In this context, the objective of this work was to study the sequence of ERK phosphorylation under two redox conditions and to analyze a putative mitochondrial contribution to this process, in LP07 murine lung cells. A time-course of H2O2 administration was used and ERK phosphorylation was analyzed in cytosol, mitochondria and nuclei. At 1μM H2O2, a proliferative redox stimulus, immunoblot revealed a fast and transient increase in cytosol pTyr and a sustained increase in mitochondrial pTyr content. The detection for pThr/pTyrERK (2pERK) showed in cytosol a marked increase at 5 minutes with a fast dephosphorylation after that time, for both H2O2 concentrations. However, at 50 μM H2O2, an anti-proliferative condition, 2pERK was gradually retained in mitochondria. Interestingly, these results were confirmed by in vivo experiments using mice treated with a highly oxidizing agent [H2O2]. By the use of two ERK2 mutant constructions, where Tyr and Thr were replaced by alanine, we confirmed that 2pERK relied almost completely on pThr183. Confocal microscopy confirmed ERK subcellular distribution dependence on the incidence of cytosolic pTyr and mitochondrial pThr at 1μM H2O2. This work shows for the first time, both in vitro and in vivo, an ERK cycle involving a cross-talk between cytosol and mitochondria phosphorylation events, which may play a significant role in cell cycle progression, proliferation or differentiation under two different redox conditions.Activation of ERK1/2 implies the phosphorylation of tyrosine (pTyr) and threonine (pThr) by MEK1/2; both reactions were thought to be cytoplasmic, promoting ERK to reach the nucleus where it activates several transcription factors. In addition, H The importance of protein kinases in proliferative or anti-proliferative cellular processes has been extensively reported. In general, the activation of classic members of the mitogen-activated protein kinase (MAPK) family ‒extracellular signal-regulated protein kinases 1/2 (ERK1/2), JNK1/2 and p38MAPK‒ involves single or double phosphorylation reactions, as given by the effects of upstream regulators like MAPK kinases (MAPKKs) called MEK 1–6 . Of greaMAPKs are unique among the Ser/Thr protein kinases, in the sense that they require both threonine (Thr) and tyrosine (Tyr) phosphorylation for full activation. ERK1/2 activation requires Ras-Raf mediated GTP-dependent double phophorylation in adjacent Tyr and Thr located in the TEY activation motif (Thr-Glu-Tyr) under epidermal growth factor (EGF) stimulus. In particular, the dual phosphorylation of Thr183 and Tyr185 in the ERK2 sequence is catalyzed by MEK1/2  and bothComplete inactivation of ERKs can be achieved by the removal of phosphates from either one of the regulatory residues, or from both of them together . Thus, p2O2) can act as second messengers, modulating the activities of molecules involved in key cellular processes, including phosphorylative cascades ss, required for tumor arrest ss in LP07 murine tumor cells is very low ‒10−11 M‒, similarly to what happens in embryonic and proliferating tissues. In these cells, mitochondria have a low H2O2 production rate but still respond to oxidative stress like normal cell mitochondria do sndria do ,21. MEK1or cells .2O2 entails the entrance of cytosolic phospho-Akt1 Ser473 to mitochondria, where it is further phosphorylated in Thr308 by constitutive kinase PI3K-dependent kinase 1 (PDK1). Moreover, the connection among mitochondrial dysfunction, H2O2 yield and activation of MAPKs still awaits further elucidation.In this regard, modulation by H2O2 conditions on the subcellular distribution of the two phosphorylating reactions of ERK structure in Tyr and Thr. For the first time, we demonstrate both in vitro and in vivo that most of the phosphorylation in Tyr is cytosolic, while phosphorylation in Thr of ERK is mainly mitochondrial, under proliferative H2O2 conditions. Although ERK2 full activity relies on the addition of phosphoric acid to both Tyr185 and Thr183 in the ERK sequence, we highlight here the relevance of one site or the other by the use of mutants for these two amino acids. Tyr185 is phosphorylated upstream by MEK and, then, Thr183 is more susceptible to phosphorylation in the mitochondrial context, perhaps allowing ERK to further translocate to the nucleus under proliferative conditions. We demonstrate that the redox status modulates the pattern of distribution of phospho-ERK providing a possible mechanistic explanation for cell cycle regulation triggered by oxidant agents such as H2O2.In this context, the objective of the present study is to establish the effect of different H2O2  for the times indicated in the figures. Sterile and plastic material for tissue culture was from Orange Scientific . All other reagents were of the highest grade available.The LP07 murine cell line was derived from P07 lung tumor spontaneously developed in a BALB/c mouse and extensively characterized ,23. CellCells were lysed in MSHE buffer , 5 μg/ml leupeptin, 5 μg/ml pepstatin, 5 μg/ml aprotinin, 25 mM NaF, and 1 mM sodium orthovanadate, pH 7.4) supplemented with a classic inhibitors cocktail , 5 mg/ml leupeptin, 5 mg/ml pepstatin, 5 mg/ml aprotinin, 25 mM NaF, and 1 mM sodium orthovanadate, pH 7.4). All reagents were purchased from Sigma-Aldrich. The homogenate was centrifuged 10 min at 1000xg (pellet = crude nuclear extract) and 20 min at 10000xg . Mitochondria were resuspended in MSHE. The crude nuclear extract was washed with buffer A  containing 0.01% NP-40, resuspended in buffer A plus 0.4 M KCl, and incubated 30 min at 4°C. The suspension was centrifuged 30 min at 105000xg and diluted with buffer A to reduce salt concentration. The purity of the fractions was assessed by western blot with antibodies against translocase outer membrane 40 (TOM40) or complex III (CIII) (mitochondria), actin (cytosol) and Polimerase II or TFIID (TBP) (nuclei). All fractions were tested against crossed contamination with antibodies for other fractions, as previously described . Proteinad libitum. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.This study was carried out in strict accordance with the recommendations and the ethical standards of the Animal Care and Research Committee of the University of Buenos Aires . This protocol was approved by CICUAL-UBA # 0037016/2012. FVB mice were housed in ventilated cages with a 12-hour light/dark cycle and controlled temperature (20–22°C), and fed with normal chow and water 2O2 or saline solution was administered intravenously (n = 3 for each treatment). After 5 minutes, mice were euthanized with an overdose of pentobarbital, and the lungs were quickly excised and placed in liquid nitrogen. Lung tissue samples were obtained by mechanical disruption in MSHE buffer using an Ultra-Turrax homogenizer and differential centrifugation: 20 min at 10000xg for the mitochondrial fraction and 1h at 105000xg for the cytosol. Samples were then subjected to western blot studies.Normal male FVB mice (25–35 g) were anesthetized with pentobarbital (90 mg/kg). Once mice were hemodinamically stable, the right yugular vein was dissected and a bolus of 6 mM HProteins separated by SDS-PAGE, were transferred onto PVDF membranes and immunoblotted with antibodies anti-double phosphorylated ERK1/2 (dilution 1:5000)  and anti-phosphoTyr ERK (dilution 1:1000) . After cell transfection, anti-V5 tag (dilution 1:1000)  was used to detect specifically recombinant over-expressed ERK2 protein. Anti-complex III or TOM40 (dilution 1:5000)  and anti-polimerase II (POLII) or anti-TFIID (or TBP)  (dilution 1:1000) were used as mitochondrial and nuclear loading control respectively, and antibodies against actin (dilution 1:4000) were used for cytosolic fraction characterization . Cells were then incubated with secondary horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibodies . Chemiluminescence was developed with enhanced ECL reagent  and bands were detected by autoradiography . Acrylamide and PVDF membranes were from Bio-Rad Laboratories Inc.LP07 cells were seeded onto a 12-well plate and grown to 80% confluency. Twenty-four hours later cells were transiently transfected with 0.4 μg of the plasmids encoding for ERK2 wild-type and ERK2 mutants Y185A or T183A in Opti-MEM Reduced serum medium (Thermo Fisher Scientific) using Lipofectamine 2000  according to manufacturer’s instructions (4μl lipofectamine/well). Cells were then placed into normal culture medium 6 h after transfection and grown for further 48 h. After transfection, cells were used as described in the respective figures. Full length ERK2 cDNA was subcloned in the eukaryotic expression plasmid pcDNA3.1 V5-His-TOPO  and directed mutagenesis was performed by a biotechnological company .2O2 as indicated in the figures, then stained with 100 nM specific mitochondrial marker MitoTracker Deep Red 633 FM  for 45 min at 37°C, fixed in 4% paraformaldehyde, blocked in 1% BSA, 0.3% Triton X-100 PBS, pH 7.4, in a humidified chamber for 1 h, and incubated with primary antibody against V5 tag (anti-V5 tag) and secondary antibody conjugated with Cy3  for 1h at room temperature in the same buffer. Cover slides were mounted in Fluorescence Mounting Media . Confocal laser scanning microscopy was performed with an Olympus FV1000 using a 6361.35 NA oil immersion objective. Excitation filters and emission detected with a PDA device were as follows: GFP, 488 nm excitation, 500–560 nm emission; Cy3, 532 nm excitation, 580±10 nm emission; MitoTracker Deep Red, 633 nm excitation, 650–750 nm emission. Images were obtained with Olympus Fluoview FV10-ASW software and analyzed with DIPimage software . MATLAB images  were analyzed by intensity correlation analysis (ICA) [Cells were grown on cover slides and transfected as mentioned above. Forty-eight hours post-transfection, cells were incubated with His (ICA) ,26. Briep< 0.05.Data are expressed as the mean ± SD and analyzed by one-way analysis of variance (ANOVA), Dunnett’s test and Scheffe’s test. Statistical significance was accepted at To analyze ERK2 protein kinetics, we incubated LP07 cells for the times indicated with 10ng/ml EGF, which is well known as a classic regulator of the MEK/ERK cascade in several cellular types . Previou2O2 concentrations.Considering that two phosphorylation events are required for ERK full activation, we next analyzed, by means of specific antibodies, the subcellular distribution of phospho-Tyr (pYERK) and the two possible phosphorylations for ERK in Thr or Tyr (2pERK), in LP07 lung tumor cells under two different redox conditions: low (1 μM) and high (50 μM) H2O2, a rapid and transient peak in pYERK (5 minutes) was detected in the cytosol, with a decrease after 15 min, almost to basal levels  in the mitochondrial fraction in mice administered 6 mM of Hted mice . Insteadstration . Some pT2O2-treated animals, which suggests 2pERK accumulation in this organelle, while lower 2pERK levels were found in the cytosol, for the same amount of protein analyzed . Results so far show that H2O2 and redox status also modulate ERK phosphorylation levels and intracellular traffic in normal murine lung tissue.In addition, high levels of mitochondrial pTyr/pThr (2pERK) were detected in lung tissue from control and Hanalyzed . Specifi2O2 challenge. For this purpose, we transiently transfected LP07 tumor cells with ERK2-V5 wt and Y185A and T183A mutant variants, in which Tyr185 and Thr183 were respectively converted to alanine, a non-phosphorylatable amino acid. It is well established that ERK mutants in Tyr phosphorylation site display reduced activity compared to wild type ERK [Next, we analyzed the capacity of residues Tyr185 and Thr183 of being phosphorylated and their distribution in mitochondria under Htype ERK .2O2. This effect was detected in the cytosol, mitochondria and nuclear fraction  are oxidized to sulfinic (–SO2H) and sulfonic acid (–SO3H), as previously described [2O2, a strong MEK-ERK association is favored and sequential phosphorylation in both Tyr and Thr residues may contribute to further addition of negative charges and to the aforementioned ERK activation cycle. On the other hand, mutation of Cys214 to alanine rendered ERK2 and MEK accumulation in mitochondria and a decrease in the mitochondrial cycle, in agreement with a lesser extent of negative charge in ERK molecule [2O2 and provide an explanation for ERK retention in mitochondria, where it could be dephosphorylated and inactivated by mitochondrial phosphatases [The findings presented here indicate that mitochondrial uptake of ERK phosphorylated in Tyr is required for sequential and immediate phosphorylation in Thr to become fully activated and released to nucleus, in redox-permissive low Hescribed . Mutatiomolecule . This isin vitro ERK activity assays [These results are completely in line with a classic study by Dr. Cobb’s group, which elegantly demonstrates that the accumulation of pTyr precedes the accumulation of pThr in y assays . Here, wMAPK signaling pathway has been extensively explored and transient and sustained ERK activation seems to be one of the most relevant events to direct cellular fate, e.g. transient activation of ERK in the cytoplasm of PC12 cells is essential for proliferation but sustained ERK activation and nuclear accumulation are both required for PC12 cell differentiation .Previous work ,19,35,36Findings regarding mitochondrial redox influence on ERK activation shown in 2O2 promotes ERK phosphorylation involving three subcellular compartments, i.e. cytosol, mitochondria and nucleus. A temporal Thr phosphorylation pattern indicates dependence on cellular redox status for the occurrence of this phosphorylation and mitochondrial contribution. It is highlighted here that two amino-acidic residues in close proximity such as Tyr and Thr in the ERK sequence can be modified in a compartmentalized manner, with direct implications for cellular fate and, in turn, cell survival or death. Given the well-established relationship between mitochondria and cancer, these studies appear to provide clues to unveil the machinery that integrates the signals for the acquisition of a malignant phenotype.


Removal of the transcriptionally activating H3K4 methylation is catalyzed by histone demethylases, including the Jumonji C (JmjC) KDM5 subfamily. The JmjC KDMs are Fe(II) and 2-oxoglutarate (2OG)-dependent oxygenases, some of which are associated with cancer. Altered levels of tricarboxylic acid (TCA) cycle intermediates and the associated metabolites D- and L-2-hydroxyglutarate (2HG) can cause changes in chromatin methylation status. We report comprehensive biochemical, structural and cellular studies on the interaction of TCA cycle intermediates with KDM5B, which is a current medicinal chemistry target for cancer. The tested TCA intermediates were poor or moderate KDM5B inhibitors, except for oxaloacetate and succinate, which were shown to compete for binding with 2OG. D- and L-2HG were moderate inhibitors at levels that might be relevant in cancer cells bearing isocitrate dehydrogenase mutations. Crystallographic analyses with succinate, fumarate, L-malate, oxaloacetate, pyruvate and D- and L-2HG support the kinetic studies showing competition with 2OG. An unexpected binding mode for oxaloacetate was observed in which it coordinates the active site metal via its C-4 carboxylate rather than the C-1 carboxylate/C-2 keto groups. Studies employing immunofluorescence antibody-based assays reveal no changes in H3K4me3 levels in cells ectopically overexpressing KDM5B in response to dosing with TCA cycle metabolite pro-drug esters, suggesting that the high levels of cellular 2OG may preclude inhibition. The combined results reveal the potential for KDM5B inhibition by TCA cycle intermediates, but suggest that in cells, such inhibition will normally be effectively competed by 2OG.Methylation of lysine-4 of histone H3 (H3K4me  •The JmjC histone demethylase KDM5B has roles in cell proliferation and tumor maintenance.•Elevated TCA cycle metabolite levels may promote tumorigenesis by inhibition of JmjC KDMs.•Crystallographic studies provide evidence for KDM5B interaction with TCA cycle metabolites.•TCA cycle metabolites can inhibit KDM5B in a 2OG competitive manner.•Oxaloacetate binds KDM5B chelating the active site metal via its C-4 carboxylate. Kinetic studies revealed a large difference in the extent of inhibition, with oxaloacetate and succinate being the most potent of the tested compounds. Crystallographic analyses support the findings suggesting competition of TCA cycle intermediates with 2OG and reveal an unexpected coordination mode for oxaloacetate. In contrast with some of the other 2OG oxygenases acting on chromatin, cellular studies on the effects of elevated TCA cycle metabolites on H3K4 methylation imply that the relatively high 2OG levels may “outcompete” the elevated TCA intermediate levels.3, H3K4me2 and (with lower activity) H3K4me1; in line with the previous work, the H3K4me2(1–21) peptide fragment was chosen for inhibition studies to avoid complications due to sequential demethylation that is observed when using the analogous H3K4me3 peptide 2 were conducted using a formaldehyde dehydrogenase (FDH)-coupled assay using 15-mer and 21-mer H3 peptide fragments, and recombinant truncated KDM5B constructs, without the PLU1, PHD2 and PHD3 domains (KDM5B(1–769) or KDM5B(1–822) Kmapp(2OG) and Kmapp(H3K4me2(1–15)) were 6 and 3.6 μM, respectively Kmapp for H3K4me2(1–21) was 0.85 μM Kmapp values may be attributed, at least in part, to different lengths of the peptide substrates as precedented in work with other 2OG oxygenases We initially investigated inhibition of purified recombinant KDM5B  by TCA cycle metabolites. KDM5B catalyzes demethylation of H3K4meKmapp values for 2OG and H3K4me2(1–21) peptide were determined for KDM5B(1–822) using MALDI-TOF-MS to identify appropriate experimental conditions for inhibition studies. Our determined values were Kmapp(2OG) = 2.6 ± 0.6 μM, Kmapp(H3K4me2(1–21)) was ~ 0.5 μM and the kcatapp was 0.015 ± 0.01 s− 1 . Thus, the results of our Kmapp(H3K4me2(1–21)) determination are in agreement with the previously reported FDH-based results Kmapp(H3K4me2) for the 21-mer peptide was lower than the MALDI-TOF-MS limit of detection, which prevented a more accurate determination.We used matrix-assisted laser desorption/ionization–time of flight–mass spectrometry (MALDI-TOF-MS) for direct analysis of methylated and demethylated peptide ratios , to avoid the possibility of FDH inhibition which could interfere with results when using the FDH-coupled enzyme assay. As a prelude to the inhibition studies, 50 values with the 2OG concentration fixed at the Kmapp(2OG) level (3 μM) and saturating levels of H3K4me2(1–21) peptide substrate (5 μM). The highest levels of inhibition were observed in the presence of oxaloacetate . D- and L-2HG inhibited KDM5B-catalyzed demethylation with IC50 values in a range of 150–200 μM, that is, less potently than for some other reported histone demethylases  The inhibition of TCA cycle metabolites was then assessed by determining the ICKi) for succinate and oxaloacetate using the MALDI-TOF-MS assay with respect to 2OG. Kmapp for 2OG was assessed in the presence of different concentrations of succinate and oxaloacetate (Kmapp for 2OG in the presence of inhibitor , while the Vmax values remained the same, revealing that both succinate and oxaloacetate inhibit KDM5B in a 2OG competitive manner. The determined Ki values were 27 ± 6 μM for succinate and 54 ± 6 μM for oxaloacetate.We then determined inhibitory constants  lacking its ARID and PHD1 domain, which we have previously successfully used in crystallization studies Nε-methyl lysine group of the substrate. The C-1 carboxylate of 2OG is further stabilized by hydrogen bonding with Ser507, which may be involved in 2OG decarboxylation/loss of CO2 during catalysis It has been previously reported that 2OG is bound at the active site of KDM5B in a manner typical for other 2OG oxygenases, including the JmjC subfamily The studied TCA cycle intermediates all bind in the 2OG pocket b–h. All,Interestingly, in the case of oxaloacetate, the ligand binds such that its C-4 carboxylate, rather than its 2-oxoacid (as observed with 2OG), ligates to the metal. This binding mode should enable discrimination between oxaloacetate and 2OG with respect to oxidative decarboxylation. It may reflect a form of negative catalytic regulation; that is, oxaloacetate binds in a catalytically non-productive manner to avoid oxidative decarboxylation. Like oxaloacetate, pyruvate binds with its carboxylate occupying the 2OG C-5 carboxylate equivalent position; however, the ketone oxygens of oxaloacetate and pyruvate project in opposite directions.The binding of L-2HG is similar to that of 2OG, with the C-2 hydroxyl group occupying a similar position to the C-2 carbonyl of 2OG and the C5 carboxylate is found in an identical position. However, in the case of D-2HG, the C-5 carboxylate is rotated out of the binding pocket to form interactions with water molecules , while its C-1 carboxylate and C-2 OH-group bind similarly as in 2OG c.Different solvation patterns relative to 2OG were observed for the studied ligands, with additional water and, sometimes, ethylene glycol molecules present within the 2OG binding pocket , likely 3 demethylation were then assessed. We used an established immunofluorescence (IF) procedure with ectopically overexpressed wild-type KDM5B (WT KDM5B) in U2-OS cells and anti-H3K4me3 antibody monitoring for changes in H3K4me3 methylation levels 3 levels were then analyzed in the transfected cells. The reported KDM5B inhibitor KDOAM-25 ((2)-((((2)-(((2)-(dimethylamino)ethyl)(ethyl)amino)-(2)-oxoethyl)amino)methyl)isonicotinamide) was used as a positive control for inhibition 3 levels in the WT transfected cells  (3 μM) for KDM5B determined with isolated recombinant KDM5B. The esters of the TCA cycle metabolites  were thus dosed up to 5–10 mM concentration. In contrast with the experiments with isolated KDM5B, no inhibition of KDM5B activity was observed  and the TCA cycle-related metabolites reveal that most of the compounds have the potential to inhibit KDM5B, and by implication other KDM5 subfamily enzymes, in a 2OG competitive manner. Although, all of the inhibiting metabolites occupy the 2OG binding site, they exhibit variations in their binding modes, which appears to correlate with the observed potencies. Thus, pyruvate was shown to be a very weak KDM5B inhibitor and did not manifest metal chelation in its binding mode. Citrate and isocitrate were also inactive as KDM5B inhibitors under the studied assay conditions, consistent with our inability to obtain crystal structures for them.2YBK and 2YBS, respectively). This property apparently enables the 2OG binding pocket in KDM5B to adapt to the different shapes of the various TCA cycle-related intermediates.The crystal structures provide evidence that the conformations of the 2OG binding pocket in KDM5B are relatively flexible  present in a L-2HG complex with KDM4A (PDB ID: 2YBS), superimposes with an ethylene glycol molecule present in all the KDM5B complexes, suggesting that the ethylene glycol occupies part of the position of the peptide substrate in the KDM5B active site.The conformations of the 2OG binding pocket residues in complexes of D- and L-2HG with KDM4A 50 values of 15 ± 10 and 62 ± 19 μM, respectively. While oxaloacetate has not been reported to accumulate under pathological conditions, increased succinate levels are established in certain cancers 50 values of 203 ± 90 μ and 150 ± 40 μM for D-/L-2HG, respectively, compared to 25 μM for KDM4A; Of the tested metabolites, succinate and oxaloacetate showed the highest potency for KDM5B inhibition, with ICKmapp values for 2OG reported for 2OG oxygenases, which likely also influence their 2OG competitive inhibition. However, treatment of U2-OS cells with increasing amounts of dimethyl-2OG did not show any effect on the activity of ectopically overexpressed KDM5B to demethylate H3K4me3, suggesting that under the tested conditions, the intracellular 2OG levels are saturating for KDM5B catalysis. This result is consistent with the relatively low Kmapp(2OG) for KDM5B (3 μM), which is likely below intracellular 2OG concentrations In contrast with the results for some other 2OG oxygenases  Thus, although there are caveats on the physiological relevance of our cellular work with KDM5B (as with that for other JmjC KDMs), the available evidence is that some JmjC KDMs and, by implication histone marks, may be more susceptible to changes in specific metabolite concentrations than others . The overall results, however, raise the possibility of regulation of KDM5 activity by competition for 2OG binding by multiple metabolites, the combined concentration of which will be high relative to 2OG. Such competition by multiple molecules has the potential to tune enzyme activity and to provide effects enabled by context- and time-dependent changes in the competing metabolic composition Finally, it is important to note that many 2OG oxygenase inhibitors, including inhibitors in clinical development and KDM5 subfamily inhibitors  Recombinant KDM5B (M1-R822 and F26-K722) was produced in Sf9 insect cells and purified as previously described 5A1F as a model). Iterative model building was done using COOT A JmjN–JmjC linked construct of KDM5B (F26-K772) was crystallized using the sitting drop vapor diffusion method as previously described 2(1–21) peptide , 10 μM Fe(NH4)2(SO4)2 and 500 μM sodium L-ascorbate. The reaction was quenched with 1% formic acid at defined time points. The samples were then mixed in 1:1 ratio with α-cyano-4-hydroxycinnamic acid matrix and spotted onto a MALDI target plate. Spectra were acquired using MALDI-TOF-MS  in reflectron positive mode and processed using MASSLYNX4.0. Time courses at each 2OG/peptide concentrations were analyzed and initial rates of demethylation were determined. The initial rates were plotted versus concentration and analyzed with the assumption of Michaelis–Menten kinetics using Origin 8.51 software.Kinetic assays were performed in 50 mM Hepes, 50 mM NaCl (pH 7.5) buffer at 25 °C. KDM5B (0.6 μM) was incubated with various amounts of 2OG and H3K4me2(1–21), 10 μM Fe(NH4)2(SO4)2 and 500 μM sodium L-ascorbate for 5 min and subsequently quenched with 1% formic acid. The samples were mixed in a 1:1 ratio with α-cyano-4-hydroxycinnamic acid matrix and spotted onto a MALDI target plate for MS analysis. The demethylation levels were normalized compared to the non-inhibitor control. The IC50 values were determined using GraphPad Prism software. All experiments were carried out in triplicate.Inhibition assays were performed in 50 mM Hepes, 50 mM NaCl (pH 7.5) buffer at 25 °C. KDM5B (0.6 μM) was incubated with 3 μM disodium 2OG, 5 μM H3K4me3 DiagenodeC15410003). The cells were then stained with secondary antibodies [Alexa Fluor 488 and Alexa Fluor 568-labeled (Life Technologies)] and 4’,6-diamidino-2-phenylindole. Image acquisition was conducted using Operetta High Content Imaging System (PerkinElmer), and image analysis was performed using the Columbus software (PerkinElmer). A minimum threshold for anti-Flag intensity was set to ensure that only cells highly producing the demethylase were analyzed.Immunofluorescent cell-based assays were performed as reported KDOAM-25 was kindly provided by Dr. Gian-Filippo Ruda and Prof. Paul Brennan  5FUP (2OG), 5FY4 (succinate), 5FY5 (fumarate), 5FYV , 5FY9 (pyruvate), 5FZ8 , 5FYS (D-2-hydroxygutarate), 5FZD (L-2-hydroxyglutarate).


The existence of a local allergic rhintis was proposed on the basis of the detection of nasal IgE in the absence of a systemic sensitization. Nevertheless, the significance of this phenomenon remains still unclear.We assessed the presence of mucosal nasal IgE in patients with ascertained allergic rhinitis, nonallergic rhinitis with inflammation and in healthy controls.Consecutive patients with a well ascertained diagnosis  underwent an immunoenzymatic measurement of specific IgE to grass, cypress, parietaria and olive in nasal scrapings.Fifteen patients with allergic rhinitis, 12 with non allergic rhinitis and 14 healthy subjects were studied. The patients with allergic and nonallergic rhinitis had higher nasal symptoms as compared to control subjects. Systemic sensitizatition (assessed by skin test and CAP-RAST) was obviously more frequent in allergic rhinitis, than in the other two groups. Allergen-specific nasal IgE could be detected in all groups , even more frequently in the control group than in nonallergic rhinitis patients. No difference among allergens was identified. Out of the 26 non-allergic patients  nasal IgE were positive in 11(42 %).According to the results, the presence of nasal IgE against allergens seems to be a non-specific phenomenon, since they can be detected also in non allergic rhinitis and in healthy subjects.It can be hypothesized that the nasal IgE production represents a form of spontaneous immune response. Rhinitis is defined as an inflammation of the nasal mucosa, usually characterized by rhinorrhoea, sneezing, nasal blockage and itching, variably associated. Those symptoms can be accompanied, expecially in the allergic form, by ocular symptoms such as lacrimation, eye itching, conjunctival hyperemia . AllergiAlso non-allergic conditions can cause symptoms of rhinitis: infections, hormonal imbalance, physical/irritant agents, anatomical abnormalities or drugs . Also thSince 1975, another pathophysiological type of rhinitis was proposed, characterized by the presence of allergen-specific IgE only in the nose, without any evidence of systemic sensitisation detectable by skin prick test (SPT) or specific IgE serum assays (CAP-RAST)  and, subAdult patients were assessed for the presence of allergen-specific nasal IgE, after the type of rhinitis was classified in detail according to the diagnostic procedure. Patients were subdivided into AR (positive SPT and CAP-RAST), and NAR . A control group of healthy subjects (no rhinitis symptoms and negative SPT/CAP-RAST) was also included for comparison. The study took place between November 2014 and January 2015 at the Rhinology Unit of the University of Bari. All subjects provided an informed consent for the management of their anonymous clinical data. The inner ethical committee was simply notified, since this was not an interventional study, no placebo was used, and the procedures were part of the standard diagnostic practice. According to the existing laws, also the healthy volunteers could be admitted, after written informed consent. The diagnostic work-up for rhinitis involved: personal clinical history, family history, symptoms , SPT, CAP-RAST assay, nasal endoscopy, nasal scraping for cytology, and assay for nasal mucosal IgE (see below). Those patients with symptoms of chronic rhinosinusitis, anatomical abnormalities , unilateral symptoms/signs or malignancies were excluded from the assessment of nasal IgE. We also excluded those patients reporting symptoms of possible acute infectious diseases (e.g. common cold) in the last month. All patients were medication-free  in the past 2 weeks.Obstruction and itching were assessed by a 10-cm visual analog scale  . RhinorrParietaria, olive, cypress, mugwort, alternaria, ragweed, cat and dog dander [It was performed using a panel of the most common aeroallergens  according to the recommendations of the European Academy of Allergy and Clinical Immunology: house dust mite, grass mix, g dander .Allergen-specific IgE antibodies against the same allergens assayed with skin test were measured by a quantitative immunoassay . The measure range of the test varies from 0.1 to 100 kU/L, and in clinical practice, 0.35 kU/L is commonly used as a the optimal lower cut-off.It was carried out by a 3,4 mm diameter flexibile-fibroscope (Vision-Sciences® ENT-2000), to assess the presence of major abnormalities, such as septal deviation, polyposis, turbinate hypertrophy, or exudation from the ostiomeatal complex.This procedure was performed by scraping the middle part of the inferior turbinate with a Rhino-Probe® device (Arlington Scientific). The sample was smeared on a slide, air-dried, then stained with the May-Grünwald Giemsa preparation. The type and cell number were examined using microscopy (Nikon® E600). Cell types were identified, and intracellular components were studied at x1000 in oil immersion. The mean number per 50 fields was calculated and reported , 15. ThiSamples for nasal IgE assay were collected by Rhino-probe® scraping, as for nasal cytology. Samples were diluted in 0.5 mL physiological solution, then stored at 4 °C. Measurement of nasal specific IgE was carried out by the Immunocap® Specific IgE tests  by a quantitative immunoassay. The test is the same routinely used on serum or plasma samples. In order to establish a lower threshold limit of detection we tested 10 negative controls , and the limit resulted to be 0.17 kU/L. Specific IgE to parietaria, olive, dust mite, cypress and grasses were assayed.Demographic and clinical characteristics were analyzed by descriptive statistics (% or mean and SD). Continuous variables were analyzed by the t test for independent samples, whereas the Chi-square test was used for qualitative measures. The 3 groups were compared by the ANOVA test. P values for the null hypothesis was set at 0.05. The dedicated software SPSS22 was employed.n = 15), NAR (n = 12) and controls (n = 14). These latter included volunteers enrolled among healthcare professionals, postgraduate students or nurses. The 3 groups were homogeneous for demographic characteristics as summarized in Table ). More in detail, it appeared that in the AR group there was a higher occurrence of positivities vs NAR and controls for cypress , olive (20 % vs 16.7 % vs 7.1 %), and grass (40 % vs 16.7 % vs 21.4 %). Local nasal IgE to Parietaria were detected more frequently in the AR patients than in NAR and controls  . Among non allergic patients  nasal IgE were detectable for parietaria (67.9 %), olive , cypress (15.4 %), dust mite (60.7 %) and grasses (38.1 %). There was no significant difference concerning each single allergen, except for Parietaria, more frequently positive in AR patients. As expected, there was a significant difference in the differential cell count at nasal cytology between the patients with and without rhinitis. This was true for each cell type  were studied between November 2014 and January 2015. All the subjects were consecutively enrolled for the nasal IgE assay, once a diagnosis was made on the basis of the procedures mentioned before. The studied population was subdivided into: AR (In general, more than 60 % of patients with NAR receive a generic diagnosis of “idiopathic” rhinitis, that is based on an exclusion criterion . AlthougIn 1975 Huggings demonstrated, in a qualitative way, the presence of house dust mite-specific IgE in the nose of subjects with symptoms of rhinitis but no evidence of systemic IgE . He asseOur results confirmed the presence of nasal IgE in AR subjects, usually in agreement with the systemic sensitization profile, revealed by IgE serum assay and SPT. Interestingly, nasal specific IgE could be detected also in a relevant percentage of patients with NAR. More importantly, as unexpected finding, nasal allergen-specific IgE could be found also in 50 % of the healthy control subjects. Although the number of patients studied is small, the percentage remains highly significant. This latter aspect would suggest, as an hypothesis, that a local secretion of IgE could be part of a spontaneous immune response to environmental agents. The identification of nasal allergen-specific IgE also in healthy subjects and in patients with non allergic rhinitis suggest that the concept of LAR should be reconsidered and re-evaluated. These observations also suggest that a more detailed diagnostic approach, involving nasal endoscopy and nasal cytology, should be carried out when the diagnosis is uncertain. In fact, nasal cytology can better define and refine the details of nasal inflammation. The technique is not invasive, does not alter the cellular profile and it is also used in electron microscopy studies for other diseaes , 20. AccIndeed, the postivity of allergen-specific nasal IgE in the absence of a detectable systemic sensitization remains unclear, needs more experimental proof , 27, andThe detectable presence of allergen-specific IgE on the nasal mucosa of subjects with nonallergic rhinitis and healthy subjects suggests that this phenomenon could be the expression of an innate response, rather than a specific local reactivity.


Screening for breast cancer has predominantly been done using mammography. Unfortunately, mammograms miss 50% cancers in women with dense breast tissue. Multi-modal screenings offer the best chance of enhancing breast cancer screening effectiveness. We evaluated the use of TAB004, an antibody that recognizes the tumor form of the glycoprotein MUC1 (tMUC1), to aid early detection of breast cancer. Our experimental approach was to follow tMUC1 from the tissue into circulation. We found that 95% of human breast cancer tissues across all subtypes stained positive for TAB004. In breast cancer cell lines, we showed that the amount of tMUC1 released from tumor cells is proportional to the cell's tMUC1 expression level. Finally, we showed that TAB004 can be used to assess circulating tMUC1 levels, which when monitored in the context of cancer immunoediting, can aid earlier diagnosis of breast cancer regardless of breast tissue density. In a blinded pilot study with banked serial samples, tMUC1 levels increased significantly up to 2 years before diagnosis. Inclusion of tMUC1 monitoring as part of a multi-modal screening strategy may lead to earlier stage diagnosis of women whose cancers are missed by mammography. Early detection of BC with regular screening increases the chances of survival. Screening for BC is predominantly done by mammography and clinical breast exams. While mammograms have resulted in early diagnosis for many women, 42-50% of cancers are missed in women with dense breasts due to lesion obscuration /2.Six – eight weeks old female, athymic nude mice purchased from Envigo Laboratories, Indianapolis, USA ((order code: 069(nu)/070(nu/+)) were injected with 5 × 10in vivo imaging system  to monitor tumor progression TAB004 antibody was conjugated with fluorophore Indocyanine green . Prior to imaging the mice, TAB-ICG in sterile saline was administered retro-orbitally (RO) in mice and imaging was conducted 24 hours post TAB-ICG injections. Mice bearing AU565 and HCC70 tumors were imaged at 21, 49 and 54 days post tumor inoculation.We used the IVIS Tumor fluorescence was analyzed using the Life Science Software Suite (Perkin Elmer) and region of interest were defined at tumor location. In parallel, the presence of tumor masses was assessed by palpation and recorded weekly.To assess tMUC1 expression, cells were harvested and stained with TAB004 conjugated to APCCy7 , or stained with APC-Cy7 mouse IgG1 isotype control . Data was collected using the BD LSR Fortessa Flow Cytometer (BD Biosciences) using filter for Cy5.5 (7-AAD) and Cy7 (TAB-004) and analyzed with FlowJo software .To measure tMUC1 protein levels in the total cell lysate of BC cell lines, we performed TAB004 Elisa. We loaded 15μg/ml of protein for each cell line. Total protein in BC cell lines was assessed by BCA assay  and followed manufacturer's recommendation to perform the assay.™ Personal Score Enzyme Immunoassay for tMUC1  is a sandwich solid phase enzyme-linked immunosorbent assay (ELISA). The standard used is the KCM cell line lysate. During this assay, the test sample is allowed to react first with the immobilized TAB004 antibody at 25° for one hour. The wells are washed with wash buffer to remove unbound antigen. The TAB004-HRP conjugate is then added for 1 hour at 25°C allowing it to react with the immobilized antigen which results in tMUC1 being sandwiched between the solid phase and the enzyme-linked antibodies. The wells are washed to remove unbound labeled antibodies. TMB reagent, which detects horseradish peroxidase (HRP) activity, was added and incubated for 20 minutes, resulting in the formation of a blue color. This color development was stopped with the addition of Stop Solution changing the color to yellow. The concentration of tMUC1 is directly proportional to the color intensity of the test sample. Absorbance was measured spectrophotometrically at 450 nm and used to calculate tMUC1 concentration to report the Agkura™ Personal score.The Agkurain vivo data (Figure Statistical analysis was done for the circulating tMUC1 data Figure  and the a Figure . Signifia Figure , the met


The evidence for systemic treatments for severe childhood eczema is limited and largely based on extrapolation of data from adult studies. Current therapies are often immunosuppressant and may be associated with both short- and long-term side effects. There is increasing in vitro and murine-model evidence for the role of IgE in the immunopathogenesis of atopic eczema. The aim of the study is to assess whether anti-IgE treatment  improves eczema, compared to placebo.The Atopic Dermatitis Anti-IgE Paediatric Trial (ADAPT) is a randomised, double-blind, placebo-controlled study assessing the role of anti-IgE in the management of severe paediatric eczema. Children with severe atopic eczema, with an objective SCORing Atopic Dermatitis (SCORAD) score of over 40 will be recruited. These children are candidates for systemic therapy, have failed systemic therapy or have experienced side effects from systemic therapy. Sixty-two patients aged between 4 and 19 years will receive anti-IgE for 6 months. The primary outcome measure will be the validated eczema score, the objective SCORAD at 24 weeks. This study has 90% power to detect a 33% relative reduction in SCORAD between active and placebo groups, with 5% significance.IgE may have a role to play in eczema, particularly in childhood. This forms the basis for the hypothesis that anti-IgE may be an effective treatment in this patient population.This will be the largest study to evaluate the efficacy of anti-IgE  versus placebo in children with severe eczema. The findings will help to clarify the role of anti-IgE as a potential treatment option in patients with severe childhood eczema.2010-020841-29. Assigned on 14 May 2010.European Clinical Trials Database (EudraCT) Number: ISRCTN15090567. Retrospectively assigned on 3 December 2014.ISRCTN Registry, Identifier: NCT02300701. First received 21 November 2014.ClinicalTrials.gov, Identifier: The online version of this article (doi:10.1186/s13063-017-1809-7) contains supplementary material, which is available to authorized users. Atopic eczema (AE) (or atopic dermatitis) is an inflammatory, chronically relapsing, pruritic skin disease. The prevalence in the United Kingdom (UK) is 16% for 6 to 7-year-olds (with 2.2% classed as severe) and 10.6% for 13 to 14-year-olds (with 1.5% classed as severe) .A detailed statistical analysis plan has been developed in conjunction with the protocol and is available [Reference from The management of AE necessitating systemic therapy is currently an area of intense interest. When systemic therapy is required in childhood AE, systemic immunosuppressants have been used with limited evidence, and in many cases as an unlicensed indication .There is increasing evidence for the role of IgE in the immunopathogenesis in AE. Omalizumab  is the only commercially available anti-IgE antibody. ADAPT is the largest randomised, double-blind, placebo-controlled study to date evaluating anti-IgE  in severe childhood AE. The aim is to definitively assess the role of anti-IgE as an alternative systemic treatment for AE.The ADAPT study received favourable ethical opinion on 7 July 2011 and local R&D approvals were obtained on 11 November 2014. Participant recruitment started on 20 November 2014 and is ongoing.Additional file 1: Figure S1.SPIRIT 2013 Checklist. (DOC 272 kb)Additional file 2: Figure S2.Schedule of enrolment, interventions, and assessments. (DOC 68 kb)Additional file 3: Figure S3.Gantt chart . (DOCX 16 kb)Additional file 4: Table S1.Primary and secondary outcomes (primary and secondary outcome measures of the study). (DOCX 20 kb)


IgG to galactose‐α‐1,3‐galactose  are highly abundant natural antibodies (Ab) in humans. α‐Gal‐specific IgE Ab cause a special form of meat allergy characterized by severe systemic reactions 3–7 h after consumption of red meat. We investigated 20 patients who experienced such reactions and characterized their α‐gal‐specific IgE and IgG responses in more detail.CAP. IgE reactivity to meat extract and bovine gamma globulin (BGG) was assessed by immunoblotting and ELISA, respectively. In some experiments, sera were pre‐incubated with α‐gal or protein G to deplete IgG Ab. α‐Gal‐specific IgG1–4 Ab in individuals with and without meat allergy were assessed by ELISA.α‐Gal‐specific IgE was determined by ImmunoBGG was the most frequently recognized meat protein. Binding of IgE and IgG to BGG was confirmed by ELISA and completely abolished after pre‐incubation with α‐gal. Neither the depletion of autologous α‐gal‐specific IgG Ab nor the addition of α‐gal‐specific IgG Ab from nonallergic individuals changed the IgE recognition of BGG of meat‐allergic patients. Meat‐allergic patients showed significantly higher α‐gal‐specific IgG1 and IgG3 Ab than nonallergic individuals, whereas the latter showed significantly higher levels of α‐gal‐specific IgG4 Ab.In immunoblots, BGG. Their enhanced α‐gal‐specific IgE levels are accompanied by high levels of α‐gal‐specific IgG1 devoid of IgE‐blocking activity. This subclass distribution is atypical for food allergies and distinct from natural α‐gal IgG responses in nonallergic individuals.Patients with delayed meat allergy display IgE and IgG Ab that selectively recognize the α‐gal epitope on  As for other food allergies, IgE responses to meat proteins have been detected in children experiencing hypersensitivity reactions upon consumption of beef We obtained sera from 20 Austrian patients collected in allergy clinics over the past 10 years who had experienced severe, delayed hypersensitivity reactions upon consumption of red meat. We confirmed their IgE reactivity to α‐gal and employed them to identify α‐gal‐carrying proteins in beef. Immunoblots with meat extract revealed BGG as most prominent protein recognized by these patients. As BGG has been shown to be recognized by IgE Ab from children with meat allergy Children sensitized to egg and/or milk but tolerating these foods have been shown to display higher levels of allergen‐specific IgG4 and higher IgG4/IgE ratios than those with allergic reactions n = 20) did not report any allergic symptoms and showed no allergen‐specific IgE Ab (data not shown). Patients with birch pollen‐related apple allergy (n = 20) were previously described A/l, ImmunoCAP, Thermo Fisher Scientific) and oral provocation tests Sera from two patients with immediate and 20 patients with delayed allergic reactions to meat were included. Characteristics of the latter are described in Table ® Emerald 300 Glycoprotein Gel and Blot Stain Kit (Thermo Fisher Scientific) according to the manufacturers’ protocol. For immunoblot experiments, the separated extract was transferred to a nitrocellulose membrane. After blocking, sera were incubated overnight at 4°C. Bound IgE was detected with 125I‐labelled anti‐human IgE antibody  and visualized by autoradiography. Buffer and sera of nonallergic donors served as negative controls.Beef was purchased at a local butcher's store, shock‐frozen with liquid nitrogen, reduced to small pieces with a mortar and stirred in PBS containing protease inhibitors  overnight at 4°C. Thereafter, the extract was centrifuged at 10 000 g for 30 min and the supernatant was filtered through filter paper , lyophilized and stored at −20°C. The protein concentration was determined by bicinchoninic acid assay . The extract (20 μg) was separated by 12% SDS‐PAGE under nonreducing conditions and stained with Coomassie brilliant blue (Bio‐Rad Laboratories). Detection of glycosylation was performed with the Pro‐Q1–4 detection) were incubated overnight at 4°C in duplicate. Buffer controls were done in sixfold replicates. After five washing steps, bound IgE and IgG Ab were determined with AP‐conjugated anti‐human IgE  and HRP‐conjugated goat anti‐human IgG , respectively. IgG1–4 Ab were analysed with anti‐human IgG1 (Sigma‐Aldrich), IgG2, IgG3 and IgG4  and visualized with HRP‐conjugated anti‐mouse IgG .Microtiter plates  were coated with either Galα1‐3Galβ1‐4GlcNAc‐BSA , BSA (5 μg/ml), BGG  or recombinant Mal d 1 . Optimal coating concentrations of each protein had been defined in preliminary experiments. Allergen‐coated plates were washed twice and saturated with 1% HSA in PBS/0.05% Tween‐20 for 6 h at room temperature. Subsequently, sera  for 4 h at room temperature. Sephadex G‐50 (Sigma‐Aldrich) served as negative control. After centrifugation, protein G Sepharose and Sephadex G‐50 were added to the supernatant and again incubated. This procedure was repeated four times. To destroy the binding of IgE Ab, sera were incubated for 30 min at 56°C.P < 0.05. Data from duplicate and triplicate experiments are shown as means with standard deviation (SD).Statistical differences were determined using SPSS 20.0 . Differences were considered statistically significant for Data on age , sex , total and specific IgE levels for beef, pork and chicken of 20 Austrian patients are summarized in Table To identify bovine proteins recognized by IgE Ab of patients with delayed meat allergy, we analysed sera from 19 patients in immunoblots Fig. . The O.DNext, we sought to evaluate whether BGG‐specific Ab of patients with delayed meat allergy were directed against α‐gal or an epitope consisting of both the carbohydrate and amino acid residues. For this purpose, sera from four meat‐allergic patients were incubated with titrated concentrations of BGG, BSA or α‐gal‐BSA and thereafter tested for IgE reactivity to BGG Fig. A. In addTo assess whether the high levels of α‐gal‐specific IgG Ab affected IgE binding to the carbohydrate, sera from seven meat‐allergic patients were either mock‐treated or depleted from IgG by repeated incubation with protein G. Thereafter, IgE binding to BGG was assessed by ELISA. Figure n = 18) with delayed meat allergy displayed significantly higher levels of α‐gal‐specific total IgG (P < 0.001), IgG1 (P = 0.002) and IgG3 (P = 0.003) than nonallergic individuals . α‐Gal‐specific IgG2 levels did not differ in both groups.We next compared the subclass distribution of α‐gal‐specific IgG Ab in meat‐allergic and nonallergic individuals. Patients . Moreover, α‐gal‐specific IgG2 and IgG3 values were significantly higher than α‐gal‐specific IgG4 values . For comparison, we tested sera from 20 patients with birch pollen‐related apple allergy P = 0.012, P < 0.001 and P < 0.001, respectively). Furthermore, Mal d 1‐specific IgG1 was significantly higher than IgG2 and IgG3 values  and α‐gal‐specific IgG (Fig. In addition, the inhibition experiments indicated that the epitope specificity of α‐gal‐specific IgG Ab differed in meat‐allergic and nonallergic individuals. In meat‐allergic patients, the IgG reactivity to BGG was concordantly blocked by α‐gal, whereas the inhibition pattern in nonallergic individuals showed high individual variability and was neither dose dependent nor significantly different from the negative control. Thus, BGG recognition by nonallergic individuals involved amino acid residues in addition to α‐gal, in contrast to the exclusive recognition of the glycan by meat‐allergic patients. We detected significantly higher levels of BGG‐specific (Finally, we were curious about the subclass distribution of α‐gal‐specific IgG Ab in patients with delayed meat allergy because α‐gal‐specific IgG1 and IgG2 and not IgG4 Ab were previously reported to accompany IgE Ab in α‐gal‐sensitized individuals In summary, patients with α‐gal‐mediated delayed meat allergy display α‐gal‐specific IgG responses atypical for food allergies and distinct from natural α‐gal responses in non‐meat‐allergic individuals. Although specific for the same epitope, the high levels of α‐gal‐specific IgG1 in meat‐allergic patients did not interfere with IgE binding to the carbohydrate. This observation may be referred to the low avidity of anticarbohydrate Ab or to the lower affinity of IgG compared to IgE Ab D.K. and B.B. designed the experiments; D.K., B.N. and C.K.; C.E., W.E., S.W. and S.V. provided patients′ samples; A.M. and H.J.A. provided experimental protocols; and D.K. and B.B. wrote the manuscript.The authors declare that they have no conflicts of interest.


This study used a pharmacological agonist of LTCC, Bay K8644, to elicit a sustained, sub-maximal contraction in VSM that mimics tone. Downstream signaling was investigated in order to determine what molecules are responsible for tone. Medial strips of swine carotid artery were stimulated with 100 nM Bay K8644 to induce a sustained level of force. Force and phosphorylation levels of myosin light chain (MLC), MAP kinase, MYPT1, CPI-17, and caldesmon were measured during Bay K8644 stimulation in the presence and absence of nifedipine, ML-7, U0126, bisindolylmaleimide (Bis), and H-1152. Nifedipine and ML-7 inhibited force and MLC phosphorylation in response to Bay K8644. Inhibition of Rho kinase (H-1152) but not PKC (Bis) inhibited Bay K8644 induced force. U0126 significantly increased Bay K8644-dependent force with no effect on MLC phosphorylation. Neither CPI-17 nor caldesmon phosphorylation were increased during the maintenance of sustained force. Our results suggest that force due to the influx of calcium through LTCCs is partially MLC phosphorylation-dependent but does not involve PKC or caldesmon. Interestingly, inhibition of MLC kinase (MLCK) and PKC significantly increased MAP kinase phosphorylation suggesting that MLCK and PKC may directly or indirectly inhibit MAP kinase activity during prolonged contractions induced by Bay K8544.Vascular smooth muscle (VSM) is unique in its ability to maintain an intrinsic level of contractile force, known as tone. Vascular tone is believed to arise from the constitutive activity of membrane-bound L-type Ca This leads to phosphorylation of the regulatory MLC at Ser19, interaction between actin and myosin filaments, and contraction  contraction is initiated by an increase in intracellular Caeticulum . Once intraction . While torganism .2+ sensitization or thin filament disinhibition. Ca2+ sensitization involves inhibition of the MLC phosphatase such that the cell can maintain levels of MLC phosphorylation without additional cytosolic Ca2+ or activation of MLC kinase. Rho kinase (ROCK), activated by GTP-bound RhoA, phosphorylates and inhibits the myosin-binding subunit of the MLC phosphatase, a protein known as MYPT1 (Vascular smooth muscle contractile force can be modulated by Caas MYPT1 . Additioas MYPT1 . Thin fi2+ concentration ([Ca2+]i) is higher than during a relaxed state but significantly lower than during a stimulated contractile event  appears to be particularly important in maintaining VSM tone  can respond to small changes in membrane potential and do not require an action potential to be opened , 5 D-glucose, and 0.02 EDTA at 4°C for no longer than 4 days. Previous studies have shown there is no loss of viability or contractile force with this storage  that contained all ingredients listed below except glucose. Arteries were immediately cleaned of excess connective tissue and fat, then stored in glucose-containing PSS composed of (in mM): 140 NaCl, 4.7 KCl, 1.2 MgSO storage . Prior to, a length within the length-range for optimal active force development , 10 mM dithiothreitol, and Protease Inhibitor Cocktail  using glass-glass homogenizers. Samples were stored at –80°C until subjected to SDS-polyacrylamide gel electrophoresis (PAGE). Protein content of homogenized samples was determined using a Lowry protein assay.For detection of CPI-17 and MAP kinase phosphorylation, 20 μg of each sample was loaded onto a 14% polyacrylamide separating gel with a 4% stacking gel. For detection of MYPT1 and caldesmon phosphorylation, 20 μg of each sample was loaded onto an 8% polyacrylamide separating gel with a 4% stacking gel. Gel electrophoresis was carried out at 200 V at room temperature for 1 h or until the dye front reached the bottom of the gel. Proteins were then transferred to a nitrocellulose membrane at 100 V for 1 h in a chilled buffer tank. Membranes were blocked in a 1:1 solution of Odyssey Blocking Buffer  and phosphate buffered saline  at room temperature for 1 h then incubated in primary antibodies diluted in a 1:1 solution of Odyssey Blocking Buffer and PBS overnight at 4°C. Membranes were washed in PBS with 0.1% Tween five times for 5 min each. Fluorescent secondary antibodies  were used at a 1:10,000 dilution in a 1:1 solution of Odyssey Blocking Buffer and PBS. Membranes were incubated in secondary antibody at room temperature for 45 min followed by five washes in PBS with 0.1% Tween for 5 min each.Membranes were imaged using a Licor FC camera system and optical densitometry of the bands was determined using Image Studio software .Thawed and dehydrated tissues were placed in sample buffer containing 8 M urea, 18.5 mM Tris (pH 8.6), 20.4 mM glycine, 0.5 M dithiothreitol, 0.05% saturated sucrose, 0.2% bromophenol blue, 0.4 M EDTA, and Protease Inhibitor Cocktail . Samples were gently rocked at room temperature for approximately 2 h during which the MLC were solubilized, then stored at 4°C until subjected to gel electrophoresis. A gel was prepared containing 18.5 % glycerol, 30% acrylamide with bisacrylamide (29:1), Tris (pH 8.6), glycine, and water. The gel was subjected to pre-electrophoresis at 400 V for 1 h. Samples were then loaded onto the gel, subjected to electrophoresis at 400 V for 90 min, then transferred to a nitrocellulose membrane at 25 V for 1 h. Western blotting was carried out as described above for SDS-PAGE.Thr185/Tyr187 , MLC , phosphorylated MLCSer19 , CPI-17 , phosphorylated CPI-17Thr38 , MYPT1 , phosphorylated MYPT1Thr850 , caldesmon , and phosphorylated caldesmonSer789 . Electrophoretic reagents were purchased from Bio-Rad Laboratories . All other chemicals were analytical grade or better and purchased from Fisher Scientific .Drugs and inhibitors were dissolved either in deionized water or dimethyl sulfoxide (DMSO) according to manufacturers’ instructions. Bay K8644 and ML-7 were obtained from Tocris . Nifedipine was obtained from Sigma . H-1152, U0126, and Bis, were ordered from Calbiochem . The following antibodies were used for Western blotting: p42/44 MAPK , phosphorylated p42/44 MAPKp-value less than 0.05 was considered significant.Results are represented as mean ± SEM with n representing the number of arteries. Statistical analysis was performed using One-way ANOVA with the Holm–Sidak Method of comparing experimental groups to Bay K8644-stimulated tissue. Time course of force development was analyzed with a One Way Repeated Measures ANOVA. A Figure 1. To study the potential role of various signaling proteins in VSM contraction, inhibitors against LTCC (nifedipine), MLC kinase (ML-7), PKC (Bis), MAP kinase kinase (U0126), and Rho kinase (H-1152) were added 20 min prior to stimulation with Bay K8644. Pre-treatment with nifedipine  or ML-7  prevented Bay K8644-induced force development. Inhibition of ROCK by H-1152  also prevented force development suggesting that ROCK activity is required for initiation of membrane depolarization-induced force. Inhibition of PKC with Bis did not significantly alter Bay K8644-stimulated force  suggesting that PKC activity is not required for force initiation or maintenance in response to opening LTCC. Pre-treatment with the MAP kinase kinase inhibitor U0126 produced greater sustained force than Bay K8644 stimulation without inhibitor  however, the rate of rise was the same . The finding that inhibition of MAP kinase kinase increases sustained force suggests that MAP kinase may have an inhibitory role in contraction, perhaps to keep the tissue from unregulated maximal contractions. As can be seen at zero time in the panels in Figure 2, basal, unstimulated force was approximately 5–10% of the maximum response to 110 mM KCl.The swine carotid artery exhibits little spontaneous tone therefore a pharmacological approach was used to mimic tone in this tissue. A dose-response curve of VSM contraction to the LTCC agonist Bay K8644  was used to determine the concentration(s) that produced sub-maximal force (data not shown). A time course experiment revealed that Bay K8644-induced force increased steadily for the first 60 min and very slowly over the next 60 min. The optimal concentration for sustained, sub-maximal force was determined to be 100 nM. The magnitude of force development was normalized to the maximum force produced by a tissue strip in response to 110 mM KCl PSS. Bay K8644-induced force was approximately 60–70% of maximal KCl-stimulated force, as shown in Figure 3 shows the force developed in response to Bay K8644 alone and in the presence of the various inhibitors at the final time point, 120 min after Bay K8644 stimulation. Consistent with the time course data presented in Figure 2, inhibition of LTCC, MLC kinase, and ROCK inhibited Bay K8644-induced force whereas inhibition of MAP kinase kinase increased force in response to Bay K8644. The statistical analysis comparing force at this single time point found the increase during MAP kinase kinase inhibition to be significant.19 was assessed by urea/glycerol-PAGE. Urea/glycerol-PAGE uses a high concentration of urea in the sample buffer to solubilize MLC and separate them from the larger heavy chain molecules. The glycerol gel is suitable for separating different phosphorylation states of the same protein. Phosphorylated and non-phosphorylated MLC have different electrophoretic mobilities within the gel and appear as discrete bands. A representative Western blot is shown in Figure 4A. Figure 4B shows quantitative results of several such Western blots and demonstrates that Bay K8644 significantly increased Ser19 MLC phosphorylation. Nifedipine and ML-7 treatment prevented any increase in Ser19 MLC phosphorylation above basal levels, as expected. The MAP kinase kinase inhibitor U0126 had no effect on Bay K8644-stimulated Ser19 MLC phosphorylation levels in contrast to significantly increased Bay K8644-induced force. The PKC and ROCK inhibitors  appeared to reduce the Bay K8644-stimulated MLC phosphorylation to some extent, but failed to bring the phosphorylation level back to the baseline. Due to the large variation in data, it is difficult to draw any meaningful correlation between the phosphorylation level and force production.Myosin light chain (MLC) phosphorylation at SerFigure 5A shows a representative Western blot of MAP kinase phosphorylation in response to Bay K8644 without inhibitor and in the presence of nifedipine, ML-7, Bis, U0126 or H-1152. Quantitation of several such blots is shown in Figure 5B. Stimulation of the tissues with Bay K8644 did not significantly increase MAP kinase phosphorylation above basal levels, represented by the dashed line. The effectiveness of U0126 to inhibit the MAP kinase kinase and therefore MAP kinase phosphorylation is demonstrated by the nearly complete abolishment of MAP kinase phosphorylation. Although Bay K8644 alone did not increase MAP kinase phosphorylation, the addition of either Bis to inhibit PKC or the addition of ML-7 to inhibit MLC kinase significantly increased MAP kinase phosphorylation levels. This was unexpected as PKC is thought to activate MAP kinase in this tissue and MLC kinase has no known role in the activation of MAP kinase. Inhibition of LTCC with nifedipine had no effect on MAP kinase phosphorylation.Activity of p42/44 MAP kinase was assessed by comparing phosphorylation levels to basal unstimulated control values. Figure 6A shows a representative Western blot of MYPT1 phosphorylation in response to stimulation with Bay K8644 without inhibitor and in the presence of nifedipine, ML-7, Bis, U0126, or H-1152. Figure 6B shows the quantitation of several such blots. The level of MYPT1 phosphorylation decreased from basal levels (dashed line) in response to Bay K8644 stimulation without inhibitor. The inclusion of ML-7, Bis, or U0126 had no effect on MYPT1 phosphorylation as compared to Bay K8644 without inhibitor. The ROCK inhibitor H-1152 abolished MYPT1 phosphorylation as expected as ROCK is the primary kinase that catalyzes MYPT1 phosphorylation. The level of MYPT1 phosphorylation in the presence of nifedipine was the same as that under baseline conditions , suggesting that MYPT1 dephosphorylation may be Ca2+-dependent consistent with the decrease in MYPT1 phosphorylation in response to Bay K8644 without inhibitor.To determine the possible involvement of the MLC phosphatase in sustaining VSM force, we measured phosphorylation of the myosin binding subunit, MYPT1. Phosphorylation of MYPT1 inhibits MLC phosphatase activity and allows for maintained MLC phosphorylation and force. 2+ sensitivity. Caldesmon is a thin filament-associated inhibitory protein that is phosphorylated by MAP kinase and potentially PKC which reverses its’ inhibitory activity  or MLC phosphorylation . Phorbol-dibutyrate (PDBu) was used as a positive control for CPI-17 phosphorylation as it directly activates PKC. PDBu produced a significant increase in CPI-17 phosphorylation demonstrating that the total lack of CPI-17 phosphorylation was not an artifact due to electrophoresis or Western blotting.The MLC phosphatase inhibitor CPI-17 is phosphorylated by PKC. CPI-17 phosphorylation is usually increased during contraction of VSM as it is one mechanism that increases myofilament Caactivity . Figure 789 . The level of phosphorylation was the same in basal unstimulated tissues as it was in Bay K8644-stimulated tissues with or without inhibitors. This suggests that thin filament disinhibition by caldesmon phosphorylation is not involved in sustained LTCC-mediated force in VSM. Inhibition of MAP kinase with U0126 had no effect on caldesmon phosphorylation.Bay K8644 stimulation alone or in the presence of the various kinase inhibitors had no effect on phosphorylation of the thin filament-associated protein caldesmon at SerThe purpose of this study was to investigate intracellular signaling involved in sustained, sub-maximal force induced by the LTCC agonist, Bay K8644. Pharmacological inhibitors of LTCC, MLC kinase, MAP kinase, PKC, and ROCK were used in order to determine their involvement in sustained force.50) that is ∼10 times lower, however, H-1152 also inhibits CaMKII, PKA, PKC, and PKG, but with IC50 values > 30 times higher than that for Rho-kinase , however, it is able to inhibit other enzymes. It has effects on cAMP-dependent protein kinases, PKC, and calcium phosphodiesterase, although the binding affinity of ML-7 for smooth muscle MLCK is ∼100 times higher than that for the other enzymes . Althougo-kinase . H-1152 o-kinase . Bis  but failed to enhance MAP kinase phosphorylation . This could be explained by assuming that MAP kinase phosphorylation is calcium dependent.Inhibition of PKC with Bis or MLC kinase with ML-7 significantly increased MAP kinase phosphorylation. The Bis effect has been previously observed in our laboratory . The sig789 relieving caldesmon’s inhibitory effect on actin-activated myosin ATPase activity  contraction induced by Bay K8644, other pathways could have come into play, notably the pathways related to the regulation of actin cytoskeletal function and the muscle’s ability to maintain force through non-cross-bridge mechanisms . Both in2+ entry through LTCC activates the MLC kinase resulting in MLC phosphorylation and force. In addition a constitutively active ROCK and potentially ROCK that is activated by Ca2+ influx via LTCC (2+-dependent. PKC catalyzed CPI-17 phosphorylation and the resultant inhibition of MLC phosphatase does not occur in response to LTCC opening by Bay K8644. Inhibition of MLCK increases MAP kinase phosphorylation suggesting that MLCK may have an inhibitory or suppressive effect on MAP kinase activity. Moreover, inhibition of MAP kinase enhances Bay K8644-dependent force. Although caldesmon phosphorylation has been shown to be increased by both membrane depolarization and agonist activation, stimulation with Bay K8644 does not increase caldesmon phosphorylation. These novel findings, especially the relationship between MLCK and MAP kinase, if confirmed, may lead to a better understanding of VSM function.In this study we have shown that opening of LTCC in response to 100 nM Bay K8644 produced a sub-maximal sustained contraction that was nifedipine-sensitive. Taken together our data suggest that Cavia LTCC , sustainHE, RM: Made substantial contributions to the conception and design of the work, the acquisition, analysis, abd interpretation of data for the work. HE, RM: Drafted the work and revised it critically for important intellectual content. CS: Made substantial contribution to interpretation of data and revising the manuscript. HE, CS: Provided final approval of the version to be published. HE, CS: Agree to be accountable for all aspects of the work and ensure that questions related to the accuracy or integrity of any part of the work can be appropriately investigated and resolved.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.The handling Editor declared a shared affiliation, though no other collaboration, with one of the authors CYS and states that the process nevertheless met the standards of a fair and objective review.


Clostridium botulinum produce botulinum toxin complex (TC), which is comprised of botulinum neurotoxin, nontoxic nonhemagglutinin, and hemagglutinins (HAs). The TC is capable of aggregating equine erythrocytes via interaction between one of the HAs, namely HA-33, and sugar chains on the cell surface. This hemagglutination is inhibited by specific sugars. In this data article, we used four TCs from serotype C and D strains. The hemagglutination-inhibiting effects of 18 sugars and 8 glycoproteins were studied. The purified TC was mixed with the sugar to enable binding of the sugar to the TC; then, the erythrocytes were added to the mixture. Specific binding between the sugar and TC resulted in inhibition of cell aggregation. Here, data illustrating the inhibitory effects of various sugars and glycoproteins against hemagglutination induced by TC of C. botulinum serotypes C and D are presented.Serotype C and D of  Specifications TableValue of the data1.C. botulinum.The data presented illustrate the specific inhibitory effect exerted by 18 sugars and 8 glycoproteins on hemagglutination by the toxin complex (TC) of serotypes C and D of 2.C. botulinum serotypes C and D strains may be distinguished via the inhibitory profiles exerted by the present sugars and glycoproteins.The results in this article indicate that two modes of hemagglutination mediated by the TCs of 3.C. botulinum serotypes C and D.The data in this article should be useful for elucidation of the mechanism underlying the recognition of sugars by the TCs of 1C. botulinum serotype C strain Stockholm (C-St), Yoichi (C-Yoichi), D strain CB-16 (D-CB16), and 1873 (D-1873).Here, we present data illustrating the inhibitory effects of 18 sugars and 8 glycoproteins  or galactose moieties (of C-Yoichi and D-1873) at their termini L-TC was purified from the culture of 33.1SDS-PAGE was performed as described by Laemmli 3.2g, 5 min, 4 °C)  and rinsed with 0.15 M phosphate buffer, pH 7.0. Suspension of the erythrocytes (1%) was prepared with the same buffer. Erythrocytes were treated with Clostridium perfringens neuraminidase (Sigma-Aldrich). Neuraminidase (0.5 U) was added to a 10% equine erythrocyte suspension, and the mix was incubated at 37 °C for 1 h. Treated erythrocytes were collected, rinsed, and suspended in 0.15 M phosphate buffer (pH 7.0) to create a 1% suspension.Erythrocytes in defibrinated equine blood  were collected by centrifugation . Samples (35 µl) of each preparation (100 µg/ml) were diluted in serial two-fold steps with 0.15 M phosphate buffer (pH 7.0) and mixed with an equal volume of a 1% equine erythrocyte suspension. After incubation at 20–25 °C for 2 h, the reciprocal hemagglutination titer was determined as 2d-glucose, d-mannose, galactose, N-acetyl-d-glucosamine, methyl-α-d-mannoside, l-fucose, xylose, fructose, rhamnose, N-acetyl-d-galactosamine, sucrose, N-acetylneuraminic acid, maltose, cellobiose, lactose, chitin oligomers, N-acetyllactosamine, and chitobiose) and glycoproteins  were used in this study. The L-TC solution  was incubated with an equal volume of sugar or glycoprotein solution , at room temperature, for 1 h. Thereafter, the solution was incubated with 20 µl of equine erythrocyte suspension (1%) at 37 °C for 1 h.For the inhibitory test, sugars (


Albedo—a primary control on surface melt—varies considerably across the Greenland Ice Sheet yet the specific surface types that comprise its dark zone remain unquantified. Here we use UAV imagery to attribute seven distinct surface types to observed albedo along a 25 km transect dissecting the western, ablating sector of the ice sheet. Our results demonstrate that distributed surface impurities—an admixture of dust, black carbon and pigmented algae—explain 73% of the observed spatial variability in albedo and are responsible for the dark zone itself. Crevassing and supraglacial water also drive albedo reduction but due to their limited extent, explain just 12 and 15% of the observed variability respectively. Cryoconite, concentrated in large holes or fluvial deposits, is the darkest surface type but accounts for <1% of the area and has minimal impact. We propose that the ongoing emergence and dispersal of distributed impurities, amplified by enhanced ablation and biological activity, will drive future expansion of Greenland's dark zone. The surface types that comprise the dark zone of the Greenland Ice Sheet, an area of bare ice with low albedo, are unknown. Here, the authors use UAV imagery to show that, during the melt-season, biologically active surface impurities are responsible for spatial albedo patterns and the dark zone itself. The dominant energy source for snow and ice melt is direct solar shortwave radiation, the absorption and reflection of which is predominantly modulated by surface albedo7. Accurately constraining spatiotemporal patterns of albedo across the ice sheet is hence fundamental to understanding and predicting surface melt and runoff along with their impact on ice sheet flow dynamics and sea-level rise. A conspicuous feature of Greenland’s ablation area is its dark zone, an area of bare ice with particularly low albedo that appears across the west and southwest sectors of the ice sheet each summer10. At the Arctic Circle, in the vicinity of the Kangerlussuaq (K-) sector, the dark zone extends between 20 and 75 km from the land-terminating margin where Moderate Resolution Imaging Spectroradiometer (MODIS) data indicate a regional albedo minimum of ~0.348. From 2000 to 2012, the spatial extent of the dark zone increased by 12% but also exhibited considerable interannual variability12. The extent of the dark zone is weakly positively correlated with air temperature and negatively correlated with solar radiation during June, July and August (JJA)12. This suggests that the ongoing albedo decrease observed during the melt season is not simply driven by melting of the winter snowpack to reveal the darker bare ice surface beneath, but, following exposure, there are changes in the nature of the bare ice itself12. However, the surface characteristics of the dark zone remain unquantified because the spatial resolution of satellite imagery is insufficient to fully resolve the specific surface types that comprise it, and how these surfaces evolve through time, distinguishing the dark zone from brighter ice surfaces adjacent to it.The Greenland Ice Sheet has become the largest cryospheric contributor to global sea-level rise predominantly through increased surface melt and runoff, which accounts for over half of its mass loss since 199115. These include features such as crevasses, fractures and foliations17; supraglacial hydrological features, including streams, rivers, ponds and lakes19; snow patches and fracture cornices; cryoconite, concentrated in holes or in supraglacial fluvial deposits21; microbes and their humic by-products24; mineral dust and aerosols from outcropping or contemporary aeolian deposition including black carbon from wildfires26 and other aerosols. While the highest resolution optical satellite imagery currently available has facilitated the examination of crevasse fields16 and surficial hydrology19, a quantitative assessment of the specific ice surface types that comprise the dark zone, and how they combine to yield observed albedo patterns across the ablation zone of the ice sheet, has yet to be made.Previous field-based, in situ observations indicate that western Greenland’s ablation zone is characterized by highly variable non-ice constituents and surface structuresHere, we utilize high-resolution (15 cm pixel size) imagery acquired from an unmanned aerial vehicle (UAV) to characterize the specific ice surfaces across the dark zone and determine their impact on the mesoscale (1–10 km) albedo distribution during peak melt season, as represented by the MODIS albedo product, MOD10A1. On 8 August 2014, a fixed-wing UAV equipped with a digital camera and upward and downward facing pyranometers was deployed from a field camp based in the vicinity of the K-transect, S6 automated weather station (AWS) on a 25 km east-west transect dissecting the dark zone  clean ice, (ii) ice containing uniformly distributed impurities, (iii) deep water, (iv) shallow water, (v) cryoconite either in holes or fluvial deposits, (vi) crevasses and (vii) snow Fig. . Distinc21. Coincident field measurements, made during UAV image acquisition, indicate that the cryoconite holes observed in our study were well developed. The implication is that once they have attained equilibrium depth, they cease to absorb additional energy (which would make them deeper) compared to surrounding ice and hence are effectively neutralized from the effects of incoming solar radiation. For these reasons, we argue that smaller cryoconite holes had a minimal net impact on MODIS-derived albedo compared to the ice surface surrounding them.Cryoconite is commonly found in holes but also in fluvial deposits near supraglacial streams and lakes Fig. . In this19 who found that surface water accounted for 1.4% of a ~5000 km2 bare ice area in the K-sector of the ice sheet. At 28 km, a small, 0.83 km2 supraglacial lake covers 32.8% of the segment , a braided meltwater channel network with a fractional area of 5.3%, intersects the transect Figs.  and 5b, ent Fig. . Crevassent Fig. . Up-glacent Fig. . Remnantα) = 0.27), with the remaining 5–20% consisting of predominately clean ice (mean α = 0.55)  Fig. . Applicaα = 0.19–0.26). The segment of the transect that corresponds to a large braided channel network at 23 km has an albedo of 0.28 : interpreted as a shoreline exposed when the lake level dropped  itself is offset by the brightness of the surrounding ice surface adjacent to it yielding minimal change in the net albedo (MOD10A1) signature.Locally, defined at the scale of a single MODIS pixel, supraglacial water, contained in both lakes and channels, has a distinct impact on albedo due to its low albedo   Fig. , with crea) Fig. . Elsewheea) Fig. 28. Cathl32. Although this mechanism means that the cryoconite hole has a very low albedo value (mean α = 0.10) when observed from directly above, the hole occupies a relatively small area and is effectively hidden at non-zenith solar illumination resulting in an increase in mesoscale albedo20. Furthermore, cryoconite holes are often covered by an ice lid, caused by the refreezing of water that has filled the hole during negative net radiation conditions31. While thin frozen lids may undergo partial or complete ablation during the day, their higher albedo acts to further moderate the impact of cryoconite that they cover and render the holes indistinguishable from the adjacent ice surface (much to the dismay of many a field campaigner with sodden feet).Increases in the fractional area of cryoconite, either in large holes or fluvial deposits, are not particularly associated with mesoscale albedo reduction across the survey transect, and surprisingly, the lowest concentration of cryoconite is actually observed within the dark zone itself Fig. . Cryocon22 report that samples of distributed impurities consist of an abundance of ice algae , cryoconite holes (<0.5%) and crevasses (<0.2%). Instead, it appears that ice containing uniformly distributed impurities draped over a relatively flat surface are the primary agent responsible for the low (MOD10A1) albedo values observed during the melt season Fig. . Near thgae Fig. , which a37. Moreover, a relatively small expansion in the spatial extent of surface water would have a disproportionate impact on mesoscale albedo and further amplify ablation. Melt rates at the base of supraglacial lakes and water bodies are double that of bare ice surfaces due to enhanced shortwave radiation absorption39. Atmospheric warming has been shown to increase the spatial extent and duration of ponded supraglacial water41. During years with higher summer temperatures, such as 2007, 2010 and 2012, supraglacial lakes formed earlier in the season and occupied a 40% larger area than in cooler summers40. It follows that increased storage of water in supraglacial lakes will play an important role in net albedo reduction across an expanding bare ice area in future.While our results attest that the variation in the fractional area of supraglacial lakes, streams, crevasses and cryoconite do not significantly affect mesoscale albedo, they still play a secondary role in determining interannual and seasonal albedo variability. For example, supraglacial water may act to consolidate or distribute sediment and impurities across the ice sheet surface43. In response to increased surface melt, GPS observations by van de Wal et al.44 report reduced net flow over the marginal zone of the K-transect, whereas Doyle et al.45 report persistent ice flow acceleration above the equilibrium line up to 140 km from the ice sheet margin in this same sector. Recent modelling46 and observations47 of new crevasses forming over 160 km from the western margin reveals that the ice sheet interior is also more dynamically sensitive to transient stress perturbations originating from downstream than a previous steady-state model suggests48. Regardless of ice dynamics, inland migration of the equilibrium line caused by atmospheric warming will drive increased bare ice extent, further exposing existing crevasses that were formerly snow and firn covered. Hence, surface crevasse extent will likely expand in future, resulting in mesoscale albedo reduction and enhanced surface absorption of incoming energy available for melt.Our analysis also demonstrates that crevasses reduce local (0.1–1 km) albedo, and hence any increase in crevasse extent will impact on mesoscale albedo patterns. Crevasses form due to localized concentration of tensile stresses which, due to highly variable subglacial conditions and longitudinal stress coupling, are spatially and temporally variable across the Greenland Ice Sheet49 showed that 53% of plot-scale (0.01–0.5 m) variation in albedo was correlated with the growth of cryoconite holes, and Chandler et al.21 report that the gradual seasonal reduction in albedo also correlates with an increase in cryoconite hole size and number. An increase in the extent of cryoconite holes may be caused by longer and warmer ablation seasons, which would increase the heat energy to the walls and base of the hole, leading to further melting and hole expansion31. On the other hand, an increase of meltwater may promote aggregation of distributed impurities and could have a surface cleaning effect, potentially raising the albedo of the surrounding bare ice31. The growth and development of cryoconite holes on mesoscale albedo is hence complex and still somewhat ambiguous.Finally, future spatial expansion of cryoconite does have the potential to significantly impact surface albedo. Hodson et al.50. In contrast, few studies have investigated the albedo of the bare ice surface types that characterize the ablation zone and they have commonly been treated as temporal and spatial constants in surface melt models53.In this study, we characterized the spatial variability of surface types across the western ablating margin of the ice sheet towards the end of the melt season when bare ice surfaces are most apparent. However, for much of the year (September to May), the ice sheet is snow-covered and it is likely that snow grain size and impurity concentration govern mesoscale albedo patterns across the K-transect and elsewhere during this period. Likewise, early melt-season albedo patterns are primarily governed by the relative proportions of snow and ice extent. Accurately determining snow melt and the timing of bare ice exposure has therefore been a priority for surface mass and energy balance models and the theoretical determinants of snow albedo and melt are relatively well established55 has motivated a new generation of surface energy balance models that assimilate spatial patterns of albedo derived from MODIS data57. Across our 25 km survey transect, the MODIS-derived surface albedo pattern is dominated by variations in the extent of uniformly distributed impurities, a result that contradicts previous research attributing it to an increased occurrence of supraglacial water18. The source, processes and drivers of distributed impurities are yet to be unequivocally established, with some studies indicating a wind-blown origin, others revealing that they are derived from melt-out of englacial dust11. Recent research has promoted the concept of bioalbedo, which argues that the melt-out and release of surface particulates and nutrients fertilizes pigmented ice surface algae, which drives albedo reduction over the duration of the melt season22. Further research though is required to determine how these factors combine to increase the spatial extent and concentration of pigmented surface algae, and their interaction with the availability of in situ and aeolian-derived nutrients, changing atmospheric forcing and enhanced ice melt and runoff.The observed spatiotemporal variability in albedo across the ablation zone58. MOD10A1 is gridded in a sinusoidal map projection and has a resolution of ~500 × 500 m or 0.25 km2. The value of each pixel represents the best single albedo observation in the day based on cloud cover and viewing and illumination angles59. We estimated that MOD10A1 has a root mean square difference (RMSD) of 7.0% in comparison to albedo measured by CNR1 or CNR4 thermopile pyranometers at the PROMICE/GAP automatic weather stations KAN-L, KAN-M and KAN-U between 2009 to 201460. This compares well to Stroeve et al.59 who estimated an RMSD of 6.7%.Albedo patterns were determined from a MOD10A1 C6 broadband  albedo product from the 8 August 2014 and available from the National Snow and Ice Data Center (NSIDC)62 The UAV has a 2.1 m wingspan and is powered by a 10 Ah, 16.8 V LiPo battery pack which, with a total weight of 4 kg, yields a 1 h endurance and 60 km range. The autonomous control system is based around an Arduino navigation and flight computer updated in real-time by a 10 Hz data stream comprising of a GPS, magnetometer, barometer and accelerometer. These data are logged along with a timestamp for each activation of the digital camera shutter which automatically triggers when a horizontal displacement threshold is exceeded. The UAV was hand launched on 8 August 2014 from a base camp at 67.08°N, 49.40°W, located at the site of the Institute for Marine and Atmospheric Research (IMAU), University of Utrecht S6 automatic weather station. It was pre-programmed to carry out a 25 km survey across the Kangerlussuaq sector of the western Greenland Ice Sheet  yielding an image footprint of ~525 × 350 m during the autonomous sortie. The width of each image is approximately similar to the pixel footprint of MODIS. The camera was preset with a fixed shutter speed of 1/1000 s, ISO 100 and F-stop of 8, and triggered every 35 m to provide a 90% forward image overlap. The relatively fast shutter speed minimizes image blur while the low ISO and F-number ensures maximum image quality where even the brightest surfaces do not saturate the image. The camera was set to record the images in RAW format, an image format that contains minimally processed data from the camera’s sensor. During the survey, ~2000 RAW images were acquired at the camera’s maximum (4912 × 3264 pixels) resolution which, once the images were corrected for barrel, or geometric, distortion, equates to a ground sampling distance of ~11 cm.http://www.agisoft.com/) following the processing sequence described by Ryan et al.60 The images were georeferenced by providing latitude, longitude and altitude data recorded by the flight controller. The orthomosaic was produced in the software’s ‘mosaic’ mode, meaning that pixels in the centre of the images were preferentially used to provide the output pixel value. The orthomosaic and DEM were nearest neighbour resampled to a ground resolution of 15 cm and 50 cm, respectively. We divided the orthomosaic into sixty 0.25 km2 segments and each segment was assigned a MOD10A1 value.The R(ed)/G(reen)/B(lue) images were used to produce an orthomosaic and DEM using Agisoft PhotoScan Pro  than for cryoconite, ice containing distributed impurities, clean ice and snow (90%). The classification of supraglacial water is relatively accurate because water has low reflectance in the red band and forms a unique cluster in the feature space Fig. . The per% than foα) of each surface type was obtained from the UAV digital imagery. An explicit description of this method can be found in Ryan et al.14, but we briefly summarize it here. Firstly, the RAW digital numbers of each proprietary Sony RAW image were preserved by converting to a 16-bit TIFF image using dcraw (http://cybercom.net/~dcoffin/dcraw/). A vignette correction mask was universally applied to compensate for image and lens distortion due to edge effects which were as high as 17.6% at the corners of some images. The correction mask was calculated from the mean vignette of all images acquired at nadir during the survey period. Barrel distortion was corrected using ImageMagick (http://www.imagemagick.org/), which utilized the coefficients stored in the image’s ancillary metadata also known as exchangeable image file format data.An estimate for the albedo (R2 = 0.96). The ratio of reflected radiation recorded by the camera and the downward radiation estimated from the calibration curve enabled the illumination-corrected reflectance of each pixel to be defined.We then corrected the images for changing illumination conditions during the survey using downward irradiance measured by a ground-based upward facing Apogee SP-110 pyranometer. To do this, images of a 25 × 25 cm Teflon white reference target were acquired every 10 min using the UAV digital camera from the ground. The relationship between the mean RGB DNs of the white reference target and the downward irradiance recorded by the upward facing pyranometer were used to construct a calibration curve using a linear least squares regression  albedo data are available from the National Snow and Ice Data Center (NSIDC) at


ANG), a member of the Ribonuclease A superfamily  are known to be associated with Amyotrophic Lateral Sclerosis   and Parkinson’s Disease (PD). In our previous studies we have shown that ANG is expressed in neurons during neuro-ectodermal differentiation, and that it has both neurotrophic and neuroprotective functions. In addition, in an extensive study on selective ANG-ALS variants we correlated the structural changes to the effects on neuronal survival and the ability to induce stress granules in neuronal cell lines. Furthermore, we have established that ANG-ALS variants which affect the structure of the catalytic site and either decrease or increase the RNase activity affect neuronal survival. Neuronal cell lines expressing the ANG-ALS variants also lack the ability to form stress granules. Here, we report a detailed experimental structural study on eleven new ANG-PD/ALS variants which will have implications in understanding the molecular basis underlying their role in PD and ALS.Mutations in Angiogenin ( Angiogenin (ANG), a 14-kDa basic protein is an angiogenic ribonuclease and member of the bovine pancreatic ribonuclease (RNase A) family 124673ANG plays important roles in various physiological and pathological processes. For a recent comprehensive review on ANG, see Sheng and XuThe mechanism of ANG action is not yet fully understood, but appears to involve different pathways, including receptor binding on endothelial cells11ANG has been shown to be a neurotropic and neuroprotective factor16et al.18ANG in ALS, both familial and sporadic. Since these first reports, several other studies worldwide have also identified ANG mutations in ALS patients202122232425262728213032Apart from ANG’s role in neovascularization, While human ANG is also shown to be upregulated especially in prostate cancers and glioblastomas373839In order to elucidate the role of ANG mutations in ALS, we have shown previously that some ANG variants have significant reduction in RNase activity which is correlated with a reduction in cell proliferation and angiogenic activitiesWe have determined the crystal structure of eleven ANG-PD/ALS variants H13R, K40R, K54R, K60E, Q77P, T80S, R95Q, F100I, V103I, H114R, R121C  and 4. W1 substrate binding site involved in phosphodiester bond cleavage; a B1 site for binding the pyrimidine whose ribose donates its 3′ oxygen to the scissile bond; a B2 site that preferentially binds a purine ring on the opposite side of the scissile bond; and additional sites for peripheral bases and phosphatesAs reported previously, the active site of ANG consists of several sub-sites : a P1 sub-site) correspond to the catalytic triad. Replacements of H13 or H114 by Ala4, 104 and 2 × 103, respectively. These early findings established that the roles of the three ANG residues are similar to those of their counterparts in RNase A.ANG residues H13, K40 and H114  in the native structure and makes direct interaction with D116. However, in the variant structure  the hydrogen-bond (2.9 Å) observed between Q77 and R21 (part of helix α2) is lost  thus creγ1 atoms of T44 and T8045γ1 atom of T80 overlaps with the equivalent position of Oγ atom of S80 in the variant structure  is obstructed by residue Q117 (part of the pyrimidine recognition site)in vitro’ model systems. Meanwhile the observations made from the present study will provide the structural basis in understanding the biological roles for these variants which have been implicated in PD and ALS.In summary, we have elucidated altered molecular interactions that underpin the abrogated catalytic efficiency or an increase in RNase activity (directly linked to ANG function) of eleven new variants of ANG implicated in PD and ALS using high resolution X-ray diffraction data. In our previous studies616et al.E. coli codon bias was inserted into pET-22b(+)(Novagen) for expression of the Met-(−1) form of the protein. The QuikChange site-directed mutagenesis method was used to introduce mutations. After DNA sequencing  to confirm the presence of the mutations, mutant plasmids were used to transform E. coli BL21(DE3) cells. Bacterial cells were grown in Terrific Broth at 37 °C to an OD600 of 0.5–0.6, after which expression was induced by addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM and incubation was continued for ~3 hours before harvesting. The target proteins from inclusion bodies were extracted, refolded and purified by SP-Sepharose chromatography followed by C4 reversed-phase HPLC−1cm−1 calculated by the method described by Pace et al.Wild-type and all ANG PD/ALS variants were prepared as reported in Holloway et al.–1 yeast tRNA (Sigma), 0.1 mg ml–1 bovine serum albumin (BSA) and 0.05, 0.1, 0.2, 0.3, 0.4, or 0.5 μM test protein in 33 mM Na-Hepes, 33 mM NaCl, pH 7.0. After 2 h of incubation at 37 °C, reactions were terminated by the addition of 2.3 vol ice-cold 3.4% perchloric acid, the mixtures were centrifuged at 13000 g for 10 min at 4 °C, and the absorbance of the supernatants were measured at 260 nm  .How to cite this article: Bradshaw, W. J. et al. Structural insights into human angiogenin variants implicated in Parkinson’s disease and Amyotrophic Lateral Sclerosis. Sci. Rep.7, 41996; doi: 10.1038/srep41996 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.