a DIG-labeled cRNA probe using peroxidase-conjugated anti-DIG antibody in DIG blocking solution  for 1 h with the Cy3-TSA Plus amplification kit (PerkinElmer) and TNT buffer overnight. On the following day, for counterstaining, sections were labeled with 0.05% NeuroTrace® 500/525 green fluorescent Nissl stain  in PBS (pH 7.2) for 10 min followed by PBS (pH 7.2) for 20 min. All of the washing and incubation solutions contained 0.0005% Tween 20. Images were taken with an FV1000 laser-scanning confocal microscope .Juvenile chum salmon were sampled at the Chitose Hatchery Serum DHP levels were measured using a time-resolved fluoroimmunoassay (TR-FIA)p < 0.05. For the downstream migration of juvenile chum salmon, statistical significance was determined using the Tukey-Kramer multiple comparison test and Scheffe’s F test. For the EOG experiments in juvenile and adult chum salmon, statistical significance was determined using one-way analysis of variance (ANOVA) followed by the Dunnett test and two-way ANOVA followed by the Tukey-Kramer multiple comparison test. For the T4 administration experiment on juvenile chum salmon, statistical significance was determined using the Steel-Dwass test and Scheffe’s F test. For adult chum salmon homing migration, statistical significance was determined using the Steel-Dwass test, Tukey-Kramer multiple comparison test, and Scheffe’s F test. For the GnRHa implantation experiment on adult chum salmon, statistical significance was determined using the Steel-Dwass test and two-way ANOVA followed by the Tukey-Kramer multiple comparison test.Differences were considered significant at This study (16-8) was approved to be conducted under the control of the committee following the “Guide for Care and Use of Laboratory Animals in Field Science Center for Northern Biosphere, Hokkaido University”, Japanese Governmental Law (No. 105) and Notification (No. 6).How to cite this article: Ueda, H. et al. Involvement of hormones in olfactory imprinting and homing in chum salmon. Sci. Rep.6, 21102; doi: 10.1038/srep21102 (2016).


Skeletal muscle regeneration and long term maintenance is directly link to the balance between self-renewal and differentiation of resident adult stem cells known as satellite cells. In turn, satellite cell fate is influenced by a functional interaction between the transcription factor Pax7 and members of the MyoD family of muscle regulatory factors. Thus, changes in the Pax7-to-MyoD protein ratio may act as a molecular rheostat fine-tuning acquisition of lineage identity while preventing precocious terminal differentiation. Pax7 is expressed in quiescent and proliferating satellite cells, while its levels decrease sharply in differentiating progenitors Pax7 is maintained in cells (re)acquiring quiescence. While the mechanisms regulating Pax7 levels based on differentiation status are not well understood, we have recently described that Pax7 levels are directly regulated by the ubiquitin-ligase Nedd4, thus promoting proteasome-dependent Pax7 degradation in differentiating satellite cells. Here we show that Pax7 levels are maintained in proliferating muscle progenitors by a mechanism involving casein kinase 2-dependent Pax7 phosphorylation at S201. Point mutations preventing S201 phosphorylation or casein kinase 2 inhibition result in decreased Pax7 protein in proliferating muscle progenitors. Accordingly, this correlates directly with increased Pax7 ubiquitination. Finally, Pax7 down regulation induced by casein kinase 2 inhibition results in precocious myogenic induction, indicating early commitment to terminal differentiation. These observations highlight the critical role of post translational regulation of Pax7 as a molecular switch controlling muscle progenitor fate. In recription –17, it ccription –20. In tcription . Nedd4 scription . It is uHere we provide evidence that Pax7 is phosphorylated by casein kinase 2 (CK2) in proliferating myoblasts. Site-directed mutagenesis and CK2 loss of function suggest that Pax7 phosphorylation prevents its down regulation via the ubiquitin-proteasome system.Procedures involving animal tissues were performed according to National Commission for Science and Technology (CONICYT) guidelines and approved by Facultad de Ciencias Biológicas of Pontificia Universidad Católica de Chile bioethics and biosecurity committee.® CRL-1772™) and C3H10T1/2 (ATCC® CCL-226™) cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Life technologies) supplemented with 10% fetal bovine serum (FBS) (Hyclone) and 1% penicillin/streptomycin (Life technologies) at 37°C with 5% CO2. C3H10T1/2 subjected to myogenic conversion, were differentiated using DMEM 2% FBS. Adult primary myoblasts were obtained as described [C2C12 (ATCCescribed  and main5’-cgtctggatgagggcgcagatgtggaatcag-3’ and 5’-ctgattccacatctgcgccctcatccagacg-3’ to Pax7 S201A (Pax7 AS) mutant; 5’-ggctcagatgtggaagcagaacccgacctc-3’ and 5’-gaggtcgggttctgcttccacatctgagcc-3’ to Pax7 S205A (Pax7 SA) mutant; 5’-ggcgcagatgtggaagcagaacccgacctc-3' and 5’-gaggtcgggttctgcttccacatctgcgcc-3' to Pax7 S201A/S205A (Pax7 AA) mutant. To mimic phosphorylation, serine was changed to aspartic acid using the following primer pairs: 5'-cgggttctgattccacatcatcgccctcatccagacggttc-3' and 5'-gaaccgtctggatgagggcgatgatgtggaatcagaacccg-3' to Pax7 S201D (Pax7 DS) mutant; 5'-gggggaggtcgggttcatcttccacatctgagccc-3' and 5'-gggctcagatgtggaagatgaacccgacctccccc-3' to Pax7 S205D (Pax7 SD) mutant; 5'-cagggggaggtcgggttcatcttccacatcatcgccctcatccagacggtt-3' and 5'-aaccgtctggatgagggcgatgatgtggaagatgaacccgacctccccctg-3' to Pax7 S201D/S205D (Pax7 DD) mutant.pCDNA3-myc-NLSPax7WT plasmid  was usedFor localization assays, pCDNA3-myc-Pax7WT, pCDNA3-myc-Pax7AS, pCDNA3-myc-Pax7SA and pCDNA3-myc-Pax7AA plasmids were generated by subcloning from pCDNA3-myc-NLS to pCDNA3-myc , using B™ H Minus M Mulv 200 U/μl and random primers at 42°C for 60 min. PCR was performed to verify reverse transcription using Taq polymerase and a 0.4 μl aliquot of cDNA with specific primer pairs. The sequences of primers used for qPCR were as follows: mouse Pax7 5’-CACCCCTTTCAAAGACCAAA-3’ (Forward) and 5’TGCTTGAAGTTCCTGCTCCT-3’ (reverse); mouse 18S 5’- GAGCGAAAGCATTTGCCAAG-3’ (Forward) and 5’- GGCATCGTTTATGGTCGGAA-3’ (Reverse). The real-time PCR reactions were conducted using SYBR Green master mix according to the manufacturer's instructions. A 7500 real-time PCR system (Applied Biosystems) was used to measure the relative RNA levels and the data were analysed using 7500 system SDS software. qPCR analysis was performed using a relative quantification mathematical model, as previously described [Total RNA was extracted using a RNASolv isolation system following the manufacturer's instructions. First, the genomic DNA was degraded using DNAse I at 25°C for 15 min. The RNAs were then quantified using Tecan equipment (with the NanoDrop module). The first strand of cDNA was synthesized using 0.5 μg of RNA in 20 μl of reaction buffer by reverse transcription using RevertAidescribed .™ anti-phospho-CK2 Substrate (pS/pT)DXE  at 1:1000. Anti-mouse IgG and anti-rabbit IgG HRP-conjugated secondary antibodies  were used at 1:5000, and HRP activity was visualized using SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Dura Extended Duration Substrate (Thermo Fisher Scientific). Relative densitometry was measured using ImageJ software.Whole cell extracts were obtained by disruption in modified RIPA lysis buffer  and incubated at 4°C for 20 min, followed by centrifugation at 15200 rpm for 10 min. Proteins (20–30 μg) were separated into 10% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific). Membranes were blocked with 5% (wt/vol) nonfat powdered milk in TBS-T  and incubated with the following primary antibodies and dilutions: mouse monoclonal anti-Pax7  at 1:10; mouse monoclonal anti-myc-tag (9B11)  at 1:1000; rabbit monoclonal anti-GFP (E385) (Abcam) at 1:10000, mouse monoclonal anti-GAPDH (EMD Millipore) at 1:10000; rabbit MultiMabCells were fixed in 4% paraformaldehyde in PBS, permeabilized with 0.5% Triton X-100 and blocked with 3% BSA in PBS. Primary antibodies and dilutions were used as follows: mouse monoclonal anti-Pax7  at 1:5; rat monoclonal anti-MyoD (5F11) (Millipore) at 1:100; mouse monoclonal anti-myogenin (F5D)  at 1:5; chicken anti-Syndecan-4  at 1:500Purified GST-Pax7WT, GST-Pax7AS, GST-Pax7SA and GST-Pax7AA (5 μg) were incubated with or without 100 ng of CK2 (New England BIolabs) in kinase buffer provided with the enzyme  supplemented with 100 μM ATP (New England BIolabs) for 30 min at 30°C. Reactions were loaded into SDS-PAGE gel and analyzed by Western blotting.Ubiquitination assay was performed as previously described . BrieflyPlasmids for bi-molecular fluorescence complementation (BiFC) were obtained as described previously . C2C12 cC3H10T1/2 cells were induced to myogenic conversion by co-transfecting pRSV-MyoD vector in all experiments. For repressing myogenic conversion, cells were co-transfected with pCDNA3-myc-NLSPax7WT, Pax7AS, Pax7SA or Pax7AA and pRSV-MyoD in 2:1 ratio as previously described . Differe6xPRS9-Luc [To evaluate Pax7-point mutants transcriptional activity, C3H10T1/2 cells were transfected with the Pax3/7 specific reporter gene PRS9-Luc  and pCDNPRS9-Luc . 24 hour2 with 2 μg trypsin . MudPIT aalysis was performed using an Agilent 1100 quaternary pump and a Thermo Velos LTQ-Orbitrap using an in-house built electrospray stage [C2C12 cells were transfected with pCDNA3-myc-NLSPax7WT using Lipofectamine 2000 (Life technologies) and were maintained in growth medium or differentiated for 48 hours before harvesting. Nuclear extracts were obtained as described  and subjay stage .http://www.integratedproteomics.com/). Tandem mass spectra were extracted from raw files using RawExtract 1.9.9 [Protein and peptide identification and protein quantitation were done with Integrated Proteomics Pipeline—IP2 . As shown in  kinases . In orden silico . Importan silico . In orden silico  in orderS201 and S205 are located between two functional Pax7 domains, the octapetide and the homeobox-DNA binding domain (HD). Based on studies evaluating deletion of specific Pax7 domains , we testTo test if CK2 activity was directly involved in the regulation of Pax7 protein levels, C3H10T1/2 cells expressing Pax7 constructs were treated with the CK2 inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) and Pax7 expression was evaluated by Western blotting. Although at different magnitudes, WT Pax7 and phospho-mutant protein levels were significantly decreased upon TBB treatment . To direAdditionally, increasing the interval of CK2 inhibition at a single TBB concentration, significantly decreased Pax7 levels as a function of incubation time .Together, these results suggest that Pax7 is phosphorylated in proliferating myoblasts by CK2 activity, inducing its stabilization.We have recently showed that Pax7 protein levels are negatively regulated in differentiating cells via ubiquitin-ligase Nedd4 and the ubiquitin-proteasome system (UPS) . It remaPax7 transcription despite the significant effect at the protein level  Pax7 phosphorylation. Based on our previous work, we specifically studied the potential effects at the levels of i) sub cellular localization, ii) ability to repress MyoD, iii) transcriptional activity and iv) protein expression. Nuclear expression pattern and transcriptional activity did not differ significantly between Pax7 and Pax7 phospho-mutants. Consistently, all Pax7 mutant proteins were capable of repressing MyoD dependent myogenic conversion of the mesenchymal cell line C3H10T1/2 . MyogeniSince we recently showed that Pax7 was down regulated by the UPS in differentiating myogenic cells, we hypothesized that CK2 inhibition resulted in ectopic Pax7 ubiquitination and proteasomal degradation. Reduction in Pax7 levels upon CK2 inhibition was prevented by concomitant proteasome inhibition , while iKey aspects of our model find strong support in the recently published work from Dick and cols. . That st2O2 . We are currently investigating the specific molecular events affected by Pax7 phosphorylation and how they affect UPS-dependent regulation, which may provide a mechanism to conciliate the molecular consequences of CK2-mediated Pax7 phosphorylation presented in both studies. Such studies will benefit from the use of genetic models, such as cell-specific/inducible CK2 knockouts mice, given the low abundance of satellite cells in the muscle tissue and the necessity of evaluating the specific role of CK2 in an inhibitor-independent manner. Additionally, uncovering the signaling controlling CK2 activity will be key to better understand the differential regulation of Pax7 in quiescent, activated and differentiating muscle progenitors. Most importantly, however, is that collectively the results presented here and work by others underscore the critical role of Pax7 post translational regulation for SC fate, and thus, muscle regeneration and long-term maintenance.Several results presented here suggest that CK2-directed phosphorylation stabilizes Pax7 primarily by decreasing its ubiquitination and proteasome-dependent degradation. Additionally, we evaluated the status of caspase-3 activity upon CK2 inhibition in in primary myoblasts maintained in proliferation culture medium, by immunofluorescence. No significant activated caspase-3 signal was detected under normal proliferating culture conditions (<3% (+)/total cells), whereas activation was evident upon apoptosis induction by increasing concentrations of H