The ST2/IL-33 pathway has been related to ulcerative colitis (UC), and soluble ST2 (sST2), to disease severity. We tested the potential usefulness of sST2 as a predictive marker of treatment response and patients’ outcome.p <0.05).Twenty-six patients with active UC were prospectively recruited and grouped according to an endoscopic score and therapy response. Colonoscopic biopsies were collected at baseline and 6 months or when patients showed clinical activity. The protocol was reinitiated in patients requiring rescue therapy. Blood and stool were collected at baseline, 1, 3, 6 and 12 months. Serum and mucosal ST2, and fecal calprotectin (FC) content were determined by ELISA and correlated to Mayo clinical and endoscopic subscore. Intestinal ST2 was evaluated by immunofluorescence. Wilcoxon signed rank test and Spearman correlations (Rs) were applied (p < 0.05), and 336.3 [211.0–403.2] to 385.3 pg/mL [283.4–517.3] in non-responders at baseline and 6 months, respectively. sST2 levels correlated with Mayo clinical and endoscopic subscore, mucosal ST2 and FC  and showed a trend similar to that of FC in responders. Non-responders revealed an increased ST2 content, restricted to the lamina propria’s cellular infiltrate.Follow-up was completed in 24 patients. sST2 levels (median and range) varied from 173.5 [136.6–274.0] to 86.5 [54.6–133.2] in responders  contains supplementary material, which is available to authorized users. The two main subtypes of inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC), are characterized by episodes of inflammatory activity and remission. To explain the multifactorial and polygenic nature of IBD, a scenario of chronic and uncontrolled activation of the mucosal immune response in genetically susceptible individuals, exacerbated by environmental factors, has been proposed .Treatment of IBD seeks to induce and maintain remission, as well as reduce inflammation, promote mucosal healing and prevent complications , such thIL1RL1 expression is regulated by distal and proximal promoters that govern expression of the ST2 membrane-anchored receptor (ST2L) and a soluble isoform (sST2) generated by alternative splicing. sST2 is identical to the ST2L extracellular domain and is a decoy receptor for IL33 , 107.1 [54.2–161.3], 119.5 [57.7–144.5], 85.4 [63.0–96.7] and 74.7 [48.9–1076.0] μg/g feces, respectively ; at baseline and 6 months, levels correlated with Mayo endoscopic subscores  and total ST2 content in mucosa   Fig. . Patientp > 0.05)  in all patients   Fig. .Fig. 3Cop < 0.01, Fig. p < 0.05; Fig. In our cohort, six patients were rescued with biological therapy. Immediately after infliximab infusions, sST2 levels were markedly increased, but protein content during the induction period gradually declined between weeks 3 and 24 of monitoring, reaching levels similar to those in patients responding to treatment  and identifying serum levels of this biomarker in a follow-up every 4 months, a protocol in which FC was shown to predict disease recurrence . FinallyThe accuracy of sST2 in endoscopic detection of UC strongly suggests its usefulness in monitoring relapse and outcome, as well as in identifying patients likely to benefit from a particular treatment.


Protein degradation plays important roles in biological processes and is tightly regulated. Further, targeted proteolysis is an emerging research tool and therapeutic strategy. However, proteome-wide technologies to investigate the causes and consequences of protein degradation in biological systems are lacking. We developed “multiplexed proteome dynamics profiling” (mPDP), a mass-spectrometry-based approach combining dynamic-SILAC labeling with isobaric mass tagging for multiplexed analysis of protein degradation and synthesis. In three proof-of-concept studies, we uncover different responses induced by the bromodomain inhibitor JQ1 versus a JQ1 proteolysis targeting chimera; we elucidate distinct modes of action of estrogen receptor modulators; and we comprehensively classify HSP90 clients based on their requirement for HSP90 constitutively or during synthesis, demonstrating that constitutive HSP90 clients have lower thermal stability than non-clients, have higher affinity for the chaperone, vary between cell types, and change upon external stimuli. These findings highlight the potential of mPDP to identify dynamically controlled degradation mechanisms in cellular systems.  •Multiplexed proteome dynamics profiling, mPDP, measures changes in proteostasis•JQ1-PROTAC degrades a key mRNA export factor and blocks protein synthesis•Raloxifene induces TMEM97 degradation dysregulating cholesterol homeostasis•Characterization of proteins dependent on HSP90 constitutively or during synthesis Tracking both protein synthesis and degradation across thousands of proteins yields insights into functional regulation by protein degradation. Protein homeostasis integrates the balanced control of protein synthesis and degradation with the regulation of mRNA metabolism. External stimuli and disease can modulate any of these processes and thus alter the proteome. Similarly to protein synthesis, the regulation of protein degradation is complex involving hundreds of proteins such as E3 ligases, deubiquitinases, and proteases to orchestrate proteasomal, lysosomal, and other degradation pathways . AlthougSmall-molecule based strategies to target specific proteins for degradation have been developed recently  and offeIn recent years methodologies have become available to measure the activity of degradation mechanisms by analyzing protein turnover , synthesHere, we present multiplexed proteome dynamics profiling (mPDP). This quantitative liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based method combines dynamic SILAC labeling with isobaric mass tagging to enable the sensitive and comprehensive simultaneous analysis of changes in protein degradation and synthesis in a single mass spectrometric experiment of biological replicates for multiple treatment conditions. To demonstrate the broad applicability of mPDP for uncovering dynamic regulation of protein degradation mechanisms, we applied this strategy to investigate proteome effects by PROTAC-mediated degradation of the BET transcription factor family, ligand-induced degradation of the estrogen receptor, and inhibition of the chaperone heat shock protein 90 (HSP90).12C614N4 and lysine 12C614N2 and switched to the heavy SILAC medium containing stable isotope-enriched amino acids arginine 13C615N4 and lysine 13C615N2 followed by different compound treatments. A biological replicate experiment is performed in parallel with cells grown in SILAC heavy medium, where the same treatments are applied after medium exchange to SILAC light medium. After cell lysis, optional affinity enrichment, e.g., with kinobeads  family inhibitor JQ1 and a JQ1-VHL PROTAC. The BET proteins BRD2, 3, and 4 and BRDT regulate gene expression by recognizing acetylated histones in open chromatin conformations. Hence, BET inhibitors and degraders alter protein levels by gene repression and potentially by indirectly affecting degradation . We rati2, on a Q-Exactive+ mass spectrometer and using the SPS MS3 approach   FiguresA and S1Gapproach  on an Orcibility A–S2D; thAfter 6 hr JQ1-Az treatment, no significant effects were observed for mature proteins but substantial regulation was detected for nascent proteins, such as the N-acetylgalactosamine transferase B3GNT7, BRD2, HEXIM1, and POLR2A in agreement with recent reports . In contNascent protein levels observed after 6 hr treatment with the JQ1-VHL PROTAC largely recapitulated the response observed with JQ1-Az, with exception of the direct PROTAC targets and of a small cluster of proteins including the BCL2-like antiapoptotic protein MCL1. However, upon 24-hr JQ1-VHL PROTAC treatment, accumulation of mature proteins typically upregulated in mitosis, such as the AURKA interactors TPX2, INCENP, and kinesins, suggested cell-cycle arrest in the G2/M transition. Further, enhanced degradation of POLR2A was observed C and S2Ein situ hybridization (FISH) and confocal microscopy  protein SOAT1 as JQ1 off-targets. The JQ1-VHL PROTAC caused ablation of FYTTD1 and led to full arrest of protein synthesis. The results indicate that off-target activities substantially contribute to molecular and phenotypic responses observed upon JQ1 and JQ1-PROTAC treatment.Next, we investigated how the estrogen receptor 1 (ESR1) agonist estradiol and the selective estrogen receptor modulators (SERMs) raloxifene and GW7604 affect proteostasis in MCF-7 breast cancer cells A. EstradWe further found that levels of the nascent estrogen-responsive protein GREB1 increased following treatment with estradiol and decreased with SERMs C and S4CTaken together, our results demonstrate the ability of mPDP to detect modest changes in protein degradation and synthesis induced by different ligands of the estrogen receptor. We found that GREB1 is an HSP90 client which is regulated through degradation in response to ER modulation and identified off-target effects contributing to the pharmacological profile of raloxifene.Next, we applied mPDP to stratify HSP90-dependent proteins based on whether they require HSP90 constitutively or only during synthesis. Jurkat and MCF-7 cells were treated with 10 μM 17-AAG, and samples were collected at different time points and processed as above A. ProteiFor both cell lines, more than 1,000 proteins were downregulated in mature and nascent states across all time points, with 511 proteins being downregulated in both cell lines . HSP90-dIn summary, mPDP identified many known HSP90 clients with a higher overlap being observed for proteins identified as constitutively dependent on the interaction with this chaperone.Next, we mapped HSP90-dependent proteins to turnover data generated by dynamic SILAC in Jurkat cells and found that they have shorter half-lives than other proteins, with constitutively HSP90-dependent proteins having shorter half-lives than those that are synthesis-dependent . To probTreatment with 17-AAG also upregulated several nascent proteins, including the heat shock response proteins HSPA6 and HSPA1A A and 5B Next, we investigated proteome homeostasis and HSP90-dependent proteins in resting, primary T cells isolated from healthy donors. In dynamic SILAC proteomics experiments, protein turnover was much lower in T cells than in Jurkat cells A, and, dSince mPDP used for cell lines requires full incorporation of stable isotope-enriched amino acids and is not applicable to non-dividing primary T cells, we adapted the strategy and combined dynamic SILAC labeling with reductive dimethylation by stable isotope encoded formaldehyde  Figure B. T cellWe then investigated how the proteome dependency on HSP90 changed upon T cell activation. Proteomic analysis of stimulated cells showed strong upregulation of markers of T cell activation, such as IRF4 , which wNext, T cells were first exposed to anti-CD3 and anti-CD28 for induction of the canonical TCR pathway for 14 hr followed by 17-AAG treatment for 24 hr and then processed with the mPDP workflow for non-dividing cells. In the nascent pool, we identified 164 regulated proteins (142 down and 22 up), of which 20 were previously described as HSP90 interactors  Figure  and S4 ade novo after a cellular treatment is attributed to (post)transcriptional regulation.We have developed mPDP, a strategy that combines dynamic SILAC  with cheFirst, we applied mPDP to study proteome effects of the bromodomain and extra-terminal domain (BET) family inhibitor JQ1 and a JQ1-PROTAC since mPDP enables distinguishing PROTAC-induced degradation of the BET proteins from BET-dependent transcriptional regulation. BET inhibitors, such as JQ1 and I-BET151, and JQ1-derived targeted degraders have been used widely as selective probes to investigate the roles of BRD2,3,4 and BRDT in transcriptional regulation and in disease mechanisms . HoweverTPP revealed additional JQ1 off-targets including the sterol acylase SOAT1. Future studies using compounds with different off-target profiles will inform to what extent off-target inhibition of the atherogenic protein SOAT1  contribuIn a second study, we investigated the effects of selective estrogen receptor modulators on protein homeostasis in MCF-7 cells and identified the estrogen responsive protein GREB1 as a constitutive HSP90 client whose degradation is indirectly induced upon ESR1 inhibition. mPDP further demonstrated different effects of the SERMs raloxifene and GW7604 on ESR1 stability consistent with a previous report  and idenThird, we studied the HSP90-dependent proteome by mPDP. Previous studies investigating proteome-wide effects of HSP90 measured changes in protein abundances but did not discern between clients requiring permanent interaction with HSP90 and those only interacting with the chaperone during maturation . Our stu2+ influx resulting from TCR stimulation, might lead to the formation of CPPs which have been reported to contain HSP90 , a primary cellular calcium sensor, 17-AAG treatment induced stabilization indicating a direct role of HSP90 for its proteasomal degradation in agreement with previous observations in cell-free systems . We alsoin HSP90 . We therin HSP90  showed iin HSP90 .In conclusion, we have established mPDP as a powerful means to elucidate the mechanism of action of bioactive molecules, thus complementing current proteome-wide target identification strategies. mPDP identifies targets of protein degraders and, more generally, enables the discovery of regulatory degradation mechanisms in biological systems.marcus.x.bantscheff@gsk.com).Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Marcus Bantscheff .Jurkat cells  were cultured in RPMI-based SILAC-L and SILAC-H medium in parallel for 14 days, and incorporation of heavy isotopes was verified by mass spectrometry. For multiplexed proteome dynamics profiling, cells were re-suspended in opposite SILAC label medium, incubated in presence of 17-AAG (10 μM) or vehicle (DMSO) for the indicated time periods at 37°C, 5% COIn activation experiments, Jurkat cells were exposed to calyculin A  and sodium orthovanadate (30 μM) for 30 min and then harvested as described above.2. For harvesting, the cells were washed in PBS, pelleted, and snap-frozen in liquid N2.THP-1 cell cultures  were established in normal growth medium (RPMI1640 + 10% FBS) or RPMI-based SILAC-L and SILAC-H medium. Cells were seeded in opposite SILAC label medium or normal growth medium and incubated in presence of compounds  or vehicle (DMSO) for the indicated time points (6 or 24 h) at 37°C, 5% COThis cell line has been authenticated using the Promega GenePrint10 kit to generate a STR Profile for comparison to the expected profile reported by ATCC. The STR Profile generated matches exactly to the expected ATCC STR Profile.2 without impact on cell viability . In combination treatments, estradiol (0.1 μM) was added 15 min after selective estrogen receptor modulator (SERM) compound treatment  with a total incubation time of 24 h. For harvesting, the cells were scraped into cold PBS, washed, and snap-frozen in liquid N2.MCF-7 cells  were cultured in RPMI-based SILAC-L and SILAC-H medium supplemented with non-essential amino acids  and sodium pyruvate  but without phenol red in parallel for 14 days, and incorporation of heavy isotopes was verified by mass spectrometry. For proteomic analysis, near-confluent (∼80%) cells were pulse labeled with ‘opposite’ SILAC label medium for 30 mins until compounds  and 0.4 μM estradiol were added. Cells were incubated for the indicated time points  at 37°C, 5% COThis cell line has been authenticated using the Promega Cell ID System to generate STR Profiles for comparison to the expected profile reported by ATCC. The STR Profile generated matches exactly to the expected ATCC STR Profile.Ethics statement: The use of buffy coat-derived peripheral blood mononuclear cells (PBMC) was reviewed and approved by the Ethical Committee of the Landesärztekammer Baden-Württemberg, Germany (#2007-036-f), based on the principles in the Declaration of Helsinki. Venous blood was collected from consented healthy adult volunteers. Pseudonymized buffy coats were obtained from the German Red Cross, Mannheim, and gender/sex, age were not disclosed.2). For proteome dynamic analysis of T cell activation (7.5 × 106 cells/data point), cells were given into washed T-flasks pre-coated with anti-CD3 , supplemented with soluble anti-CD28 , and cultured for 14 h. Then, cells were pulse-labeled by transfer into SILAC heavy medium, re-stimulated with anti-CD3/anti-CD28, cultured for 1.5 h (lag time), and exposed to 17-AAG (10 μM) or vehicle (DMSO) for 24 h. Resting T cells were incubated similarly (for 24 h or 48 h) but without exposure to anti-CD3/anti-CD28, and the lag time between pulse labeling and 17-AAG/vehicle treatment was 4 h. For expression proteomic analysis of activated T cells (5 × 106 cells/data point), freshly purified cells were adapted to SILAC light medium for 4 h, then treated with 17-AAG or vehicle for 16 h and transferred into washed T-flasks pre-coated with anti-CD3 and supplemented with anti-CD28 as described above. Cells were further incubated for 7 h, washed, pelleted, and snap-frozen in liquid N2. The viability of resting and anti-CD3/anti-CD28-activated T cells treated with 17-AAG or vehicle was not significantly altered after up to 48 h of culture. The ATP levels of vehicle- anti-CD3/anti-CD28-treated cells were significantly increased compared to resting cells, indicating successful T cell activation (data not shown). Experiments were performed on two individual donors and no statistical assessment of the sample size was done.Primary human CD3+ T cells were isolated by negative selection (StemCell Technologies) from PBMCs derived from buffy coats and accustomed to SILAC light medium , both at 100 mg/L, instead of the “light” amino acids.Compounds JQ1, I-BET151, estradiol, 17-AAG, GW7604 and raloxifene were either available from the GSK compound collection or sourced commercially with purity of 98% or higher. Reagents and media were purchased from Sigma  unless otherwise noted. SILAC “light” (L) medium for Jurkat and primary human T cells was made of basal SILAC RPMI medium supplemented with 10% dialysed fetal bovine serum (FBS), glucose (2 g/L), L-glutamine (2 mM), phenol red  as well as L-Arginine and L-Lysine . The corresponding SILAC “heavy” (H) medium contained stable isotope-labeled 254). Proton (1H) and carbon (13C) NMR spectra were recorded on a Bruker Avance spectrometer (400 MHz) using the indicated deuterated solvent. Chemical shifts are given in parts per million (ppm) .All commercial solvents, reagents and building blocks are of reagent grade and were used as received without further purification unless otherwise specified. For separation of dichloromethane and aqueous layers during sample workup Radleys Phase Separation 6 mL Columns (RR99821) were used. Flash column chromatography was performed using a Biotage Isolera four purification system using Biotage flash silica cartridges. Preparative mass directed high performance liquid chromatography (preparative HPLC) was done on a XBridge BEH C18 OBD 5μm Prep Column (19 × 150 mm) at a flow rate of 30 mL/min, eluting with acetonitrile in water (0.2% formic acid as modifier). Purifications were conducted on a Waters autopurification system [detectors: 3100 Mass Detector and a 2996 Photodiode Array Detector]. Analytical UPLC (LCMS Method A) was done on a Waters Acquity BEH C18 1.7 μm Column (2.1 × 50 mm) at 40°C and a flow rate of 0.5 mL/min with a linear gradient over 4 min . The instrument used for analysis was a Waters Acquity system [detectors: Acquity SQD and Acquity PDA]. Analytical UPLC (LCMS Method B) was done on a Waters Acquity BEH C18 1.7 μm Column (2.1 × 50 mm) at 40°C and a flow rate of 1 mL/min with a linear gradient over 1.5 min . The instrument used for analysis was a Waters Acquity system . The purity of all final compounds was 95% or higher as determined by LCMS (UVS)-4-(4-Chlorophenyl)-2,3,9-trimethyl-6H-thienotriazolodiazepin-6-yl)acetic acid  and 2-(2-azidoethoxy)ethanamine  were dissolved in 2 mL DMF at room temperature. Then DIPEA  was added, followed by HATU . The reaction mixture was stirred for 16 hr and then diluted with ethyl acetate (25 mL). This was then washed with 25 mL each of water (twice) and Brine. The organic layer was dried over sodium sulfate, filtered and evaporated. Flash chromatography (cyclohexane to ethyl acetate to 20% methanol in ethyl acetate) gave JQ1-Az as an off-white solid . 1H NMR  δ 8.29 , 7.49 , 7.42 , 4.51 , 3.62 , 3.50 , 3.41 , 3.35–3.16 , 2.59 , 2.41 , 1.62 . 13C NMR  δ 169.69, 162.98, 155.09, 149.79, 136.76, 135.20, 132.26, 130.68, 130.13, 129.81, 129.55, 128.44, 69.00, 68.97, 53.82, 49.95, 38.53, 37.52, 14.03, 12.67, 11.28. LCMS Method A (Rt = 2.43 min) m/z 513 [M+1]+.2--1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride  and 2,5-dioxopyrrolidin-1-yl 3-(prop-2-yn-1-yloxy)propanoate  were dissolved in 1 mL DMF at room temperature. Then triethylamine  was added. The reaction mixture was stirred for 3 hr and then directly purified by mass directed preparative HPLC. This gave VHL-alkyne as a white solid . 1H NMR  δ 8.88 , 7.51–7.38 , 4.65 , 4.60–4.48 , 4.36 , 4.16 , 3.90 , 3.84–3.72 , 2.85 , 2.60 , 2.53–2.44 , 2.22 , 2.08 , 1.04 . 13C NMR  δ 174.46, 173.49, 172.20, 152.84, 149.05, 140.28, 133.35, 131.53, 130.38, 128.98, 80.42, 76.05, 71.09, 66.92, 60.82, 59.03, 58.91, 57.99, 43.71, 38.91, 37.12, 36.72, 27.02, 15.79. LCMS Method A (Rt = 1.84 min) m/z 541 [M+1]+.(2tert-butanol and then a premixed solution of tris(3-hydroxypropyltriazolylmethyl)amine  and copper (II) sulfate  was added. At last, a freshly prepared solution of sodium L-ascorbate  was added and the reaction mixture was stirred at room temperature overnight. The solution was diluted with water (5 mL) and extracted with 5 mL each of dichlormethane (twice) and ethyl acetate (twice). The organic layers were dried using a hydrophobic frit and sodium sulfate, combined and evaporated. Flash chromatography (0%–20% MeOH in dichloromethane) gave JQ1-VHL-PROTAC as a white solid . 1H NMR  δ 8.71 , 7.98 , 7.63–7.55 , 7.46–7.29 , 4.76 , 4.70–4.47 , 4.46–4.41 , 4.35 , 4.15 , 3.89–3.71 , 3.70–3.63 , 3.59 , 3.54–3.22 , 2.65 , 2.59–2.47 , 2.46–2.32 , 2.19 , 1.65 , 0.96 . LCMS Method A (Rt = 2.32 min) m/z 1053.5 [M+1]+.JQ1-Az  and VHL-alkyne  were mixed, dilute with 409 μL R)-1-(pyridin-2-yl)ethyl)-1H-imidazoquinolin-2(3H)-one  was dissolved in DMF (10 mL) and 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine  was added, then the mixture was stirred for 30 min before addition of 2-(2-bromoethyl)isoindoline-1,3-dione . The suspension was stirred for 6 hr and then poured onto a strong cation exchange cartridge . This was then washed with methanol (50 mL) and eluted with 2 M methanolic ammonia (50 mL). The eluant was evaporated. Flash chromatography (0%–10% 2 M methanolic ammonia in dichloromethane) gave 2--8-methoxy-2-oxo-1-((R)-1-(pyridin-2-yl)ethyl)-1H-imidazoquinolin-3(2H)-yl)ethyl)isoindoline-1,3-dione  as pale yellow gum. This was used in the next step without any further purification. LCMS Method B (Rt = 0.92 min) m/z 589.4 [M+1]+.7--8-methoxy-1-((R)-1-(pyridin-2-yl)ethyl)-1H-imidazoquinolin-3(2H)-yl)ethyl)isoindoline-1,3-dione  was dissolved in ethanol (10 mL) and hydrazine monohydrate  was added, then the mixture was heated at reflux under nitrogen for 2 hr. The mixture was cooled, diluted with water (20 mL) and evaporated to half volume. Then it was extracted with ethyl acetate (2 × 20 mL) and the combined organic layer was washed with brine, dried over sodium sulfate and evaporated. This gave 3-(2-aminoethyl)-7--8-methoxy-1-((R)-1-(pyridin-2-yl)ethyl)-1H-imidazoquinolin-2(3H)-one  as colorless glass. 1H NMR  δ 8.74 , 8.66 , 7.82 , 7.63 , 7.32–7.21 , 6.85 , 6.47 , 4.22 , 3.54 , 3.25 , 2.30 , 2.19–2.12 . LCMS Method B (Rt = 0.62 min) m/z 459.3 [M+1]+.2--8-methoxy-2-oxo-1- δ 8.82 , 8.62 , 7.85–7.77 , 7.57 , 7.37 , 6.78 , 6.43 , 4.31 , 3.82–3.63 , 3.57–3.47 , 3.46–3.42 , 3.19 , 2.30 , 2.28 , 2.15 , 2.09 . 13C NMR  δ 174.14, 168.06, 160.87, 160.59, 156.89, 155.97, 150.62, 141.35, 139.24, 133.29, 132.31, 129.79, 125.55, 124.29, 123.14, 122.76, 117.24, 113.63, 102.38, 70.78, 67.82, 56.57, 51.48, 42.65, 38.94, 37.58, 18.18, 11.45, 10.59. LCMS Method A (Rt = 4.78 min) m/z 600.4 [M+1]+.3-(2-aminoethyl)-7--8-methoxy-1-((R)-1-(pyridin-2-yl)ethyl)-1H-imidazoquinolin-2(3H)-one  and 2,5-dioxopyrrolidin-1-yl 3-(2-azidoethoxy)propanoate  were dissolved in DMF (1 mL) and then triethylamine  was added. The reaction mixture was stirred at room temperature for 3 hr. It was then directly purified by mass directed preparative HPLC. This gave IBET151-Az  as a white solid. tert-butanol (260 μL) and then a premixed solution of THPTA  and copper (II) sulfate  was added. Finally, a freshly prepared solution of sodium L-ascorbate  was added. The reaction mixture was stirred at room temperature for 3.5 hr and then it was directly purified by mass directed preparative HPLC. This gave IBET151-VHL-PROTAC formic acid salt  as a white solid. 1H NMR  δ 8.84 , 8.66 , 8.63 , 7.83 , 7.81 , 7.66 , 7.61 , 7.38–7.29 , 7.23 , 7.20–7.13 , 7.04 , 6.71 , 6.44–6.33 , 4.73–4.43 , 4.38 , 4.34–4.19 , 4.07 , 3.82–3.45 , 2.50–2.44 , 2.39 , 2.27 , 2.23–2.06 , 0.95 . 13C NMR  δ 171.87, 171.50, 171.47, 171.18, 166.60, 159.74, 155.74, 150.44, 149.52, 144.54, 138.42, 137.53, 131.01, 130.55, 129.55, 128.11, 124.37, 122.92, 121.57, 112.37, 99.79, 97.13, 70.19, 69.04, 66.90, 66.45, 64.84, 59.01, 57.72, 57.36, 55.89, 50.17, 43.26, 41.88, 38.48, 36.90, 36.72, 35.52, 26.56, 16.20, 11.85, 10.93. LCMS Method A (Rt = 1.83 min) m/z 570.9 [M+2]2+ .IBET151-Az  and VHL-alkyne  were mixed, diluted with in situ hybridization (FISH) was performed according to a published procedure . Additionally, staining with Hoechst 33258 (Sigma) and Wheat Germ Agglutinin (WGA) Alexa Fluor 647 Conjugate (Life Technologies) was performed to enable quantification of the data.THP-1 cells have been cultured as described above. Cells were treated with either vehicle, JQ1-Az (10 μM), JQ1-VHL PROTAC (1 μM), JQ1-VHL PROTAC (10 μM) or IBET151-PROTAC (10 μM) for 6 h and afterward RNA fluorescence rocedure  using a +RNA probe) was then measured at single cell level using the previously defined regions of nucleus and cytosol, and the ratio between both compartments was calculated. On average, 500-700 cells were quantified for each condition.Images were acquired with a Zeiss LSM 780 NLO microscope  with excitation at 633, 514 and 405 nm using a 63x oil objective and the auto-focus function. Images were then analyzed using ImageJ (1.45 s) and CellProfiler (2.1.1). In short, a Hoechst staining was used to identify the nucleus and a WGA staining to identify the cell border via the outer cell membrane. The cytosol area was then identified as a difference between cell border and nucleus. The mean fluorescence intensity of the Cy3 probe For the enrichment of GREB1 500 μL MCF-7 cell extracts corresponding to 1 mg protein were prepared by lysing the cells in 50 mM Tris–HCl, pH 7.5 (5% (w/v) glycerol, 1.5 mM MgCl2, 150 mM NaCl, 25 mM NaF, 1 mM sodium vanadate detergent (IGEPAL CA-630) at a concentration of0.8% (w/v), EDTA-free tablet, protease inhibitor cocktail (Roche Diagnostics) per 25 mL of lysis buffer) and incubated for 1 h at 4°C with 10 μM 17-AAG or vehicle (DMSO) followed by incubation with the according antibodies  for two additional hours. Antibodies were captured by incubation with Protein G beads (Sigma) for 1 h and bound proteins were eluted with 80 μL 2x SDS sample buffer and subjected to SDS gel electrophoresis. Samples were further processed for LC-MS/MS analysis.Jurkat cells treated with 17-AAG (10 μM) or vehicle for 6 h were lysed in SDS-based sample buffer and analyzed by SDS gel electrophoresis. Western blots were probed with antibodies against GLUL (BD Biosciences), HSP70 , or α-tubulin (Sigma), and the fluorescence signal of the IRDye secondary antibodies  was analyzed quantitatively (Odyssey software). Statistics were calculated using a one-sample t test.T) was normalized to RPL7, yielding ΔCT, and the data are displayed as 40-ΔCT.mRNA of Jurkat cells treated with 17-AAG (10 μM) or vehicle for 3 or 7 h was purified using the RNAquous extraction kit (Life Technologies), quantified using Qubit kit (Life Technologies), reverse transcribed , and analyzed by qPCR  using primers targeting GLUL  and RPL7 (VIC) and the TaqMan master mix (Thermo Fisher) in 96-well reaction plates (Invitrogen). The amplification of GLUL cycle time  in 20 mM HEPES (pH 7.0), 150 mM NaCl, 5% glycerol and 0.02% Tween20. DMSO (vehicle) or JQ1 (100 μM) was added. Heat treatment using a temperature range from 37°C to 70°C was done as described before  and aggrGel lanes were cut into three slices covering the entire separation range (∼2 cm) and subjected to in-gel tryptic digestion . PeptideTMT labeling was performed using the 10-plex TMT reagents, enabling relative quantification of 10 conditions in a single experiment . The lab2O and NaBH3CN while vehicle (1) and 17-AAG (3) treated samples subjected to heavy SILAC medium switch were labeled with 13CD2O and NaBH3CN to yield dimethylated N-termini and lysine residues containing (13CD2H)2 (1), (CH3)2 (2), (13CD2H)2 (3) respectively. Subsequently labeled peptide extracts from (1) was mixed with an aliquot of (2), and (3) was mixed with another aliquot of (2) and both samples were analyzed separately by LC-MS/MS. The analysis of sample (1-2) provided data for new synthesis and degradation of proteins under vehicle treatment, while the analysis of sample (2-3) represented 17-AAG treatment. Presence of vehicle controls (2) in both experiments enabled cross comparison of synthesis and degradation rate changes between vehicle and 17-AAG treatment conditions.Reductive dimethylation was perfomed as described in -1. Separation was done at 40 μL min-1 on a reversed-phase-column ]) with a gradient of 115 min length ranging from 97% buffer A (1.25% ammonia in water) to 60% B (1.25% ammonia in 70% acetonitrile in water). Fractions were collected for 120 s (9 fractions) or 60 s (25 fractions). After reaching the desired number of fractions (n) the next fraction (n+1) was pooled with the first fraction and so on until the gradient has finished.Lyophilized samples were re-suspended in 1.25% ammonia in water. The whole sample was injected onto a pre-colum ]) at a flow rate of 15 μL minSamples were dried in vacuo and resuspended in 0.05% trifluoroacetic acid in water. Half of the sample was injected into an Ultimate3000 nanoRLSC  coupled to a Q Exactive (Thermo Fisher Scientific). Peptides were separated on custom-made 50 cm × 100 μm (ID) reversed-phase columns (Reprosil) at 55°C. Gradient elution was performed from 2% acetonitrile to 40% acetonitrile in 0.1% formic acid and 3.5% DMSO over 2 hr. Samples were online injected into Q-Exactive mass spectrometers operating with a data-dependent top 10 method. MS spectra were acquired by using 70.000 resolution and an ion target of 3E6. Higher energy collisional dissociation (HCD) scans were performed with 35% NCE at 35.000 resolution (at m/z 200), and the ion target settings was set to 2E5 so as to avoid coalescence . The ins3 experiments were performed on an Orbitrap Fusion Lumos operated with fixed cycle time of 2 s. MS spectra were acquired at 60.000 resolution and an ion target of 4E5. Tandem mass spectra were acquired in the linear ion trap following HCD fragmentation performed with 5% stepped collision energy around 38% NCE and an ion target 1E4. MS3 were performed synchronously on the 5 most abundant MS2 signals in the range of 400-2000 m/z using HCD at 70% NCE in the Orbitrap at a resolution of 30.000 and an ion target of 1E5. The instrument was operated with Tune 2.1.1565.23 and Xcalibur 4.0.27.10.Synchronous precursor selection (SPS) MSMascot 2.4  was used for protein identification by using a 10 parts per million mass tolerance for peptide precursors and 20 mD (HCD) mass tolerance for fragment ions. The search database consisted of a customized version of the International Protein Index protein sequence database combined with a decoy version of this database created by using scripts supplied by MatrixScience.Carbamidomethylation of cysteine residues and TMT modification of lysine residues were set as fixed modifications. Methionine oxidation, and N-terminal acetylation of proteins and TMT modification of peptide N-termini were set as variable modifications.13C615N2, and arginine 13C615N4, and methionine oxidation and N-terminal acetylation of proteins were set as variable modifications.Carbamidomethylation was set as fixed modification whereas lysine 13C615N2 with TMT, and arginine 13C615N4 were set as fixed modifications in the heavy SILAC search. Methionine oxidation, N-terminal acetylation of proteins and TMT modification of peptide N-termini were set as variable modifications in both searches.Searches for light and heavy SILAC were perfomed independently. Carbamidomethylation of cysteine residues and TMT modification of lysine residues were set as fixed modifications for the SILAC light search. Carbamidomethylation, lysine 3)2, Lysine 13C615N2 with dimethyl (CH3)2, lysine 13C615N2 with dimethyl (13CHD2)2, arginine 13C615N4, methionine oxidation and N-terminal acetylation of proteins as variable modifications.Searches were perfomed with Carbamidomethylation as fixed modifications and Dimethyl  For single-spectrum to sequence assignments, we required this assignment to be the best match and a minimum Mascot score of 31 and a 10 × difference of this assignment over the next best assignment. Based on these criteria, the decoy search results indicated < 1% false discovery rate (FDR). (ii) For multiple spectrum to sequence assignments and using the same parameters, the decoy search results indicated < 0.1% FDR. Quantified proteins were required to contain at least 2 unique peptide matches. FDR for quantified proteins was < 0.1%.Reporter ion intensities were read from raw data and multiplied with ion accumulation times (the unit is milliseconds) so as to yield a measure proportional to the number of ions; this measure is referred to as ion area . Spectrahttps://github.com/protcode/isob/archive/1.1.0.zip: further details on the processing will be published separately. Unique quantified peptides associated with each protein group were selected and filtered according to the settings given in the isobarQuant configuration file (proteinquantfication.cfg): Mascot score > 15, peptide length ≥ 6, FDR at Mascot score < 1%, maximum least-squares fit of both peptides in light/heavy pair ≤ 0.1 and maximum prior ion ratio of both peptides in light/heavy pair ≤ 0.2. Protein fold changes were calculated using the median of all valid quantified peptide ratios (the fitted intensity of heavy SILAC to light SILAC) linked to the protein group. For protein groups where there was a minimum of one peptide with a positive ratio, any peptides with an undetermined ratio were excluded from the median calculation. In some cases, due to the slow protein turnover in T cells, proteins that were robustly quantified in the vehicle condition were not identified in the 17-AAG condition due to strong downregulation. In such cases we used half of the smallest detected relative protein abundance across the experiment as a proxy for the abundance of the non-identified proteins in order to be able to calculate the fold change and to capture that the protein was downregulated by 17-AAG. Proteins were filtered out in case the effect between the 2 donors in either vehicle control or 17-AAG differed by more than 8-fold or if the quantification was based on less than 3 quantified spectrum sequence matches. Changes in nascent and mature protein concentrations were calculated in the vehicle and 17-AAG experiments and compared to each other in order to determine 17-AAG induced synthesis and degradation changes. Experiments were repeated for 2 independent donors in order to rule out donor to donor variations.All acquired raw data were processed using a modified version of isobarQuant available from the Github code repository Experiments were analyzed as described in •2, which indicates how well the fold changes fit the melting curve.The coefficient of determination R•m) of the protein under the corresponding condition, which is given by the temperature at which the value of the melting curve is 0.5.The melting point .•The plateau of the fitted melting curve, which is given by its lower horizontal asymptote.Following LC-MS/MS analysis, protein fold changes were computed relative to the protein abundance at the lowest temperature. These fold changes represent the relative amount of nondenatured protein at the corresponding temperature. After a global normalization procedure, melting curves were fitted to the fold changes of each individual protein according to the formulaA change in the thermal stability of a protein is indicated by a compound-induced shift of its melting curve, which becomes apparent in a difference between the melting points derived under vehicle- and compound-treated conditions.2 of both the vehicle- and the compound-treated melting curves to be > 0.8 and the plateau of the vehicle curve to be < 0.3 in our analysis. The slope of the melting curve has a substantial impact on the reproducibility of the melting point difference. Thus, the slope has a central role in our data quality–dependent significance assessment. We divided the considered set of proteins into bins, starting with the shallowest slope, so that each bin contained at least 300 proteins. Then, we used z-tests to assess the statistical significance of melting point differences for the proteins one bin at a time, before performing the Benjamini-Hochberg correction for multiple testing on the full dataset. For a compound-induced change in a protein’s thermal stability to be regarded as significant, it was required to fulfill the following criteria:•One of the p values for the two replicate experiments is < 0.05 and the other one is < 0.1.•The melting point shifts in the two vehicles versus treatment experiments have the same direction •Both melting point differences in the two pairs of control versus treatment experiments are greater than the melting point difference between the two vehicle controls.•The minimum slope in each of the control versus treatment experiments is < −0.06.For a protein to be considered in the statistical evaluation, we required the RFor each of the experiments that underwent heat treatment at one of the 12 different temperatures (see above) we performed the following analysis: Following LC-MS/MS analysis, fold changes were computed to the protein abundance at the lowest compound concentration (vehicle). These fold changes represent the protein’s apparent stability at the corresponding compound concentration. If a protein’s fold change at the highest compound concentration was > 3/2, we regarded the protein to be potentially stabilized by the compound. Conversely, if a protein’s fold change at the highest compound concentration was < 2/3, we regarded the protein to be potentially destabilized by the compound. Otherwise, we did not consider the protein for further analysis. Subsequently, the fold changes were transformed such that with increasing compound concentration they range from 0 to 1 for stabilized proteins and from 1 to 0 for destabilized proteins. A sigmoidal curve according to the formula2 of the curve fit was > 0.8, we regarded the observed stabilization or destabilization to be a compound-induced effect as opposed to random fluctuations. In that case, we derived the pEC50 as the compound concentration at which the value of the fitted curve is 0.5. If the derived value was below the lowest (nonvehicle) compound concentration ), this was reported in the output and that pEC50 was not considered valid.If the Rkdp was computed according tom is the number of time points (ti) considered and rti was the fold change ratio (nascent proteins / mature proteins) of a specific protein at each time point. If required kdp was corrected for the cell cycle time tcc by extending the formula toT1/2) of a protein is then calculated bye(rti+1)] and the coefficient of determination (R2) for the linear regression was recorded. The QC value was set to ‘weak’ if it was possible to determine a fold change in at least three out of the four time points, ‘good’ if the protein fold changes at three out of the four time points were based on a minimum of three quantified peptides and ‘poor’ for the remainder.Protein half-lives were determined according to a modified version of the protein decay rate method described by All experiments have been performed in biological duplicates if not stated otherwise.2 transformation median normalization was applied to ensure that the data was centered around zero. For cases where a substantial number of the protein population was regulated, as was the case for the nascent proteins upon JQ1-VHL PROTAC treatment, normalization factors calculated for the mature proteins were applied to the nascent proteins. After the normalization proteins were divided into bins according to the number of quantified spectrum sequence matches. Each bin consisted of at least 300 proteins. This data quality–dependent binning strategy is analogous to the procedure described previously (2-transformed fold changes (FC) of the same proteins from two biological replicates divided by the square root of two:2 fold changes of proteins between two biological replicates the reproducibility between the replicates is taken into account in the statistical test. This enables to assess biologically relevant regulation upon perturbation without having to adhere to the assumption that the majority of measured proteins are not regulated. The basis for the p value calculations is the same as for the MaxQuant  in at least one time point. When a protein was significantly downregulated in its mature state at any time point it was classified as constitutively HSP90 dependent irrespective of the effect on nascent protein. Significant downregulation at only nascent state classified the protein as HSP90 dependent during synthesis.Fisher’s exact test was used to evaluate the significance of enrichment of annotated HSP90 interactors among proteins dependent on constitutively or during synthesis compared to all other identified proteins.PXD008637, Thermal proteome profiling of JQ1: PRIDE: PXD008638, 2 Dimensional Thermal proteome profiling of JQ1: PRIDE: PXD008626, Multiplexed proteome dynamics profiling of Estradiol and SERMs: PRIDE: PXD008636, Expression proteomics combination treatment of Estradiol and SERMs: PRIDE: PXD008628, Multiplexed proteome dynamics profiling of HSP90 inhibitor 17-AAG: PRIDE: PXD008633, Immunoaffinity enrichment of GREB1 to co-enrich HSP90: PRIDE: PXD008631, Thermal proteome profiling of Jurkat cells: PRIDE: PXD008639, Kinobeads affinity enrichment from lysates derived from stimulated cells: PRIDE: PXD008632, Jurkat protein half live: PRIDE: PXD008629, T cell protein half live: PRIDE: PXD008630, Multiplexed proteome dynamics profiling of HSP90 inhibitor 17-AAG in T cells: PRIDE: PXD008635, Multiplexed proteome dynamics profiling of HSP90 inhibitor 17-AAG in anti-CD3 and anti-CD28 stimulated T cells: PRIDE: PXD008634 and Expression profiling of HSP90 inhibitor 17-AAG in anti-CD3 and anti-CD28 stimulated T cells: PRIDE: PXD008627.Mass spectrometry raw data are available via PRIDE using the following accession numbers for the individual datasets Multiplexed proteome dynamics profiling of JQ1-PROTAC: PRIDE: https://doi.org/10.17632/8pzhg2tdyb.1.Full datasets for individual figures containing e.g., fold changes and p values were uploaded to Mendeley Data and can be retrieved using the following link


Hepatitis C virus (HCV) therapy should be considered without delay in all patients with significant (SIGFIB) or advanced liver fibrosis (ADVFIB). We aimed to investigate the rates of treatment initiation with interferon-free regimens within a screening program for SIGFIB/ADVFIB in human immunodeficiency virus/HCV coinfected patients (HIV/HCV).The FIB-4 was calculated in all HIV/HCV from 2014–2016. HIV/HCV were counselled by the HIV clinic and referred to the Division of Gastroenterology and Hepatology for transient elastography (TE) and evaluation for HCV therapy. Patients were stratified by FIB-4 of </≥1.45 (established cut-off for ruling out ADVFIB) and SIGFIB/ADVFIB were defined by liver stiffness >7.1 kPa/>9.5 kPa, respectively.Among 1348 HIV+ patients, 16% (210/1348) had detectable HCV-RNA. One hundred HIV/HCV had a FIB-4 ≥1.45. Among these, 57% (57/100) underwent TE. The majority of these patients had SIGFIB  or ADVFIB , however, interferon-free treatment was initiated in only 56% (24/43).In addition, fifty-two percent (57/110) of HIV/HCV with FIB-4 <1.45 underwent TE. Interestingly, 40% (23/57) and 18% (10/57) of these patients showed SIGFIB or even ADVFIB, respectively, and 78% (18/23) finally received interferon-free treatment. Overall, only 20% (42/210) of HIV/HCV received interferon-free treatment.FIB-4 was not useful for ruling out SIGFIB/ADVFIB in our cohort of HIV/HCV. Treatment was initiated only in a small proportion (20%) of HIV/HCV during the first 2 years of interferon-free treatment availability, although the observed proportion of patients with SIGFIB (assessed by TE) was considerably higher (58%). Thus, it requires the ongoing combined efforts of both HIV and HCV specialists to increase treatment uptake rates in this special population.The online version of this article (doi: 10.1007/s00508-017-1231-x) contains supplementary material, which is available to authorized users. Worldwide, approximately 80 million people suffer from chronic hepatitis C (CHC) . In low The FIB-4, an index based on simple laboratory parameters, e.g. age, aspartate aminotransferase (AST), platelet count (PLT), alanine aminotransferase (ALT) has been developed to rule out  or include  advanced fibrosis  in HIV/HCV coinfected patients  and has Within this systematic screening project, HIV/HCV coinfected patients were counselled by the HIV clinic and referred to the Division of Gastroenterology and Hepatology for TE and evaluation for HCV therapy.The study aimed to explore whether FIB-4 index is an accurate tool to identify patients who are at considerable risk for SIGFIB or ADVFIB, and thus, might have an urgent need for antiviral therapy. Moreover, the proportions of patients with DDIs between DAA-based regimens and antiretroviral therapy (ART) as well as the rates of treatment uptake in a cohort of HIV/HCV coinfected patients were assessed.Within this systematic screening project, HIV/HCV coinfected patients were counselled by the HIV clinic and referred to the Division of Gastroenterology and Hepatology for TE and evaluation for HCV therapy between 2014 and 2016. All HIV-positive patients with chronic hepatitis C were included in this retrospective analysis of the screening program.−1.Epidemiological characteristics were assessed from patient medical history. The HCV genotype was determined using the VERSANT® HCV Genotype 2.0 Assay Line Probe Assay  and HCV-RNA was assessed using the Abbott RealTime HCV assay  with a lower limit of quantification and detection of 12 IU ml9/l) × ALT (U/l)1/2)−1 [Measurement of liver stiffness was performed by TE , as previously described , 21. Thel)1/2)−1 :The SIGFIB and ADVFIB were defined by liver stiffness values >7.1 and >9.5 kPa, respectively . PatientStatistical analyses were performed using IBM SPSS Statistics 23 . Initially, normal distribution of continuous variables was tested by applying the Kolmogorov-Smirnov test to both the entire study population and to each individual subgroup. Continuous variables were reported as mean ± standard deviation or median (interquartile range), while categorical variables were reported as number of patients with/without (proportion of patients with) the certain characteristics.2-test and Fisher’s exact test. A P value of ≤0.05 was considered statistically significant.Student’s t‑test was used for group comparisons of continuous variables when applicable. Otherwise, the Mann-Whitney U-test was applied. Group comparisons of categorical variables were performed using the χ−1). The vast majority received at least one nucleoside reverse transcriptase inhibitor (N(t)RTI, 93%), followed by HIV protease inhibitor  and non-nucleoside reverse transcriptase inhibitor  treatment. Of the patients one quarter (25%) received either an integrase inhibitor or an entry inhibitor (II/EI) as part of their ART.Among the 1348 HIV-positive patients counselled by the HIV clinic, 33% (439/1348) were HCV-antibody positive and 16% (210/1348) of patients had detectable HCV-RNA. The majority of HIV/HCV coinfected patients were male (65%) and the median age was 37.9 (SD±16.61) years. The main route of transmission was IVDA (75%) followed by heterosexual intercourse (14%) and 5% of patients were MSM. More than half of the patients were coinfected with HCV genotype 1 (57%), while HCV genotypes 2, 3, and 4 were observed in 1, 10, and 32% of patients, respectively. Among HCV genotype 1 patients the subtype 1a (76%) was more common than subtype 1b (24%). The vast majority of the patients were on ART (91%), with 63% of HIV/HCV-coinfected patients having suppressed HIV-RNA (<50 copies mlP < 0.001) and patients who underwent TE showed a better immune status, i. e. a higher CD4+ T‑lymphocyte count (511 (414) vs. 352 (380) cells μl−1; P = 0.001), proportion of patients with HIV-RNA <50 copies ml−1 (74% (84/113) vs. 49% (46/94); P < 0.001) and HIV-RNA <400 copies ml−1 (89% (100/113) vs. 61% (57/94); P < 0.001). Moreover, TE patients were more likely to receive an II/EI (35% (38/108) vs. 11% (9/82); P < 0.001). The opposite applied for HIV PI (44% (48/108) vs. 73% (60/82); P < 0.001). The proportion of patients with FIB-4 index ≥1.45 was comparable between patients with (50% (57/114)) and without (45% (43/96)) information on TE (P = 0.452).Of the HIV/HCV coinfected patients 114 (54%) underwent TE, while 96 (46%) patients did not  among patients with a FIB-4 index <1.45. Moreover, bilirubin was lower among patients with a FIB-4 index <1.45 (0.43 (0.34) vs. 0.54 (0.46) mg dl−1; P < 0.001). The proportion of patients who underwent TE was comparable between patients with a FIB-4 index <1.45 (52% (57/110)) and ≥1.45 (57% (57/100); P = 0.452).Of the patients 110 had a FIB-4 index <1.45 and 100 patients presented with a FIB-4 index ≥1.45  and ADVFIB in 37% .A total of 57 patients 52%) with a FIB-4 index <1.45 as well as 57 patients (50%) with a FIB-4 index ≥1.45 were evaluated by TE (Table % with a P = 0.026).Among the patients who underwent TE, treatment was initiated in 37% 42/114) (Table 14 (TableP = 0.001) and ADVFIB (45% (19/42) vs. 17% (12/72); P = 0.001) were higher in patients in whom treatment was initiated.Unsurprisingly, the prevalence of SIGFIB (88% (37/42) vs. 40% (29/72); Sofosbuvir (SOF)/ribavirin (RBV) – 5% (2/42)SOF/daclatasvir (DCV) – 55% (23/42)SOF/ledipasvir (LDV) – 29% (12/42)Ombitasvir/paritaprevir/dasabuvir (3D) ± RBV – 12% (5/42)Overall, treatment was initiated in only 20% (42/210) of a total of 210 HIV-positive patients with CHC. The following interferon-free regimens were prescribed:The majority of HIV/HCV coinfected patients received ART (91%). According to current data on drug-drug interactions (DDI) between DAA and ART , the comSince HIV positive persons are at substantial risk of being coinfected with HCV due to the shared routes of transmission , the EurWe observed a significant difference in HIV infection parameters such as CD4+ T‑lymphocyte count and HIV-RNA between patients who underwent TE compared to the ones who did not. According to previous studies , 27, immSimple non-invasive indices have good diagnostic accuracy when combined with TE . The FIBA FIB-4 cut-off of 1.45 had a sensitivity of 68%  and negative predictive value (NPV) of 82% (95%CI: 70–90%) for ruling out ADVFIB, which is numerically lower than the sensitivity (70%) and NPV (90%) reported in a previous study by Sterling et al. . SimilarOur analysis revealed that treatment was initiated in only 20% of all HIV/HCV coinfected patients. Modern IFN-free regimens have shown excellent results in clinical trials with SVR rates exceeding 95% –14. AlthPatients coinfected with HIV/HCV are no longer considered as difficult to treat population ; howeverAlthough IFN-free regimens are now reimbursed for most HIV-positive patients with CHC regardless of the severity of liver fibrosis, considerable compliance issues remain. Thus, it is unclear whether extending the reimbursement of IFN-free treatment to patients without SIGFIB will lead to the anticipated increase in treatment uptake rates. Additional studies are needed to investigate the underlying factors hindering liver fibrosis assessment and treatment uptake in order to promote HCV elimination in this special population.In conclusion, FIB-4 was not useful for ruling out ADVFIB in our cohort of HIV/HCV. Treatment was initiated only in a small proportion of HIV/HCV during the first 2 years of IFN-free treatment availability, although the observed proportion (20%) of patients with SIGFIB (assessed by TE) was considerably higher (58%). Thus, it requires the ongoing combined efforts of both HIV and HCV specialists to increase treatment uptake rates in this special population.Supplementary Table 1 Comparison of all HIV/HCV coinfected patients with FIB-4 <1.45 and ≥1.45


Moderate exercise reduced tissue damages, improved functional scores, and restored mitochondrial respiration and calcium retention capacity in the ischemic limb . Exercise also enhanced the transcript levels of PGC1α, PGC1β, NRF1, as well as SOD1, SOD2, and catalase. Moderate exercise restores mitochondrial respiration and calcium retention capacity, and it has beneficial functional effects in chronic CLI, likely by stimulating reactive oxygen species-induced biogenesis and anti-oxidant defenses. These data support further development of exercise therapy even in advanced peripheral arterial disease.Whether and how moderate exercise might allow for accelerated limb recovery in chronic critical limb ischemia (CLI) remains to be determined. Chronic CLI was surgically induced in mice, and the effect of moderate exercise (training five times per week over a 3-week period) was investigated. Tissue damages and functional scores were assessed on the 4th, 6th, 10th, 20th, and 30th day after surgery. Mice were sacrificed 48 h after the last exercise session in order to assess muscle structure, mitochondrial respiration, calcium retention capacity, oxidative stress and transcript levels of genes encoding proteins controlling mitochondrial functions  and anti-oxidant defenses markers . CLI resulted in tissue damages and impaired functional scores. Mitochondrial respiration and calcium retention capacity were decreased in the ischemic limb of the non-exercised group (V Peripheral arterial disease (PAD) is linked to stenosis or occlusion of the arteries that are responsible for decreased perfusion of the lower limbs. PAD symptoms range from intermittent claudication  to critical limb ischemia . French laws for animal use and care were respected. Twenty Swiss mice  were handled. Surgery was performed under general anesthesia and post-operative analgesia was obtained using buprenorphine .General anesthesia was conducted in an airtight ventilated chamber  and air) and anesthesia maintenance was obtained by spontaneous ventilation through a mask .The surgery included two phases. First, the right femoral artery and its first three collateral vessels were ligated under microscope. Ligation of the femoral artery was standardized. It was located midway between the superficial epigastric artery and the bifurcation of the popliteal and saphenous arteries. Second, the right common iliac artery was ligated 0.5 cm distal to its origin, 4 days after the first phase, as described in our established CLI model ; and one group to be subjected to exercise and sacrificed 30 days after surgery .Animals were divided into two groups, one group to be sacrificed 30 days after surgery . Exercise was performed on a motorized treadmill . Each session was preceded by a warm-up phase of 2 min . During Week 1, animals run in the treadmill with a 10° inclination and a belt speed set at 25 cm/s. Length of exercise was 30 min for Day 1 and Day 2, 45 min for Day 3 and 60 min for Day 4 and Day 5. During Week 2, the inclination remains at 10° but the belt speed was set at 30 cm/s. Length of exercise was 60 min for Day 1 and Day 2, 75 min for Day 3 and Day 4 and 90 min for Day 5. During Week 3, animals run for 90 min with the treadmill set at 10° 30 cm/s for Days 1–5. Animals were sacrificed at Day 30 (48 h after the last exercise session).In vivo clinical follow-up was assessed using already-established clinical scores  were applied to skinned fibers of gastrocnemius muscle. The mitochondrial calcium uptake was thereafter assessed after the addition of a single calcium pulse (20 μM), by measuring the decrease of the extramitochondrial calcium concentration. The measure was monitored using a fluorescent probe . When mitochondrial calcium release started, that is when the calcium uptake showed an inflection point, it was considered that mPTP opening was observed. Mitochondrial calcium retention capacity, which is a reliable index of mPTP sensitivity, was calculated as the total amount of calcium taken up by mitochondria before calcium release.Gastrocnemius muscles (1 mm3 fragments) were incubated with a 200 μM CMH molecular probe , which is oxidized in the presence of unpaired electrons of ROS. The amount of oxidized CMH, and thus the related amount of free radicals produced, was measured by the intensity of the resonance signal .Tibialis muscles were immersed in liquid nitrogen and stored at −80°C. Muscles were embedded in paraffin. A cryostat microtome was then used in order to obtain 10 mm thick sections which were mounted onto glass slides. Hematoxylin and eosin stain was applied to the specimens which were examined under bright-field microscopy. Specimens were also incubated with 2.5 mM dihydroethidium (DHE), which produces red fluorescence when oxidized to ethidium bromide (mainly by superoxide anion). After staining, sections were examined under epifluorescence microscope (Nikon Eclipse E800) and emission signals recorded.Tibialis muscles were immersed in liquid nitrogen and stored at −80°C. Transcripts encoding antioxidative enzymes  and proteins involved in mitochondrial function  were analyzed. Two micrograms of RNA, isolated with TRIzol Reagent (Invitrogen), were converted to cDNA with SuperScript II reverse transcriptase  and hexamer primers according to the supplier's protocol. Quantitative RT-PCR was performed using the QuantiTectTM SYBR Green PCR kit (Roche) according to the supplier's protocol. Primers were for Sod1, 5′-CCAGTGCAGGACCTCATTTT-3′ (sense) and 5′-TTGTTTCTCATGGACCACCA-3′ (antisense); for Sod2, 5′-ACCCAAAGTCACGCTTGATAG-3′ (sense) and 5′-GGACAAACCTGAGCCCTAAG-3′ (antisense); for Catalase, 5′-CACTGACGAGATGGCACACT-3′ (sense) and 5′-TGTGGAGAATCGAACGGCAA-3′ (antisense); for Pgc-1α, 5′-AAGTGTGGAACTCTCTGGAACTG-3′ (sense) and 5′-GGGTTATCTTGGTTGGCTTTATG-3′ (antisense); for Pgc-1β, 5′-TGCGGAGACACAGATGAAGA-3′ (sense) and 5′-GGCTTGTATGGAGGTGTGGT-3′ (antisense); for Nrf-1, 5′-TGGAGTCCAAGATGCTAATG-3′ (sense) and 5′-AGAGCTCCATGCTACTGTTC-3′ (antisense).t-tests. Other parameters were compared using a one-way ANOVA design.Statistical analysis was performed with GraphPad Prism 5®  under the supervision of the Department of Statistics of our university. Clinical follow-up scores of groups were compared using p < 0.05 were considered as indicative of statistical significance.Results are expressed as means and standard deviations. At Day 30, among the 10 mice in the non-exercised group, ischemic limb showed auto-amputation of a toe in two mice, toe necrosis in seven mice and toe cyanosis in one mouse. Mean tissue damage score increased progressively from Day 4 to Day 30 with scores of 3.6, 3.8, 3.9, 4.0, and 4.1 at Days 4, 6, 10, 20, and 30, respectively. Concerning limb function, 8 mice dragged the ischemic limb and 2 could not achieve plantar flexion at Day 30. Mean functional damage score increased progressively from Day 4 to Day 30 with scores of 2.0, 2.6, 2.7, 2.8, and 2.8 at Days 4, 6, 10, 20, and 30, respectively . The same applied to Vmax ; Vamytal ; and Vtmpd  in control limb and 2.39 ± 0.19 mmol 02/min/g dw in ischemic limb  compared to control limbs , 0.32 ± 0.16 vs. 0.11 ± 0.07 for SOD2 (p < 0.05) and 0.38 ± 0.04 vs. 0.22 ± 0.11 for catalase (p < 0.05) in control and ischemic muscles respectively; Figure p < 0.05), 0.87 ± 0.41 vs. 0.68 ± 0.39 for PGC1β (p < 0.05) and 0.34 ± 0.14 vs. 0.24 ± 0.13 for NRF1 (p < 0.05) in control and ischemic muscles respectively; Figure Finally, ischemia decreased mRNA expression of antioxidant enzymes , 0.32 ± 0.16 vs. 2.90 ± 0.84 for SOD2 (p < 0.001) and 0.39 ± 0.16 vs. 1.61 ± 0.49 for catalase (p < 0.05) in non-exercised and exercised muscles respectively]. Exercise also increased significantly the transcripts of anti-oxidant enzymes in ischemic muscles (0.10 ± 0.06 vs. 1.73 ± 1.03 for SOD1 (p < 0.01), 0.11 ± 0.07 vs. 1.33 ± 1.01 for SOD2 (p < 0.01) and 0.22 ± 0.11 vs. 1.29 ± 1.18 for catalase (p < 0.05) in non-exercised and exercised muscles respectively; Figure Exercise increased significantly mRNA levels of anti-oxidant enzymes in control muscles , as well as in ischemic muscles [0.52 ± 0.39 vs. 0.93 ± 0.42 for PGC1α (p < 0.01), 0.68 ± 0.39 vs. 1.79 ± 0.83 for PGC1β (p < 0.01) and 0.24 ± 0.13 vs. 0.66 ± 0.34 for NRF1  ligation  responsible for clinical and functional symptoms lasting for more than 2 weeks (Norgren et al., Preclinical or clinical studies examining the effect of exercise in the setting of intermittent claudication are numerous (Gardner et al., Exercise therapy is proposed in first intention for patients presenting with infrainguinal lesions and intermittent claudication (Norgren et al., Our data demonstrate that moderate-intensity exercise training reduces significantly tissue damages. Indeed, confirming the functional improvement observed after exercise, HE staining highlights the fact that the CLI-related myopathic features disappear after training.Further analyzing skeletal muscle integrity, we also observed a normalization of the mitochondrial respiration; all complexes of the mitochondrial respiratory chain showing increased oxidative capacities. This is a key result since, besides reduced oxygen supply, mitochondrial dysfunction participates in CLI pathophysiology (Dopheide et al., In addition, we observed that moderate exercise restored the mitochondrial calcium retention capacity, delaying mPTP opening and therefore likely apoptosis. Indeed, the mPTP is a non-selective channel located through the inner mitochondrial membrane which is predominantly in a closed state during physiological conditions. A major consequence of mPTP opening is dissipation of the proton electrochemical gradient, leading to the inhibition of ATP production and finally, mitochondrial swelling, and rupture, initiating the apoptotic processes (Tran et al., Interestingly, moderate exercise delays mPTP opening, mitigating therefore CLI damages, and favoring increased functional abilities. This highlights the potential of exercise therapy in CLI, particularly knowing that improved exercise performance generally results in quality of life improvement and thus better prognosis.Exercise could be achieved despite flow limitation and mitochondrial function impairment. Indeed, one might suggest that strong flow limitation should be associated with “no” exercise capacity. In fact, although significant, mitochondrial impairment is relatively moderate and our model induces hypoperfusion rather than complete ischemia. Additionally, muscle susceptibility to ischemia reperfusion depends on their metabolic phenotype and thus some muscles—mainly oxidative—might be less impaired (Charles et al., We aimed to challenge the hypothesis that ROS signaling might participate in muscle protection through mitochondrial functions and anti-oxidative pathways enhancement. Although, ROS production is thought to be a key event during ischemia-reperfusion-induced deleterious effects, its kinetic is poorly studied and particularly, whether there is ROS production during ischemia remains a debated question (Paradis et al., It has been demonstrated that exercise induces ROS production in skeletal muscle (Sakellariou et al., Our results suggest that ROS signaling is, at least in part, responsible for mediating exercise-induced increases in mitohormesis within skeletal muscle, as well as antioxidant capacity (He et al., Accordingly, exercise increases the expression of PGC1-α and NRF1. PGC1-α is considered as one of the most important regulator of mitohormesis. Specifically in skeletal muscle, a large body of evidence suggests that PGC1-α regulates mitochondrial function, mitochondrial respiration and biogenesis. In particular, PGC1-α interacts with NRF-1 to activate genes involved in the respiratory chain and to stimulate mitochondrial biogenesis (Ljubicic et al., Similarly, PGC1-β was shown to promote the expression of genes involved in mitochondrial structure and function, but also to be significantly involved in anti-oxidant defense (Charles et al., This study presents several limitations. Clearly, moderate exercise promotes recovery of the ischemic limb in CLI, but experiments of contraction strength would have been useful to further assess muscle function. Moreover, our data do not allow differentiating between increased mitochondrial number and/or capacities. Finally, further studies at the protein levels should further reinforce the implication of mitochondria and ROS in the mechanisms involved.In conclusion, moderate exercise training in CLI provides beneficial effects allowing accelerated limb recovery. Mechanisms involved in this protective effect could be activation of antioxidant defenses and mitohormesis, supporting that moderate exercise should be taken into account as a potential therapeutic option in patients with CLI.AL: Study design, data collection, data analysis, writing. GL, SD: Data analysis. SP, AS, FS, EL, VW: Writing. AC: Data collection, data analysis. JZ: Study design, data collection. FT, FF: Study design. DM: Data analysis, writing. NC: Study design, writing. BG: Study design, data analysis, writing.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Xan was added to the HNE- or oligomeric Aβ42-treated SK-N-SH human neuroblastoma cells and then levels, oxidative modification and enzymatic activities of NEP protein were measured. Increased HNE levels on NEP proteins and reduced enzymatic activities of NEP were observed in the HNE- or oligomeric Aβ42-treated cells. Xan reduced HNE levels on NEP proteins and preserved enzymatic activities of NEP in HNE- or oligomeric Aβ42-treated cells. Xan reduced Aβ42 accumulation and protected neurones against oligomeric Aβ42-induced neurotoxicity through preservation of NEP activities. These findings indicate that Xan possesses therapeutic potential for the treatment of neurodegenerative diseases, including AD, and suggest a potential mechanism for the neuroprotective effects of antioxidants for the prevention of AD.Activity of neprilysin (NEP), the major protease which cleaves amyloid-β peptide (Aβ), is reportedly reduced in the brains of patients with Alzheimer’s disease (AD). Accumulation of Aβ generates reactive oxygen species (ROS) such as 4-hydroxynonenal (HNE), and then reduces activities of Aβ-degrading enzymes including NEP. Xanthorrhizol (Xan), a natural sesquiterpenoid, has been reported to possess antioxidant and anti-inflammatory properties. The present study examined the effects of Xan on HNE- or oligomeric Aβ Alzheimer’s disease (AD) is a progressive and irreversible neurodegenerative disorder characterized by cognitive decline ,2. AD isin vitro [Increasing evidence has shown that Aβ accumulation enhances its production and decreases its degradation through the actions of several Aβ-degrading enzymes, including neprilysin (NEP), insulin-degrading enzyme, and endothelin-converting enzyme . NEP, a in vitro . These rOxidative stress has long been recognized as an important factor in the early development of AD . Aβ induCurcuma xanthorrhiza RoxB, has been reported to possess antibacterial and anti-inflammatory activity [42-treated neuroblastoma cells, along with N-acetyl-l-cysteine (NAC) that has been reported to reduce Aβ42-mediated oxidative modification [Antioxidants have been reported as promising treatments for protecting neurones against oxidative stress ,32. Xantactivity . It is aactivity . Therefofication .2 at 37°C. Cells were subcultured twice per week and had undergone four to eight passages prior to the experiments.Human neuroblastoma SK-N-SH cells were obtained from the American Type Culture Collection  and maintained in essential medium supplemented with 1 µM non-essential amino acids, 100 UI/ml penicillin, 100 µg/ml streptomycin, and 10% (v/v) FBS  in a humidified atmosphere with 5% CO42 , or thiorphan , a specific NEP inhibitor, was added to the cultured cells according to experimental design. Xan and NAC were dissolved and diluted in Dulbecco’s PBS . HNE and TP were freshly prepared in DMSO and diluted in PBS prior to the experiment. To induce oxidative modification of NEP, cells were kept in 2% serum-reduced medium for 16 h, and HNE (10 µM) or oligomeric Aβ42 (1 µM) were then added to the cultured cells for 12 h. The same volume PBS was added to the cultures to serve as untreated controls.A combination of HNE , Xan , NAC , oligomeric Aβ42 were prepared as described previously [42. For oligomeric Aβ42, lyophilized Aβ42 aliquots (0.3 mg) were dissolved in 0.2 ml of 1,1,1,3,3,3-Hexafluoro-2-propanol  and then added to 0.7 ml H2O. Samples were loosely capped and stirred on a magnetic stirrer under a fume hood for 48 h, and used within 36 h. Monomeric Aβ42 was prepared immediately before use by rapidly evaporating the HFP via gentle bubbling of nitrogen gas into the solution. The quality of Aβ42 preparations was checked by immunoblot with anti-A-11  and anti-6E10  antibodies.Monomeric and oligomeric Aβeviously , from al2, 1 mM MgCl2, and 1× complete protease inhibitor cocktail ) for 1 h at 4°C. Total lysates (1 mg) were immunoprecipitated with an anti-NEP antibody  at 4°C overnight, and protein/antibody immunocomplexes were purified with protein A-magnetic beads and a magnetic separator . After washing, immunocomplexes were separated by SDS-PAGE using 10% gels (Invitrogen), transferred on to nitrocellulose membranes, and incubated with the primary rabbit polyclonal anti-HNE or rabbit polyclonal anti-NEP (both from Millipore) antibodies at room temperature overnight. After incubation with the secondary horseradish peroxidase (HRP)-conjugated goat anti-rabbit or mouse IgG  antibodies, the membranes were developed with ECL and exposed to X-ray film (Thermo Scientific). As a control, membranes were stripped and re-probed with the primary mouse anti-NEP  or mouse anti-β-actin  antibodies followed by the secondary HRP-conjugated goat anti-mouse IgG  antibody. Immunoreactivity was assessed by densitometric analysis of films using an HP Scanjet densitometer  and ImageJ image analysis software  as described previously [Cultured cells were lysed in cold lysis buffer  coated coverslips  were fixed with 4% paraformaldehyde at room temperature for 20 min and permeabilized with 0.2% Triton X-100 in 1× PBS (pH 7.4). After pre-blocking for 1 h at room temperature with 1% normal goat serum/1× PBS, cells were incubated overnight at 4°C in a humidified chamber with the primary mouse anti-NEP  or rabbit anti-HNE  antibodies. At the end of the incubation period, the cells were rinsed three times with 1× PBS containing 0.05% Tween-20 (PBS-T) and then incubated with the secondary Alexa 488-conjugated goat anti-mouse or Alexa 568-conjugated goat anti-rabbit IgG  for 60 min at room temperature. All primary and secondary antibodies were diluted in PBS-T with 2% normal goat serum. After rinsing with 1× PBS, the coverslips were mounted using ProLong Gold antifade reagent with DAPI (Invitrogen), and viewed and photographed on a Zeiss LSM 710 laser scanning confocal microscope . Immunofluorescence staining was repeated at least three times.42 (1 µM) without or with Xan  or NAC  for 12 h, collected, lysed in 1× PBS (pH 7.4) with 0.1% Triton X-100, and the lysates were placed on ice for 30 min. NEP activity in cell lysates was analyzed using a synthetic NEP fluorogenic peptide substrate  in the presence/absence of 500 nM TP. Samples were dissolved in 50 mM HEPES buffer (pH 7.5) and pre-incubated with TP or 1× PBS for 20 min prior to adding the NEP fluorogenic peptide substrate (dissolved in HEPES). Fluorescence was read at 320 nm excitation and 405 nm emission on a fluorescent microplate reader .To determine NEP activity in whole cell lysates, cells were incubated with HNE (10 µM) or oligomeric AβThe activity of membrane-bound NEP in intact cells was measured as previously described . BrieflyVmax and Km values) for NEP activity were determined by non-linear least squares fitting to the Michaelis–Menten equation using GraphPad Prism 6 software .FRET assay was performed for kinetic analyses of NEP activity. Cell lysates prepared by the method described above were incubated with increasing concentrations of NEP fluorogenic peptide substrate (0–20 µM) at room temperature. Fluorescence was measured over a 1-h period. NEP activity was determined as the difference in fluorescence in the presence/absence of 500 nM TP. Kinetic isotherms  (Invitrogen), blotted, and probed with anti-6E10 antibody  as described in ‘Immunoprecipitation and immunoblot analysis’ section. Cleavage of Aβ42 was discerned by the disappearance of protein bands corresponding to intact monomeric and oligomeric Aβ42. The densities of the remaining Aβ42 bands were quantitated using an HP Scanjet densitometer and ImageJ image analysis software, and plotted using GraphPad Prism 6 software.SK-N-SH cells were incubated with Xan  or NAC , in the presence/absence of 500 nM TP for 1 h. NEP proteins were isolated by immunoprecipitation from cells. For the 42 for 24 h. Another batch of cells were co-treated with Xan or NAC in the presence/absence of 500 nM TP. After 24 h, a cell viability assay was performed as described previously [°C in 5% CO2. The culture medium was collected and detected with a microplate reader at a wavelength of 450 nm. The difference in optical density (OD) relative to untreated controls was taken as a measure of cell viability, and the percentage of viable cells was calculated by comparing the OD at 450 nm for the Aβ42-treated and control wells.Cells were cultured in 48- or 96-well plates at a density of ~3000–5000 cells/well in complete growth medium for 24 h. Growth medium was replaced with fresh culture medium (~100–200 µl/well) containing 2.0% FBS and 1 µM oligomeric Aβeviously ,36 using42-treated cells were examined using a t test. One-way ANOVA was conducted to see the effects of Xan and NAC on conditions with these experimental treatments. Subsequent post hoc test (Tukey’s multiple comparison) was followed. P-values less than 0.05 were considered significant, unless specified otherwise.Data were expressed with a percentage of untreated controls. All data are presented as mean ± S.E.M. from three or more independent experiments, unless otherwise indicated. Differences between untreated cells and HNE- or oligomeric AβP<0.001; P<0.01); 10 and 100 µM NAC also reduced 36 and 49% HNE levels on NEP proteins, respectively, in the HNE-treated cells  =11.17, P<0.01; Effects of Xan and NAC on the HNE-treated SK-N-SH cells were examined by measurements of oxidative modification of NEP . Any treFurthermore, we observed that HNE-induced HNE increases on NEP proteins using double immunofluorescence staining. As shown in 42-treated SK-N-SH cells was examined by measurements of oxidative modification of NEP (42 treatments markedly increased HNE levels on NEP proteins (t(10) =9.04, P<0.001; 42-treated cells, respectively  =11.83, P<0.01); 10 and 100 µM NAC treatments also reduced 42 and 70% HNE levels on NEP proteins in the oligomeric Aβ42-treated cells, respectively  =8.57, P<0.01; Effects of Xan and NAC on the oligomeric Aβn of NEP . Any tren of NEP A. Howeve42-induced HNE increases on NEP proteins using double immunofluorescence staining. As shown in 42-treated cells. Both 10 µM Xan and 100 µM NAC treatments reduced the HNE-positive signals in the oligomeric Aβ42-treated cells.Furthermore, we observed oligomeric Aβ42-induced NEP modification would preserve NEP activity. The present experiment examined the changes in NEP activity in HNE- or oligomeric Aβ42-treated SK-N-SH lysates and intact cells, using a fluorometric peptide substrate. Exogenous treatment of 10 µM HNE into cells led to a significant loss of NEP activity, by 51% in whole cell lysates (t(20) =5.45, P<0.05; P<0.01; P<0.05). In addition, 1 and 10 µM Xan treatments increased 41 and 60% NEP activities in the HNE-treated intact cells, respectively  =16.26, P<0.01). Similar patterns were observed in the NAC experiments: 10 and 100 µM NAC treatments increased 37 and 52% NEP activities in the HNE-treated cell lysates, respectively,  =6.81, P<0.05; P<0.01; HNE treatment induces NEP modification, resulting in inactivation of NEP protein. Therefore, it is predicted that prevention of either HNE- or oligomeric Aβ42 increases endogenous generation of HNE and subsequently leads to HNE-induced modifications in NEP, we measured NEP activity in oligomeric Aβ42-treated cells (42 decreased 53% NEP activities in whole cell lysates (t(18) =5.86, P<0.05; P<0.01; 42-treated whole cell lysates  =11.74, P<0.05); 36 and 59% in the Aβ42-treated intact cells  =17.05, P<0.01); 10 and 100 µM NAC treatments also recovered 23 and 40% NEP activities in the Aβ42-treated whole cell lysates, respectively  =3.93, P<0.05); 15 and 57% in the Aβ42-intact cells, respectively  =9.18, P<0.01).Since oligomeric Aβed cells C,D. SimiVmax of NEP activity was decreased in the HNE- or oligomeric Aβ42-treated cells; 1 µM Xan pre-treatment cells slightly normalized the Vmax in the HNE- or oligomeric Aβ42-treated cells, but it was not statistically significant, compared with the HNE- or oligomeric Aβ42-treated cells; however, 10 µM Xan completely recovered the Vmax (Vmax of NEP activity (mK) for the enzyme in all samples. The NEP Km values were decreased by HNE or oligomeric Aβ42 treatment and recovered by Xan or NAC pre-treatment  in SK-N-SH cells and then measured NEP activity using a FRET assay. The measurement of NEP activity was saturable and followed Michaelis–Menten kinetics in all cell samples . The Vmathe Vmax A,B; 100 activity A,B. Linereatment C.42; 1 and 10 µM Xan treatments decreased 54 and 72% monomeric Aβ42 levels, respectively, compared with the untreated control  =69.57, P<0.01). Similarly, 10 and 100 µM NAC treatments decreased 52 and 71% monomeric Aβ42 levels, respectively, compared with the untreated control  =74.81, P<0.01); 500 nM TP, an NEP inhibitor, almost completely abolished the effect of Xan (t(10) =9.739, P<0.01) and NAC (t(10) =13.61, P<0.01) on Aβ42 degradation  =57.96, P<0.01). And 10 and 100 µM NAC reduced 37 and 56% oligomeric Aβ42 levels, respectively, compared with the untreated control  =47.18, P<0.01); 500 nM TP completely abolished the effect of Xan or NAC on oligomeric Aβ42 cleavage (The present experiment examined the effect of Xan though NEP action on the degradation of Aβradation A. Notablcleavage B.42-induced neurotoxicity via their NEP action. Oligomeric Aβ42-treated SK-N-SH cells were incubated with Xan or NAC in the presence/absence of TP. The viabilities of neuroblastoma cells were significantly decreased by the oligomeric Aβ42 treatment (t(14) =9.28, P<0.01). But Xan and NAC treatments increased the viabilities of the oligomeric Aβ42-treated cells in a dose-dependent manner; 1, 5, and 10 µM Xan increased 5, 30, and 40% viabilities, respectively  =11.94, P<0.01), and 10, 50, and 100 µM NAC increased 5, 16, and 36% viabilities  =7.73, P<0.01). NEP inhibition by TP almost completely abolished the protective effect of Xan (t(14) =4.63, P<0.01) or NAC (t(14) =3.41, P<0.01) on cell viability  on substrate concentration. HNE or oligomeric Aβ42 decreased Vmax and Km of NEP activity, whereas Xan or NAC restored Vmax and Km of NEP activity in cells with HNE or oligomeric Aβ42 treatments. These results suggest that a reduction in NEP activity by HNE could be reversed by antioxidants. Furthermore, the present experiment demonstrated that Xan or NAC treatments degraded both monomeric and toxic oligomeric Aβ42 and protect neuronal cells against oligomeric Aβ42-induced toxicity via enhancing NEP activity.Aβ peptide plays a pivotal role in the pathogenesis of AD ,12. Aβ- ed cells . Thus, red cells ,40. On ted cells ,22. In tNumerous studies have demonstrated that direct antioxidants, such as flavonoids, indirect antioxidants, such as NOS inhibitors, and metabolic antioxidants, such as NAC, can prevent neurodegeneration in AD ,42. The in vitro as well as in vivo in the brains of AD animal models with several pathological features of AD, with research to reveal its molecular and cellular mechanism.However, amyloid plaque composed of Aβ is one of two major pathological features in AD. Even though if it is limited to the amyloid cascade hypothesis, aggregation of Aβ evokes oxidative damage, inflammation, and neurotoxicity. Oxidative damage contributes to inflammation in AD and Aβ-induced neurotoxicity is exacerbated under inflammation dysregulation , 23. The


Chrozophora tinctoria and identified as amentoflavone (1), apigenin-7-O-β-d-glucopyranoside (2), apigenin-7-O-6′′-E-p-coumaroyl-β-d-glucopyranoside (3), acacetin-7-O-β-d-[α-l-rhamnosyl(1→6)]3′′-E-p-coumaroyl glucopyranoside (4), apigenin-7-O-(6′′-Z-p-coumaroyl)-β-d-glucopyranoside (5), and rutin (6). An extensive review of the literature as well as NMR and mass spectral techniques was employed in order to elucidate the compound structures. Proliferation was enhanced in MCF7, MG-63, and SAOS-2 cells after exposure to subcytotoxic levels of the tested flavonoids. Rutin was chosen for subsequent studies in SAOS-2 cells. Rutin was not found to cause any alteration in the index of proliferation of these cells, when examining the cell cycle distribution by DNA flowcytometric analysis. Rutin was, however, found to increase osteocyte and osteoblast-related gene expression and lower the expression of RUNX suppressor and osteoclast genes. When examining the influence of rutin on vitamin D levels and the activity of alkaline phosphatase enzyme, it was found to enhance both, while decreasing acid phosphatase which is a marker of osteoporosis. Thus, rutin enhances proliferation and ossification markers in bone cells.Osteoporosis is a chronic disease in which the skeleton loses a weighty proportion of its mineralized mass and mechanical pliability. Currently available antiosteoporotic agents suffer adverse effects that include elevated risk of thrombosis and cancer. Phytochemicals may constitute a safer and effective option. In the current work, six flavonoids were obtained from  Osteoporosis is a chronic disease in which the skeleton loses a weighty proportion of its mineralized mass and mechanical pliability . It occuFlavonoids, which are widely found in fruit and vegetables, are bioactive polyphenols with anti-inflammatory and antioxidant properties. Bone health has been associated with the intake of flavonoids. Intake of flavonoids increases bone mass density (BMD) in the neck and spine and decreases bone resorption in perimenopausal women . MoreoveChrozophora tinctoria (L.) A. Juss. has several other names including turnsole, Dyer's croton or giradol. It is an annual plant that belongs to the Euphorbiaceae family 3′′-E-p-coumaroyl glucopyranoside (4) [O-(6′′-Z-p-coumaroyl)-β-d-glucopyranoside (5) [There is an immense need for the development of novel drugs to treat osteoporosis which are devoid of potentially life-threatening side effects, namely, stroke and carcinogenesis , 26. We ne cells . Flavonone cells , 28–31. acacetin , 20, we acacetin . Our stuanalysis . Cochromside (2) , apigeniside (3) , acacetiside (4) , apigeniside (5) , and rutside (5) .A. meleguea seeds, where we noted an accelerated proliferation [μM concentrations. Exposing the cells to a higher concentration (100 μM) of amentoflavone for 72 h (1) lead to a 78.8% viability of the control untreated cells  to MG-63 cells for 72 h had no more than a 20% change in the viability of the cells. However, exposure to 1 μM of 1 and 5 reduced cell viability to 74.9% and 78.1% of the control cells, respectively. The addition of a greater concentration (10 μM) of all flavonoids under investigation induced 20–40% killing effect at 72 h of exposure. Further exposure to 100 μM of all compounds under investigation dropped cell viability of MG-63 cells to 40–70% of the control untreated cells .The cytotoxicity of these compounds was studied using the SRB viability assay in various estrogenic cell lines, namely, MCF-7, SAOS-2, and MG-63 cells. We have previously used these cell lines to study the effects of paradol on feration . In the ed cells . As in tectively . Interesed cells .μM) from all compounds under investigation on doubling times of MCF-7, SAOS-2, and MG-63 cells was tested. A concentration of 0.1 μM E2 served as a positive control. Compounds 1, 3, and 6 caused a significant lowering of the doubling times of MCF-7 cells from 16.6 ± 1.4 h to 9.3 ± 0.2 h, 8.6 ± 0.4 h, and 7.2 ± 0.5 h, respectively. The addition of E2 to MCF-7 cells resulted in a lowering of the doubling time to 8.5 ± 0.4 h (2 decreased the doubling time to 20.1 ± 1.2 h (2 (2 which might be attributed to the higher concentrations used (more than 10-fold). When comparing all tested flavonoids, rutin (6) showed promising proliferative properties with minimal expected mutagenic influence. This is in line with reports by Hyun et al. regarding its osteoclast activating properties [It has been previously reported that several flavonoids and related phytochemicals interact with estrogen receptors , 37. The ± 0.4 h . When st ± 1.2 h . In addi1.2 h . Treating SAOS-2 cells with E2 caused no significant changes of cells in G0/G1 or S-phases  might indicate uncontrolled cell proliferation. Interestingly, after SAOS-2 exposure to rutin, cells in supra-G2 phase were much lower than control cells , rutin and E2 caused an increase in expression of all the tested osteocyte activity gene markers by 1.9- to 2.9-folds and 2.7- to 3.5-folds, respectively , rutin downregulated the expression of HES1and TWIST1 mRNA to 0.7 ± 0.1- and 0.4 ± 0.07-folds of the control cells, respectively. On the other hand, E2 was found to suppress HES1, STAT1, TWIST1, and HAND2 gene expression to 0.4- to 0.6-folds of control (HES1and TWIST1 (RUNX suppressors) by rutin supports its osteogenic effects. All these data are in alignment with our previous publication on paradol [CALCR expression.Several studies have reported that targeting ossification is a more fruitful approach than targeting proliferation, when looking at developing new drug targets to treat osteoporosis. , 45. Theectively . The rolon of E2 . Comparein or E2 . These f control . Studies paradol . HoweverμM rutin for 48 h on the levels of four essential osteoporosis-related markers. The results were compared with treatment of the cells with 0.1 μM E2 as a positive control. Rutin produced a significant increase in the activity/concentrations of all ossification markers, ALP enzyme, OCN hormone, and active Vit-D3 concentration, by 7.1-, 3.4-, and 1.1-folds, respectively. Similarly, E2 increased the activity/concentrations of ALP enzyme, OCN hormone, and active Vit-D3 concentration, by 6.9-, 3.9-, and 1.2-folds, respectively (Figures 2-treated cells (C. tinctoria extract led to the isolation of various flavonoids showing antiosteoporosis influence. Rutin was especially promising as it showed ossification in bone cells as well as possessing punctuate proliferative activity with minimal influence to cell cycle distribution. In other words, rutin might be further studied as a potential antiosteoprotic agent with minimal expected mutagenic effects.In the present study, we determined the effects of treating SAOS-2 cells with 1  Figures . On the ed cells . The bio


Low 25-hydroxyvitamin D (25OHD) levels (< 75 nmol/l) are inversely associated with anemia prevalence. Since anemia and low 25OHD levels are common in patients with heart failure (HF), we aimed to investigate whether vitamin D supplementation can reduce anemia prevalence in advanced HF.3 daily or a matching placebo for 36 months. A total of 172 patients  were investigated in this pre-specified secondary data analysis. Hemoglobin (Hb) and other hematological parameters were measured at baseline and study termination. Assessment of between-group differences in anemia prevalence and Hb concentrations was performed at study termination, while adjusting for baseline differences.EVITA  is a randomized, placebo-controlled clinical trial in patients with initial 25OHD levels < 75 nmol/l. Participants received either 4000 IU vitamin DP = 0.19). At study termination, the proportion of patients with anemia in the vitamin D and placebo groups was 32.2% and 31.8%, respectively (P > 0.99). There was no between-group difference in change in the Hb concentrations . Results regarding anemia risk and Hb concentrations were similar in the subgroup of patients with chronic kidney disease . Moreover, results did not differ substantially when data analysis was restricted to patients with deficient baseline 25OHD levels.In the vitamin D and placebo group, baseline proportions of patients with anemia  were 17.2% and 10.6%, respectively .The study was registered at EudraCT (No. 2010–020793-42) and The online version of this article (doi:10.1186/s12937-017-0270-5) contains supplementary material, which is available to authorized users. The measuring range for 25OHD lies between 10 and 375 nmol/l. Values < 10 nmol/l were considered 9.9 nmol/l. The limit of 1,25(OH)2D quantitation is 12 pmol/L and we considered values below this limit as 11 pmol/L. C-reactive protein (CRP), brain natriuretic peptide (BNP), calcium, and creatinine values were analyzed by the Architect Autoanalyzer . Estimated glomerular filtration rate (eGFR) was calculated using the Modification of Diet in Renal Disease formula . Skewed variables were log and no pleasure to participate (n = 24). Baseline Hb levels were significantly higher in survivors than in nonsurvivors  but were similar in patients who dropped out and did not drop out . Baseline characteristics of the included study participants are shown in Table P = 0.19). Causes of anemia in the vitamin D and placebo group were iron deficiency in 0 and 1 patients, folate or B12 deficiency in 3 and 2 patients, CKD in 3 and 5 patients, inflammation in 0 and 0 patients, and unexplained in 3 and 7 patients, respectively. Of the 172 patients, 47.1% and 37.9% in the vitamin D group and placebo group, respectively, had vitamin D deficiency. In addition, 30.6% and 22.4% in the vitamin D group and 41.4% and 20.7% in the placebo group had insufficient and borderline 25OHD levels, respectively.The numbers of patients who died, dropped out or were lost to follow-up in the vitamin D and placebo groups were 39 and 36, 56 and 57, and 17 and 18, respectively. Main causes of drop out were poor health condition (P < 0.001). The corresponding values of circulating 1,25(OH)2D were + 17.1 pmol/L . Compared with placebo, vitamin D supplementation also resulted in significantly higher in-study plasma calcium levels. Prevalence of anemia increased by 15.0% in the vitamin D group (P = 0.003) and by 21.6% in the placebo group . Fig. Table  P = 0.00 and by 2n = 26; placebo group: n = 23) anemia prevalence at baseline was in the vitamin D and placebo groups 23.1% and 21.7%, respectively (P > 0.99). At study termination, anemia prevalence was 50.0% and 47.8%, respectively (P > 0.99). Moreover, vitamin D had no effect on mean change difference in Hb levels  stage 3 or 4  for 12 weeks [2D levels were presented in this study. 1,25(OH)2D has been shown to stimulate erythroid progenitor cell proliferation via increased erythropoietin sensitivity [2D levels > 70 pmol/L, the multivariable-adjusted odds ratio for anemia was 2.35 to 4.08 in the categories of circulating 1,25(OH)2D levels < 40 pmol/L [2D was on average + 17.1 pmol/L. The increment is in line with a recent meta-analysis reporting that vitamin D supplements increase circulating 1,25(OH)2D on average by + 18.8 pmol/L (95% CI: 9.2 to 28.4 pmol/L) [2D level of 81.6 pmol/L was already above the threshold for stimulating erythropoiesis. It is also noteworthy that anemia risk is associated with the severity of HF [2, where EPO production and circulating 1,25(OH)2D levels are reduced, vitamin D treatment had no effect on anemia prevalence and Hb levels. Although vitamin D treatment decreased MCHC values, median values remained in the reference range. We therefore conclude that this vitamin D-associated between-group difference is not of clinical relevance.Our data do not support results of a recent meta-analysis of observational studies . This me12 weeks . Unfortusitivity . Some ea0 pmol/L , 12. In  pmol/L) , but wasty of HF , 33, 34.2D, in addition to circulating 25OHD levels. One limitation is that only surrogate parameters were available on nutritional risk factors that may contribute to the development of anemia such as iron, folate or vitamin B12 deficiency, whereas data on ferritin concentrations and/or transferrin saturation for the assessment of iron deficiency were missing. Considering that iron therapy is increasingly used in HF patients [Our study has several strengths, and a number of limitations. Strengths are the design of an RCT, the study duration of 3 years, and the measurement of the active form of vitamin D, 1,25(OH)patients , we havepatients , a proteIn summary, the present study found no significant effects of a daily vitamin D supplementation with 4000 IU for 36 months on the prevalence of anemia and Hb values in patients with advanced HF. Our data challenge the clinical relevance of vitamin D supplementation to increase Hb levels.