We analysed the in vivo phosphoproteome, proteome and metabolome and found that activation of SnRK1 is essential for repression of high energy demanding cell processes such as protein synthesis. The most abundant effect was the constitutively high phosphorylation of ribosomal protein S6 (RPS6) in the snrk1α1/α2 mutant. RPS6 is a major target of TOR signalling and its phosphorylation correlates with translation. Further evidence for an antagonistic SnRK1 and TOR crosstalk comparable to the animal system was demonstrated by the in vivo interaction of SnRK1α1 and RAPTOR1B in the cytosol and by phosphorylation of RAPTOR1B by SnRK1α1 in kinase assays. Moreover, changed levels of phosphorylation states of several chloroplastic proteins in the snrk1α1/α2 mutant indicated an unexpected link to regulation of photosynthesis, the main energy source in plants.Since years, research on SnRK1, the major cellular energy sensor in plants, has tried to define its role in energy signalling. However, these attempts were notoriously hampered by the lethality of a complete knockout of SnRK1. Therefore, we generated an inducible amiRNA::SnRK1α2 in a  SnRK1α1  and SnRK1α2 , encode the catalytic α-subunit of the SnRK1 complex. In the mammalian systems, AMPK was shown to sense the cellular energy status via the AMP/ATP ratio678911As sessile organisms, plants have to cope with ever changing environmental conditions. Hence, they have developed strategies to sense and to acclimate to unfavourable circumstances. Such stress situations are often linked to the availability of energy. When energy is not limited, plants produce energy-rich compounds and direct resources to the synthesis of storage compounds and growth. In contrast, under stressful conditions nutrient remobilization and growth arrest, as part of a vast metabolic reprogramming, are the dominating processes. Sucrose non-fermenting related kinase 1 (SnRK1) and its orthologs, the AMP-dependent protein kinase (AMPK) and sucrose non-fermenting 1 (SNF1) kinase in mammals and yeast, respectively, are conserved signalling components that significantly contribute to the maintenance of cellular energy homeostasis1in vitro, nowadays, mass spectrometry (MS) based large-scale phosphoproteomics experiments enable the detailed and comprehensive analysis of in vivo phosphorylation of hundreds of putative targets in a single measurement.A pivotal role of SnRK1 in linking stress, development and sugar signalling has been described on the level of gene expression, indicating a crucial regulatory influence on global plant metabolism, energy balance and growth141520449 of S6K1 demonstrating further the link between S6K and TOR30rps6 mutant displayed reduced cell size and delayed growth and flowerings6k1s6k2/++ mutant had a higher proportion of smaller cells compared to the wild type while the cell number remained unalteredLike SnRK1, target of rapamycin (TOR) is a central regulator of energy metabolism in eukaryotic organisms2223242223262728Based on studies in mammalian cells, it is known that the TOR pathway is inhibited by energy depriving conditions and by AMPK-driven phosphorylation of the regulatory-associated protein of TOR (RAPTOR)Arabidopsis thaliana phosphoproteome. For this purpose, we measured the phosphoproteome in snrk1α1 single knockout (ko), overexpressor (OE) and snrk1α1/α2 double knockdown (kd) lines and identified and quantified more than 1000 phosphoproteins and 2000 phosphorylation sites. An intriguing observation was the upregulation of RPS6 in vivo phosphorylation in snrk1α mutants. The molecular link between SnRK1 and TOR pathways in plants was so far only speculative. Based on the observation of high RPS6 phosphorylation in snrk1α mutants and the reports from mammalian systemssnrk1α1/α2 mutant during the low energy syndrome (LES). Metabolomics analysis of the snrk1α1/α2 mutant revealed a strong effect on mitochondrial metabolism. Altogether the data suggests an early and late SnRK1 control on a highly complex network of pathways in starvation conditions.In summary, AMPK-/SNF1-/SnRK1- and TOR-related signalling networks affect a multitude of central processes involved in embryogenesis, growth and development of various eukaryotes. Hence, it is not surprising that the metabolic output as well as the identification of involved signalling compounds remains a complex task, which demands a combination of comprehensive experimental and theoretical methods for its conclusive analysis. The aim of the present study was to depict a comprehensive picture of SnRK1-induced dynamics in the snrk1α1 T-DNA knock-out mutant (snrk1α1-3)snrk1α1-3 mutant expressing a β-estradiol inducible artificial micro (ami) RNA for SnRK1α2 (hereafter referred as snrk1α1/α2 mutant) as a way to deplete SnRK1 activity. With this approach we focused on the dynamics of SnRK1-dependent metabolic reprogramming, by comparing the phosphoproteome, the proteome and the metabolome of Col-0 and snrk1α1/α2 plants at early and late time points during an extended night of increasing length   and SnRK1α2 (lower band 58.7 kDa)  snrk1α1/α2 plants were accumulating anthocyanins and their growth was significantly reduced after knock down of SnRK1α2 , a 360 min) . The expOE lines . In the 2 mutant  and seco8.7 kDa) . Furthere plants . Given t SnRK1α2 . SimilarArabidopsis thaliana wild type and mutant plants to an extended night treatment that induces SnRK1 signalling. Metal-oxide affinity chromatography (MOAC) enrichment and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis of phosphopeptides according to established protocols in the lab were applied to leaf extracts of the treated plants4041424345in vitro experiments, HMG, NRs, SPSs, F2KP, and TPSs are potential targets for SnRK1α1718snrk1α1 and in snrk1α1/α2 mutants. At most of the extended night time points the phosphorylation status of these proteins was higher in wild type than in mutant lines, and the highest difference is about 2-fold. The typical target motif for SnRK1 phosphorylation is [MLVFI]x[RKH]xx[S]xxx[LFIMV], while a less preferred motif is [MLVFI][RKH]xxx[S]xxx[LFIMV]. This motif is nevertheless very similar to the phosphorylation motif of calcium dependent protein kinases (CDPKs) and has to be interpreted with care. When the early and late phases of extended night were examined, NR2 and F2KP were phosphorylated immediately after the onset of the treatment but have decreased phosphorylation level after 80 min of extended darkness in the wild type. In snrk1α1/α2 mutant F2KP phosphorylation was decreased in the early phase (see Discussion). TPS5 and TPS7 were the most affected proteins. The phosphorylation of these proteins was about 2-fold higher in the early time points but also later at 120 min after extended night. Phosphopeptides corresponding to several transcription factors were also identified and differentially regulated in the OE1 and OE2 mutant compared to the wild type snrk1α1/α2. Altogether 33 proteins were identified with high stringency SnRK1 motif and 40 proteins with lower stringency (To identify putative phosphorylation targets of SnRK1 which should be downregulated in the ringency .snrk1α1/α2 and wild type at least at one time point were standardized to zero-mean-unit-variance (referred to as z-scores), to allow the comparison of relative changes of different phosphopeptides, and subjected to hierarchical cluster analysis (HCA) based on Spearman correlation , which was found to be phosphorylated much stronger at Thr80 in the wild type. This phosphorylation pattern could already be observed in the light, but in darkness the relative phosphorylation status of CPN20 at Thr80 was found to be significantly lower in the mutant. In addition to these chloroplastic proteins, also phosphorylation of CHLOROPLAST UNUSUAL POSITIONING 1  and thylakoid soluble phosphoprotein of 9 kDa  were affected by SnRK1  was observed to be even lower in the mutant than in the wild type  and eIF5A3 (AT1G69410) of snrk1α1/α2 when compared to the wild type  in energy stress by phosphorylating one of the complex members, namely RAPTOR cytosol . Also main vivo. To this end, leaves of N. benthamiana were transiently co-transformed with plasmids containing RAPTOR1B plus the N-terminal part of VENUS (VENUSN173) and SnRK1α1 plus the C-terminal part of VENUS (VENUSC155). As negative control we used the cytosolic fructose-1,6-bisphosphatase (FBPase) fused to the VENUSC155 in combination with VENUSN173-RAPTOR1B. As shown in C155 and VenusN173-RAPTOR1B induced a fluorescence signal indicative for an interaction of both proteins in the cytosol while the control combination yielded no signal. To test weather SnRK1α1 can phosphorylate RAPTOR1B, we performed an in vitro kinase assay. The phosphorylation of RAPTOR1B by SnRK1α1 is demonstrated in in vivo interaction and phosphorylation of RAPTOR1B by SnRK1α1 suggest a mechanism of SnRK1 control over TOR signalling by phosphorylation of RAPTOR.We also performed bimolecular fluorescence complementation (BiFC) assays to investigate whether SnRK1α1 and RAPTOR1B are interacting snrk1α1/α2 plants in the accumulation of sugars, sugar-alcohols, organic acids, amino acids and polyamines  were found to be significantly affected in the snrk1α1/α2 mutant. In particular, during the first 180 min of extended night a linear increase of proteins related to the mETC could be observed for the wild type (R2 = 0.94) while the response in snrk1α1/α2 was found to be less linear (R2 = 0.49).Relative protein abundances revealed a significant perturbation of the C/N-metabolism in rk1α1/α2 . This insnrk1α1/α2, also abundances of related phosphorylated peptides indicated a contrasting reprogramming in both genotypes. In particular, phosphopeptide abundance of thioredoxin H-type 9  was slightly elevated in snrk1α1/α2 during extended night, indicating an interaction of SnRK1 signalling with REDOX regulation.In agreement with the observation that protein components of cellular redox metabolism behaved differently in the very early phase of extended night in wild type and in vivo. Heterotrimeric SnRK1 kinase is activated in scarce energy conditions such as in extended darkness. This is the first phosphoproteomics report of a SnRK1 alpha subunit double knockdown Arabidopsis mutant  in extended darkness. An overview of the changes in the in vivo phosphoproteome is shown in Protein phosphorylation is an important post-translational modification that controls e.g. protein activity, interaction with other proteins, protein turnover, and their subcellular localization. Phosphoproteomics technology allows the examination of thousands of phosphorylation events snrk1α1/α2 mutant this inactivation was lost and it led to constitutively high phosphorylation of RPS6  is inhibited by energy depriving conditions and by AMPK-driven phosphorylation of one of the complex members, namely RAPTORein vivo . To testin vitro . Becausesnrk1α1/α2 mutant than in Col-0 wild type  OseIF5A1 and OseIF5A1 in suspension cell culture are strongly affected by carbohydrate starvation and the amount of mRNA is reduced when sugars are not availableTrypanosoma cruzi TceIF5A is highly phosphorylated at the same N-terminal Ser2 as in ArabidopsisSaccharomyces cerevisiae) rapidly affects the protein translation rate impairing it by 2–3-fold74The eukaryotic translation factor eIF5A , appears to be highly conserved in all eukaryotes70ild type . Additioin vitro and here we showed that they represent also in vivo targets of SnRK1 35S::SnRK1α1 overexpressor lines 1 and 2  was used. Plants were grown in a 12/12 light/dark cycle with irradiance of 100 μmol m−2 s−1. After 4 weeks they were sprayed with 10 μM β-estradiol, 0.1% DMSO, 0.005% Silwet for 6 days. On day 7 plants were exposed to extended night treatment and harvested at 0, 20 40, 60, 80, 100, 120, 180, and 360 min after start of the treatment. Also samples in the middle of the day (6 h light) were collected. At this time, plant leaf rosettes were fully grown but not yet flowering.For the first experiment, a SnRK1α1 T-DNA knockout mutant Protein for Western blot analysis was extracted with buffer containing 175 mM Tris-HCl (pH 8.8), 5% SDS, 15% glycerol and 300 mM DTT. After vortexing, samples were centrifuged with 21 000 g and the protein was precipitated from the supernatant with four volumes of ice-cold acetone. Protein precipitates were collected and resuspended in Laemmli buffersnrk1α1, 35S:SnRK1α1 overexpressor lines 1 and 2 and their corresponding Columbia (Col-0) and Landsberg erecta (Ler) wild type backgrounds as well as from snrk1α1/α2 mutant were extracted with TCA precipitation and phenol extraction404142Leaves of Phosphopeptide pellet was resolved in 8 μL of 5% (v/v) acetonitril (ACN), 0.5% (v/v) formic acid (FA) and 5 μL were loaded on C18 LC column. The samples of the first experiment were measured on monolithic Chromolith C18 CapRod column with a length of 15 cm and an inner diameter (ID) of 0.1 mm . Peptides were eluted using water/ACN gradient from 2% to 35% mobile phase B with 500 nL/min flow rate generated with nanoLC (Eksigent) within 150 min linear gradient. Mobile phase A: 0.1% (v/v) FA, mobile phase B: 0.1% (v/v) FA in 90% ACN. In the second experiment UltiMate 3000 RSLCnano system with EASY-Spray PepMap C18 column with a length of 50 cm and ID of 0.075 mm with 2 μm particles (Thermo Scientific) was used. Same mobile phases were used and the gradient was 150 min from 2% to 40% mobile phase B with 300 nL/min flow rate.5, the maximum injection time (max IT) was set to 500 ms. Orbitrap online calibration using internal lock mass calibration on m/z 371.10123 from polydimethylcyclosiloxane was used. Multistage activation/pseudoMS3 was enabled with a neutral loss mass list of 24.49, 32.66, 48.999, 97.97, 195.94, and 293.91 Da for neutral loss of phosphoric acid [H3PO4] at charge states +1, +2, +3 and +4, respectively, 195.94, 97.97 and 48.999 Da for neutral loss of two phosphoric acids [2H3PO4] at charge states +1, +2 and +4, respectively, and 293.91 Da for neutral loss of three phosphoric acids [3H3PO4] at charge state +1. The AGC for MS/MS acquisition in the LTQ was set to 1 × 104, the max IT was set to 100 ms.The samples were measured with an LTQ-Orbitrap mass spectrometer (Thermo Electron). Orbitrap mass analyser settings: ion transfer capillary temperature 180 °C, full scan range 350–1800 m/z, resolution 30 000. Each FTMS full scan was followed by up to five data dependent (DDA) CID tandem mass spectra (MS/MS spectra) in the linear triple quadrupole (LTQ) mass analyser. Dynamic exclusion was enabled with list size of 500 with exclusion width ±10 ppm for 60 s. Charge state screening was enabled and unassigned and +1 charged ions were excluded from MS/MS acquisition. For injection, control automatic gain control (AGC) for full scan acquisition in the Orbitrap was set to 5 × 10ProteomeDiscoverer 1.3 (Thermo), SEQUEST search algorithmThe data from experiment 2 were analysed with MaxQuant 1.4 and Andromeda search algorithm86A. thaliana reverse transcribed total cDNA (from rosette leaf) and cloned into a modified pBIN19 vectorN. tabacum (cv. petit Havana SR1) leaves. After 36 h infiltrated tobacco leaves were subjected to microscopy. Microscopy was performed on a Leica TCS SP5 confocal platform. General microscope settings: objective - HCX PL APO CS 63.0×1.30 GLYC 21 °C UV; emission light source - white light laser Ex1 515 nm (for RAPTOR1B-YFP) Ex2 687nm (for SnRK1α1-mCherry). Detection bandwidth: PMT1 520–560 nm (RAPTOR1B-YFP) PMT2 605–630 nm (SnRK1α1-mCherry) PMT3 687–752 nm (plastid autofluorescence). Images were analysed with the LAS AF Lite software from Leica (pictures and diameter blots) and FijiRAPTOR1B (AT3G08850) cDNA was PCR amplified from A. tumefaciens C58C1 and transiently expressed by Agrobacterium-infiltration in N. benthamiana. The BiFC-induced YFP fluorescence was detected by confocal laser scanning microscopy  48 h post infiltration. The specimens were examined using the LD LCI Plan-Apochromat 253/0.8 water-immersion objective for detailed images with excitation using the argon laser (458 or 488 nm line for BiFC and chlorophyll autofluorescence). The emitted light passed the primary beam-splitting mirrors at 458/514 nm and was separated by a secondary beam splitter at 515 nm. Fluorescence was detected with filter sets as follows: on channel 3, 530–560 band pass; and on channel 1, 678–743 for red autofluorescence of chlorophyll. All samples were investigated using the same microscope settings. Images were processed using the ZEN software package (Zeiss).For BiFC experiments constructs were transformed into A. thaliana rosette leafs), cloned into pGEX-4T which contains an IPTG inducible promoter  and transformed into Escherichia coli (strain ER2566) for expression. E. coli cells carrying the expression plasmids were grown in LB at 37 °C under vigorous shaking until an OD600 of 0.4 was reached. At this point cultures were transferred to room temperature and SnRK1α1 expression was induced by addition of IPTG to a final concentration of 1 mM. After 4 h cells were harvested. The protein containing an N-terminal GST tag was purified using Glutathione Sepharose 4B  according to the manufacturer’s instructions. In brief, cells were homogenized in GST binding buffer  by sonification, followed by centrifugation. Proteins were purified by incubating the supernatant with Glutathione Sepharose beads at 4 °C for 1 h, followed by washing with GST binding buffer and elution with GST elution buffer . Proteins were stored at −80 °C in GST elution buffer containing 20% glycerol. RAPTOR1B (AT3G08850) cDNA was PCR amplified from total cDNA and cloned into a modified pQE80L-YFP vector resulting in a 6xHIS-YFP-RAPTOR1B fusion ORF under the control of an IPTG inducible promoter  according to manufacturer’s instructions. Briefly, protein was homogenized and sonicated in homogenization buffer  to obtain RAPTOR1B protein. HiTrap columns were charged with 100 mM NiSO4 and equilibrated with biding buffer . Protein was loaded into the column and then washed with washing buffer . Finally, protein was eluted with elution buffer  and stored at −80 °C in elution buffer containing 10% of glycerol.SnRK1α1 cloning/expression was done as described previouslyin vitro kinase assays, recombinantly expressed GST-SnRK1α1 and YFP-Raptor1b were incubated together in kinase reaction buffer  for 35 min at room temperature. The reaction was stopped by adding 4x Leammli buffer, samples were boiled at 94 °C for 4 min and separated by SDS-PAGE. Dried gels were exposed on a Storage Phosphor Screen .For ®, Santa Clara, CA, USA) coupled to a LECO Pegasus® 4D GCxGC-TOF mass spectrometer . Compounds were separated on an Agilent HP5MS column . Deconvolution of the total ion chromatograms was performed using the LECO Chromatof® software. For absolute quantification, calibration curves were recorded comprising 5 different substrate concentrations within the linear range of detection.Frozen leaf rosettes were homogenized with pestle and mortar. Primary metabolites were extracted and derivatized as described previouslyhttp://www.celldesigner.org/). Linkage of covariance data with the network structure follows equation 16893The functional connection of GC-MS metabolomics data with causal biochemical network information was performed, as previously describedJ represents the Jacobian matrix and D is a fluctuation matrix which integrates a Gaussian noise function simulating metabolic fluctuations. In context of a metabolic network, entries of the Jacobian matrix J represent the elasticity of reaction rates to any change of metabolite concentrations which are characterized by equation 2:4 Jacobian approximations for each genotype and time point, and normalized the medians to the squared interquartile distances. All calculations of Jacobian matrices were performed based on a modified version of the toolbox COVAIN® (V8.4.0 R2014b).N is the stoichiometric matrix or a metabolic interaction matrix if reactions and reactants have been modified in the original network. r represents the rates for each reaction and M represents metabolite concentrations. As stated before, the Jacobian approximation comprises the stochastic term D. Therefore, we performed 10How to cite this article: Nukarinen, E. et al. Quantitative phosphoproteomics reveals the role of the AMPK plant ortholog SnRK1 as a metabolic master regulator under energy deprivation. Sci. Rep.6, 31697; doi: 10.1038/srep31697 (2016).


The aim of the present study was to investigate whether the inhibition of HSP27 phosphorylation, which affects certain cellular functions, modulates sensitivity to 5-fluorouracil (5-FU) in colorectal cancer cells. Exposure to 5-FU in HCT116 and HCT15 cells expressing high levels of HSP27 with a low 5-FU sensitivity caused a minimal change in HSP27 expression, but induced the upregulation of HSP27 phosphorylation, particularly at Ser78. By contrast, exposure to 5-FU in HT29 cells expressing a low level of HSP27 with a high 5-FU sensitivity marginally increased HSP27 expression, with minimal phosphorylation. Treatment with a selective inhibitor, p38 mitogen-activated protein kinase , caused the dose-dependent suppression of HSP27 phosphorylation, which was upregulated by 5-FU, reducing the half maximal inhibitory concentration values of 5-FU in the HCT116 and HCT15 cells. However, treatment with SB203580 exhibited no significant effect on cell growth or survival. In conclusion, this study indicated that the inhibition of HSP27 phosphorylation by a selective inhibitor of p38 MAPK promotes 5-FU sensitivity without causing cytotoxicity in colorectal cancer cells. A number of studies have reported the association between the response to or toxicity of the drug and 5-FU metabolism-associated factors, including thymidylate synthase, dihydropyrimidine dehydrogenase, folate cofactors and orotate phosphoribosyltransferase –4. Howevin vitro  such as HSP90, HSP70, HSP60 and small HSPs 15–30 kDa), including HSP27, are present in numerous organs and are considered as molecular chaperones in protein-protein interactions, including folding, unfolding and assembly, as well as anti-apoptotic proteins and contributors to cell survival –30 kDa, . In coloin vitro ,13, and in vitro –16. Our ft model ,18.A variety of stimuli induce the phosphorylation of serine residues 15, 78 and 82 in HSP27 using various protein kinases, such as mitogen-activated protein kinase (MAPK) activated protein kinase 2 (MAPKAPK-2), via p38 MAPK –24. ThisThe present study aimed to clarify whether HSP27 expression or phosphorylation was modulated by 5-FU exposure and to investigate whether the inhibition of HSP27 phosphorylation by a specific kinase inhibitor affected the sensitivity of colorectal cancer cells to 5-FU.2 incubator at 37°C. Prior to the experiments, the cells were incubated without FBS for 24 h.The anticancer drug 5-FU was purchased from Kyowa Hakko Bio Co., Ltd. . A selective p38 MAPK inhibitor, SB203580 , and a total of 20 μg lysates were resolved in Ready Gel (Bio-Rad) and transferred to an Immuno-Blot polyvinylidene fluoride membrane (Bio-Rad). The membrane was blocked with phosphate-buffered saline  containing 5% skimmed milk powder for 2 h at room temperature, then incubated at 4°C overnight with anti-human HSP27 mouse monoclonal antibody , anti-human phospho-HSP27 (Ser15) rabbit polyclonal antibody , anti-human phospho-HSP27 (Ser78) mouse monoclonal antibody , anti-human phospho-HSP27 (Ser82) rabbit polyclonal antibody  or anti-human β-actin mouse monoclonal antibody . The membranes were incubated for 30 min with a horseradish peroxidase-conjugated anti-mouse immunoglobulin G (IgG)  or anti-rabbit IgG (Promega Corporation). Bound complexes were detected using the ECL-Plus reagent  according to the manufacturer’s instructions. Each experiment was performed in triplicate.Subconfluent cells were exposed or not exposed to 5-FU (1.28 μg/ml) with SB203580 treatment  in culture medium for 48 h. The total cell lysates were then extracted using lysis buffer [20 mM Tris/HCL (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% TritonX-100, 2.5 mM sodium pyrophosphate, 50 mM NaF, 50 mM HEPES, 1 mM Na50) value, which corresponded to the concentration of the drug required to inhibit cell growth by 50% relative to the untreated cells.A cell proliferation assay was performed using the 3--2,5-diphenyl bromide (MTT) assay, as described previously . BrieflyA cell count of viable cells was also performed to evaluate cell proliferation. Viable cells were counted four days after plating in triplicate dishes in various concentrations of SB203580  in RPMI-1640 containing 5% FBS.Data are expressed as the mean ± standard deviation. The statistical analysis was performed using Student’s t-test or the Mann-Whitney U test. P<0.05 was considered to indicate a statistically significant difference.The effect of 5-FU exposure on HSP27 expression or phosphorylation was examined in colorectal cancer cells. Colorectal cancer cells expressing high levels of HSP27 with a low sensitivity to 5-FU (HCT116 and HCT15) and those expressing a low level of HSP27 with a high sensitivity to 5-FU (HT29)  were useThe effect of SB203580, a p38 MAPK selective inhibitor . These r50 value. The HCT116 and HCT15 cells expressing high levels of HSP27 exhibited high IC50 values, while the HT29 cells expressing a low level of HSP27 exhibited a low IC50 value  in RPMI-1640 containing 5% FBS . As a rein vitro . In the colorectal cancer cells with a low level of HSP27 expression (HT29), HSP27 expression was marginally increased, with a small amount of phosphorylation of HSP27 at the three serine residues. The phosphorylated residues of HSP27 have been reported to differ in response to different inducers . In thesin vitro. This result indicates that the dephosphorylation of HSP27 via the inhibition of p38 MAPK by SB203580 exerted little toxicity in these colorectal cancer cells.SB203580, a p38 MAPK selective inhibitor, clearly inhibited the phosphorylation of the Ser78 residue of HSP27 induced by 5-FU exposure. Furthermore, the inhibition of HSP27 phosphorylation by SB203580 significantly promoted the sensitivity of the HCT116 and HCT15 cells to 5-FU. This effect may be caused by a reduced anti-apoptotic effect as a result of the inactivation of HSP27. In addition, SB203580 had a minimal effect on cell growth or survival In conclusion, the present study indicated that the inhibition of HSP27 phosphorylation promotes 5-FU sensitivity in colorectal cancer cells with high levels of HSP27 expression. If the agents that specifically inhibit HSP27 phosphorylation exhibit minimal toxicity, such agents may present a novel strategy for the treatment of colorectal cancer when combined with current chemotherapy using 5-FU. Further investigations into HSP27 regulation are important and are required for the development of novel treatments targeting HSP27 in colorectal cancer.


We used a chemical structure-similarity search to identify another anti-inflammatory drug, diflunisal, that inhibits p300 more potently than salicylate. At concentrations attainable in human plasma after oral administration, both salicylate and diflunisal blocked the acetylation of lysine residues on histone and non-histone proteins in cells. Finally, we found that diflunisal suppressed the growth of p300-dependent leukemia cell lines expressing AML1-ETO fusion protein in vitro and in vivo. These results highlight a novel epigenetic regulatory mechanism of action for salicylate and derivative drugs.Salicylate and acetylsalicylic acid are potent and widely used anti-inflammatory drugs. They are thought to exert their therapeutic effects through multiple mechanisms, including the inhibition of cyclo-oxygenases, modulation of NF-κB activity, and direct activation of AMPK. However, the full spectrum of their activities is incompletely understood. Here we show that salicylate specifically inhibits CBP and p300 lysine acetyltransferase activity DOI:http://dx.doi.org/10.7554/eLife.11156.001 People have been using a chemical called salicylate, which was once extracted from willow tree bark, as medicine for pain, fever and inflammation since ancient Greece. Aspirin is derived from salicylate but is a more potent drug. Aspirin exerts its anti-inflammatory effect by shutting down the activity of proteins that would otherwise boost inflammation. Aspirin achieves this by releasing a chemical marker, called an acetyl group, to be added to these proteins via a process known as protein acetylation. However, salicylate cannot trigger protein acetylation and so it was not clear how it reduces inflammation.An anti-diabetes drug that is converted into salicylate in the body reduces inflammation by inhibiting a protein called NF-κB. In 2001, a group of researchers reported that NF-κB becomes active when an enzyme called p300 adds an acetyl group to it. This raised the question: does salicylate reduce inflammation by blocking, instead of triggering, protein acetylation.Now, Shirakawa et al. – who include a researcher involved in the 2001 study – show that salicylate does indeed block the activity of the p300 enzyme. Shirakawa et al. then searched a database looking for drugs that have salicylate as part of their molecular structure. The search led to a drug called diflunisal, which was even more effective at blocking p300 in laboratory tests.Some cancers, including a blood cancer, rely on p300 to grow; diflunisal was shown to stop this kind of cancer cell from growing, both in the laboratory and in mice. Together, the experiments suggest that salicylate and drugs that share some of its structure might represent useful treatments for certain cancers, as well as other diseases that involve the p300 enzyme.DOI:http://dx.doi.org/10.7554/eLife.11156.002 One of its widely used derivatives, acetylsalicylic acid (Aspirin), inhibits prostaglandin biosynthesis by irreversibly inactivating cyclooxygenases via non-enzymatic acetylation of a single serine residue . InteresHowever, salicylic acid blocks cyclooxygenase expression at the transcriptional level thereby explaining its anti-inflammatory properties . In addiSalsalate, a salicylate precursor, is an effective therapy for type 2 diabetes , a metabInterestingly, examination of the chemical structure of anacardic acid, a previously reported p300 inhibitor, revealed that it contained a salicylic acid moiety linked to a long alkyl chain . Previouin vitro acetylation assays with purified histones and a recombinant p300 catalytic domain. Salicylate effectively inhibited p300 dependent acetyltransferase activity (IC50 = 10.2 mM) and CBP-mediated acetyltransferase activity (IC50 = 5.7 mM) in vitro, but did not detectably inhibit PCAF or GCN5 acetyltransferases . Thermal melt curves were obtained by heating the protein from 20–95°C and monitoring fluorescence at 590 nm. This experiment revealed that the thermal unfolding temperature of p300/acetyl-CoA was 48.6°C, while treatment with 10 and 25 mM salicylate reduced the unfolding temperature to 46.1°C and 40.8°C, respectively  bearing an inactivating Tyr1467Phe mutation to facilitate purification of homogeneously hypoacetylated p300 was expressed and purified with an N-terminal 6-His tag from ectively . Kineticectively  and histectively  substratK5/K9, H2BK12/K15, and H3K56 in a dose-dependent manner  was close to the IC50 of CBP measured in vitro and to the plasma concentrations of salicylate (1–3 mM) in humans after oral administration  p300, catalytically inactive p300 Y1503A and F1504A mutants , the immunosuppressant mycophenolic acid (IC50 = 664 μM), and diflunisal, an older nonsteroidal anti-inflammatory drug (IC50 = 996 μM)  ; 2008 ident IC50 . Three o 996 μM) .10.7554/50 of 374 μM is irrelevant to its antidiabetic activity. We therefore selected diflunisal for further analysis. Diflunisal induced deacetylation of specific histone residues, H2AK5, K9, H2BK12/K15, H3K56  after daily oral administration  translocation in acute myelogenous leukemia, is regulated by p300-mediated acetylation of lysine 43 of the fusion protein . To invein vivo, we inoculated SCID mice with Kasumi-1 cells. Starting 3 weeks after inoculation, the mice were treated daily with diflunisal  or vehicle. Diflunisal reduced tumor volumes in a dose-dependent manner  translocation, an exciting potential application of our observations..This study shows that salicylate inhibits CBP/p300 acetyltransferase activity by directly competing with acetyl-CoA, and it down-regulates the specific acetylation of histones and non-histone proteins in cells. We also found that diflunisal, an FDA-approved drug containing a salicylic acid substructure, inhibited CBP/p300 more potently than salicylate. Both drugs inhibited p300-dependent AML-ETO leukemic cell growth In plants, from where it was originally isolated, salicylate acts as an immune signal to induce systemic acquired resistance. It specifically activates the transcription cofactor NPR1 (nonexpressor of PR genes 1) by binding to its paralogs NPR3 and NPR4 . Since pIn animals, salicylate is an extensively studied small compound widely used as an anti-inflammatory drug. Many mechanisms of action have been proposed for the anti-inflammatory effects of salicylate in mammalian cells, including weak inhibition of cyclooxygenase , inhibitin vitro. In terms of what is observed for the other histone aceylated sites, the situation is more complex. Indeed, many histone modifications are regulated by multiple HAT enzymes. For example, acetyl H4K5 is regulated by HAT1, CBP, p300, Tip60, HB01 whereas acetyl H3 K14 is regulated by CBP, p300, PCAF, gcn5, ScSAS3 (We observed that the acetylation of both histones and non-histone proteins (NF-κB) is suppressed in cells treated with either salicylate or diflunisal. We identified histone AcH3K56 as the most sensitive histone acetyl mark to inhibition by both drugs. This results is highly consistent with the literature showing that CBP  in flies and CBP and p300 in humans acetylate H3K56 . We also, ScSAS3 . We therWe also observed inhibition of NF-κB acetylation by both salicylate and diflunisal  and 3D. Overall, our findings suggest that many of the pleiotropic effects of salicylate on different cell types and in diseases, including leukemia, are mediated by specific inhibition of CBP/p300 acetyltransferase activity, leading to deacetylation of histones and non-histone proteins.50 for p300 inhibition by both salicylic acid and diflunisal was significantly lower in HEK293T cells  inhibit both COX-1 and COX-2, although they vary in their relative potencies against the two isozymes . Howeverin vitro   range between 1–3 mM, a concentration at which partial inhibition of p300/CBP is observed. These data are therefore consistent with the proposed model that partial or complete inhibition of p300 by salicylate represents one of its relevant biological targets. It is important to note that a small molecule the size of salicylic acid used at such high concentrations is expected to interact with a number of cellular proteins and that our discovery that salicylic acid targets p300 does not imply that previous targets are not also part of the cellular response to these drugs.Several other FDA-approved drugs with substructures similar to that of salicylate also directly inhibited p300 acetyltransferase activity . The salin vitro and in vivo . Importantly, NSAIDs that lack p300 inhibitory activity failed to inhibit Kasumi-1 cells growth (data not shown).Recently, p300 has emerged as a potential therapeutic target for respiratory diseases, HIV infection, metabolic diseases, and cancer . Indeed, in vivo . We haveThese results identify a novel epigenetic therapeutic target for salicylate, the epigenetic regulator CBP/p300. Further efforts will focus on unraveling the relative roles of different cellular targets of salicylate, such as CBP/p300, cyclooxygenases, IKKβ, and AMPK. Our results also suggest that salicylate may be useful for treating inflammation, diabetes, neurodegenerative disease, and other pathologies in which CBP/p300 has a critical role.14C acetyl-CoA at 30°C for 60 min. Reactions were stopped by adding 6x sample buffer and analyzed by SDS-PAGE. The gels were dried, and signals were obtained by autoradiography and quantified with Image J software. To quantify acetylated histone levels, we generated a standard curve from signals of lanes loaded with 2.5, 5, and 10 g of 14C labeled histones.Recombinant HAT (1 mg), either p300, CBP, PCAF, or GCN5 (Enzo Life Sciences), and 10 µg of histones (Sigma) were incubated with sodium salicylate (Sigma) in reaction buffer  at 30°C for 30 min and then with 0.1 mCi of E. coli cells. Cells were grown at 37°C until they reached an optical density (600 nm) of 0.8, incubated with 0.5 mM IPTG  overnight at 18°C to induce protein expression, harvested, and lysed by sonication in lysis buffer . The lysate was cleared by centrifugation and applied to a Ni-NTA affinity column. The protein was eluted from the column with increasing concentrations of imidazole in lysis buffer (20–250 mM) and treated overnight with TEV protease to cleave the 6-His tag. The protein was further purified by passage through a HiTrap SP HP ion-exchange column and a size-exclusion Superdex 200 column equilibrated with 25 mM HEPES, pH 7.5, 150 mM NaCl, and 5 mM -mercaptoethanol.A thermal stability assay was used to assess the binding of salicylate to the p300 HAT domain. A p300 HAT domain construct (residues 1279–1666) bearing an inactivating Tyr1467Phe mutation to facilitate purification of homogeneously hypoacetyalted p300 was cloned into a pET-DUET vector with an N-terminal 6-His tag and expressed in BL21 (DE3) X-ray crystallography showed that the purified p300 HAT domain protein binds to acetyl-CoA or CoA   In thermSalicyl-CoA and diflunisal-CoA were synthesized from their parent carboxylic acids and HPLC purified according to previously reported methods . The purHEK293T cells (ATCC) were maintained in DMEM supplemented with 10% FCS. The viability of Kasumi-1 cells and AE9a mouse leukemia cells was assessed in triplicate by trypan blue exclusion. The Kasumi cell line was isolated and characterized by one of the coauthors (S. Nimer) . All celpCi-p300 and pCi-PCAF are described elsewhere . pcDNA3/K5 , acetyl histone H2AK9 , acetyl histone H2BK12/K15 , acetyl histone H3K9 , acetyl histone H3K14 , acetyl histone H3K27 , acetyl histone H3K36 , acetyl histone H3K56 , acetyl histone H4K5 , acetyl histone H4K8 , acetyl histone H4K12 , acetyl histone H4K16 , histone H2A , histone H2B , histone H3 , histone H4 , p300 , PCAF , tubulin , acetyl NF-κB p65K310 , NF-κB p65 , and acetyl lysine .HEK293T cells were treated with sodium salicylate for 24 hr and lysed in lysis buffer . For Western blot analysis, we used antibodies against acetyl histone H2ApCSII-CMV-MCS vectors and packaging plasmids were transfected to HEK293T cells, supernatant were collected and ultracentrifuged 48 hr after transfection. Same amount as p24 levels of lentiviruses contain empty or p300 expression vectors were transduced to Kasumi-1 cells.Kasumi-1 cells were treated with sodium salicylate or diflunisal for 24 hr and lysed in RIPA buffer. AML1-ETO protein in the lysate was immunoprecipitated with an anti-ETO antibody (Santa Cruz Biotechnology). Antibodies against AML1 and acetylated AML1-ETO K24/K43 (generated in the Nimer lab) were used for Western blotting.Apoptosis was analyzed with an Annexin V-APC/7AAD Apoptosis kit (Becton-Dickinson) according to manufacturer’s instructions. To assess the distribution of nuclear DNA content in the cell-cycle analysis, cells were collected, washed in PBS, fixed overnight in 75% ethanol at –20°C, treated with 1% RNase A for at least 15 min at 37°C, and stained with 50 mg/ml propidium iodide. To monitor CD34 expression, the cells were stained with an allophycocyanin-conjugated anti-CD34 antibody (Becton Dickinson). To monitor CD11b expression, the cells were stained with a phycoerythrin-conjugated anti-CD11b antibody (Beckman-Coulter). To monitor C-kit and Mac-1 expression, the cells were stained with allophycocyanin-conjugated anti-C-kit and phycoerythrin-conjugated anti-Mac-1 antibodies (Becton-Dickinson). Cells were sorted with a Becton-Dickinson FACSCalibur, and the data were analyzed with FlowJo software.Severe combined immunodeficiency (SCID) mice were injected with 30 million Kasumi-1 cells in 100 μl of PBS and 100 μl of Matrigel. Three weeks after inoculation, when tumors can be detected, mice were treated daily with oral diflunisal (50 or 100 μg/kg) or vehicle. Tumor volume and body weight were recorded every 2 days. After 3 weeks of treatment, the mice were killed and tumor size and body weight were recorded. All mice were maintained according to National Institutes of Health guidelines and all animal use protocols were approved by the Institutional Animal Care and Use Committee. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. I am happy to report that your manuscript has been reviewed by three experts in the field and by a member of the Board of Reviewing Editors (BRE) who are unanimously excited about the findings reported in this manuscript. The evaluation was overseen by Randy Schekman as the Senior Editor.Thank you for submitting your study titled, "Salicylate and diflunisal are CBP/p300 inhibitors with anticancer activity" for consideration by Furthermore, the reviewers have discussed the reviews with one another, and the BRE member and we hope that you will be able to address their comments included below and to submit the revised version within two months. We have also included at the bottom of this letter the entire reviews provided by the reviewers. Please let us know if you have any questions in this regard.We urge you to consider the following additional efforts:50 in vivo and in vitro as this information would make the paper much stronger.1) Perform further studies on the analysis of diflunisal to help explain the differences observed in its IC2) Perform further studies to rule out off-target effects perhaps by characterizing one or two of the commonly known targets of diflunisal.Reviewer #1:In this manuscript, Shirakawa and coworkers characterized salicylate and diflunisal as novel CBP/p300 inhibitors. This finding is surprising but also important for several reasons. First, salicylate is widely known for its anti-inflammatory property by targeting mainly COX1/2 and other proteins. The finding that this molecule also acts on CBP/p300 adds another functionally important target to appreciate the role of salicylate. Second, the Nimer lab previously showed the importance of CBP/p300 for the survival of ETO AML and herein demonstrated that salicylate and diflunisal are effective against ETO AML at the dose relevant to depletion of several acetylation marks. The therapeutic implication of the two compounds is important given their ready use, although the two compounds may hit multiple other targets at the present high doses.The main concerns of the reviewer are:1) Although the role of salicylate against CBP/p300's acetylation activity was rigorously examined by the key rescue experiment shown in 2) For the efficiency of the two compounds against ETO AML, it is not clear whether the effect is achieved by targeting CBP/p300 alone or other targets alone or multiple targets in a combined manner. If the latter are the case, the flow of the work will be disrupted and the impact of the work will be significantly dampened  of the therapeutic outcome of the two compounds were not CBP/p300). The reviewer strongly feels that the rescue experiments with wild-type and catalytically dead p300 should be carried out at a cellular level in parallel with the experiments in Reviewer #2:50 in the 10 mM range. They show that they can inhibit some, but not other histone modifications that are known to be a result of p300/CBP in cells. They go on to identify the small molecule diflunisal as a Hat inhibitor and show that it can inhibit acetylation of AML1-ETO and growth in mice. The data are of interest but there are significant issues regarding specificity of mechanism.Shirakawa et al. assess salicylate derivatives as potential p300/CBP HAT inhibitors based on the fact that other recently developed HAT inhibitors contain a salicylic acid moiety. They first show that salicylate inhibits p300/CBP Hat activity in vitro with an IC50 of 10mM for salicylate seems high. Can the authors put this into some context as to the concentrations at which salicylate works against other targets?1) The IC2) The fact that salicylate inhibits acetylation of specific histone residues is interesting, but the fact that not all p300/CBP dependent modifications are inhibited is concerning in terms of mechanism. The authors explain this by potential redundant enzymes. Anything that can be done here to help explain this result would be very helpful.50 for diflunisal is that the in vitro IC50 is 1mM and the cellular half-maximal inhibition is 100µM. Can this be explained? It seems strange that the IC50 in cells would be less than the IC50 in vitro.3) A significant concern in 4) The data on inhibition of AML1-ETO cell growth in vitro and in vivo is of interest, however there still remains a specificity issue. The authors need to test growth inhibition on other cell lines without AML1_ETO rearrangement or show that indeed inhibition of AML1-ETO acetylation is the reason for the antiproliferative effect. Also, there needs to be assessment of inhibition of acetylation in the leukemia cells in vivo.Reviewer #3:50 of salicylate for p300/CBP seems to be too high to be specific for HAT/KATs. The authors should show the specificity, using in vivo acetylation of nonhistone substrate, such as p53 acetylation by p300/CBP and PCAF .The ICIs it really inhibiting KAT activity in the animal system? The authors may consider to feed the animal (mice) and do immunohistochemistry (IHC) analysis of liver tissue.Inhibition of cancer progress has been claimed by the authors. The data is quite convincing. Is it possible to show that it is due the inhibition of p300/CBP KAT activity by salicylate?eLife. Your revised article has been favorably evaluated by Randy Schekman as the Senior Editor, a Reviewing editor, and three reviewers. Please note that the reviewers have requested modifications in the text that need to be implemented before the manuscript can be accepted. We have included the reviewers' comments below for you and look forward to receiving your revised manuscript and the rebuttal letter fully addressing the reviewers' comments.Thank you for resubmitting your work entitled, "Salicylate and diflunisal are CBP/p300 inhibitors with anticancer activity," for further consideration at Reviewer #1:The revised paper addressed the most of the previous concerns. In particular, the new model of the formation of "salicyl-CoA" and "diflunisal-CoA", if they are indeed the cases, may explain many conflicting data in vitro. However, for such situations, the in vitro biochemical data may not be relevant anymore to evaluate these compounds because they will act differently upon the formation of CoA-adduct. In contrast, the weak interaction of salicyl or diflunisal only argues that HATs contain binding pockets to accommodate these moieties. The reviewer strongly suggests revising the wording of "salicyl or diflunisal as inhibitors of HATs)" into "HATs contain the binding pockets of salicyl or diflunisal to engage the corresponding interaction in vitro and in cellular contexts". By this way, it leaves the possibility that salicyl or diflunisal may be subject to the formation of CoA-adduct and thus act as pro-drugs inside cells. The paper can even be further strengthened by showing the formation of "salicyl-CoA" and "diflunisal-CoA" in a cellular setting.Reviewer #2:The manuscript is improved as a result of the new experiments provided. However, while the authors respond to all of the concerns by the reviewers, there is no evidence that these discussions made it into the manuscript. It would be helpful if the authors added some discussion in the manuscript that focus on the reviewers’ questions given it is likely readers will have the same questions.Also, it is not clear why the authors did not expand their assessment of cell lines beyond Kasumi/AML1-ETO. This is not difficult and thus makes one wonder if the compound it toxic to many cell lines.Reviewer #3:The authors have addressed most of reviewers’ concerns. I am satisfied with the revised version of the manuscript. It can be accepted for publication. We urge you to consider the following additional efforts:50 in vivo and in vitro as this information would make the paper much stronger.1) Perform further studies on the analysis of diflunisal to help explain the differences observed in its ICK12/K15 acetylation , inhibits CBP/p300 with 28-fold increased potency in comparison with salicylate: IClate new . A similisal new . Althoug2) Perform further studies to rule out off-target effects perhaps by characterizing one or two of the commonly known targets of diflunisal.As described above, during the revision of this manuscript, we have conducted additional experiments with overexpressed p300 to demonstrate that the effects of diflunisal are dependent on p300 when examining histone acetylation. We have also tested the effect of p300 overexpression on the anticancer effect of diflunisal on Kasumi cell lines using lentiviral expression vectors for p300 or empty control new . Cells tWith respect to the suggestion of other possible targets of diflunisal, a review of the literature indicates that the only other reported targets of diflunisal is prostaglandin synthetase, also known as cyclooxygenase  Although the role of salicylate against CBP/p300's acetylation activity was rigorously examined by the key rescue experiment shown in As requested by the reviewer, we repeated the p300-overexpression rescue experiments using diflunisal and found that overexpression of p300 also suppressed diflunisal-mediated histone H2B deacetylation as shown in our revised new 2) For the efficiency of the two compounds against ETO AML, it is not clear whether the effect is achieved by targeting CBP/p300 alone or other targets alone or multiple targets in a combined manner. If the latter are the case, the flow of the work will be disrupted and the impact of the work will be significantly dampened  of the therapeutic outcome of the two compounds were not CBP/p300).50 of 113µM : 765-9). We have tested whether other NSAIDs, including acetaminophen and indomethacin, also inhibit p300 acetyltransferase in vitro, but did not detect any inhibitory activity (not shown). Diflunisal is a known COX-1/2 inhibitor with ICci USA96 . Cells tci USA96 . In contci USA96 , and exhci USA96  and sub-ci USA96 . These rThe reviewer strongly feels that the rescue experiments with wild-type and catalytically dead p300 should be carried out at a cellular level in parallel with the experiments in As we described above, we have performed these experiments and found that overexpression of p300 but not catalytically inactive mutants reduced diflunisal-mediated histone H2B deacetylation new . We alsoReviewer #2:50 in the 10 mM range. They show that they can inhibit some, but not other histone modifications that are known to be a result of p300/CBP in cells. They go on to identify the small molecule diflunisal as a Hat inhibitor and show that it can inhibit acetylation of AML1-ETO and growth in mice. The data are of interest but there are significant issues regarding specificity of mechanism.Shirakawa et al. assess salicylate derivatives as potential p300/CBP HAT inhibitors based on the fact that other recently developed HAT inhibitors contain a salicylic acid moiety. They first show that salicylate inhibits p300/CBP Hat activity in vitro with an IC1) The IC50of 10mM for salicylate seems high. Can the authors put this into some context as to the concentrations at which salicylate works against other targets?Most of the NSAIDs inhibit both COX-1 and COX-2, although they vary in their relative potencies against the two isozymes  J. Physiol. Pharmacol. 48, 623–631). However, salicylate does not, unlike its acetylated derivative aspirin, inhibit COX-1 and COX-2 activity in vitro  Nature New Biol. 231, 232–235; Vargaftig, B. B. (1978) J. Pharm. Pharmacol. 30, 101– 104; Proc Natl Acad Sci USA. 1993 Dec 15; 90(24):11693-7; Cromlish, W.A., and Kennedy, B.P. (1996) Biochem.Pharmacol. 52, 1777–1785). However, salicylate has been shown to exert a comparable analgesic and anti-inflammatory action as aspirin. Salicylates have been proposed to exert their pharmacological effects via inhibition of the transcription factor nuclear factor NF-κB and other targets. In these experimental systems, the concentrations used were in the same range as used in the experiments described in our paper (5-20 mM)  Science265, 956–959 17; Pierce, J. W., Read, M. A., Ding, H., Luscinskas, F. W., and Collins, T. (1996) J. Immunol. 156, 3961–3969 18; Schwenger, P., Bellosta, P., Vietor, I., Basilico, C., Skolnik, E. Y., and Vilcek, J. (1997) Proc. Natl. Acad. Sci. USA.94, 2869 –2873; Oeth, P., and Mackman, N. (1995) Blood86, 4144–4152 40; Schwenger, P., Skolnik, E. Y., and Vilcek, J. (1996) J. Biol. Chem. 271, 8089 – 8094 41. Schwenger, P., Alpert, D., Skolnik, E. Y., and Vilcek, J. (1999) J. Cell. Physiol179, 109–114).50 (IC50 ~5mM for H2BK12/15) and new data show that a salicylic acid metabolite, salicyl-CoA inhibits CBP/p300 with 28-fold increased potency in comparison with salicylate: IC50 =220 μM for salicyl-CoA vs 6.12 mM for salicylate  range between 1-3 mM, a concentration at which partial inhibition of p300/CBP is observed. In addition, our inhibition data in cell lines show a lower IClate new . These d2) The fact that salicylate inhibits acetylation of specific histone residues is interesting, but the fact that not all p300/CBP dependent modifications are inhibited is concerning in terms of mechanism. The authors explain this by potential redundant enzymes. Anything that can be done here to help explain this result would be very helpful.K5 is regulated by HAT1, CBP, p300, Tip60, and HB01 whereas acetyl H3 K14 is regulated by CBP, p300, PCAF, gcn5, and ScSAS3 (Cell128 (In 3) A significant concern in  IC50for diflunisal is that the in vitro IC50is 1mM and the cellular half-maximal inhibition is 100µM. Can this be explained? It seems strange that the IC50in cells would be less than the IC50in vitro.50 =20 µM for diflunisal-CoA vs. 1.05 mM for diflunisal  The data on inhibition of AML1-ETO cell growth in vitro and in vivo is of interest, however there still remains a specificity issue. The authors need to test growth inhibition on other cell lines without AML1_ETO rearrangement or show that indeed inhibition of AML1-ETO acetylation is the reason for the antiproliferative effect. Also, there needs to be assessment of inhibition of acetylation in the leukemia cells in vivo.50 of 113µM (Proc Natl Acad Sci USA96 (13): 7563-8), but other NSAIDs that lack p300 inhibitory activity failed to inhibit Kasumi-1 cells growth (data not shown). Further, we have also tested the effect of p300 overexpression on the anticancer effect of diflunisal on Kasumi cell lines using lentiviral expression vectors for p300 or empty control : 765-9). We have tested whether other NSAIDs, including acetaminophen and indomethacin, also inhibit p300 acetyltransferase in vitro, but did not detect any inhibitory activity (not shown). Diflunisal is a known COX-1/2 inhibitor with ICtrol new . Cells ttrol new . In conttrol new , and exhtrol new  and sub-trol new . These rReviewer #3:50 of salicylate for p300/CBP seems to be too high to be specific for HAT/KATs. The authors should show the specificity, using in vivo acetylation of nonhistone substrate, such as p53 acetylation by p300/CBP and PCAF .The ICAs suggested by the reviewer, we have examined the effect of salicylate and diflunisal on a non-histone target and show in Is it really inhibiting KAT activity in the animal system? The authors may consider to feed the animal (mice) and do immunohistochemistry (IHC) analysis of liver tissue.Inhibition of cancer progress has been claimed by the authors. The data is quite convincing. Is it possible to show that it is due the inhibition of p300/CBP KAT activity by salicylate?Direct demonstration that inhibition of p300/CBP KAT activity in the context of tumor growth in vivo would have required the generation of multiple clonal cell lines overexpressing p300 and control cell lines with the empty vector, their characterization (for stability of p300 expression etc…) and a large number of xenograft experiments showing that overexpression of p300 in vivo suppresses the ability of diflunisal to inhibit tumor growth. Given the short time allowed for resubmission, we elected to conduct this experiment on Kasumi cells in vitro. The results presented in our revised Reviewer #1:The revised paper addressed the most of the previous concerns. In particular, the new model of the formation of "salicyl-CoA" and "diflunisal-CoA", if they are indeed the cases, may explain many conflicting data in vitro. However, for such situations, the in vitro biochemical data may not be relevant anymore to evaluate these compounds because they will act differently upon the formation of CoA-adduct. In contrast, the weak interaction of salicyl or diflunisal only argues that HATs contain binding pockets to accommodate these moieties. The reviewer strongly suggests revising the wording of "salicyl or diflunisal as inhibitors of HATs)" into "HATs contain the binding pockets of salicyl or diflunisal to engage the corresponding interaction in vitro and in cellular contexts". By this way, it leaves the possibility that salicyl or diflunisal may be subject to the formation of Co-A adduct and thus act as pro-drugs inside cells. The paper can even be further strengthened by showing the formation of "salicyl-CoA" and "diflunisal-CoA" in a cellular setting.and their metabolites inhibit CBP/p300 and exhibit anticancer activity”. This change acknowledges our observation that the metabolites also exhibit inhibitory activity against p300 and CBP and that we cannot distinguish whether the primary compound or the metabolite is important in the cellular context.We have modified the title of the manuscript in the following manner: “Salicylate, diflunisal Reviewer #2:The manuscript is improved as a result of the new experiments provided. However, while the authors respond to all of the concerns by the reviewers, there is no evidence that these discussions made it into the manuscript. It would be helpful if the authors added some discussion in the manuscript that focus on the reviewers’ questions given it is likely readers will have the same questions.Also, it is not clear why the authors did not expand their assessment of cell lines beyond Kasumi/AML1-ETO. This is not difficult and thus makes one wonder if the compound it toxic to many cell lines.We have added new discussion points in the manuscript as recommended by the reviewer.


The International Society for Peritoneal Dialysis recommends the regular application of topical antibiotic-containing preparations in addition to a routine exit site care to reduce the risk of exit site infection (ESI). Among these prophylactic antimicrobial preparations, topical gentamicin is one of the widely used and effective antibiotics for prevention of ESI and peritonitis in peritoneal dialysis (PD) patients. Overall, topical gentamicin is well tolerated; however, its use can be associated with the development of allergic contact dermatitis (ACD). We describe a first reported case of PD catheter exit site contact ACD due to topical gentamicin mimicking ESI. The patient in this report developed worsening violaceous in color and pruritic rash surrounding the PD catheter exit site that appeared 3 weeks after the initiation of gentamicin cream. The association between development of rash and initiation of topical gentamicin led to a suspicion of local reaction to gentamicin rather than ESI. Skin biopsy confirmed ACD. Discontinuation of the provoking agent and subsequent treatment with topical hydrocortisone application led to a resolution of the exit site rash. Any rash at a PD catheter exit site should be considered infectious until proven otherwise. However, it is important to be aware of noninfectious etiologies of exit site rashes as the treatment of these 2 conditions differs. We describe for the first time a clinical presentation and diagnostic approach to gentamicin-induced contact dermatitis at the PD catheter exit site that mimicked ESI.The development of rash at the peritoneal dialysis (PD) exit site is never trivial for nephrologists and is always concerning for PD exit site infection (ESI). ESI occurs in up to 20% of PD patients and is associated with a 6-fold increase in incidence of PD-associated peritonitis in the subsequent 60 days even with appropriate treatment of ESI.A 54-year-old African American female was undergoing continuous cycling PD for end-stage renal disease due to diabetes. She presented to clinic with 2.5-week history of a worsening pruritic rash around her PD catheter exit site. Three weeks earlier she began topical gentamicin sulfate 0.1% cream for ESI prophylaxis. The patient reported no fever or abdominal pain. On examination, she had normal vital signs. The PD catheter exit site had an 8.5 × 4.5 cm ovoid crusted plaque, violaceous in color with a peripheral rim of erythema and without granulation . There wClinical diagnosis of allergic contact dermatitis (ACD) due to gentamicin was made and gentamicin cream was discontinued. Skin biopsy performed the following day demonstrated psoriasiform spongiotic dermatitis with eosinophils, consistent with ACD  and C. TACD due to gentamicin cream35 The most common pathogens causing ESI are Staphylococcus aureus and Pseudomonas aeruginosa. Additional pathogens leading to ESI include Staphylococcus epidermidis, Escherichia coli, diptheroids, streptococci, nontuberculous mycobacteria, and fungi.4 The ISPD recommends cleaning of PD catheter exit site with antiseptic agent and application of topical antimicrobials, such as gentamicin or mupirocin, for the prevention of ESI.3 Topical gentamicin has been shown to reduce ESI and peritonitis due to gram-positive and gram-negative organisms;7 while, topical mupirocin mainly reduced ESI due to gram-positive organisms.8 ESI is typically diagnosed clinically based on the finding of purulent or bloody drainage from PD catheter exit site, surrounding erythema, tenderness, and swelling. However, the presence of skin rash and erythema without drainage at the PD catheter exit site can be also due to early infection, allergic reaction to PD catheter material,9 or to mechanical trauma.4 Additionally, allergic reactions to PD catheter exit site care products such as antibiotic preparations (mupirocin and polysporin)10 and antiseptic agents12 can manifest as skin rash around PD catheter exit site.ESI should be considered with the development of rash at the PD catheter exit site. ESI is a major risk factor for the development of PD-associated peritonitis; therefore, prompt diagnosis and treatment of ESI are essential.Erythema and rash around the exit site can be mistaken for ESI; however, absence of drainage, tenderness, and swelling may be clues for contact dermatitis. The diagnosis of contact dermatitis is usually established on clinical grounds based on characteristic appearance of rash, negative Gram stain and culture of exit site, and favorable response to withdrawal of suspected agent along with supportive measures such as topical steroid preparations. Skin biopsy can be used to confirm the diagnosis, and in our patient it demonstrated psoriasiform spongiotic dermatitis with eosinophils  and C.13 In a randomized controlled trial comparing effectiveness of topical gentamicin sulfate 0.1% cream and mupirocin 2% cream, only minor exit site irritations developed in 10.5% of patients in both treatment arms with no reported cases requiring discontinuation of antimicrobial preparations.6 ACD is a cell-mediated type 4 delayed hypersensitivity reaction.14 ACD is observed more frequency in patients with atopic eczema, nickel sensitization, stasis dermatitis, and chronic actinic dermatitis.15 Patients who develop ACD from gentamicin are at an increased risk for generalized eczematous eruption following parenteral administration of gentamicin.16 Contact dermatitis from gentamicin can also result in cross-sensitivity to other aminoglycosides like neomycin.17 Of note, allergy to topical neomycin—the most commonly used topical antibiotic in the United States18—has been reported to occur in up to 13.1% of the general population.19Topical gentamicin  has been previously linked to periocular ACD.ACD due to topical gentamicin is not uncommon and can occur at the PD catheter exit site. It is important to be aware of this association to avoid incorrect diagnosis of PD catheter ESI and inappropriately continued antibiotic use in cases of ACD. In contrast, the routine exit site care, including topical antimicrobials, is continued during PD catheter ESI. The failure to discontinue provoking allergens in ACD can lead to the worsening of exit site rash, incorrect diagnosis of refractory ESI infection, and potentially result in PD catheter removal.


PLOS Biology, Shiwen Wang, Yue Xiong, Dan Ye, Kun-Liang Guan, and colleagues elucidate in detail a regulatory pathway that begins with insulin and ends with removal of an acetyl group on PGK1, opening up the enzyme’s active site to bind ADP  plays a pivotal role. After a couple of preliminary steps that prime the pump, it is PGK1 that creates the first molecule of ATP from the energy stored in glucose, by swinging a molecule of ADP in close to pick a phosphate off an energetic intermediate. While much is known about transcriptional control of PGK1, relatively little is known about how extant PGK1 is regulated. In a new study in bind ADP .Acetylation is perhaps less well known as a regulatory modification than phosphorylation, but it is a ubiquitous method of changing a protein’s structure and function. PGK1 is acetylated in multiple locations, and the authors showed that inhibiting a family of deacetylases led to a decrease in PGK1 activity, indicating the biochemical relevance of the modification. They identified five highly conserved lysine residues on the protein that could serve as a site of acetyl group attachment, and showed that mutating one, K220, reduced activity by over 80%. Modeling suggested, and experiments confirmed, that an acetyl group attached to this site blocked binding of ADP, thus preventing ATP production.Knockdown of a set of acetyltransferase enzymes, which add acetyl groups, revealed one, KAT9, whose loss decreased PGK1 acetylation and whose overexpression increased it. Conversely, overexpression of the deacetylase HDAC3 decreased PGK1 acetylation, and its depletion increased it.Because of its central role in regulating glucose, the authors tested the effect of insulin on PGK1 acetylation. They found that insulin reduced acetylation at K220, an effect that was accompanied by an increase in enzyme activity. Insulin did not change the expression or activity of KAT9. Nor did it change the expression of HDAC3; instead, it enhanced the association of PGK1 and HDAC3 in a dose-dependent manner, indicating the central importance of deacetylation, and therefore activation, of the enzyme as a rapid response to changing energy needs.How was HDAC3 itself regulated? Its ability to deacetylate PGK1 could be inhibited by mutating a critical serine residue, preventing its phosphorylation, and insulin promoted the phosphorylation of that same site. The fraction of HDAC3 bound to PGK1 was highly phosphorylated, while the free enzyme was not, all suggesting that serine phosphorylation was central to regulation of HDAC3.Previous work had identified phosphoinositide-3-kinase (PI3K) as a regulator of HDAC3; here, inhibiting PI3K prevented HDAC3-PGK1 association, leading to a rise in the acetylation level of PGK1. Inhibition of other participants in the pathway, including the central growth controller mTOR, had a similar effect, preventing the upstream insulin signal from triggering the downstream activation of PGK1. Without normal levels of active PGK1, cells could not consume glucose or make ATP at the proper rate, and they displayed multiple signs of metabolic stress.The identification of this important regulatory modification on a metabolically important enzyme is likely to have several important consequences. A full understanding of cell metabolism is still elusive, but modeling improves with every new discovery. The other (non-ATP) product of PGK1 is 3-phosphoglycerate, critical for producing serine, among other cell building blocks, and the authors also showed that PGK1 is involved, in a way not yet clear, in regulating production of NAPDH, a key source of chemical reducing power for anabolic reactions. Since there are several human disorders involving PGK1 deficiency, the discovery of a way to activate the remaining enzyme may have direct clinical implications.


The procedure for removing a broken pedicle screw shouldideally be technically easy and minimally invasive, as anydamage to the pedicle, during removal of the broken screw,may weaken the pedicle, thus compromising on the successof re-instrumentation. We describe the case of a 32-year oldman who had undergone surgery for traumatic third lumbarvertebral body fracture three years prior to current admissionand had developed the complication of pedicle screwbreakage within the vertebral body. The patient underwentre-exploration and removal of the distal screws. Through aparavertebral incision and muscle separation, the screws androds were exposed and the implants were removed.Implant failure, spine surgery, pedicle screws, spinalfracture, spinal instrumentation At the same time it may be associated with ahigher rate of complications and so the safety and theeffectiveness of pedicle-screw instrumentation have beenquestioned 3, 6. We describe a rare complication of pediclescrew breakage within the vertebral body and discus themanagement including the technique of screw removal.It has been recognized that pedicle screw fixation in spinalsurgery produces a higher rate of fusion compared with othermethodsA 32-year old man had undergone pedicle screw surgery for-third lumbar vertebral body fracture three years previously.Following surgery, he had been apparently well, until sixmonths prior to the current presentation, he had complainedof local pain at the site of surgery which increased with stressand work. Bowel and bladder functions were normal. Motorpower and sensation in the lower limbs were normal.Lumbar spine x-ray showed breakage of both distal screws atthe neck of the screws 3, 5. This implantfailure can be a result of pseudarthrosis and can lead topedicle screw or rod breakage5. By using the finite elementmodel Chen et al demonstrated that screws on the caudal endwere subjected to larger axial stress than those on thecephalic end, supporting the clinical finding that 75% ofpatients had screw breakage caudally (as in present case. Inaddition, screws show fatigue striations and ductile fracturearound the edge1. Appropriate radiographs can demonstratethe screw breakage and revised spinal surgery is themainstay of treatment when there is a broken pedicle screw1.Removal of the broken screw can be a difficult task andmany techniques have been described to remove deeplyembedded broken pedicle screw fragments1, 3. The methodsdescribed to remove broken screws include creating a deeppilot hole in the center of the fractured screw and engagingthe screw driver to reverse and rotate the screwcounterclockwise, whereby the retained screw fragment isbacked out of the hole, preserving the bone interface6.Alternatively, using a high-speed drill with a long bit - tomake a slot in the top surface of the broken screw toaccommodate a standard flathead screwdriver to remove thescrew3. Both of these techniques have been used successfullyto extract broken pedicle screws, while maintaining theintegrity of the pedicle3. These procedures can be limited bythe availability of drill and other equipment and also deepdrilling inside the broken screw for the application of screwextractor is usually difficult and the additional effort in tacking of the screw extractor towards the broken screwcarries the risk of moving the broken screw deeper4. Inanother method, wide bone removal of the pedicle at the periphery of the broken screw can help to grasp the brokenscrew by retrieval instruments and remove the screw bycounter clockwise movement with the help of friction andgrasping, respectively2. These methods may necessitate theremoval of a large amount of bone around the broken screwtip for application of clamp, and clamp failure duringretrieving can be a major problem4. After successful removalof the broken screws larger diameter screws can be replaced4.Pedicle screw breakage is reported to occur in 1-11.2% ofinserted screws and in 0.4-24.5% of patientsIt has been suggested that many complications can beprevented by careful attention to the application ofestablished surgical techniques, an understanding of thedynamic stabilization system, and proper selection ofpatients. The removal process of a broken embedded pediclescrew should be technically easy and noninvasive to thepedicle, as any damage to the pedicle, during removal of thebroken screw, may weaken the pedicle, thus compromisingon the success of re-instrumentation.


The United States system of immigrant detention centers has been the subject of considerable scrutiny with respect to health care of detainees. We sought to characterize the rates and types of deaths that have occurred within this system between the years 2003–2015. We analyzed a file of detainee deaths released by the U.S. Department of Homeland Security as part of a freedom of information request. Between 2003 and 2015, 150 deaths were recorded. During this time period, the annual rate of death among detainees dropped dramatically, whether measured by annual admissions or by person years of exposure. The most common causes of death were cardiovascular, cancer and suicide. More research is needed to adequately account for the contributors to these declining rates of death in immigration detention settings. Over the past ten years the media, governmental, and nongovernmental organizations have raised concerns about health care provided to detainees held in the custody of U.S. Immigration and Customs Enforcement (ICE), a department under the US Department of Homeland Security (DHS) ,2,3. TheThe United States detains noncitizen immigrants to guarantee court appearance for immigration proceedings. ICE is responsible for the oversight of medical care and conditions of detention in the three types of facilities in the United States where immigrants are detained. These include service processing centers, which are owned and operated by ICE; contract detention facilities, which are managed by private companies; and state and local jails that enter intergovernmental service agreements (IGSAs) with ICE. The ICE Health Service Corps (ISHC) is responsible for providing and coordinating health care services for detained immigrants. Over the past decade, ICE has worked to concentrate the number of detainees who are held in sites with ISHC staff on site. In addition, ICE has promulgated several rounds of new medical policies for detention settings, including in 2000, 2008 and 2011. Our experience in advocating for patients, visiting detention centers and meeting with ICE has been that health practices have improved for many detainees, however we sought to quantify changes in death rate among ICE detainees, and also to provide context for any changes we perceived.Data for this analysis were obtained from the ICE freedom of information act (FOIA) library, which is publically accessible on the internet . ICE posOverall, the yearly number of deaths occurring in ICE facilities has dropped dramatically between 2004 and 2014 . In 2004When cause of deaths was categorized, the most common category was cardiovascular (33.3%), followed by cancer (16.1%), suicide  and other 12.8%, . Deaths When facility type was analyzed for all 150 deaths, 54.5% of cases occurred in Inter-governmental Service Agreement  settings, while 15.2% occurred in Federal Bureau of Prisons (BOP) settings, 11.0% in Contract Detention Facilities (CDF), and 9.7% in service processing centers (SPC). Among the suicide cases, 16 out of 20, or 80%, occurred in IGSA settings. Of note, 5 of the suicides occurred in the Eloy Federal Contract Facility, while no other facility had more than one suicide. Both overdose deaths occurred in holding facilities.These data reflect a significant decline in the number of persons who have died in ICE custody over the past 11 years, as well a corresponding decrease in the rate of death, whether calculated by person years of exposure or number of admissions. The rate of decline in deaths in ICE custody exceeds any decline reported in jail or prison systems during a similar time ,6 and maA second demographic shift among ICE detainees may be linked to the increasing reliance on programs such as the Secure Communities program and the Priority Enforcement Program that direct immigrants arrested for crimes into ICE custody from jails and prisons. These programs rely on coordination between local criminal justice systems and ICE, so that persons who enter custody for a criminal charge may be discharged directly into ICE custody. From a health standpoint, this arrangement may result in local jails doing much of the initial screening and treatment of ill patients and some of the highest risk patients may either have died while in criminal justice detention or been diverted away from ICE detention. Accordingly, persons arriving in ICE custody through this path may be lower risk than others who are detained directly from the community, especially as relates to common causes of early admission death such as alcohol withdrawal and untreated seizure disorder. Since 2008, ICE deported more than 283,000 convicted immigrants as a result of Secure Communities. The percentage of detainees identified through the Secure Communities program grew from 4% in FY 2008  to 21% iversus other facilities, we do not have a direct measure of the change in IGSA death rates. Rather, we can only note that over the time of this analysis, fewer detainees are being held in IGSA settings, where health services are less robust. From a health standpoint, housing fewer persons in small county jails and increasing the utilization of dedicated ICE facilities, which have health staff provided by the U.S. Public Health Service, may account for some amount of mortality reduction.Another potential contributor to decreasing mortality in ICE detention is improvement in ICE medical policies and practices. ICE introduced a set of performance based national detention standards for all detention facilities in 2008, and again in 2011. ICE implemented these standards with considerable exemptions in IGSA settings because of the small scale and lower level of resources in many of these county jails. Over time, ICE has both increased the compliance demands on IGSA’s and also increased the utilization of U.S. Public Health staff to provide direct care. These staff receive specialized training in delivering care to immigrants and they now number over 900, providing care to almost half of detainees  while also involved in oversight of care for the remainder  of detainees. Because we were unable to obtain the yearly breakdowns of detainee census in ISGA The causes of death described in this analysis are similar to those reported in U.S. jails ,11. The Overall, the rate of death in ICE detention settings has declined significantly. It appears as though suicide may be overrepresented as a cause of death in IGSA settings. Important limitations exist in this analysis, including the lack of information about mortality among persons who are briefly detained and removed outside of the ICE detention system. More analysis is needed to provide reliable interpretations of the reasons for these decreases in mortality rates among detained immigrants. In particular, more analysis is needed to quantify rates of death among those briefly detained by the U.S. Border Patrol, but not ICE, as well as those who have been removed to other nations and die shortly after arrival. Finally, because an increasing share of those who ultimately come into ICE custody start in criminal justice detention, more focused analysis of deaths in those settings could be undertaken.


Proactive and reactive inhibitory processes are a fundamental part of executive functions, allowing a person to stop inappropriate responses when necessary and to adjust performance in in a long term in accordance to the goals of a task. In the current study, we manipulate, in a single task, both reactive and proactive inhibition mechanisms, and we investigate the within-subjects effect of increasing, by means of anodal transcranial direct current stimulation (tDCS), the involvement of the right inferior frontal cortex (rIFC). Our results show a simultaneous enhancement of these two cognitive mechanisms when modulating the neural activity of rIFC. Thus, the application of anodal tDCS increased reaction times on Go trials, indicating a possible increase in proactive inhibition. Concurrently, the stop-signal reaction time, as a covert index of the inhibitory process, was reduced, demonstrating an improvement in reactive inhibition. In summary, the current pattern of results validates the engagement of the rIFC in these two forms of inhibitory processes, proactive and reactive inhibition and it provides evidence that both processes can operate concurrently in the brain. A key aspect of executive functions is behavioral inhibition, the ability to control inappropriate or unwanted responses At the cognitive level, the stopping performance has been described as a mechanism in relation with the appearance of a signal –reactive inhibition–, and with the preparation to stop an upcoming response tendency in accordance with the goals of a person –proactive inhibition– At the neural level, the inhibitory function in the brain is proposed to be implemented by a specific fronto-basal-ganglia circuit At the experimental level, response inhibition is often assessed using the Stop-Signal task (SST) or the Go/Nogo task (GNG). In the SST, subjects are instructed to stop responding immediately after the appearance of a specific signal, and its underlying inhibitory process is reactive inhibition. The effectiveness of a correct performance in the SST is understood as an independent In fact, several authors have challenged the idea of response inhibition as a “unitary” and isolated function in the last decade  together with the manipulation of the difficulty in discriminating the stimuli and implemented anodal tDCS to the rIFC. We hypothesize that if both inhibitory processes are governed by rIFC, an increase in both reactive (reduction of the SSRT) and proactive inhibitory processes (increase of RT and/or reduction of commission and omissions) should be achieved, providing direct evidence that these two processes can operate simultaneously. Moreover, by contrasting how signal detection process affects the different outlined measurements on the inhibitory function, we expect to provide new data on the interrelation of response inhibition with other aspects of the executive function.Comissió de Bioètica of the University of Barcelona. Written informed consent was obtained from each participant before the experiment.Twenty-two healthy volunteers  participated in the experiment. All participants were right handed according to the Edinburgh handedness inventory test We implemented a new experimental design that aimed to observe at within-subject level both proactive and reactive inhibitory processes. We adapted a choice-reaction GNG task Each subject participated in two experimental sessions  counterbalanced across participants and conducted at least one week apart. The experiment began with a practice block that consisted of 64 trials to familiarize the participants with the task. To guarantee that participants began the task aware of the difference between “l” and “1”, the practice block was repeated until the number of commission and omission errors were equal or less than 10. The practice block lasted for ∼4 min, and it was repeated for all participants except for 3. Only 1 subject needed 3 practices blocks to achieve the defined criterion. In the second session, a single practice block was administered, with the only purpose of reminding participants of the task procedure. After every 29 trials, a short break of 5 sec was included to allow participants to rest, and after 144 trials, a 10 sec break was given. Each experiment session lasted for approximately 23 min.In both experimental sessions the location of the tDCS electrodes was established following the Jacobson et al. 2; current density 0.16 mA/cm2) and delivered by a battery, constant-current stimulator  for 18 min. An automatic on and off ramp of 10 sec was used. In the sham condition, the intensity of the current was the same, but the duration of the stimulation was limited to the duration of the current being ramped on and off (over 20 sec) at the beginning and at the end of the 18 min period. By following this protocol in the sham tDCS session, we ensured that participants felt the same sensations that they felt in the anodal stimulation session.In the anodal condition, a direct current of 1.5 mA was induced by two square saline-soaked surface sponge electrodes .Importantly, participants were not informed about the different stimulation protocols until the end of the entire experiment, and they could not distinguish between the anodal and the sham tDCS, as assessed by subject responses on a questionnaire completed at the end of each session For all the analyses the same two factors were introduced in separated repeated measures ANOVAs with two within-subjects factors: tDCS-session  and discriminability (easy vs. hard).p>0.2; anodal-easy: t(21) = −2.6; p<0.02; anodal-hard: t(21) = −3.7; p<0.01]. ANOVA results revealed a main effect of tDCS, denoting that participants inhibited a significantly larger number of trials in the anodal than in the sham tDCS-session . A trend towards a significant difference was observed for the discriminability factor , which indicated a propensity toward responding to more trials for the hard than for the easy condition.Participants inhibited approximately half of the stop trials in both tDCS-session, indicating a correct implementation of the tracking algorithm . Only in the anodal session the p was significantly less than expected (50%) , reflecting a behavioral inhibitory improvement caused by the anodal stimulation. No significant effects were found for the discriminability factor or interaction term . However, and for the purpose of further exploring the data, a subsequent post-hoc analysis was conducted which revealed that a significant effect of tDCS-session was observed in the easy-discriminability condition . For the hard-discriminability condition, a trend towards significance was found . Comparable results were obtained when analyzing the SSRT using the mean method  = 8.7; p<0.01]. The discriminability factor was also found to modulate the SSD, as revealed by the significant effect  and a trend, albeit non-significant, towards an interaction of discriminability by tDCS-session . Post-hoc analysis showed a larger increase of the SSD in the easy (41 msec) than in the hard (29 msec) condition caused by anodal tDCS  = 61.8; p<0.001], which indicated that this manipulation worked as expected. Importantly, a significant main effect of tDCS-session was found for Go RT . The interaction was not significant . The analysis of commission errors revealed only a main effect of discriminability , although a significant reduction of commission errors was observed for the easy discriminability condition during the anodal tDCS condition . Finally, the analysis of omitted responses revealed again only a main effect of discriminability . Thus, participants did not increase omission rates during the anodal tDCS condition, but a significant reduction of commission errors was observed for the easy discriminability condition, as it could be predicted by the speed-accuracy trade-off, yielding a slight improvement in response accuracy and indicating that tDCS applied over the rIFC could modify both reactive and proactive processes simultaneously.When analyzing the RT for Go trials, a strong main effect of discriminability was found , while the factor discriminability  and the interaction of tDCS-session by discriminability  did not reach statistical significance.In order to further testing whether discriminability modulated performance in a considered general inhibitory function, we conducted a Multivariate Analysis of Variance (MANOVA) in which Z-scores of RT and SSRT values were entered into the analysis as dependent variables and discriminability and tDCS-session as fixed factors. The results of this analysis revealed a main effect of tDCS-session [p-values>0.5), thereby indicating certain level of independency between the two studied inhibitory processes.Finally, we conducted a set of Pearson correlation analyses between RT, Commission and Omission and SSRT values with the aim to explore a possible relation between reactive and proactive inhibitory behavior. These analyses were computed separately for easy and hard discrimination condition and considering only the effects of the tDCS . The results did not reveal any significant correlation  and proactive  processes. Anodal tDCS slowed down the RT on Go trials, and this result was accompanied by a significant reduction in commission errors when the discriminability of stimuli was easy, although a significant interaction discriminability per tDCS-session did not reach statistical significant levels. These results point out to a speed-accuracy trade-off, thereby indicating a possible increase in task control, which was interpreted as an index of a proactive inhibitory process. Additionally, under tDCS stimulation, the SSRT, a covert index of the inhibitory process, was reduced, demonstrating an improvement in reactive inhibition. Thus, although the activity of the rIFC is directly related in the SST and GNG tasks, the neural mechanisms through which the rIFC plays a role in their execution differs, engaging proactive and reactive inhibitory processes concurrently when needed. Therefore, our data supports a view in which the rIFC is characterized as a key area for behavioral inhibition, and supports the view that the same brain structure could be engaged in a distinct and adaptive manner to inhibit behavioral responses A variety of tasks has been used for the assessment of response inhibition in a large set of studies. However, none of these studies explored whether two typically differentiated processes like proactive and reactive inhibition can be manipulated concurrently through the influence of the same brain structure, namely the rIFC. In fact, previous studies have noted that such methodological difficulty resided in the high degree of functional similarity of these two processes Although the GNG and SST are the most commonly used tasks for assessing behavioral inhibition, we believe that in the current study they engage different cognitive operations in their execution. In the current GNG task, subjects select the response strategy at the beginning of the block, thereby requiring the subjects to constantly adjust their responding threshold such that a motor response can be withheld before it is initiated In terms of cognitive control and under reactive control, goal representations are only retrieved at the time they are needed, giving the advantage of its computational efficiency due to the fact that resources are freed up after the inhibitory command is executed The recruitment of inhibitory and control mechanisms may explain the extensive activation encountered within the rIFC in most of the fMRI studies using variants of SST that increase decision-making demands and strategy adjustments In addition, we found that the characteristics of the stimuli did not differently affect the execution of reactive and proactive inhibitory processes, although a small but significant effect was favored in the condition where the difficulty of task was reduced. Hence, in the condition in which the stimulus was easy to discriminate, participants committed less number of errors. Noteworthy, the SSD was significantly larger for the hard than for the easy condition, leaving the possibility that the categorization of the stimulus may be related to the inhibitory response process. Previous neuroimaging studies have investigated the distinct cognitive processes that are involved in stopping, such as response inhibition, attentional capture of the stopping stimulus, and error monitoring One important aspect of the present study was the opportunity to employ tDCS to modulate a cognitive function. The mechanism underlying the neuromodulatory effects induced by tDCS are well established. Several studies using animal models have suggested that tDCS can promote effective excitation or inhibition of neurons in a polarity-specific manner Some results of stop-signal studies have shown that subjects trade speed in Go trials for success in stopping after stop-signal trials Reactive and proactive inhibitory processes are considered to be an integral part of cognitive control Data S1Data summary. Average data for all subjects on the different measurements obtained in the task are provided, as well as data used for computing the SSRT with the integration method  and mean method (SSD and mean RT).(XLSX)Click here for additional data file.