In vitro, CID613034 inhibited mTORC2 kinase activity at submicromolar concentrations and in cellular assays specifically inhibited phosphorylation of mTORC2 substrates, including AKT (Ser-473), NDRG1 (Thr-346) and PKCα (Ser-657), while having no appreciable effects on the phosphorylation status of the mTORC1 substrate S6K (Thr-389) or mTORC1-dependent negative feedback loops. CID613034 demonstrated significant inhibitory effects on cell growth, motility and invasiveness in GBM cell lines and sensitivity correlated with relative Rictor or SIN1 expression. Structure-activity relationship analyses afforded an inhibitor, JR-AB2-011, with improved anti-GBM properties and blocked mTORC2 signaling and Rictor association with mTOR at lower effective concentrations. In GBM xenograft studies, JR-AB2-011 demonstrated significant anti-tumor properties. These data support mTORC2 as a viable therapeutic target in GBM and suggest that targeting protein-protein interactions critical for mTORC2 function is an effective strategy to achieve therapeutic responses.A small molecule which specifically blocks the interaction of Rictor and mTOR was identified utilizing a high-throughput yeast two-hybrid screen and evaluated as a potential inhibitor of mTORC2 activity in glioblastoma multiforme (GBM).  GBM are highly malignant and invasive tumors, properties which prevent total surgical resection, and render these neoplasms refractory to chemotherapeutic interventions . PrognosThe mechanistic target of rapamycin (mTOR) kinase is the major mediator of phosphatidylinositol 3-kinase (PI-3K) signaling and is an important target for molecular therapeutics in GBM , 5. mTOR473-AKT levels -2escribed . Cells wescribed . TUNEL sructions .All animal experiments were performed under an approved Institutional Animal Care and Use Committee protocol and conformed to the guidelines established by the Association for the Assessment and Accreditation of Laboratory Animal Care. Xenografts of LN229 cells were performed in female C.B.-17-scid (Taconic) mice as previously described .t test and ANOVA models using Systat 13 . P values of less then 0.05 were considered significant.Statistical analyses were performed with Student’s in vitro kinase activity. CID613034 also blocked the association of Rictor with mTOR in immunoprecipitates of mTOR from U87 cells treated with the inhibitor , no significant improvements in mTORC2 signaling blockade, as determined by the degree of p-S473-AKT inhibition relative to values obtained with the parent inhibitor, were observed. Additionally, no alterations in IC50 or apoptosis values were observed with these analogs suggesting that this region of the molecule was not critical for inhibition of mTORC2. Within the analogs containing modifications to the right-side of the molecule (R2), JR-AB2-005 displayed a significant increase in IC50; however p-S-473-AKT levels and the degree of apoptosis in cells following exposure were unaffected. The analog JR-AB2-011 however, markedly reduced mTORC2 signaling ; however none of these significantly improved mTORC2 signal blockade or anti-GBM properties as compared to the parent inhibitor. Moreover, we determined the in vitro cytotoxicities of analogs JR-AB2-011 and JR-AB2-030 relative to JR-AB2-000 in human neurons (not shown). JR-AB2-011 displayed the least toxicity to normal neurons with no significant cytotoxic effects for concentrations up to 10 mM and was chosen for further study.A series of thirty analogs were synthesized based on the structure of CID613034  to explore the SAR properties of the inhibitor. Modifications were made to all functional groups of the molecule as shown in see also  and IC50d was determined from steady-state binding associations and was calculated at 1 μM. Contrastingly, JR-AB2-000 was unable to bind mTOR or mSIN1, another regulatory subunit of mTORC2. We were unable to observe mTOR binding to immobilized Rictor, however in the reverse configuration immobilized mTOR bound Rictor with a calculated Kd of 257 nM  in comparison with the parent compound  , mice were randomized into treatment groups receiving vehicle, JR-AB2-011 (4 mg/kg/d) and JR-AB2-011 (20 mg/kg/d). As shown in in vitro with the parent compound  either directly involved in mTOR binding or at a site which allosterically prevents mTOR association. The improved ability of the SAR optimized analog JR-AB2-011 to block mTORC2 signaling may be due to the greater apparent affinity of JR-AB2-011 for Rictor. This is supported by SPR experiments in i for JR-AB2-011 was found to be 6–7 fold lower relative to JR-AB2-000 in competitive binding assays.There is increasing evidence to support dysregulation of mTORC2/AKT signaling in the pathogenesis and progression of several cancers , 36. ThuMammalian TORC2 is ~1.3 MDa and contains two copies of each of six subunits: mTOR, mLST8, Rictor, mSIN1, Deptor and PRR5 or its paralog PRR5L . Besides389-S6K, as a readout. Additionally, we noted that phospho-S312 and total IRS1 levels were unchanged suggesting that at least the S6K/IRS1/PI-3K feedback loop remained intact in the presence of CID613034 ; however none of these inhibitors are specific. All suffer from the potential limitation of blocking mTORC1 and inactivating negative feedback loops resulting in the activation of AKT, even in the context of mTORC2 inhibition . The inhID613034 . MAPK acID613034 .Our data demonstrated that an important determinant of sensitivity to CID613034 is relative Rictor or SIN1 expression. This is most likely a result of increased mTORC2 formation, as increased expression of these subunits would be expected to promote the nucleation of signaling competent mTORC2 kinases. Since our data demonstrated that these inhibitors bind Rictor, it is possible that post-translational modifications of Rictor, such as phosphorylation or acetylation may additionally be critical determinants of sensitivity for these compounds. Indeed, recent experiments show that glucose-dependent acetylation of Rictor promotes resistance to EGFR, PI-3K or AKT targeted therapies in GBM . It is cet al., engineered an allele of TOR2 which accepted an orthogonal kinase inhibitor that did not inhibit TOR1 [et al., characterized the downstream effectors of TORC2 by engineering TORC1 to be resistant to the ATP-competitive TOR inhibitor NVP-BHS345 and subsequent treatment with the compound specifically blocked TORC2 [Recent studies have described the development of strategies to specifically inhibit TORC2 acutely in yeast utilizing reverse chemical genetics . To invebit TOR1 . These sed TORC2 . Phosphoin vitro and in xenograft experiments. SPR and mTOR-bead pull-down experiments suggest that CID613034 and JR-AB2-011 specifically bind to Rictor and block association with mTOR. These results demonstrate that mTORC2 is a compelling target in GBM and support the use of these compounds in further dissection of mTORC2-mediated signaling processes.In summary, we have identified inhibitors of mTORC2 activity which target the regulatory subunit Rictor. We have demonstrated that these inhibitors have broad anti-GBM effects S1 Figpdr1and pdr3 loci via homologous recombination. Following transformation, the presence of the cassette was confirmed via amplification using a gene-specific primer and a primer within the kanMX ORF (primer arrows shown on schematic and arrow next to gel shows appropriate sized PCR products amplified from the indicated transformant). Subsequently, the Cre recombinase was expressed to remove the kanMX cassette. (B) Following knockout of the Pdr genes, HA-tagged versions of Hxt9p and Hxt11p under control of the Cup1 promoter were stably integrated and induced by the presence of copper in the media. (C) Drug sensitivities of the parental and modified drug sensitive yeast two-hybrid strain on solid (above) or liquid (below) media in the presence of the indicated inhibitor and copper to induce the Hxt transporters. Open circles denote values for the parental strain, while closed circles denote growth in the presence of the indicated inhibitor.(A) Schematic diagram of kanMX PCR disruption cassettes used to knockout the (TIFF)Click here for additional data file.S2 FigData shown are mean +S.D., n = 3.(TIF)Click here for additional data file.S3 Fig(A) LN229 cells were treated with CID613034 (1 μM) for the indicated time points and lysates immunoblotted for the indicated proteins. (B) LN229 cells were treated with CID613034 at the indicated concentrations for 2.5 h and protein lysates immunoblotted for the indicated proteins.(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 FigData are expressed as percent of control mice that received vehicle only and assigned 100%.(TIF)Click here for additional data file.S1 File(PDF)Click here for additional data file.


Hemodiafiltration with on-line endogenous reinfusion (HFR) is an extracorporeal dialytic method that combines diffusion, convection and adsorption. HFR-Supra (HFR-S) is a second-generation system with increased convective permeability and adsorption capability. Previous studies suggested that HFR reduces oxidative stress compared to standard haemodialysis. The principal aim of the present study was to compare antioxidant vitamins behavior and oxidative status of hemodialysis patients treated with HFR and HFR-S.The study was designed as a multicenter, randomized, crossover trial. Forty-one patients were recruited from 19 dialysis centers and after a 4-month washout stabilization period in on-line hemodiafiltration (ol-HDF), each patient was randomized to a sequence of treatments (HFR-S followed by HFR or viceversa) with each treatment applied over 6 months. Plasma levels of Advanced Oxidation Protein Products, Total Antioxidant Status, vitamins C, A and E and their ligands  were measured at baseline and at the end of each treatment period.Results show that the higher convective permeability of HFR-S with respect to HFR did not produce additional beneficial effects on the patients’ oxidative status, a slight decrease of both Vitamin A and Retinol Binding Protein being the only difference registered in the long-term. However, as compared to ol-HDF, both the re-infusive techniques allowed to reduce the intradialytic loss of Vitamin C and, in the long-term, improve the patients’ oxidative status and increase Retinol Binding Protein plasma values. No significant differences were found between the Vitamin C concentration of pre- and post cartridge UF neither in HFR-S nor in HFR showing that the sorbent resin does not adsorb Vitamin C.HFR-S and HFR are almost equivalent in term of impact on antioxidant vitamins and oxidative status of hemodialysis patients. Nonetheless, as compared to ol-HDF, both treatments produced a sensible sparing of Vitamin C and may represent a new approach for reducing oxidative stress and related complications in dialysis patients. Long-term effects of re-infusive treatments on patients’ cardiovascular morbidity and mortality need to be evaluated.NCT01492491, retrospectively registered in 10 December 2011.ClinicalTrials.gov Identifier  Oxidative stress and inflammation are common occurrences in end stage renal disease (ESRD) and are believed to play an important role in the excess cardiovascular morbidity and mortality of these patients . OxidatiThe enhanced oxidative capacity in uremia is due, at least in part, to systemic microinflammation and up-regulation of superoxide-producing enzymes . This chHemodiafiltration with endogenous reinfusion (HFR) is a kind of HDF that utilizes separated convection, diffusion and adsorption Fig.  17]. Th. Th17]. Finally, we evaluated the sieving coefficient (SC-like) of vit C through the convective membranes and its adsorption on the SDVB resin cartridges at both the start and the end of HFR-S and HFR single sessions.2 PES HF membrane, each patient was randomized to a sequence of treatments (HFR-S followed by HFR or viceversa) with each treatment applied over 6 months. The randomization procedure was stratified by Center. Twenty-nine patients completed the study: two patients withdrew during the run-in period, further seven and three patients during the first and second treatment period, respectively . Exclusion criteria were: residual diuresis > 300 mL/day; significant acute or chronic inflammatory comorbidities; non-renal related anemia; alcohol or drugs abuse; malignant neoplasms; hemoglobinopathy or mielopathy; pregnancy. The causes of ESRD were diabetes (21 %), glomerulonephritis (16 %), interstitial nephrits (9 %), angionephrosclerosis (30 %), polycystic kidney disease (5 %), undeterminated etiology nature (19 %). The majority of patients followed a drug regimen for the control of blood pressure, anemia and secondary hyperparathyroidism. All the subjects were warned to avoid antioxidant supplements thorough all the study.The extracorporeal dialysis treatment was performed three times per week. All 19 participating Centers made use of ultrapure, sterile and pyrogen-free dialysis solutions checked once monthly by cultural and endotoxins confirmation. Dialysate fluids were produced by a bi-osmosis and purified by daily nocturnal thermal disinfection of the piping (≥90 °C) –34.ex vivo degradation of Vit C, a reducing agent  was added in advance to the collection tubes. 10X DTE in physiological saline solution was prepared just before use, made sterile by filtration (0.22 μm pore size) and added to the tubes for blood and UF withdrawal . An insulin syringe was used to preserve the vacutainer void. The entire procedure was performed in a sterile environment and was highly reproducible (CV = 1.6 %). The tubes with the additive were stored at 4 °C until use (max 30 days). Preliminary experiments showed that the amount of DTE added did not affected blood cells integrity nor blood rheology up to at least 60 min from the collection, effectively preserving the Vit C content of either plasma or UF up to at least 90 days when specimens were stored at −25 °C [To prevent the t −25 °C .Blood and pre- and post-cartridge UF samples were collected at any time of the study concomitantly with routine blood tests, both at the beginning and at the end of a first weekly dialysis session. Blood was collected in heparin gel-containing tubes (for vitamins’ assays) and EDTA tubes  and centrifuged within 30 min from the collection. Gel-containing centrifuged tubes, separated EDTA plasma and UF samples were then immediately frozen (−20 °C). Within 10 days from the collection, the samples were transferred in dry ice to the laboratory where they were stored at −80 °C until analysis (30 days max).Plasma AOPP levels were measured by spectrophotometry on a microplate reader  calibrated with chloramine-T solutions  in the presence of potassium iodide . AbsorbaPlasma TAS levels were measured by a commercial colorimetric assay .s C, A and E was carried out by reverse-phase high performance liquid chromatography  with UV and FL detection, according to previously published methods with some modifications [s A and E assay, plasma aliquots were supplemented with the internal standard (6 μmol/L retinyl acetate) and extracted with an equal volume of a mixture of ethyl acetate-butanol (1:1). The separation of organic extracts was then performed on a fully end-capped C18  using 100 % methanol as mobile phase. Chromatographic runs were performed isocratically at a flow rate of 1.0 mL/min and the column temperature was kept constant at 28 °C.The determination of Vitications , 38. ForThe eluted compounds were detected spectrophotometrically at 245 nm (Vit C), 292 nm (Vit E) and 325 nm (retinyl acetate) and fluorometrically at 325 nm/475 nm excitation/emission wavelengths (Vit A and retinyl acetate). Chromatograms were analysed by the Galaxie Chromatography Workstation software . Sample concentrations were calculated from peak areas by a linear calibration model. The limits of detection, calculated as the analyte concentration corresponding to a signal-to-noise (S/N) ratio of 3, were 0.6 μmol/L, 0.1 μmol/L and 2 μmol/L for Vits C, A and E, respectively. A correction factor was applied to Vit C results to correct for dilution by DTE. The chemicals used were of the highest purity available (Sigma-Aldrich) and the water was ultrapure .Plasma RBP was determined by a commercial nephelometric assay . Cholesterol and triglycerides were determined by local clinical chemistry laboratories by means of commercial enzymatic methods. Total lipids were calculated as the sum of cholesterol and triglycerides.t-test  were used for comparison within arms and between arms, respectively. As for the acute effects, the percent reduction ratios  of the different treatments were compared by either ANOVA or ANCOVA, the latter being used in case of significant correlation between pre-dialysis levels and intra-dialytic losses. Only values ≥ LOD were entered into RR% calculation. Associations were assessed by Spearman rank correlation test. Extreme outliers (more than 3 inter-quartile ranges) were excluded from the statistical tests. P < 0.05 were considered significant and all tests were 2-tailed. Statistical analysis and data plotting were performed by using SigmaPlot ® 12.0 (Systat Software Inc.).The statistical power was verified by the Authors and Biostatistic Specialist of Ethic Committee. All blood values at the end of the dialysis session were corrected for hemoconcentration. Results are expressed as means and standard deviations - or medians and interquartile range depending on the distribution’s normality - for continuous data and as frequency for categorical data. The assumption of normality was checked by the Shapiro-Wilk test. Vitamin levels below the method LOD were assigned a value of ½ LOD. For all the variables but AOPP and TAS, the long-term effects of the treatments were compared by either CROS-analysis  , and, as expected, ii) Vit.s A and E levels were highly correlated to their respective plasma carriers, RBP and total lipids  Vit. C levels were negatively related to the patients’ age and positively related to the vit. A levels  and the lack of measurement of inflammation and uremic retention biomarkers. Further studies overcoming the above limitations will allow to strengthen and expand on the present results.Kidney failure is associated with oxidative stress. Hemodialysis may contribute to increase oxidative stress of ESRD patients due to the activation of inflammatory pathways and to the loss of small water-soluble antioxidants. Convective dialysis modalities can reduce the burden of oxidative stress of hemodialysis patients by allowing the removal of large pro-oxidant uremic solutes from plasma. Unlike ol-HDF, which is a mixed convective-diffusive method, HFR-S and HFR exploit in sequence convection, adsorption and diffusion. Thanks to the adsorption step, UF is purified by toxic molecules and it can be re-infused to the patient. Since other UF components such as albumin, amino acids and Vit C are not adsorbed, HFR architecture allows to increase convective permeability without the risk of losing useful substances.In the present study, we show that the higher convective permeability of HFR-S with respect to HFR did not produce additional beneficial effects on the oxidative status of ESRD patients, the only significant difference being a long-term decrease of Vit. A due to the more efficient removal of its plasma carrier, RBP. Further studies are needed to investigate whether the more efficient removal of large molecules by HFR-S produces additional beneficial effects over HFR in term of (micro)inflammation and uremic toxins retention.In comparison to ol-HDF, both the re-infusive techniques allow to significantly reduce the intradialytic loss of Vit. C and, in the long-term, improve the patients’ oxidative status and increase RBP plasma values, all effects which might have a potentially positive impact on patients’ cardiovascular morbidity and mortality.Nutrient supplementation is often used to provide malnourished haemodialysis patients with essential aminoacids, vitamins and minerals and/or to counteract the loss of useful substances upon high-convection dialysis techniques. HFR-S and HFR, by returning his own plasmatic water back to the patient, can be a way to restore a more physiological status without additional therapies. Moreover, both the financial and technical costs of nutrient supplementation make HFR definitely more advantageous over other convective techniques with similar depurative capacity. Finally, HFR architecture paves the way for potential, further increase of convective permeability, an improvement otherwise entailing severe loss of plasma proteins which must then be restored by additional infusional therapies.Ol-HDF has been reported to produce clinical and survival benefits with respect to HD. Nonetheless ESRD patients on convective dialysis still show high levels of inflammation, oxidative stress and high cardiovascular morbidity and mortality. HFR-S and HFR could represent a new biotechnological response potentially able to reduce side effects and complications. The long-term impact of these new techniques on patients’ survival and cardiovascular risk need to be evaluated.AOPP, advanced oxidation protein products; DTE, dithioerythritol; ESRD, end stage renal disease; HFR, hemodiafiltration with on-line endogenous reinfusion; HFR-S, HFR-Supra; ol-HDF, on-line hemodiafiltration; PES, polyethersulfone; RBP, retinol binding protein; RR, reduction ratio; SDVB, styrene divinylbenzene; TAS, total antioxidant status; UF, ultrafiltrate; Vit A, vitamin A; Vit C, vitamin C; Vit E, vitamin E


Reductions in Arctic sea ice may promote the negative phase of the North Atlantic Oscillation (NAO−). It has been argued that NAO-related variability can be used an as analogue to predict the effects of Arctic sea ice loss on mid-latitude weather. As NAO− events are associated with colder winters over Northern Europe, a negatively shifted NAO has been proposed as a dynamical pathway for Arctic sea ice loss to cause Northern European cooling. This study uses large-ensemble atmospheric simulations with prescribed ocean surface conditions to examine how seasonal-scale NAO− events are affected by Arctic sea ice loss. Despite an intensification of NAO− events, reflected by more prevalent easterly flow, sea ice loss does not lead to Northern European winter cooling and daily cold extremes actually decrease. The dynamical cooling from the changed NAO is ‘missing', because it is offset (or exceeded) by a thermodynamical effect owing to advection of warmer air masses. It is proposed that Arctic sea ice loss may be a cause of colder European winters, by promoting the negative phase of the North Atlantic Oscillation (NAO). Here, the author shows that despite an intensification of negative NAO events, sea ice loss does not lead to cooling, and cold extremes actually decrease. The Intergovernmental Panel on Climate Change Fifth Assessment report236781011121314The response of the large-scale Northern Hemisphere atmospheric circulation to Arctic sea ice loss has proven hard to elucidate, owing to its inherent nonlinearity151618192022272829303132333435192024383940414243444517212547The negative phase of the NAO is associated with cooler winter temperatures over Europe495051185354555657This study presents evidence from model simulations that strongly support the notion of a negative NAO response to Arctic sea ice loss. This atmospheric circulation change would be expected to lead to cooling over Europe, if the NAO is a good analogue for the expected temperature response to Arctic sea ice loss. However, such a cooling response is ‘missing' in these model simulations, because it is offset (or exceeded) by a thermodynamical effect owing to advection of warmer air masses.This study makes use of large-ensemble atmospheric model simulations with perturbed sea ice conditions to isolate the influence of Arctic sea ice loss on the negative phase of the NAO (NAO−). It focuses on the NAO− for two key reasons. First, the climatological winter mid-tropospheric circulation response to sea ice loss in these simulations projects onto the NAO− , prompti183336462 in early winter; −10.2 versus −5.2 million km2 in midwinter). However, in some regions, the differences in sea ice concentration are larger between LI and HI than between C21 and C20  may not simply follow changes in mean climate and society is arguably more sensitive to the former. This study focuses on seasonal-scale (January–February mean) NAO− events, which are associated with prolonged periods of anomalous weather and significant impacts on society. Simulated NAO− events  are characterized by raised mid-tropospheric (500 hPa) geopotential heights centred over Greenland and lowered heights over the North Atlantic (P<0.001) increase in the s.d. of the surface NAO index ) but no significant (P=0.96) change in its mean (0.02 hPa (−0.64 to 0.68); 19To estimate the influence of Arctic sea ice loss on NAO− events, the difference is taken between a composite mean of NAO− events in LI and that in HI. Mid-tropospheric height differences between NAO− events in LI and HI , shadingThe temporal evolution of polar cap (>65° N) height (PCH) is a commonly used metric to infer the evolution of the NAO (or AO) through time19201920As large differences in stratospheric PCH emerge in November preceding midwinter NAO− events , attenti596033371920−1 compared with an average of 1.98 m s−1 (Focus now shifts to the effects of NAO− events on near-surface (1.5 m) temperature. The NAO explains the largest percentage of midwinter temperature variance over a region covering 15° W–40° E 50–65° N , encompa98 m s−1 . Figure P=0.06) warming despite a highly significant (P<0.001) decrease in zonal wind .There is a strong linear relationship between Northern European daily zonal wind and temperature during NAO− midwinters, evident in both LI and HI . As ment−1. These are more frequent in LI relative to HI and are associated with cold temperature anomalies; hence, they act to lower the midwinter temperature in LI relative to HI. It is noteworthy that although days of zonal wind less than −4 m s−1 are also increased, these are few in number so contribute less to the midwinter mean change. There is a smaller dynamical cooling contribution from days of zonal wind in the range 4–8 m s−1, owing to fewer of these typically warmer days in LI compared with HI. The dynamical contribution is small for days of zonal wind in the range 0–3 m s−1. In contrast, the thermodynamical contribution (−1) and on days of strong (>5 m s−1) westerly flow, and warming  on days of moderate (−5 to 5 m s−1) easterly and westerly flow. Summed over all days in midwinter NAO− events , the cooling effect of intensified NAO− is missing, owing to a larger and opposite warming effect.The temperature difference between NAO− events in LI and HI (0.68 °C (−0.02 to 1.39)) can be partitioned into contributions coming from days of differing daily zonal wind strength ; −1 (−0.88to −0.27); 1516P=0.81) mean temperature response (0.10 °C (−0.66 to 0.87)). Despite an increase in frequency (as well as intensity) of NAO− midwinters in C21 relative to C20 (see Methods), there is no evidence of Northern European cooling in the climatological temperature response to sea ice loss  model HadGEM2-ES4445σ) of observed sea ice concentration and sea surface temperature (SST), 1979–2013, was calculated at each grid point from the UK Met Office Hadley Centre Ice and SST data set (http://www.metoffice.gov.uk/hadobs/hadisst). In the HI experiments, a sea ice concentration anomaly of +2σ was applied to the climatological mean and for the LI experiments an ice concentration anomaly of −2σ was applied to the climatological mean. At grid points where a sea ice anomaly was imposed, an SST anomaly was also imposed to account for SST changes linked to sea ice changes, adapting the approach of ref. σ was applied to the climatological mean and in LI an SST anomaly of +2σ was applied to the climatological mean. At grid points where sea ice is never present or always has the same concentration, the climatological sea ice concentration and SST was used. Specific ice-related anomalies are applied in each calendar month, but only in the northern hemisphere. Sea ice concentrations were restricted to being between 0 and 100%, to avoid unphysical values. Sea ice thickness was calculated empirically within the model code from the prescribed sea ice concentrations. Previous work has shown that the atmospheric response to sea ice loss can be sensitive to the background SST22http://www.esrl.noaa.gov/psd/data/climateindices/list/), 1948–2013, was regressed against detrended annual mean global SST, to yield an SST anomaly per 1σ change in the PDO index (β). For one background state, an SST anomaly of +2β was applied and the other an SST anomaly of −2β was applied. The SST anomalies were applied globally at all ice-free grid points, with the same SST anomalies applied in each calendar month. SSTs were restricted to no lower than −1.8 °C  to avoid unphysical values. An analogous procedure was applied for the AMO to yield two additional background states. In both LI and HI (to ensure no net difference in SST between these), the two PDO background states were each applied in 150 members and the two AMO background states were each applied in 101 members.Two ensemble experiments were performed with either positive or negative sea ice anomalies. Both experiments consist of 502 ensemble members, with each member being 1 year in duration and having the same surface boundary conditions, but starting from a different atmospheric initial condition. For sea ice boundary conditions, the monthly mean climatological mean and s.d. (N=4). For the C21 experiment, sea ice concentrations were taken from the CMIP5 RCP8.5 simulations of HadGEM2-ES, averaged for the period 2080–99 and across all available ensemble members (N=4). SSTs in C21 were the same as C20, except at grid boxes where sea ice was lost, where the climatological SST of the late twenty-first century was used. This procedure accounts for the local SST warming associated with reduced sea ice cover. The RCP8.5 simulations are forced by a continuous increase in greenhouse gas concentrations and are often viewed as a ‘business-as-usual' scenario, with limited mitigation strategies applied. This scenario was chosen to maximize the signal-to-noise ratio. Further details on these simulations can be found in ref. Two 260-member ensembles were performed with either sea ice conditions representative of the late twentieth century (C21) or those projected for the late twenty-first century (C20). For the C20 experiment, sea ice concentrations and SSTs were taken from the CMIP5 historical simulations of HadGEM2-ES, averaged for the period 1980–99 and across all available ensemble members  weighted area-average mean sea level pressure over the domain 0–80° W 30–50° N minus that over the domain 0–80° W 60–80° N. Sensitivity tests confirmed that the results were robust for alternative NAO definitions, for example, based upon the Principal Component time series of the leading Empirical Orthogonal Function of sea level pressure over the Atlantic sector . The NAO−1 with an interval of 1 m s−1. The dynamical and thermodynamical contributions were estimated from:The methodology to decompose the dynamical and thermodynamical contributions was adapted from ref. T is the total change in temperature between LI (C21) and HI (C20), Ti is the bin-averaged temperature in HI (C20), fi is the frequency of occurrence of bin i in HI (C20), Δfi is the change in frequency of occurrence for bin i between LI (C21) and HI (C20), ΔTi is the change in bin-averaged temperature between LI (C21) and HI (C20), and N is the total number of bins . The first term, TiΔfi, relates to changes in the frequency of occurrence of particular wind regimes and provides an estimate of the dynamical contribution. The second term, fiΔTi, relates to changes in temperature averaged over all days that belong in each bin, which provides an estimate of the thermodynamical contribution. The third term, ΔTiΔfi, represents the contribution from the interaction of both changing wind regime and bin-averaged temperature. It is included in the net contributions shown (for completeness), but is not presented as an individual term, as it was found to be small compared with the other two terms.where ΔAll graphics were produced using IDL version 8.2.2.All relevant data are available from the corresponding author on request.How to cite this article: Screen, J. A. The missing Northern European winter cooling response to Arctic sea ice loss. Nat. Commun.8, 14603 doi: 10.1038/ncomms14603 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures


Chronic hyperglycemia enhances the formation of advanced glycation endproducts (AGEs) and reactive oxygen species (ROS), contributing to diabetic complications. Thus, controlling blood glucose levels, inhibiting the formation of AGEs and reducing ROS are key therapeutic targets in early stage type 2 diabetes.p-nitrophenyl-α-D-glucopyranoside solution, respectively. Antiglycation activity was determined using the formation of fluorescent protein-bound AGEs. Total phenolic and flavonoid content and antioxidant properties  and ferric reducing antioxidant power (FRAP)) were determined for correlation among these components and inhibitory activities.The inhibitory effects of seven commercial liquid nutritional supplements against carbohydrate hydrolysing enzymes, α-amylase and α-glucosidase, was determined by dinitrosalicylic (DNS) reagent and 2 = 0.766; p < 0.01) and FRAP  whereas no correlation was observed for α-amylase inhibition. All supplements inhibited fluorescent protein-bound AGEs, with the greatest effect exerted by Olive Leaf Extract, Blood Sugar Support (IC50 = 0.5 mg/ml). The IC50 values negatively correlated with TPC  and DPPH scavenging activities .Samoan noni juice showed the greatest inhibitory effects against α-amylase, whereas chlorophyll extracts showed the greatest inhibitory effect against α-glucosidase. Inhibition of α-glucosidase correlated with TFC (rThe findings of this study highlight the potential of liquid nutritional supplements in managing and treating type 2 diabetes mellitus. The early stages of type 2 diabetes mellitus (T2DM) is characterised by postprandial hyperglycemia due to increased breakdown of starch by α-amylase, absorption of glucose by α-glycosidase, insulin resistance and defects with insulin secretion from beta cells of the pancreas , 2. If uImproving glycaemic control and preventing postprandial hyperglycemia is one of the main targets for the treatment and management of the early stage of T2DM. Salivary and pancreatic α-amylase and intestinal α-glucosidase are responsible for the digestion and absorption of glucose from the diet. By inhibiting either of these enzymes the amount of glucose absorbed into the blood following a meal can be reduced, thus preventing the postprandial surge in glucose and maintaining glucose homeostasis , 7. AcarLiquid nutritional supplements, a class of neutraceuticals including herbal extracts and plant-based extracts, claim to contain high levels of polyphenols and may be a suitable and cost-effective alternative to synthetic drugs. Very little scientific evidence exists concerning dietary supplements and their health benefits, and none to our knowledge on commercial liquid nutritional supplements on their potential role in the management of hyperglycemia and T2DM. Thus, this study was aimed to investigate the inhibitory effect of commonly consumed liquid nutritional supplements against α-amylase, α-glucosidase, and AGE formation using in vitro model. Total phenolic and flavonoid content, and antioxidant properties were evaluated for correlation among these components and inhibitory activities.Chemicals and solvents were purchased from Sigma-Aldrich  unless otherwise specified. All chemicals were either analytical or HPLC grade.Liquid nutritional supplements, purchased from local retailers and online included: Acai Power, Acai Juice Blend ; Chlorophyll, Detox Health Drink ; Olive Leaf Extract, Natural High Strength ; Olive Leaf Extract, High Strength, ; Olive Leaf Extract, Blood Sugar Support ; Noni Juice, Samoan ; Fijian Noni .control – Asample)/Acontrol) × 100.The α-amylase inhibitory action was determined as described previously with some modifications . Samplesp-nitrophenyl-α-D-glucopyranoside solution ), was added to the reaction mixture and incubated at 37 °C for 30 min. Sodium carbonate solution  was added and the reaction mixture was further incubated at 37 °C for 20 min. Absorbance was measured at 450 nm. Acarbose was used as a positive control. Inhibitory activity of α-glucosidase was expressed as the % inhibition = (Acontrol – Asample)/Acontrol) × 100.The α-glucosidase inhibitory action was determined as described previously with some modifications . Diluted50 values were calculated as % inhibition = ((Fnegative control – Fsample)/Fnegative control) × 100.Fluorescent protein-bound AGEs were measured according to a previous method with modification . BrieflyTotal phenolic content was determined using Folin–Ciocalteu assay and total flavonoid content was determined by aluminium chloride colorimetric technique as described previously .control – Asample)/Acontrol × 100).The radical scavenging activity was measured according to an assay described previously with modifications . BrieflyThe reducing power of the samples was determined as described previously . Ascorbi50 values were calculated by non-linear regression using Graphpad Prism Version 6.0 for Windows. One-way analysis of variance (ANOVA) was determined using Tukey’s or Dunnet’s multiple comparison tests and linear relationship was determined with Pearson’s correlation coefficient (r2). Difference were considered to be significant at p < 0.05.Results were expressed as the mean ± standard deviation (SD) or standard error of the mean (SEM) from three independent experiments. ICNatural products containing polyphenols and antioxidants may be an effective way of reducing oxidative stress and delaying the onset of diabetic complications. Dietary or nutritional supplements are commonly used for general wellness and prevention of disease. Nutritional supplements including herbal extracts and fruit juices may contain pharmacoactive components, and whilst manufacturers claim that these components provide health benefits, very little scientific evidence is available to-date to support these claims. In this study we aimed to investigate the antiglycation, α-amylase and α-glucosidase inhibitory potential of selected liquid nutritional supplements.p < 0.001) whereas APNG and CHC showed no difference when compared to acarbose. Olive leaf supplements  showed similar level of inhibition and were significantly higher (p < 0.01) than acarbose. Our study supports the findings of Komaki and co-workers [One of the primary targets for the management of early stage T2DM is to maintain steady blood glucose levels and prevent the postprandial surge of glucose, and an effective way of achieving this is by inhibiting α-amylase or α-glucosidase . In our -workers  who foun-workers .Fig. 1ThInhibition of α-glucosidase ranged from 10.33 ± 0.25 to 35.29 ± 0.93 %, with CHC showing the highest inhibition , inhibiting fluorescent AGE formation [OLE03 showed to be the most effective inhibitor with the lowest ICormation . The aut2 = 0.6329, p = 0.0021) but not with TFC. Interestingly, FRAP results showed a strong positive correlation with TPC  and TFC . These relationships show that the total phenolic content defines their antioxidant activities.TPC ranged from 0.48 ± 0.03 to 3.00 ± 0.36 mg GAE/ mL whereas TFC ranged from 0.06 ± 0.03 to 1.52 ± 0.97 mg QE/mL in liquid nutritional supplement  and FRAP   and herbal teas (r2 = 0.371) has been reported [50 values for fluorescent AGE inhibitions negatively correlated with TPC  and DPPH scavenging activity . Positive relationships were observed with wild berries, where % AGE inhibition correlated with TPC (r2 = 0.760) and DPPH (r2 = 0.452) [Various levels of correlation between TPC, TFC and antioxidant activities and enzyme inhibition are reported in the literature. For example, no linear relationship was identified between α-glucosidase inhibitory activities of native Australian herb fractions and their FRAP values . A moderreported . In the Commercial nutritional supplements analysed in this study exhibited different levels of inhibitory activity against α-amylase, α-glucosidase and protein-bound AGEs. While some supplements were more effective than others these inhibitory activities did not always correlate with phenolic content, flavonoid or antioxidant activities, suggesting that the inhibition may occur via different pathways. This study highlights promising potential of liquid nutritional supplements as a therapeutic approach to managing T2DM. As this study was the first to investigate antiglycation potential of liquid nutritional supplements, it provides opportunity to explore more neutraceutical supplements and their relevant metabolites in cells, animal or humans to determine the bioavailability and the mechanism of action.


The role of reactive oxygen species (ROS) in smooth muscle contraction is poorly understood. We hypothesised that G-protein coupled receptor (GPCR) activation and hypoxia induce Rho-kinase activity and contraction in rat intra-pulmonary artery (IPA) via stimulation of ROS production and subsequent Src-family kinase (SrcFK) activation.20 phosphorylation and contraction in IPA, and all these responses were inhibited by antioxidants . Contraction and SrcFK/MYPT-1/MLC20 phosphorylations were also inhibited by combined superoxide dismutase and catalase, or by the SrcFK antagonist PP2, while contraction and MYPT-1/MLC20 phosphorylations were inhibited by the Rho guanine nucleotide exchange factor (RhoGEF) inhibitor Y16. H2O2 and the superoxide-generating quinoledione LY83583 both induced c-Src oxidation, SrcFK auto-phosphorylation and contraction in IPA. LY83583 and H2O2-induced contractions were inhibited by PP2, while LY83583-induced contraction was also inhibited by antioxidants and Y16. SrcFK auto-phosphorylation and MYPT-1/MLC20 phosphorylation was also induced by hypoxia in IPA and this was blocked by mitochondrial inhibitors rotenone and myxothiazol. In live PASMC, sub-cellular translocation of RhoA and the RhoGEF ARHGEF1 was triggered by both U46619 and LY83583 and this translocation was blocked by antioxidants and PP2. RhoA translocation was also inhibited by an ARHGEF1 siRNA. U46619 enhanced ROS-dependent co-immunoprecipitation of ARHGEF1 with c-Src.The T-type prostanoid receptor agonist U46619 induced ROS production in pulmonary artery smooth muscle cells (PASMC). U46619 also induced c-Src cysteine oxidation, SrcFK auto-phosphorylation, MYPT-1 and MLCOur results demonstrate a link between GPCR-induced cytosolic ROS or hypoxia-induced mitochondrial ROS and SrcFK activity, Rho-kinase activity and contraction. ROS and SrcFK activate RhoA via ARHGEF1.  •GPCR-induced contraction of intra-pulmonary artery (IPA) is ROS-dependent.•Endogenous and exogenous ROS induce RhoA translocation and Rho-kinase activity in IPA.•Endogenous and exogenous ROS also activate Src-family kinases (SrcFK) in IPA.•SrcFK and ARHGEF1 are required for ROS-induced RhoA and Rho-kinase activity.•SrcFK may induce ROS-dependent activation of RhoA via ARHGEF1. Regardless of the initial stimulus, Rho-kinase activity usually requires prior activation of monomeric G-proteins of the Rho family (including RhoA), which in turn normally requires prior activation of guanine nucleotide exchange factors selective for the Rho family of G-proteins (RhoGEFs) Increased Rho-kinase activity contributes to acute hypoxic pulmonary vasoconstriction 22.13, 1 MgSO4, 4 KCL, 5.56 D-glucose, 0.434 NaH2PO4, 1.8 CaCl2, pH 7.4). Small intra-pulmonary arteries  were dissected free of surrounding parenchyma and either used for protein extraction and immunoblotting, mounted on a myograph for measurement of contractile force, or used for preparation of cultured pulmonary artery smooth muscle cells (PASMC). PASMC were dispersed by enzymatic digestion , grown to passage 3–4 in DMEM and serum-starved for 24 h prior to use. Each batch of cells was verified as smooth muscle by immunostaining for smooth muscle α-actin, calponin and desmin, as shown previously This study conforms with UK Home Office regulations and Directive 2010/63/EU of the European Parliament. Adult male Wistar rats were killed by lethal overdose of pentobarbital (~50 mg/kg i.p.). The lungs were excised and placed in cold physiological saline solution  at 37 °C. Vessels were stretched and pre-conditioned by stimulation with repeated exposures to 80 mmol/l K+ PSS , with resting tone being set at 1–3 mN, as previously described IPA rings were mounted on a Mulvany-Halpern wire myograph (DMT.dk) bathed in PSS and gassed with 95% air/5% CO2.32/95% air) or hypoxic conditions , IPA tissue samples were snap frozen in liquid nitrogen, homogenised and protein extracted for IP and/or SDS-PAGE and Western blot. For IP followed by WB, protein was extracted in TRIS-buffered saline with triton (1%), whereas for immediate WB, protein was extracted in TRIS-buffered saline with SDS (5%). IP was performed using the Pierce™ Crosslink Magnetic IP/Co-IP Kit (8805) according to the manufacturer instructions. Briefly, lysates were incubated with anti-c-Src antibody  and then covalently bound to protein A/G Magnetic Beads (Pierce) overnight at 4 °C. Following appropriate washing steps and elution, immuno-precipitates were re-suspended in TRIS-SDS sample buffer.Following isolation and treatment with U46619 under normoxic conditions  then probed with primary antibodies overnight at 4 °C (in 5% milk/TBS-T). Dilutions were optimised for each primary antibody, typically 1:1000, followed by horseradish peroxidase-conjugated secondary antibodies for 1hr at room temperature (1:3000 in 5% milk/TBS-T). Membranes were first probed with anti-phospho-antibodies, then stripped for 1hr (Pierce stripping buffer), re-blocked and re-probed with anti-total antibodies. Protein bands were visualised using either SuperSignal West Femto chemiluminescent substrate (Thermo Scientific) or ECL Prime  in a Biorad ChemiDoc XRS+ Gel Imaging System. Band intensity was expressed as a ratio of phospho/total for each protein band of interest, and for each treated sample these ratios were then expressed as a percentage of the control (untreated) samples run on the same gel. Each control value was taken as the average of 2–3 identically treated samples on each gel.2.4d]pyridazine-1,4--dione  2O2 production in IPA. Changes in luminescence induced by acute drug treatments were detected using a Promega GloMax Multi+ plate reader. Background measurements using ROS probe plus drug, but in the absence of cells/tissue, were subtracted from all equivalent test readings in the presence of cells/tissue. Each final value for each treatment for each batch of cells was the average of measurements from at least 8 identically treated wells in each plate.PASMC were seeded into 96-well plates, grown to 80–90% confluence and serum-starved for 24hrs. Cell were then incubated with the luminol-derived superoxide probe 8-amino-5-chloro-7-phenylpyridopyrimidine (PP2), the antioxidants ebselen and hydroxy-tempo (Tempol), the intracellular superoxide generator 6-anilo-5,8-quinoledione (LY83583) and the thromboxane A2 mimetic U46619 were all obtained from Merck Millipore. Superoxide dismutase (SOD), catalase, maleimide, polyethyleneglycol maleimide 5000, dithiothreitol, the nitric oxide synthase inhibitor L-nitro-arginine methyl-ester (L-NAME) and the inhibitor of RGS-domain containing RhoGEFs (Y16) were obtained from Sigma. Anti-Src, anti-phospho-Src family kinases (Tyr416), anti-MLC20, anti-phospho-MLC20 (Ser19), anti-MYPT1, anti-ARHGEF1, anti-phospho-tyrosine (PY100) and anti-GAPDH were all obtained from Cell Signalling (UK). Anti-phospho-MYPT1 (Thr850) was from Upstate (UK). Anti-mouse IgG and anti-rabbit IgG were from Sigma.The Src-family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo in PASMC, it does not influence MYPT-1 phosphorylation 2O2 contribute to this kinase activity and contraction in response to GPCR stimulation. One potential implication of this result is that endogenously (intracellular) generated H2O2 has different actions to that applied exogenously. This may be related to the normal compartmentalisation of cellular ROS generators, ROS effectors and cellular antioxidant systems being disrupted by the application of exogenous H2O2The fact that individually, Tempol, ebselen and PP2 all completely prevent U46619-induced phosphorylation responses suggests that ROS and SrcFK may be acting as part of the same signalling pathway, resulting in activation of Rho-kinase and MLCK. To test this assumption and to examine the relative contribution of superoxide and H2O2 and prevention of this intensification by Tempol, suggest that both exogenous and endogenous ROS are oxidising a fraction of cellular c-Src in IPA. Intensity of the main non-pegylated c-Src band was not significantly reduced by any of the treatments, suggesting that most of the cellular c-Src remains in the non-oxidised state. It may be that the relatively small fraction of total cellular c-Src that is being oxidised under these conditions fully accounts for the subsequently observed ROS-dependent downstream signalling events, since broadly similar order of magnitude increases in c-Src oxidation and SrcFK auto-phosphorylation were recorded in response to U46619 and LY83583. There is previous evidence in other tissues for activation of c-Src by direct cysteine oxidation, particularly Cys487 and Cys235, which when oxidised, trigger auto-phosphorylation Having established that U46619-induced SrcFK activity is ROS-dependent, we sought to determine whether this occurred via direct oxidation of c-Src. Our cysteine oxidation data suggest that at least a component of the ROS-dependent activation of c-Src occurs via this route. The appearance of additional c-Src bands following pegylation of oxidised cysteine residues, particularly one approximately 5 kDa larger than the main band, suggests oxidation of a single cysteine residue on c-Src 20 phosphorylation induced by hypoxia. The relative selectivity of rotenone and myxothiazol for mitochondrial over GPCR-induced ROS are confirmed by the lack of significant effect of these inhibitors on U46619-induced SrcFK/MYPT1/MLC20 phosphorylation. Together these results are consistent with the concept that SrcFK are activated by mitochondrial ROS and mediate the subsequent activation of Rho-kinase during HPV. Notably, as rotenone and myxothiazol block HPV SrcFK and Rho-kinase are also both activated by hypoxia in pulmonary artery and are major contributors to hypoxic pulmonary vasoconstriction (HPV), and as with G-protein coupled receptor signalling, we have shown that SrcFK is acting upstream of Rho-kinase in response to hypoxia 4.2or instead of interaction with Gα12/13Having established that SrcFK contribute to ROS-mediated Rho-kinase activation in response to U46619 stimulation and hypoxia, we attempted to further clarify the link between SrcFK and Rho-kinase by identifying molecular targets/binding partners for SrcFK. Rho-kinase requires prior activation of the monomeric G-protein RhoA 12/13We specifically chose to examine ARHGEF1 in more detail because it has previously been implicated in AngII-induced hypertension and shown to be tyrosine phosphorylated by Janus kinase-2 (JAK2) in mesenteric artery as an essential step in its activation by AngII 2αOur observation that co-immunoprecipitation of ARHGEF1 and c-Src in IPA is enhanced by U46619 suggests that a close association between the two proteins may occur. Furthermore, the co-immunoprecipitation was prevented by Tempol, suggesting that the association is ROS-dependent. This is also supported by the observation that LY83583 (this study) and PGF5Our study characterises a contractile signalling pathway in pulmonary artery that incorporates ROS, SrcFK, ARHGEF1, RhoA and Rho-kinase. As outlined in the graphical summary , we idenBritish Heart Foundation [grant number FS/12/43/29608 to G.K.] and Wellcome Trust [grant #087776 to J.P.W].This work was supported by None.


SIRT2 is potently and selectively inhibited by in situ-generated KPM-2 (36)-ADP ribose conjugate. +-dependent histone deacetylase family, has recently received increasing attention due to its potential involvement in neurodegenerative diseases and the progression of cancer. Potent and selective SIRT2 inhibitors thus represent desirable biological probes. Based on the X-ray crystal structure of SIRT2 in complex with a previously reported weak inhibitor (6), we identified in this study the potent mechanism-based inactivator KPM-2 (36), which is selective toward SIRT2. Compound 36 engages in a nucleophilic attack toward NAD+ at the active site of SIRT2, which affords a stable 36-ADP-ribose conjugate that simultaneously occupies the substrate-binding site, the “selectivity pocket” and the NAD+-binding site. Moreover, 36 exhibits antiproliferative activity in cancer cells and remarkable neurite outgrowth activity. This strategy for the selective inhibition of SIRT2 should allow further probing of the biology of SIRT2, and promote the development of new disease treatment strategies.Sirtuin 2 (SIRT2), a member of the NAD SIRT5 exhibits desuccinylase enzymatic activity.6 The catalytic region of sirtuins consists of a Rossmann-fold domain, a zinc-binding domain and the loops connecting them.7 The interface between the loops and the two domains form both NAD+ (subdivided by the pockets A–C) and Ac-Lys substrate-binding sites. This region is highly conserved in SIRT1–7 and mutations can drastically reduce the enzymatic activity. Even though the sirtuin subcellular localization is strongly connected to the cell type, stress conditions and interactions with other proteins, SIRT1, 6 and 7 are mainly localized in the nuclei, SIRT3–5 are mitochondrial sirtuins, and SIRT2 is classified as a cytoplasmic isoform.8 The ability of sirtuins to deacetylate histones, transcription factors and nuclear receptors reflects their involvement in many physiopathological processes. The activity of sirtuins has been related to metabolic disorders,9 cancer progression10 (in which they play a Janus-faced role) and neurodegenerative diseases.11Human sirtuins (SIRT1–7) are currently the only known class of histone deacetylases that require NADi.e., it can regulate genomic stability through DNA repair. In cancer cells, its overexpression leads to the negative regulation of p53, FOXO3a,14 HIF1-α15 and a blockage of Bax translocation to the mitochondria.16 Thus, SIRT1 induces cancer cell survival, accumulation of DNA mutations and drug resistance. While it is undisputed that the activity of SIRT1 is correlated to the growth of several kinds of cancer cells, the exact function of SIRT2 within cancer cells was the subject of strong controversy until recently. SIRT2 has been reported to act as a tumor suppressor and as an oncogene.17 SIRT2 levels are reduced in various types of cancer such as glioma,18 liver cancer,19 esophageal adenocarcinoma20 and breast cancer,21 while they are increased in neuroblastoma,21 acute myeloid leukemia22 and prostate cancer.23 However, recent reports strongly suggest that SIRT2 plays a key role in the invasion and metastasis formation of malignancy by increasing the cell motility of cancer cells.23 On the other hand, the function of SIRT2 in neurons remains the subject of controversy. While it has been reported that SIRT2 inhibition impairs neurogenesis and results in depression-like behaviors,24 a SIRT2 inhibitor has been reported to induce antidepressant-like action. Thus, potent and selective SIRT2 inhibitors are desirable as biological probes.Of the seven isoforms, SIRT1 is considered the genome “guardian angel” in normal cells, e.g. salermide (1),27 AC93253 (2),28 tenovin-6 (3), TM (4),31 SirReal2 (5), 3′-phenethyloxy-2-anilinobenzamide (6),34 AGK2 (7),35 AK-1 (8)36 and EX-527 (9).37 These compounds have shown antiproliferative activity when tested in cancer cells and some of these have shown beneficial effects in neurological disorder models, but in many cases, the lack of potency and isoform specificity suggests their effect is not solely related to SIRT1/2 inhibition. Therefore, the development of more potent and specific compounds is currently of high interest.So far, several SIRT1 and SIRT2 inhibitors have been developed , includiet al., who discovered that the novel drug-like small molecule SirReal2 (5) is able to induce a rearrangement of the SIRT2 active site by creating a binding pocket, called the “selectivity pocket”, which is responsible for isoform specificity, suggesting a potential common mode of action for selective SIRT2 inhibitors. In fact, the “selectivity pocket” is the pocket that accommodates the long chain fatty acyl groups.In the field of SIRT2 inhibitor design, a milestone on the elucidation of the mechanism behind the isotype selectivity of SIRT2 inhibitors has recently been reported by Rumpf 34 as selective SIRT2 inhibitors, in which a phenylethyl ether moiety acted as the optimal fragment to improve both potency and isoform selectivity. Herein, with the aim to elucidate the binding mode of this prototype and to further improve the inhibitory activity, we initially solved the single-crystal X-ray diffraction structure of SIRT2 in complex with 3′-phenethyloxy-2-anilinobenzamide (6),34 which revealed that 6 is located in the selectivity pocket. Then, inspired by this result, we applied structural modifications to the amide moiety in order to target the acetyl–lysine-substrate-binding site and NAD+-binding pockets, which finally led to the identification of KPM-2 (36) as a novel class of mechanism-based SIRT2 inactivators. Moreover, 36 shows highly potent and selective SIRT2 inhibition with antiproliferative activity in breast cancer cells and potent neurite outgrowth activity in neuro-2a (N2a) cells.Previously, we proposed a novel class of 2-anilinobenzamides6 , whereby the main divergences occur at the cofactor binding loop (Gly92-Leu112), which is not well defined, and at the downward-shifted connection loop at β8–α10  scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>O) and aromatic amine (–NH) moieties. This partially locked conformation renders the speculation feasible that there might be an additional structural requirement to achieve SIRT2 inhibition, as previously observed for SIRT1.41To gain insight into the binding mode of 2-anilinobenzamides, we determined the single-crystal X-ray diffraction structure of SIRT2 in complex with  Table S1. In genet β8–α10   and SIRT2/SirReal1/AcLys-OTC peptide  crystal structures demonstrate that inhibitors 6, SirReal1 and 5 occupy the same binding site . The screening of SIRT1–3 and 5 . A methylene bridge was used to link 6 with the acetyl derivative of UKU10363,42 which is a previously identified pan-SIRT1–3 inhibitor  by 26  to test whether replacement of the acyl group in 26 with a thioacyl group could lead to an improvement of the SIRT2 inhibitory activity by generating a novel trapped intermediate  with an IC50 of 55 nM, which is by a factor of ∼32 lower than that of 6, and by a factor of ∼11 lower than UKU10363. Notably, the SIRT2-inhibitory activity of 36 was much greater than representative previously reported SIRT2 inhibitors 3 and 5–9. The moderate SIRT1 and SIRT3 inhibition by 36 is not surprising as SIRT1 and SIRT3 are also known to be a demyristoylase and have a pocket similar to the SIRT2 selectivity pocket that can accommodate the long chain fatty acyl group.3Closer evaluation of the superimposed SIRT2 structures revealed that the amide group of ubstrate . This re1 and 42 . All com1 and 42  were scrUKU10363  and 5 we–3 and 5  revealedulations . The glyulations , locatedh Val233  and a sunhibitor . UKU1036M) by 26 , which w7 and 28 . As expe by ∼20% , while fver rate  and S3B†y pocket . At 1 μM41 and 42, thioamide analogues of 18 and 19, respectively. As shown in 41, no improvement was achieved as compared with 18. Furthermore, the elongation of aliphatic chain (19) resulted in a loss of potency. These results suggest that compounds 41 and 42 do not react with NAD+ because the short alkyl chain of 41 and 42 is too flexible to fix the thioamide group in proximity to the nicotinamide moiety of NAD+.In addition, we examined the SIRT-inhibitory activity of compounds 36-induced SIRT2 inhibition mechanism, a substrate competition analysis was carried out  mass spectrometric analysis was conducted, in order to determine whether the 36-ADP-ribose conjugate could be detected after simultaneous incubation of 36 with SIRT2 and NAD+ -NAD+, which is catalyzed by SIRT2. In their entirety, these results suggest that 36 is a substrate-competitive inhibitor. Moreover, they indicate that 36 specifically reacts in a time-dependent manner with NAD+ exclusively in the presence of SIRT2, which affords a Michaelis adduct intermediate, supporting the hypothesis that 36 is a SIRT2-mechanism-based inhibitor.Once proven that  Fig. S3C and 6C. 2 Fig. S7. In orde26 and 36 toward the reduction of MDA-MB-231 and MCF-7 cancer cell proliferation and the selective intracellular inhibition of SIRT2 was examined. As reference compounds, 5, 6 and EX-527 (9)37 were included in the assay as a potent SIRT2-selective inhibitor, a weak SIRT2-selective inhibitor and a potent SIRT1-selective inhibitor, respectively. After 72 h of incubation, GI50 values were determined. Neither the potent and selective SIRT1 inhibitor 9, nor the weak SIRT2-selective inhibitor 6 showed significant antiproliferative activity against MDA-MB-231 cells . 36, i.e., the most potent SIRT2 inhibitor, showed the highest activity (GI50 = 8.3 μM). We also examined the effect of 6, 9, 26 and 36 in MCF-7 cells , which is a SIRT1 substrate, in MDA-MB-231 cells. In their entirety, these results suggest that 36 selectively inhibits SIRT2 over SIRT1 in the cellular context.So far, valuable antiproliferative activity in cancer cells has been established for several SIRT1/2 inhibitors. However, on account of their questionable potency of action and selectivity profile, a possible off-target activity could not be ruled out. In this study, the ability of the highly potent and selective SIRT2 inactivators s Fig. S8. The antblotting . Treatme the function of the catalytic activity of SIRT2 in neurogenesis is still unclear.24 Therefore, we investigated the effect of the SIRT2-selective inhibitor 36 on neurite outgrowth. Mouse neuroblastoma neuro-2a (N2a) cells, which have previously been validated for the study of neurite morphology in neuronal differentiation, were used in this study. Treatment of N2a cells with 36 (0.5 and 1 μM) for 72 h induced neurite outgrowth (36vs. vehicle; 23.09 ± 3.21% and 25.73 ± 1.3% vs. 14.22 ± 2.9%; 36 0.5 μM vs. vehicle, p < 0.05, 36 1 μM vs. vehicle, p < 0.01; Although it has been suggested that SIRT2 inhibition by small molecules might be beneficial in the context of neurological disorders,utgrowth  and sign6 on SIRT2 was elucidated using X-ray crystallography. On the basis of these results, a new molecular design was developed around the scaffold of this compound. Herein, we demonstrated that targeting of both the SIRT2 selectivity pocket and the substrate-binding site with 26 represents an efficient strategy to achieve potent and selective inhibition. Furthermore, the molecular conjugation of two distinct sirtuin inhibitors, UKU10363 and 6, through a simple methylene bridge led to the new potent SIRT2 inhibitor 36. Kinetic and mass spectroscopic analyses supported the hypothesis that 36 is a mechanism-based inhibitor that affords in situ occupation of the substrate-binding site, the selectivity pocket, and the NAD+-binding site. Thus, 36 should be a valuable tool for the detailed investigation of the effect of SIRT2 inhibition in the cellular context. In MDA-MB-231 and MCF-7 breast cancer cell cultures, 36 showed antiproliferative activity with selective acetylation the SIRT2 substrate α-tubulin. Moreover, 36 showed neurite outgrowth activity in N2a cells, suggesting the possibility of SIRT2-selective inhibitors as therapeutic agents for neurological disorders.Since 2012, 2-anilinobenzamides have been considered as one of the most promising classes of drug-like SIRT2 inhibitors. In this study, the binding mode of +-binding site provides a new approach to the design of mechanism-based selective sirtuin inhibitors, which could be potentially extended to explore and probe the limits of the SIRT1–7 active sites.Our strategy to simultaneously target the substrate-binding site, the selectivity pocket and the NADThe authors declare no competing financial interest.


Oral cancers are easily accessible compared to many other cancers. Nevertheless, oral cancer is often diagnosed late, resulting in a poor prognosis. Most oral cancers are squamous cell carcinomas that predominantly develop from cell hyperplasias and dysplasias. DNA damage is induced in these tissues directly or indirectly in response to oncogene-induced deregulation of cellular proliferation. Consequently, a DNA Damage response (DDR) and a cell cycle checkpoint is activated. As dysplasia transitions to cancer, proteins involved in DNA damage and checkpoint signaling are mutated or silenced decreasing cell death while increasing genomic instability and allowing continued tumor progression. Hyperphosphorylation of Replication Protein A (RPA), including phosphorylation of Ser4 and Ser8 of RPA2, is a well-known indicator of DNA damage and checkpoint activation. In this study, we utilize S4S8-RPA phosphorylation as a marker for cancer development and progression in oral squamous cell carcinomas (OSCC). S4S8-RPA phosphorylation was observed to be low in normal cells, high in dysplasias, moderate in early grade tumors, and low in late stage tumors, essentially supporting the model of the DDR as an early barrier to tumorigenesis in certain types of cancers. In contrast, overall RPA expression was not correlative to DDR activation or tumor progression. Utilizing S4S8-RPA phosphorylation to indicate competent DDR activation in the future may have clinical significance in OSCC treatment decisions, by predicting the susceptibility of cancer cells to first-line platinum-based therapies for locally advanced, metastatic and recurrent OSCC. Oral cancer is the sixth most common cancer with a poor five year survival rate, less than 50%. Genetic alterations are common in OSCC, including inactivation of tumor suppressors such as p53 and activation of oncogenes, including H-ras and cyclin E . OverexpIt is clear that there are enormous biological consequences of a mutated or incapacitated DDR. Through better understanding of the DDR and differential expression of DDR factors in health and disease, there is promise of not only improving the diagnosing and prognosticating of various cancers but also in guiding treatment decisions and developing new therapies to target these differences between malignant and healthy tissues. This is especially pertinent when it comes to the treatment of cancer since the mechanism of action of non-surgical treatment methods such as radiation therapy (RT) and many types of chemotherapies focus on inflicting DNA damage. Current first-line treatments for OSCC patients with locally advanced, metastatic and recurrent cancers primarily involve single agent platinum-based therapies or in combination with mitotic and/or EGFR inhibitors. Current oral cancer treatment protocols call for chemotherapy with cancer stages III through IVC. These cancers can have tumors graded T1 to T4. Therefore, knowing the different responses to chemotherapy based on tumor grade would be important in determining treatment outcome and, inevitably, treatment success or failure. Although great progress has been made in recent years towards understanding the DDR, there is still a great deal that needs to be investigated to achieve a better understanding of health and disease management.Though there are hundreds of proteins that play a role in the DDR, there are apical kinases that work together to coordinate the response: Ataxia telangiectasia and Rad3 related protein (ATR), Ataxia telangiectasia mutated (ATM) and DNA-dependent protein kinase (DNA-PKcs) –7. ATR pIn this study, we determine that immunohistochemical detection of S4S8-RPA phosphorylation can act as a surrogate to determine DDR activation status in human tissues. The resultant amount of phosphorylation is correlative in determining whether a tissue is normal, dysplastic, or cancerous, as well as the grade of tumor. These results suggest that S4S8-RPA phosphorylation may act as a useful clinical indicator of the DDR in oral dysplastic and tumor tissue, and would be clinically beneficial in determining therapeutically efficacious strategies for treatment in stage III–IVC OSCC.n = 15), no samples rated high for S4S8-RPA phosphorylation, while 60% of samples rated as moderate and 40% rated as low , 40% of samples rated high for S4S8-RPA phosphorylation, while 45% were rated as moderate, and 15% rated as low. In OSCC samples (n = 58), 15.5% of tissues were rated high for S4S8-RPA phosphorylation, while 44.8% were rated as moderate, and 39.7% rated as low. The high S4S8-RPA phosphorylation proportion difference between normal and dysplasia samples was determined to be highly statistically significant (p < 0.01). The low S4S8-RPA phosphorylation proportion difference between dysplasia and OSCC samples was also statistically significant (p < 0.05).Immunohistochemical staining with pS4S8 and RPA antibodies was used to determine S4S8-RPA phosphorylation and RPA expression levels. Samples were grouped according to whether they came from normal, dysplastic or OSCC tissues Figure . In normw Figure . In dyspn = 15) rated 33.3% high, 40% rated moderate, and 26.7% rated low. In dysplasia (n = 20), 15% rated high, 65% rated moderate, and 20% rated low. In OSCC (n = 58), 44.8% rated high, 39.7% rated moderate, and 15.5% rated low . Representations of low, moderate and high S4S8-RPA phosphorylation and RPA expression are shown in OSCC samples in For overall RPA expression, normal tissues   was 60%; for the moderate S4S8-RPA phosphorylation group (n = 23), 67.41%; for the low S4S8-RPA phosphorylation group (n = 21), 74.24%. Survival time was lowest for the high S4S8-RPA phosphorylation group between 2 and 9 years in the study. The difference between the survival probabilities of the groups was not significant (Logrank p = 0.9205).The determination that S4S8-RPA phosphorylation greatly increased in dysplastic tissues, then generally decreased in OSCC tissues, is consistent with the model that proposes the DDR acts as a barrier to tumorigenesis in precancerous lesions . Retaini) Figure . The 5-yn = 25), 75.6% (n = 17), and 57.14% (n = 9) respectively .The 5-year survival rate for our high, moderate, and low RPA expression groups were 67.83%  , 85.71% (n = 15) and 77.92% (n = 11) respectively . However, this result trends to the possibility that high S4S8-RPA phosphorylation in OSCC leads to poorer prognosis possibly due to resistance to DNA damaging agents.Since cancers with low S4S8-RPA phosphorylation, and therefore low DDR signaling, would likely be more susceptible to therapies that involved DNA damage agents, we evaluated a subgroup of tissues from patients receiving chemotherapy and/or radiation therapy in addition to surgery  Figure . The 5-yy Figure . Due to n = 17), 85.74% (n = 8), and 50% (n = 4) respectively .The 5-year survival probabilities of high, moderate, and low RPA-expression groups within the chemotherapy/radiation subgroup were 92.86%  between the relative low and high S4S8-RPA phosphorylation group tumor stage grouping indicates that relative S4S8-RPA phosphorylation is higher in early stage tumor progression.To further assess 5-year survival rate and RPA hyperphosphorylation status, we created subgroups of relative-high hyperphosphorylating tissues (having S4S8-RPA phosphorylation amounts high relative to RPA expression), and relative-low hyperphosphorylating tissues (having S4S8-RPA phosphorylation amounts low relative to RPA expression). We examined the relationship between relative-high S4S8-RPA phosphorylation and relative-low S4S8-RPA phosphorylation in relation to tumor stages, T1-T2 and T3-T4  tumors. This pattern of S4S8-RPA phosphorylation is consistent with previous studies which demonstrated down-regulation of DDR expression in advanced-stage tumors versus early stage tumors . The preIn this study, we identified increased S4S8-RPA phosphorylation in dysplastic tissues which may indicate the activation of a barrier to tumor progression. In addition, we provide evidence of down-regulation of the DDR in advanced-stage tumors compared to early-stage tumors using S4S8-RPA phosphorylation. Our data suggest analyzing levels of S4S8-RPA phosphorylation in untreated tumors may have limited advantages as compared to analyzing levels in tumors exposed to chemotherapy. Results of S4S8-RPA phosphorylation did not predict survival in the untreated tumor groups; however, trends observed in 5-year survival rates of those receiving chemo- and radiotherapy suggest promise of S4S8-RPA phosphorylation analysis as potentially useful in further chemotherapy treatment choices. This initial study demonstrates promising results and merits larger scaled studies to examine the role of S4S8-RPA phosphorylation in OSCC in the future.Primary antibodies used for Immunohistochemistry (IHC) are mouse anti-RPA2  and rabbit anti-S4S8 Phospho RPA32 . Secondary antibodies and IHC for detection of RPA32 was carried out using Anti-mouse Ig HRP Detection Kit . For pS4S8-RPA2 detection, anti-rabbit Bethyl IHC Accessory Kit was used.Normal (non-dysplastic or cancerous) and dysplasia human biological material (HBM) tissues used for immunohistochemistry were obtained from archived paraffin-embedded, formalin-fixed tissue samples from the University of Nebraska Medical Center College of Dentistry Oral Pathology biopsy service in Lincoln, NE. Oral, laryngeal, and hypopharyngeal squamous cell carcinoma (OSCC) tissues were obtained in the form of paraffin-embedded slides from the University of Nebraska Medical Center College of Medicine Department of Pathology and Microbiology in Omaha, NE. When applicable, biographical data and disease-specific medical history was obtained from the UNMC College of Medicine Department of Otolaryngology/Head & Neck Surgery in Omaha, NE. Institutional Review Board (IRB) approval was obtained for the use of collected HBM and relevant medical data. 15 normal, 20 dysplasia, and 58 OSCC tissues were examined in this study.2O2) to block endogenous peroxidase activity. Antigen retrieval was performed by a 20 minute water bath in Tris EDTA buffer (pH 9.0) between 96–99 degrees Celsius. Slides were allowed to cool at room temperature for 30 minutes. Slides were washed with IHC solution wash (Bethyl). Primary antibody incubation was performed at 1:100–125 dilution overnight in a condition-controlled cold room. After washing slides, biotinylated anti-mouse secondary antibody was incubated at 1:100 dilution and according to manufacturer's recommendations for the pre-diluted anti-rabbit Ab for one hour at room temperature. Following slide washing, RPA2 antibody-treated slides were treated with streptavidin-HRP for 30 minutes and washed. S4S8-RPA antibody treated slides had HRP conjugated directly to secondary antibody. Diaminobenzene (DAB) exposure for 2–5 minutes was then followed by distilled water wash (3 × 3′). Slides were counterstained with Meyer's hematoxylin for 2 minutes, and washed in a basic bluing solution or running tap water. Slides were then dehydrated with increasing concentrations of ethanol  and xylene immersion (3 × 5′) to prepare for slide mounting.From paraffin-embedded tissue blocks, 4–6 μm thick sections were prepared on slides for IHC. Negative control slides were processed without primary antibody. High-staining tissues were used as positive controls. Slides were deparaffinized and rehydrated with three stages of xylene immersion (5′) and decreasing concentrations of ethanol from 100%(2 × 5′), 95% (2 × 3′), 80% (3′), 70% (3′), 50% (3′). Slides were then treated for 10 minutes with 3% hydrogen peroxide (HSlides were independently evaluated by two calibrated examiners and rated as high, moderate, or low expression based on the relative number of cells stained per section as well as the intensity of staining . A thirdRelevant treatment outcome history was obtained for OSCC tissue donors when applicable, including TNM stage, histological grade, 5-year survival probability, and tumor recurrence. 5-year survival was determined for all possible OSCC tissues. Tumor TNM stage and histological grade were determined by a certified pathologist.p-value < 0.05. Logrank scores were computed to test for the difference in survival probabilities between high, moderate, and low S4S8-RPA phosphorylation as well as RPA expression. Survival probability statistics and Kaplan-Meier curves were generated with GraphPad Prism 4.Chi-squared tests for independence was performed to evaluate differences in S4S8-RPA phosphorylation proportions and RPA expression proportions between groups. Statistical significance was considered at a