Commiphora myrrha (Nees) Engl.), coffee charcoal (Coffea arabica L.), and chamomile flower dry extract (Matricaria chamomilla L.) in the therapy of diarrhea and inflammatory bowel disease. Mast cells seem to play a key role in the symptom generation of irritable bowel syndrome (IBS). To evaluate the use of the TMP in IBS treatment, the effects of the herbal extracts on the release of mast-cell mediators from stimulated RBL-2H3 cells were investigated. Therefore, degranulation was induced by phorbol-12-myristate-13-acetate (PMA) and calcium ionophore A13187 (CI) or IgE stimulation, and the amounts of released β-hexosaminidase and histamine were quantified. The extracts showed no effect on the mediator release of PMA- and CI-stimulated RBL-2H3 cells. Myrrh and chamomile were able to reduce the β-hexosaminidase release of IgE-stimulated cells, while myrrh showed stronger inhibition of the mediator release than chamomile, which reduced only IgE-stimulated histamine release. Thus, these results indicate a mechanistic basis for the use of the herbal combination of myrrh, coffee charcoal, and chamomile flower extract in the symptom-oriented treatment of IBS patients with diarrheal symptoms.Recent clinical evidence supports the efficacy of a traditional medicinal product (TMP) containing a combination of myrrh ( Irritable bowel syndrome (IBS) affects about 11% of the global population . While t® is a traditional herbal medicinal product containing a combination of myrrh (Commiphora myrrha (Nees) Engl.), coffee charcoal (Coffea arabica L.), and chamomile flower dry extract (Matricaria chamomilla L.), which is used for the treatment of diarrhea and gut disorders. Clinical efficacy and safety have been shown in diarrhea and IBS patients [In severe IBS cases, patients cannot fulfil their daily tasks or participate in social activities, which drastically decreases their quality of life ,16. Accopatients .Previous pharmacological in vitro studies demonstrated spasmolytic, anti-inflammatory, and intestinal-barrier-stabilizing effects of the plant compounds ,20,21,22To support the hypothesis that the antidiarrheal effects of the herbal combination might be related to the compounds inhibiting the mediator release from mucosal mast cells, the potential effects were investigated using the cell line RBL-2H3. The cell line can be used as a model for mucosal mast cells, releasing different mediators upon degranulation, induced by a variety of compounds as well as IgE sensitization followed by antigen stimulation ,24,25. DAll extracts used in the following in vitro studies have been previously characterized, mainly via liquid chromatography and subsequent mass spectrometry ,21,22.HPLC-DAD analysis of the chamomile extract revealed seven main peaks . UV-specMass spectrometry alongside 1D and 2D NMR of isolated compounds from the myrrh extract showed the presence of several sesquiterpenes , peak 7 Analysis of the coffee charcoal extract via HPLC, reference standards, and literature data led to the identification of caffeine  peak 4 ap < 0.05). Based on these results, concentrations were chosen for further experiments.Before conducting any experiments, the extracts were tested for cytotoxicity using an XTT assay . Only my2+ concentration through CI, as a pore-forming agent or carrier [Upon stimulation with a combination of CI (1 µM) and PMA (40 nM), the plant extracts were unable to exhibit an effect on the release of β-hexosaminidase and histamine from RBL-2H3 cells compared to the stimulated control . Since t carrier , this mep < 0.05) and chamomile flower extract down to 80.4%   . The effp < 0.0001). IC50 is reached at a concentration of 12.12 µg/mL. This result suggests inhibitory properties from mast-cell mediator release upon IgE stimulation, which is underlined by the findings of a study with HMC-1 cells showing Commiphora myrrha (Nees) Engl. extract reducing histamine release and IL-31 production [Myrrh resin extract shows a concentration-dependent inhibitory effect on IgE-induced β-hexosaminidase release, reducing it by about one-third compared to the stimulated control at the highest concentration of 50 µg/mL . The inhibition might be induced by flavonoids such as apigenin and luteolin, as previous studies suggested [For further investigation of these results, the histamine concentration was determined in the supernatant of IgE/HSA-stimulated cells treated with myrrh and chamomile flower extract . Contraruggested ,28,34.These results indicate that the inhibition of β-hexosaminidase release is independent of the amount of histamine released in IgE-stimulated RBL-2H3 cells. Studies on mice bone marrow mast cells showed that they form various secretory granules, which all store β-hexosaminidase; however, some do not contain histamine ,36. Furt2·2H2O; MgCl·2H2O; citric acid monohydrate), Carl Roth , Fluka , Th. Geyer , or Sigma ; phorbol-12-myristate-13-acetate (PMA); calcium ionophore A13187 (CI); di-nitrophenyl human serum albumin (DNP-HSA); mouse anti-DNP-HSA-IgE; 4-nitrophenyl-n-acetyl-ß-d-glucosaminide (pNAG)). Cell culture supplies MEM, fetal bovine serum (FBS), penicillin-streptomycin (P/S), glutamine stable, trypsin-EDTA and Dulbecco’s phosphate-buffered saline (DPBS) were provided by Biowest .Ethanol for extraction was purchased in HSL quality from CSC Jäckelchemie  and purified by evaporation. Dimethyl sulfoxide (DMSO) for solving plant extracts was purchased from VWR . Chemicals (p.a. grade) were supplied by Merck . For IgE stimulation, 5 × 105 cells/mL were seeded into 96-well plates, before plant extracts and 500 ng/mL anti-DNP-HSA-IgE were added and incubated overnight (18 h) for sensitization. Cells were washed with HEPES buffer and incubated with 100 µL of 1 µg/mL DNP-HSA solution in HEPES for 30 min.RBL-2H3 cells, purchased from DMSZ , were cultured with MEM supplemented with 10% FBS, 1% P/S, and 1% glutamine stable. All cell lines were cultivated under standard cell-culture conditions. For chemical stimulation, RBL-2H3 cells were seeded into a sterile 96-well plate ) at 1 × 10m/m; DER: 4–6:1, batch-no. HC0070) as well as powdered myrrh  and powdered coffee charcoal  were provided by Lomapharm .Ready-to-use chamomile flower dry extract  pH = 4.5) for 90 min at 37 °C without CO2. The enzymatic reaction was stopped by the addition of 50 µL glycine buffer pH = 10.7 (glycine 400 nM). Extinction was measured at 405 nm (reference 620 nm) with an Infinite® M200 plate reader .The β-hexosaminidase release was analyzed similarly to Kuehn et al. . BrieflyThe histamine concentration in the supernatants was determined using the Histamine ELISA kit (cat no A05890), provided by Bertin , according to the manufacturer’s instructions.The measurement of the metabolic activity for the RBL-2H3 cells after overnight incubation in the presence of the plant extract was performed with the XTT-Vitality-Assay Kit provided by Roche , according to the manufacturer’s instructions.p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.Microsoft Excel (Office 365) was used for data capturing and processing of the extinction measurements. For statistical evaluation, a mixed-model one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test against the stimulated control was conducted with GraphPad Prism 8.0.2 (* The RBL-2H3 cell line was used as a model for mucosal mast cells to investigate the effects of myrrh, coffee charcoal, and chamomile flower extracts on degranulation. In IgE-stimulated cells, myrrh and chamomile flower extracts were able to inhibit the release of the protease β-hexosaminidase, while chamomile flower extract exhibited a moderate effect on histamine release, probably driven by flavonoids.In this context and with the available clinical evidence suggesting the antidiarrheal properties of the herbal combination of myrrh, coffee charcoal, and chamomile flower, this study indicates a potential target on mucosal mast cells and builds a foundation for further investigation into the mechanism of action, as well as supporting its rational use in IBS. It also underlines the multi-target approach of herbal treatments in complex diseases such as IBS, to improve the quality of life of patients.


A low-value by-product of cold-pressed sesame oil is defatted black sesame cake (DBSC). The remaining protein and essential amino acids may be utilized as a renewable biological source to produce bioactive products. The bioactivities of the protein hydrolysate from black sesame cake and its peptide fractions were examined in this study for in vitro antioxidant activity and inhibition of DPP-IV, ACE, α-amylase, α-glucosidase, and pancreatic lipase. By using Flavourzyme to hydrolyze DBSC, followed by ultrafiltration, fractions with peptide sizes of <3, 3–10, and >10 kDa were obtained. According to the findings, the products of DBSC could neutralize free radicals and prevent ferric ion redox reactions. The highest inhibitory effects were shown with low Mw peptides (<3 kDa) against ACE, DPP-IV, α-amylase, and α-glucosidase. DBSC has demonstrated potential as a nutraceutical or functional ingredient for preventing and treating disorders associated with free radicals, such as diabetes, hypertension, and hyperglycemia. Enzymatic hydrolysis is useful for enhancing the nutritional and functional qualities of protein-rich foods. Better solubility, foaming, emulsifying properties, and the release of physiologically active peptides from proteins have benefited from this method . It provBioactive peptides (BP) are protein fragments produced by hydrolyzing protein-rich sources with nutritional benefits and positive health impacts . They arSesamum indicum L.) is a widely cultivated crop for cooking and producing edible oil. Sesame seeds offer a high nutritional value with up to 50% oil content and significant levels of minerals, vitamins, and omega-3 fatty acids. The oil has a higher percentage of unsaponifiable matter (2%) than other vegetable oils, including triterpene alcohols, phytosterols, tocopherols, and lignans (primarily sesamin and sesamolin). This increases its oxidative stability and provides several advantageous physiological effects  × 100, where A0 is the absorbance of the control, and AS is the absorbance of the sample or the positive control.Free radical scavenging ability was determined by DPPH radical scavenging assay , with sl•+) was assessed using the technique previously described  × 100, where A0 is the absorbance of the control, and AS is the absorbance of the sample or the positive control.Radical scavenging activity  in the ratio 10:1:1 (v/v/v). In this experiment, 180 µL of the FRAP reagent and 20 µL of the samples were added to 96-well plates and incubated for 5 min at room temperature in the dark to complete the reaction. A microplate reader was used to measure the reacting samples at 595 nm. As a positive control, Trolox (1 mg/mL) was utilized. Instead of the sample, distilled water was used to prepare the blank control. An increase in absorbance at 700 nm indicated that protein hydrolysates had more reducing power.The ferric-reducing antioxidant power (FRAP) was assessed using a slightly modified version of the FRAP assay described in a prior study. In brief, the FRAP reagent was freshly achieved by combining 10 mM 2,4,6 tripyridyl-s-triazine (TPTZ) in 40 mM HCl, 20 mM FeClNC − AS)/ANC] × 100, where ANC is the absorbance of the control (no inhibitor), and AS is the absorbance of the sample or the positive control.A modified approach was used to execute the suppression of α-glucosidase activity protocol . A 96-we0 − As) × 100/(A0 − ANC)], where A0 is the absorbance of the blank (without enzyme), ANC is the absorbance of the control (without inhibitor), AS is the absorbance of the sample or the positive control.The α-amylase inhibitory activity of samples was measured using a slightly modified approach as previously described . Pre-incNC − AS)/ANC] × 100, where ANC is the absorbance of the control (no inhibitor), and AS is the absorbance of the sample or positive control.An in vitro inhibition of pancreatic lipase activity was used to determine the anti-hyperlipidemia assay as described in a prior study with slight modification . A reactblank1 − Asample)/(Ablank1 − Ablank2)] × 100, where Ablank1 is the absorbance of the positive control (without ACE inhibition), Asample is the absorbance of the sample, and Ablank2 is the absorbance of the reagent blank. The IC50 value was determined using nonlinear regression analysis. To identify statistical differences, the one-tailed Welch’s t-test was used. p-values < 0.05 were regarded as significant. The information was presented as means of the IC50 value.The ACE-inhibitory activities of the different sample concentrations were investigated using an ACE kit-WST . The assay was carried out following the manufacturer’s instructions. A microplate reader was used to measure the reactions’ absorbances at 450 nm. The following equation calculated the ACE-inhibitory activities of the samples: ACE-inhibitory activity (%) = [ was used to assess the DPP-IV inhibitory effect of the oligopeptide fractions. The assay was carried out following the manufacturer’s instructions. The cleavage of the substrate to produce a fluorescent product is the basis for measuring the DPP-IV activity (λex = 360 nm/λem = 460 nm), depending on the amount of enzymatic activity in the test sample. A standard DPP-IV inhibitor (Sitagliptin), which was included with the kit, was used to compare the efficiency of the tested inhibitors. The measurement was deployed with kinetic mode (reading every minute) for 15–30 min at 37 °C, and the DPP-IV inhibition activity of the samples was given as a percentage of relative inhibition  to the manufacturer’s protocol. The results expressed the ICp < 0.05 was used as the criterion of significance.The mean and standard deviation of triple experiments expresses the results. SPSS 21.0  was used for the multivariate analysis of variance. Duncan’s multiple range test was used to find significant discrepancies between mean values. According to our research, an evaluation of the antioxidant, anti-diabetic, anti-obesity, and antihypertensive properties of defatted sesame seed cake protein hydrolysate and its ultrafiltered peptide fractions was conducted. The sesame cake products’ abilities to scavenge free radicals and inhibit the redox reaction of ferric ions were variable, which may be explained by changes in their size, solubility, or content of amino acids. The low Mw peptide fraction (<3 kDa) showed the highest α-amylase, α-glucosidase, ACE, and DPP-IV inhibitory activities, while larger peptides (>3 kDa) exhibited higher inhibition against pancreatic lipase. According to the findings, sesame hydrolysate and its peptides could be used as ingredients in the treatment of free radical-related disorders, the prevention of atherosclerosis in blood vessels, and the inhibition of various enzymes involved in the progression of hypertension and hyperglycemia. The in vitro qualities mentioned in this paper must be confirmed in subsequent in vivo investigations utilizing animal models.


Protein phosphorylation is a major regulatory mechanism of cellular signalling. The c-JUN proto-oncoprotein is phosphorylated at four residues within its transactivation domain (TAD) by the JNK family kinases, but the functional significance of c-JUN multisite phosphorylation has remained elusive. Here we show that c-JUN phosphorylation by JNK exhibits defined temporal kinetics, with serine63 and serine73 being phosphorylated more rapidly than threonine91 and threonine93. We identify the positioning of the phosphorylation sites relative to the kinase docking motif, and their primary sequence, as the main factors controlling phosphorylation kinetics. Functional analysis reveals three c-JUN phosphorylation states: unphosphorylated c-JUN recruits the MBD3 repressor, serine63/73 doubly-phosphorylated c-JUN binds to the TCF4 co-activator, whereas the fully phosphorylated form disfavours TCF4 binding attenuating JNK signalling. Thus, c-JUN phosphorylation encodes multiple functional states that drive a complex signalling response from a single JNK input. Phosphorylation at multiple sites is a major regulatory mechanism in cellular signalling. Here, the authors show that multisite phosphorylation of the c-JUN transcription factor by the JNK kinase exhibits intrinsic kinetics that allow a precise and timed regulation of the transcriptional output. Deregulation of phosphorylation pathways commonly underlies disease aetiology4. Proteins often are phosphorylated not only at one but at multiple sites by a single kinase. Multisite phosphorylation is a key regulatory mechanism in signalling that controls many cellular processes, and has been suggested to provide a precise tool to generate complex functional outputs, often from a single kinase input9. This is often achieved via interactions between the multisite phosphorylated protein and partner proteins, and these phosphorylation-dependent interactions determine the biological output10. Transcription is an important biological process regulated by multisite phosphorylation12. We have previously provided a mechanistic understanding of the temporal dynamics of multisite phosphorylation of the Ternary Complex Factor (TCF) Elk-1 transcriptional coactivator by the ERK kinase13. Phosphorylation at eight S/T-P (Ser or Thr-Pro) sites of Elk-1 occurs with a defined kinetics, leading to intrinsic temporal phosphorylation events that effectively trigger and subsequently limit its transcriptional response by promoting and then inhibiting the recruitment of the Mediator complex13. Thus, understanding the intrinsic kinetics of multisite phosphorylation is key to gain insights on how collectively multiple phosphorylated sites by a kinase within a domain can shape the transcriptional response.Protein phosphorylation is the most abundant and important post-translational protein modification14. AP-1 consists of heterodimeric complexes formed by members of the JUN family  together with the FOS proteins  and members of the ATF and CREB families. The proto-oncoprotein c-JUN is a major component of the AP-1 transcription factor that controls the expression of many genes important for cell proliferation and tumorigenesis16.AP-1 (activator protein 1) activity is strongly induced in response to numerous signals including growth factors, cytokines and extracellular stress17. The JNKs have three isoforms: JNK1, JNK2 and JNK3. JNK1 and JNK2 are expressed in almost all tissues, whereas the expression of JNK3 is restricted to neuronal tissues. Both JNK1 and JNK2 actively contribute to c-JUN phosphorylation, and are at least partly redundant17.An important mechanism to stimulate AP-1 function is the N-terminal phosphorylation of the c-JUN transactivation domain (TAD) at multiple S/T-P sites by the c-JUN N-terminal kinase (JNK) group of mitogen-activated protein kinases (MAPK)20. The unphosphorylated c-JUN TAD interacts with MBD3, a subunit of the nucleosome remodelling and histone deacetylation (NuRD) repressor complex, which in turn recruits NuRD to AP-1 target genes to mediate gene repression. This repression is relieved by c-JUN phosphorylation following JNK activation21. Phosphorylated c-JUN at its TAD by JNK interacts with the TCF4 HMG-box transcription factor activating the c-JUN promoter16.JNKs phosphorylate the c-JUN TAD at four S/T-P residues, S63, S73, T91 and T93 Fig. , which mJunAA/JunAA) develop normally but show specific defects in oncogenic transformation22. Moreover, in the intestine both MBD3 and TCF4/b-catenin control cellular proliferation and tumorigenesis via JNK/c-JUN N-terminal phosphorylation. Mice lacking MBD3 (mbd3ΔG/ΔG) show increased intestinal progenitor proliferation, which can be rescued upon deletion of one c-jun allele 21. In a mouse model of intestinal cancer that is heterozygous for a nonsense mutation at codon 850 of the Apc gene (ApcMin/+), mice that are crossed to JunAA mice  exhibit greatly reduced intestinal tumors comparing to the ApcMin/+ mice16. However, although the biological significance of JNK c-JUN S63 and S73 N-terminal phosphorylation and its role in tumour development have been clearly demonstrated, the roles of the four individual phosphorylation sites and the interplay among them, and particularly how they might cooperate to regulate the temporal cofactor recruitment and thus transcriptional output, have not yet been explored.The multisite N-terminal phosphorylation of c-JUN by JNK is a key event linked to tumorigenesis. Mice in which the c-JUN phosphorylation sites S63 and S73 are mutated to alanines  containing all four phosphorylation sites, S63, S73, T91 and T93 and the MAPK binding motif  which controls phosphorylation of these sites27 32 32 Fig. . These p)32 Fig. .33. However, both residues, S63 and S73, despite now being further away from the D-motif than T91 and T93, were still phosphorylated with higher rates than T91 and T93, whose modification rate remained at similar levels than the wild-type  alone, we generated a c-JUN TAD variant in which serine and threonine residues were exchanged (‘TTSS’ variant: S63T/S73T/T91S/T93S), and measured phosphorylation rates by time-resolved NMR Fig. . The TTSc-jun luciferase reporter gene assay using constitutively active MEKK as an upstream activator of c-JUN phosphorylation (pFC-MEKK), which specifically activated JNK and not the other MAPK kinases (p38 or ERK)  exhibited increased reporter activity comparing to the wild-type. Lastly, alanine substitution of all four phosphorylation sites (4A) showed decreased reporter activity relative to wild-type protein, similar to that seen with the 2SA variant  and slow  phosphorylation sites, we next explored the biological function of the kinetics of the c-JUN phosphorylation by testing the effect of each phosphorylation kinetic group of the c-JUN TAD on its ability to activate transcription. For that purpose, we first analysed the effect of each individual c-JUN phosphorylation site with a ant Fig. . Taken tMBD3 or TCF4 expression with small interfering RNAs (siRNAs)  pull-down experiments using recombinant GST-c-JUN TAD phosphorylated for different times such that phosphorylation was either mainly at S63 and S73 or complete Fig. , and tesTo examine the contribution of the different kinetic phosphorylation groups of c-JUN TAD to the TCF4/phosphorylated c-JUN interaction, we performed co-immunoprecipitation assays using HCT116 cells, transiently expressing Flag-tagged c-JUN or mutants , to recover binding of endogenous TCF4 from cell extracts treated either with JNK inhibitor (JNKi) or with anisomycin to induce partial or complete c-JUN phosphorylation Fig. . JNK phoE. Coli, and performed glutathione S-transferase (GST) pull-down experiments using recombinant unphosphorylated or fully phosphorylated wild-type GST-c-JUN TAD and phosphorylated 2TA mutant, and tested the recovery of recombinant MBD3 and TCF4 respectively. Recombinant MBD3 bound to unphosphorylated c-JUN TAD and this interaction was abrogated by phosphorylation  each generating distinct phosphorylation kinetics13. Such a competitive mechanism appears more flexible, allowing for different phosphorylation patterns depending on the kinase. In the case of c-JUN, which is exclusively phosphorylated by JNK family kinases, substrate site preference appears to be a more conservative mechanism that would not only ensure accurate timing of transcriptional activation and subsequent elimination but potentially filter binding of other MAPK kinases41.A comparison of the mechanisms by which the MAP kinases JNK1 and ERK2 recognise their substrate phosphorylation sites on c-JUN and Elk-1 TAD respectively, to generate the kinetics of multisite phosphorylation shows significant differences. The similar phosphorylation rates observed for residues in different c-JUN alanine variants provide insight into the mechanism of multisite phosphorylation by JNK1, indicating that the JNK1 active site cannot be completely saturated by the c-JUN phospho-sites, as the elimination of one phosphorylation site does not lead to a redistribution to other sites. This appears to contrast with the competitive mechanism of multisite phosphorylation observed in Elk-1, in which its active site is saturated by substrates such that elimination of a certain kinetic group of phospho-sites results in a redistribution of the kinase to the remaining sites and hence more rapid phosphorylation43. JNK plays an important role in various normal biological functions and its direct inhibition could have undesired side effects. Inhibitors targeting specific JNK-mediated downstream substrates and cellular events, such as phospho-c-JUN or the interaction of phospho-c-JUN binding partners, may show increased tumour specificity and efficacy. Our results suggest that in the case of c-JUN, the active doubly phosphorylated pS63/pS73 c-JUN form, and its interaction with activators, would be the most promising candidates for more personalised therapeutic intervention.The identification of three functional states associated with c-JUN N-terminal phosphorylation may have implications for the development of novel therapeutic cancer strategies. The JNK signalling pathway has been a desirable drug target for years, but so far JNK inhibitors have not been translated into clinical use. The main reason is the lack of specificity of the current inhibitors and cellular toxicityIn summary, in this study we have shown that c-JUN multisite phosphorylation by JNK exhibits an intrinsic kinetics that leads to a precise and timed regulation of the transcriptional output of the JNK signalling pathway, where both positive and negative outputs are generated by a single kinase input. The concept of the temporal phosphorylation pattern and its translation to a self-eliminating transcriptional mechanism thus appear to be more general in the MAPK signalling. This mechanism contrasts the dogma of the classic ‘on and off switch’ of the transcriptional response via the dual role of kinases and phosphatases in MAPK signalling and perhaps more general in signalling pathways, revealing that the temporal dimension of multisite phosphorylation of a protein domain allows for more elaborate signalling responses. We speculate that the intrinsic kinetics of post-translational modifications across multiple sites might provide a simple and universal mechanism for the generation of a complex temporal biological response to a kinase signalling event.Plasmid constructions, protein analysis by SDS-PAGE and immunoblotting used standard methods. c-JUN point mutations were generated by PCR-based mutagenesis using the site-directed mutagenesis kit (New England Biolabs). cDock, mDock and swap c-JUN, MBD3 and TCF4 constructs were chemically synthesized by ATCC and GeneArt (Thermofischer).pS63 c-Jun (#9261), pS73 c-Jun (#9164), JNK (#9252), pT183pY185 JNK (#9251), pT202pY204 ERK [20G11] (#4376), ERK [L34F12] (#4696), pT180pY182 p38 (#9211), p38 (#9212), and TCF4/TCF7l2 [C48H11] (#2569) from Cell Signalling; pT91 c-Jun [EPR2236] (#ab247509), pT93 c-Jun (#ab28854), and MBD3 [EPR9913] (#ab157464) from Abcam; c-Jun [3/Jun] (#610326) from BD Biosciences; Vinculin [hVIN-1] (#V9131), GST (#G7781) and Flag [M2] (#F3165) from Sigma-Aldrich; Peroxidase AffiniPure Goat anti-Mouse IgG (H + L) (#115-035-146), Peroxidase AffiniPure Goat anti-Rabbit IgG (H + L) (#111-035-144) (Jackson ImmunoResearch). Primary antibodies were used at 1:1000 dilution, except for Vinculin that was used 1:10000 and GST 1:20000. Secondary antibodies were used at 1:10000 dilution.13 and expressed as GST-(His)6 fusion proteins. Protein expression was induced in E. coli Rosetta(DE3) (Novagen) with 0.5 mM IPTG for 3 h at 37 °C for c-JUN TAD, and in E. coli BL21(DE3) (Novagen) with 0.5 mM IPTG overnight at 18 °C for MBD3. Cells expressing c-JUN TAD or MBD3 were lysed in 50 mM Tris pH 8.0, 300 mM NaCl, 5 mM DTT, 5 mM MgCl2, 1 mM PMSF supplemented with EDTA-free protease inhibitor cocktail (Merck) and traces of Deoxyribonuclease I from bovine pancreas (Sigma). Cleared lysate was applied to glutathione sepharose resin (Cytiva), incubated for 1 h with the resin, and then washed extensively with lysis buffer. C-JUN proteins were either eluted with 20 mM reduced glutathione in 50 mM Tris pH 8.0, 150 mM NaCl, 5 mM DTT, or after adsorption to glutathione sepharose the c-JUN TAD proteins or MBD3 were released by incubation with 1:50 (w/w) GST-3C protease on the resin overnight at 4 °C, and further purified on a Superdex 200 HR10/30 size exclusion column (Cytiva) in 10 mM K2HPO4 pH 6.8, 50 mM NaCl for c-JUN TAD or in 25 mM Hepes pH 7.5, 100 mM NaCl, 5 mM DTT, 10% Glycerol for MBD3. For 15N- and 15N-/13C-labelling, cells were grown in M9 minimal medium containing 15NH4Cl and 13C-glucose as required and c-JUN was expressed for 3 h at 37 °C after induction with 0.5 mM IPTG. Human full-length TCF4 (residues 1-619) was inserted between the NcoI and EcoRI sites of a modified pET-30a vector (kind gift form Dr. Phil Robinson) carrying an N-terminal 6xHis tag and a TEV protease cleavage site. Protein expression was induced in E. coli BL21(DE3) (Novagen) with 0.5 mM IPTG overnight at 18 °C. Cells were lysed in 50 mM Tris pH 7.5, 300 mM NaCl, 2 mM β-mercaptoethanol, 5 mM MgCl2 and 5% glycerol, supplemented with 0.25 mg/ml lysozyme, EDTA-free protease inhibitor cocktail (Merck) and traces of Deoxyribonuclease I from bovine pancreas (Sigma), using an EmulsiFlex-C3 homogeniser (Avestin). Cleared lysate was loaded onto a 5 ml HisTrap column (Cytiva) equilibrated in the lysis buffer using an AKTA purifier (Cytiva). After extensive washing, the protein was eluted using an imidazole gradient 10–400 mM. The fractions containing purified TCF4, as confirmed by western blot analysis, were combined and imidazole was removed by dialysis.Wild-type or mutant human c-JUN sequences encoding residues 1-151 or full-length human MBD3 (residues 1-291) were inserted between the BamHI and NotI sites of modified pET-41a1H Larmor frequency). A 150 μM sample of uniformly 15N,13C-labelled sample of c-JUN was prepared in 10 mM K2HPO4 pH 6.8, 150 mM NaCl, 5 mM MgCl2, 10% (v/v) D2O and 0.01% (w/v) DSS. BEST-HNCO, HNCACO, HNCACB and HNCOCACB experiments44 were acquired, processed using nmrPipe45 and analysed using CCPN Analysis (v2.5.1)46. Secondary structure populations were calculated from HN, N, CO, CA and CB chemical shifts using δ2D28. c-JUN phosphorylation kinetics were measured at 20 °C using a Bruker Avance III NMR spectrometer with TXI cryoprobe operating at 950 MHz (1H Larmor frequency) and running Topspin 3.5pl6. 391 μL samples of 15N-labelled cJun (WT or variants) were prepared in Shigemi tubes without plungers, at concentrations of ca. 75 μM in 10 mM K2HPO4 pH 6.8, 50 mM NaCl, 5 mM MgCl2, 2 mM DTT, 1 mM ATP, 5% (v/v) D2O. Reference 2D 1H,15N SOFAST-HMQC experiments47 were acquired using a 50 ms recycle delay, with 1536 complex points and a sweep width of 16 ppm in the direct dimension (100 ms acquisition time), and 64 complex points and a sweep width of 20 ppm in the indirect dimension (33.2 ms acquisition time). Phosphorylation was then initiated by rapid mixing with 290 U recombinant active JNK1 or JNK2 (Proqinase), and a series of SOFAST-HMQC experiments were acquired over a period of several hours, with 4 scans per increment (corresponding to 93 s per spectrum). This kinase concentration was optimised following initial estimates based on the specific activity, substrate concentration and the number of phosphorylation sites24 in order to obtain measurable levels of phosphorylation within hours, but not so fast that kinetics could not accurately be resolved by NMR. Spectra were processed in nmrPipe (v10.9)45 using linear prediction and cosine-squared window functions, then imported into Julia (v1.6) for analysis with NMRTools.jl (v0.0.1). Following previous studies24 box integrals were calculated around phosphorylated resonances and fitted to single exponential functions as a function of time. Phosphorylation progress was reported by normalising phosphorylated peak integrals according to the average of several well-resolved resonances (coefficient of variation (standard deviation/mean) of ca. 20%) in the N- and C-termini of the sequence, located far from phosphorylation sites.NMR data for assignment of c-JUN backbone resonances were acquired at 283 K using a Bruker Avance III NMR spectrometer with TXI cryoprobe operating at 700 MHz (NIH3T3 and HCT116 were provided by the Francis Crick Institute Cell Services and cultured in Dulbeccos’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cell lines were tested for mycoplasma and authenticated by short-tandem repeat DNA profiling by the Francis Crick Institute Cell Services. Cells were treated when indicated with 50 µM JNK inhibitor (JNKi)  or 15 ng/ml anisomycin (Sigma). HCT116 cells were plated at subconfluence and transfected with lipofectamine 2000 (Invitrogen) when indicated.To silence MBD3 or TCF4, ON-TARGETplus MBD3  or TCF4/TCF7L2  siRNAs and a non-targeting control  were used. Cells were reversed-transfected with 25 nM small interfering RNAs (siRNAs) using Lipofectamine RNAiMAX (Invitrogen) for 48–72 h. Where indicated, cells were further treated with JNK inhibitor or anisomycin for 30 min.15N isotope-labeled protein version, 75 μM of substrate was incubated with 290 U recombinant active JNK1 (Proqinase) in 10 mM K2HPO4 pH 6.8, 50 mM NaCl, 1 mM ATP, 2 mM DTT, 5 mM MgCl2 at 20 °C for the indicated times. For phosphorylation of endogenous JNK and c-JUN, cells were treated with 50 μM JNKi for 2 h followed by 15 ng/ml anisomycin treatment for the indicated times at 37 °C. Lysates for SDS-PAGE were prepared in 300 μl RIPA buffer (Thermo Fisher) supplemented with protease inhibitor (Sigma) and phosphatase inhibitor . Protein quantification was performed with the BCA kit (Thermo Scientific) and densitometry analysis of the immunoblot with the Image Studio Lite Software (Licor).For phosphorylation of GST-c-JUN TAD, or the trans-Reporting System from Agilent Technologies), or the indicated pFA2-c-JUN mutants or pFC2-dbd (empty vector); pFR-Luc; pFC-MEKK; and tk-Renilla, using Lipofectamine 2000 (Invitrogen). Next day, medium was replaced with starvation medium (0.3% FBS) for 24 h. Luminescence activity was measured 48 h post-transfection using Dual-Luciferase Reporter Assay System following manufacturer’s protocol (Promega) in a FLUOStar Omega Plate reader (BMG Labtech). Data were expressed as fold induction after being normalized using tk-renilla luciferase.NIH3T3 cells were transiently transfected with pFA2-c-JUN WT (PathDetect c-JUN RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. One microgram of total RNA was reverse-transcribed using the iScript cDNA synthesis kit (BioRad) according to the manufacturer’s instructions. cDNA was diluted five-fold and 1 μL was used per RT-qPCR reaction. PowerUp SYBRGreen (Applied Biosystems) was used for real-time quantitative PCR (qPCR). Assays were performed on an ABI Applied Biosystems 7500 or QuantStudio Real-Time PCR System. RT-qPCR primers are listed in Supplementary Table g at 4 °C. Lysates were incubated with 20 μl of anti-FLAG M2 magnetic beads (Sigma) for 16 h at 4 °C. Beads were washed 3 times with Cell Lysis Buffer and proteins were eluted with 20 μl of 2x SDS loading buffer or with 3X FLAG peptide (Sigma).HCT116 cells were transiently transfected with pCMV-Tag2-Flag-cJUN (provided by Axel Behrens) wild-type or mutants, and were treated with JNKi or additionaly with anisomycin. Cells were lysed in Cell Lysis Buffer  with protease inhibitor (Sigma), phosphatase inhibitor  and 1 mM PMSF, and cleared by centrifugation for 15 min at 10,000 × g at 4 °C. 10 μl of gluthatione sepharose beads (Cytiva) were incubated with 10 μg GST c-JUN TAD fusion protein for 1 h at 4 °C. After binding beads were washed 3 times with lysis buffer, or 25 mM Hepes pH 7.5, 100 mM NaCL, 5 mM DTT, 10% Glycerol (for recombinant purified protein pulldowns) and were next incubated with the lysate or purified recombinant protein for 3 h at 4 °C. Beads were washed 3 times with Wash Buffer  (for endogenous MBD3) or Cell Lysis Buffer (for endogenous TCF4) or 25 mM Hepes pH 7.5, 100 mM NaCL, 5 mM DTT, 10% Glycerol for recombinant MBD3 and TCF4. Proteins were eluted with 20 μl of 1x SDS loading buffer.HCT116 cells were lysed in Cell Lysis Buffer  with protease inhibitor (Sigma), phosphatase inhibitor  and 1 mM PMSF, sonicated and cleared by centrifugation for 15 min at 10,000 × p-values are provided in the figure panels. A p-value of <0.05 was regarded as statistically significant for all datasets. The exact sample sizes (n) used to calculate statistics are provided in the figure legends. All biochemical and cell-based experiments were reproduced with similar results a minimum of three times. Time-resolved NMR measurements were performed on at least two biological independent replicates (n ≥ 2). Immunoblots images are representative of a minimum of three biologically independent experiments and uncropped scans of all representative immunoblots contained in the manuscript are shown in the Source Data file.Results are represented as mean or mean ± standard error of the mean (SEM) for bar graphs and scatter plots. Statistical significance was determined as described in the figure legends and calculated using GraphPad Prism 8. Briefly, one-way analysis of variance followed by Dunnett’s or Tukey’s correction for multiple comparisons was used. Further information on research design is available in the Supplementary InformationReporting Summary


L = 5.8) and the Pohang earthquake  occurred in Korea, thereby increasing social concern about earthquakes. Because Korea is not located near the Circum-Pacific Belt, also referred to as the “Ring of Fire”, people in Korea are not used to earthquake disasters. Coastal areas in Korea are lined with multiple mega-cities and major industrial facilities. Most of them constructed on landfill, they have a high threat level to the damage to populations and structures leading to complex disasters in the event of an earthquake. However, studies of seismic hazards in ports is incomplete compared with those of onshore sites. Improving the understanding of seismic hazards and characterizing them catching up with related research from various perspectives being actively conducted. In this study, the site-specific response characteristics of the Pohang International Container Terminal in Pohang Yeongil New Port were analyzed using the S-wave energy of 10 sets of ground motions recorded by seismic accelerometers operated by the Ministry of Oceans and Fisheries based on the horizontal-to-vertical spectral ratio (H/V ratio) method. As a result of analysis, the H/V ratio curves show that the peak frequency values for the target site averages 9 Hz, and the natural period values of the site were preliminarily predicted to average 0.11 s. In addition, site amplification characteristics are different based on the seismic wave calculation method. The result can be used as data for identifying ground dynamic characteristics and verifying the site amplification coefficients and design spectrum in the seismic design.Recently, medium-sized earthquakes such as the Gyeongju 9.12 earthquake (September 12, 2016, M Since the beginning of these instrumental observations, the largest magnitude EQ was occurred on September 12, 2016 , which raised awareness that the Korean Peninsula is no longer safe from EQs2. Then, when the Pohang EQ that occurred the following year  caused enormous social and economic damage, it further raised public concern about the dangers of EQ disasters. The Korean Peninsula is in a situation where 99.7% of all imports and exports are transported by shipping3. Thus, national trade ports are representative infrastructures and are highly significant for securing the stability of national logistics and transportation against natural disasters. Ports are one of the most vulnerable coastal utilities and need to be protected against their devastating effects of EQs. Unlike other natural disasters that can be partially predicted and prepared for, such as typhoons, heavy rains, heavy snowfall, cold waves, and landslides, seismic disasters cannot realistically be predicted in time and space. Therefore, it is necessary to develop a method that can minimize the loss of lives and property damage through rapid response by applying observation technology through monitoring.For instrumental earthquakes (EQs), the seismic data can be analyzed from data recorded by seismometers. In the case of the Republic of Korea, the Korea Meteorological Administration (KMA) started officially seismic measurement in 1978. In the 1990s, digital instrument observation began to be conducted mainly by the Korea Institute of Geoscience and Mineral Resources4. This system was used to assess and respond to emergency caused by the PH EQ in 2017. Significant damage occurred on the major facilities, which are the quay walls and cranes, of the Pohang International Container Terminal at Pohang Yeongil New Port  in Korea is operating the port alert system for seismic response by installing seismometers at national trade ports across the country. To establish an EQ database in ports, continuous efforts are being made to develop data processing techniques and to strengthen the information delivery service on seismic response in ports7. Differential settlement occurred at the foundation owing to the large amplification in the low-frequency band. This can also be found in San Francisco Bay, during the Mw = 6.9 Loma Prieta earthquake EQ of 17 October 19898. Also, an extensive soil liquefaction was observed during the 1995 Kobe earthquake9. Lateral spreading in the storage yards at Port-au-Prince port occurred by sediment-induced amplification during the Mw 7.0 Haiti earthquake in 201010. Such characteristics of ground amplification have been mainly studied in the field of natural sciences for seismological observation, which improves the accuracy of seismic sources by reducing local site-effects11. From the engineering perspective, it is also important to investigate ground motion attenuation and ground amplification effects according to the ground properties essential for seismic design.In the soft ground, depending on the site-effect, even an EQ of the same magnitude can be amplified at a specific frequency, leading to a major disaster. The most famous instance is the Michoacan EQ with a moment magnitude of 8.0, which occurred in Mexico on September 19, 198515. Research related to seismic amplification or liquefaction on port area sites is still at an early stage. This is because the history of strong ground motion data in the Korean Peninsula is small owing to the low incidence of strong EQs, and it has been less than 10 years the digitized database of seismic measurement data in the port area began to be constructed. The first damage case in a port area since records began with instrumental observations in Korea was reported for the 2017 PH EQ. Thus, studies have been conducted to analyze the damage mechanism and the behavior of port structures17, and research on the earthquake-induced liquefaction is also being actively conducted18. In this study, based on the accelerograph record measured by the Port Alert System for Seismic Response operated by the MOF, the site response characteristics were analyzed around the port measuring station. A spectral ratio method was used, which is the horizontal component at the surface to the vertical component in the bedrock referred to as the “H/V ratio”. The response characteristics at Pohang Yeongil New Port, where the measuring station is located, were analyzed using 10 seismic motion data of the foreshock, mainshock and aftershocks during the 9.12 and PH EQs. The site-specific response for characteristics of seismic motion recorded at measuring stations of the surface and bedrock (borehole) was investigated.There was a lack of real EQ records to be reflected in the domestic seismic design standards, particularly in Korea. For a more rational seismic design, the necessity of a design response spectrum suitable for domestic ground characteristics has been revealed in literature reviews19. Based on waveform types, in addition to the S-wave energy, background noises (microtremors) and the coda wave are used to analyze the spectrum. In particular, the horizontal-to-vertical spectral ratio (H/V ratio) method, which is widely used because of its speed and convenience, was employed to estimate the site effect evaluation21. A method that considers the H/V ratio of the target site to the reference site with well-developed outcrops as the amplification ratio of that site has been studied22. The method of using the H/V ratio by employing the three components observed at arbitrary sites24 has recently been applied to nuclear power plants, dams, reservoir, and major observatory sites in Korea32. In practice, it is often determined by the H/V ratio technique in which the peak frequency  and can be divided into the spectral, inverse calculation. and unknown term separation method33. The aforementioned two seismic stations were separated by approximately 8.6 km in a straight line, and specific details are summarized in Table As of June 2021, the MOF in Korea has been operating the Port Alert System for Seismic Response in 28 stations in 13 ports, centered on trade ports across the country, such as Busan Port and Incheon Port. Among the other stations, the seismic motion data were collected from two measuring stations (PHF and PHT) installed in Pohang Port to understand the characteristics of the port site-specific response and to analyze the amplification quantitatively. A borehole seismometer is installed in the bedrock, which is less affected by the site amplification effect by the upper sedimentary layer or the background noise of the surface; therefore, it is possible to observe the seismic observations accurately compared with the surface-type seismometer. However, unlike in bedrock areas, in soils with a large site amplification, it is necessary to consider the site effect characteristics4. In addition, the surface-type accelerometer at PHT as the target station for site amplification analysis is installed in the basement of the machine room in Pohang Yeongil New Port.The seismic accelerometer at PHF as a reference station in this study is installed in the bedrock, and the ground layer formations are as follows. The ground layer consists of a reclamation layer with fine sand up to 1.5 m below the surface; from 1.5 to 38 m, it is composed of a marine sedimentary layer with silty clay, containing small amounts of fine gravel and shells in the lower part. The 38–42 m section is a soft rock layer composed of mudstone, and cracks and joints are developed. Finally, the 42–48 m section is composed of muddy hard rock, and a borehole accelerometer was installed nearly 48 m below34.Table The seismic motion data used in this study were stored in the format of mini-SEED (Standard for the Exchange of Earthquake Data) obtained from the three-axis accelerometer, and the physical factor (pf) was applied to convert the acceleration values. The Nyquist frequency was set to 50 Hz because 100 samples per second (0.01 s) were sampled. Approximately 15 s to less than 20 s of the S-wave energy window were selected for analysis. Table 29. Ninety sets of data for each component and waveform type were verified from 10 EQ records. Here, the S-wave energy window was selected for approximately 15–20 s from the arrival time of the S-wave, and the coda wave window was sampled for 15–20 s after the point when the S-wave travel time was approximately double. In addition, the background noise was used for 15–20 s before the arrival of the P-wave. At the two stations (PHF and PHT) as shown in Fig. Prior to the site-specific response analysis, the consistency of the energy magnitude according to the frequencies of acceleration waveforms was verified by analyzing from S-wave energy, coda waves, and background noise on the seismic motion data for each measurement stationE, N and Z . Figures 34. In the case of PH EQ#1, which was relatively close to the epicenter, the vector summation of three components measured at the PHF station reached maximum of 454.839 gal, as summarized in Table Seismic motion waves were measured in three directions: E-component in a horizontal direction for each seismic motion, Figs. N-component in the horizontal direction, and Figs. Z-component in the vertical direction in the FFT result. The 9.12 EQ#2 had a large high-frequency energy, and, for the PH EQ#1, relatively low-frequency energy predominated. In particular, in the band below1.0 Hz, the amplitude of the horizontal components  of PH EQ#1 was approximately twice as large as that of the 9.12 EQ#2. In addition, for 9.12 EQ#2 and PH EQ#1, the peak frequency band of the Z-component was different from that of the E- and N-components. The high-frequency (short-period) component of the Z-component was predominant in the PH EQ#1 compared to the 9.12 EQ#2.Figures E, N, Z). In particular, for the vertical direction of the PH EQs, shown in Fig. 35.Figures 31: (1) the first method  on the bedrock of Pohang Yeongil New Port. In Fig. 5 g on th39.For the seismic wave from the El-centro Observatory on the 1940 Imperial Valley EQ, which is typically used for seismic design and response analysis, the strong motion duration was approximately 24.44 s, whereas the EQs used in this study had the form of shock waves with a short strong motion duration. It can be observed that the seismic wave characteristics recorded for ground motion are different and not compatible with the design response spectrum. Therefore, to generate the ground motion using these measuring records, it should be converted as a virtual ground motion appropriately according to synthesis conditions. In order to adequately model the standard deviation of the surface response spectra, at least 10 motions (and preferably 20) are required to obtain stable average spectral results40. In addition, the engineering characteristics of vertical ground motion had emphasized in the seismic analysis and design41.In the recent study of the response spectrum based on acceleration measurement data, it was found that the natural frequency, epicenter distance, and soil type of the site strongly influenced on the characteristics of the response spectrum, and the fault movement type of the seismic source, magnitude of the EQ, and depth of the sedimentary layer were less affected42 using an acceleration response spectrum with a damping ratio of 5%. The vertical axis represents the amplification ratio of the normalized response spectrum with respect to the maximum acceleration. The measured acceleration values of the seismic ground motion were calculated using geometric mean values. In the case of the 9.12 EQs, the response spectral ratio occurred up to three times in the short period range at approximately 0.1–0.3 s, and in the case of the PH EQs, it also occurred up to seven times in a short period of approximately 0.06–0.2 s. It was similar to the peak period derived from the H/V Fourier spectra ratio in Figs. Figure 43. Therefore, the measured data of the seismic accelerometers located in the port area may have been reflected in greater amplification.For reference, background noise sources in the sub-1.0-s band include natural vibration sources, such as wind and waves, and artificial vibration sources, such as transportation and factories. In the period band of 1.0–10.0 s (0.1–1.0 Hz), the main source is a microseism such as the wave activity on the coast and tidesThe response spectra on seismic waves showed that the high-frequency (short-period) component of the vertical direction was predominant in the PH EQ#1 compared to the 9.12 EQ#2.As a result of analyzing the H/V Fourier spectra ratio of the PHT site, the peak frequency was observed from 6 to 12 Hz for Method 1, 7 to 18 Hz (0.056–0.143 s) for Method 2, and 5 to 8 Hz for Method 3.As a result of analyzing the H/V response spectra ratio of the PHT site by Method 2, the amplitude occurred up to three times in the short period of approximately 0.1–0.3 s and up to seven times in the similar short period of approximately 0.06–0.2 s for the 9.12 EQs and PH EQs, respectively. This was similar to the peak period in the H/V Fourier spectra ratio analysis. However, the site amplification might have been reflected more greatly in the measured data of the seismic accelerometers located in the port because of the characteristics of the background noise with a specific period band.In terms of an emergency response system and flexible decision-making support in the event of an EQ, the scientific and reasonable safety assessment technology underlying port areas, which is a representative infrastructure, is of major importance. This can cause significant secondary damage caused by downtime beyond damage to the facility itself. In Korea, the recent 9.12 and the PH EQs caused severe socio-economic damage, prompting concern about EQs. In countries near the Ring of Fire, such as the United States, Japan, and Chile, which have a high probability of EQ occurrence, infrastructure facilities were already managed based on probabilistic seismic hazard analysis and the ground motion prediction equation to prepare for and respond to EQ disasters. Research on the site effects has not been systematic in Korea, and it is necessary to study the local site-specific amplification to analyze seismic sources and an attenuation in crusts-related values to improve the assessment of seismic hazards and risks. In this study, the amplification response characteristics of the site in Pohang Yeongil New Port based on the H/V ratio technique were analyzed in an exemplary manner using the S-wave energy of the acceleration response signal obtained from the seismic accelerometer operated by the MOF, and it can be summarized as follows.This result of site responses can be used to build a database suitable for each geological condition and improve the regional and site coefficients. Furthermore, it can be used to estimate an advanced seismic hazard and develop a safety assessment technology utilizing of seismic accelerometers. After the outbreak of a large EQ, it is important to estimate strong ground motions at the site of damaged port structures to analyze the damage mechanism and determine the restoration policy. In particular, for a port composed of reclaimed ground, more reasonable and reliable characteristics of the port site can be obtained through the site amplification response analysis by also using measured data from a sand layer.Supplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.


Trypanosoma cruzi. The only therapies are the nitroheterocyclic chemicals nifurtimox and benznidazole that cause various adverse effects. The need to create safe and effective medications to improve medical care remains critical. The lack of verified T. cruzi therapeutic targets hinders medication research for Chagas’ disease. In this respect, cytochrome bc1 has been identified as a promising therapeutic target candidate for antibacterial medicines of medical and agricultural interest. Cytochrome bc1 belongs to the mitochondrial electron transport chain and transfers electrons from ubiquinol to cytochrome c1 by the action of two catalytic sites named Qi and Qo. The two binding sites are highly selective, and specific inhibitors exist for each site. Recent studies identified the Qi site of the cytochrome bc1 as a promising drug target against T. cruzi. However, a lack of knowledge of the drug mechanism of action unfortunately hinders the development of new therapies. In this context, knowing the cause of binding site selectivity and the mechanism of action of inhibitors and substrates is crucial for drug discovery and optimization processes. In this paper, we provide a detailed computational investigation of the Qi site of T. cruzi cytochrome b to shed light on the molecular mechanism of action of known inhibitors and substrates. Our study emphasizes the action of inhibitors at the Qi site on a highly unstructured portion of cytochrome b that could be related to the biological function of the electron transport chain complex.Chagas’ disease is a neglected tropical disease caused by the kinetoplastid protozoan  Trypanosoma cruzi, which is prevalent in low-income African, Asian, and American countries and spread through the rest of the world through migrations infecting about 8 million people and causing 10 000 deaths per year. Currently, the only drugs approved for treating Chagas’ disease have various side effects. Identifying proteins of the parasite that can limit its activity can help the development of new and more effective drugs. In this context, the cytochrome bc1 of Trypanosoma cruzi has been identified as a promising drug target. However, it is unclear how certain drugs affect cytochrome bc1 biological activity. Molecular dynamics simulations have been used to analyze the impact of specific small molecules on protein conformational changes that may be related to cytochrome bc1’s biological function. This manuscript presents a computational approach that could identify small-molecule lead compounds and distinguish between true and false cytochrome b inhibitors.Chagas’ disease is caused by the parasite  Trypanosoma cruzi causes Chagas’ disease, a neglected tropical disease that is endemic in South and Central America. Chagas’ disease has also spread through migration across North America, Europe, and the Western Pacific region, infecting about 8 million people worldwide and causing 10 000 deaths annually [The protozoan parasite annually . The treannually ,4. New eT. cruzi drug targets. In this context, cytochrome bc1 has been identified as a drug target for antimicrobial agents of medical and agricultural interest [T. cruzi [Drug discovery for the treatment of Chagas’ Disease is hampered by the scarcity of validated interest ,6 and asT. cruzi . The cytT. cruzi –10. In dT. cruzi .2 of complex III after incubation of GNF7686 [T. cruzi epimastigotes and intracellular amastigotes. GNF7686 is one the most promising Qi site inhibitors, also effective against Leishmania donovani and T. brucei [T. cruzi epimastigotes were cultured in vitro with GNF7686 until drug resistance appeared, finding the L197F mutation in the cytochrome b Qi site [T. cruzi undergoes when the Qi site is occupied by Qi inhibitors (antimycin A and GNF7686), Qo inhibitors (myxothiazol and strobilurin A), and substrates (ubiquinol and ubiquinone), comparing them to the apo-form.Inhibition of the activity of the bc1 complex blocks the generation of ATP  and seve GNF7686  in T. cr. brucei . However. brucei . Clonal  Qi site . In this Qi site . In this Qi site ,22. HereT. cruzi cytochrome b was not available in the literature and homology modelling techniques were employed to address this point. First, the Cytochrome b genes of T. cruzi were extracted from Silvio X10-1 genome  with maxi-circle [T. cruzi cytochrome b, the RNA editing within the coding region before translation was necessary [T. cruzi cytochrome b homology model was built using the crystallographic structure of the avian Gallus gallus (PDB ID: 1BCC)[The 3D structure of .1, NCBI). In ordeecessary . The T. ID: 1BCC) selectinID: 1BCC). The seqID: 1BCC) (see S1 ID: 1BCC) and restID: 1BCC).T. cruzi homology model built, highlighting a good match of the two structures. The quality of the obtained structural model was evaluated by PROCHECK [PROCHECK  observinCHEMPLP scoring function was used in combination with search speed 1, 20 aco ants, and 25 aco evaporation factor [Molecular docking was carried out by PLANTS software . The lign factor . The Qi T = 1.0 ps; time step = 1 fs)[-1·nm-2) were performed using Berendsen semi-isotropic pressure coupling scheme [T = 1.0 ps), together with the semi-isotropic Parrinello-Rahman barostat [The cytochrome b–ligand complexes were inserted in a lipid bilayer of about 160 molecules of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using CHARMM-Builder with 3 nm of water thickness and 150 mM NaCl to neutralize the net charge system. Each resulting molecular system was minimized by 1000 steps using the steep descent algorithm. Two 400 ps position restrained simulations with decreasing force constants to all heavy atoms (2000 and 1000 kJ mol-1·nm-2) were performed under NVT ensemble at 300 K using v-rescale thermostat (τp = 1 fs). Then, t= 5.0 ps) at a ref= 2.0 ps). The sim= 2.0 ps). The par= 2.0 ps) was used= 2.0 ps), assigni= 2.0 ps). Protein= 2.0 ps). All MD = 2.0 ps). The Vis= 2.0 ps) package In order to increase the statistical power, three replicas of each molecular system have been carried out. An overview of the molecular systems investigated is reported in the T. cruzi evaluating three ligand classes bound in the Qi site: Qi inhibitors (antimycin A and GNF7686), Qo inhibitors (myxothiazol and strobilurin A), and substrates (ubiquinol and ubiquinone)  . All molMolecular docking was used to predict the starting pose of each ligand at the Qi site. Each molecular docking configuration was used as starting point for MD simulations. To characterize the effect of ligand-cytochrome b interactions on protein conformational dynamics and the cytochrome b dynamics itself, three independent MD replicas have been performed for each molecular system. During the MD simulations, the cytochrome b’s Qi pocket undergoes deep conformational changes, and each ligand interacted with the Qi site in a different way. Each class of ligands generates a conformational response of the Qi pocket that affects the conformation of a specific area of the protein. In order to highlight the large-scale and low frequency modes of cytochrome b, we performed the Principal Component Analysis (PCA). First, the Cα atoms of the cytochrome b were aligned, then the covariance matrix of the atomic fluctuations was calculated and diagonalized for each molecular system. The molecular trajectories were filtered along the first eigenvector obtaining the low frequency protein motion. T. cruzi.The combined analysis of the RMSF and PCA suggests that the Qi inhibitors’ presence changes the rigidity in the proximity of the active site triggering the conformational changes of the cytochrome b of T. cruzi due to the lack of well-validated molecular targets that limits the medical treatment [T. cruzi [T. cruzi. Two Qi-site inhibitors (antimycin A and GNF7686), two Qo-site inhibitors (myxothiazol and strobilurin A) were chosen and compared with two forms of substrate (ubiquinone and ubiquinol). Membrane proteins are the most accessible targets for therapies in receptor-based techniques, although structural information is limited. In this context, no crystal structure for the cytochrome bc1 of T. cruzi is available and homology modelling techniques have been employed. We have considered the cytochrome b heterodimer in order to focus on the Qi site. In particular, the homology model was built starting from the crystallography of Gallus gallus. In order to reproduce the physiological environment and dynamics of the receptor, the bL-heme and bH-heme molecules of the Qo and Qi catalytic sites were modelled, and the complex was placed in a neutral lipid bilayer model of POPC. The starting configuration of each ligand was predicted by molecular docking and the pose of the antimycin A ligand was used as a control for the docking protocol, obtaining RMSD values of less than 2 Å compared to the pose present in the crystallography with PDB ID 3H1I[In the last decade, there has been renewed attention on Chagas’ disease caused by reatment ,42–45. Creatment . Recent T. cruzi ,46,47. TT. cruzi . OverallB ID 3H1I. The binB ID 3H1I–50. The B ID 3H1I,22. The B ID 3H1I,16. FurtB ID 3H1I–55 and cB ID 3H1I. We haveB ID 3H1I by compeB ID 3H1I,16. In tB ID 3H1I. ExperimB ID 3H1I. A similB ID 3H1I. To summT. cruzi parasite. However, the lack of verified T. cruzi pharmacological targets is a major roadblock in the development of new treatments for Chagas’ disease. In this context, the cytochrome bc1 is a promiscuous drug target candidate for T. cruzi and has been identified as a target for antibacterial drugs of medicinal and agricultural interest. As part of the electron transport chain (ETC), cytochrome bc1 connects electron transport and proton translocation across the inner mitochondrial membrane via the Q cycle. Cytochrome b has two catalytic sites, the Qo site and the Qi site, and numerous hypotheses have been made on the mechanism of cytochrome bc1 inhibition by drugs targeting the Qo binding site [T. cruzi. The observed fluctuations might be a clear sign of the stability of the binding site associated with a given molecule, emphasizing the inhibitors’ competitive effect against substrates and that Qo inhibitors are not suitable for inhibiting cytochrome bc1 activity by binding at the Qi site. Providing detailed mechanistic information about the mechanism of action of inhibition of the Qi site is important if we consider that development of new drugs is often hindered by a lack of information about the mechanism of action. The recent failure of posaconazole, an established CYP51 inhibitor, in phase II clinical trials for Chagas’s Disease highlights the need to better understand the mechanisms of action of drug candidates [T. cruzi cytochrome b and the influence of inhibitors and substrates is critical for drug development and optimization. Our work allows to observe the conformational changes occurring near the Qi site when occupied by true and false inhibitors. We propose that the fluctuation between a ’closed’ and an ’open’ state detected between residues L200 and D230 is responsible for enhancing the protons’ entry into the intermembrane space. The considered inhibitors are specific for a determined catalytic site [T. cruzi cytochrome b induce the same molecular changes in the protein target described in the present manuscript.In recent years, there has been growing interest in discovering new pharmacological treatments for diseases caused by the ing site . The cyting site . For thindidates ,20. Undetic site  and act tic site ,16. In cS1 Table(XLSX)Click here for additional data file.S1 FigSequence alignment of cytochrome b of T. cruzi, and G. gallus.(TIFF)Click here for additional data file.S2 FigPsi-phi angle in degree of the cytochrome b homology model built.(TIFF)Click here for additional data file.S3 FigA) Representative complex model built and B) an enlargement of the ligand and heme groups. The cytochrome b is shown in orange, the POPC lipid bilayer in violet, the heme groups in cyan and the ligand in the Qi site in green.(TIFF)Click here for additional data file.S4 FigMolecular docking pose validation of the antimycin A in the Qi site.(TIFF)Click here for additional data file.


The dynamic mass loss of ice sheets constitutes one of the biggest uncertainties in projections of ice-sheet evolution. One central, understudied aspect of ice flow is how the bulk orientation of the crystal orientation fabric translates to the mechanical anisotropy of ice. Here we show the spatial distribution of the depth-averaged horizontal anisotropy and corresponding directional flow-enhancement factors covering a large area of the Northeast Greenland Ice Stream onset. Our results are based on airborne and ground-based radar surveys, ice-core observations, and numerical ice-flow modelling. They show a strong spatial variability of the horizontal anisotropy and a rapid crystal reorganisation on the order of hundreds of years coinciding with the ice-stream geometry. Compared to isotropic ice, parts of the ice stream are found to be more than one order of magnitude harder for along-flow extension/compression while the shear margins are potentially softened by a factor of two for horizontal-shear deformation. This study uses radio-echo sounding measurements, ice-core data and models to map the spatial variation in ice-crystal orientation in the northeast Greenland Ice Stream and shows how it potentially affects the ice-flow dynamics in this region. In the absence of strain, the crystal orientation fabric (COF) of snow and ice tends to be close to isotropic, so the c-axes point in random directions. In many parts of ice sheets, however, deformation causes an apparent rotation of c-axes towards the maximum shortening direction4, resulting in an anisotropic COF that can be overprinted by subsequent deformation events5. In other words, the COF reflects past ice deformation while simultaneously affecting the present-day mechanical properties.The orientation of the crystals that comprise glacier ice exerts an important physical control over its bulk mechanical properties6 or infer isotropic enhancement factors that subsume some effect of anisotropy8. These enhancement factors are often tuned as model parameters or based on experimental values. In both cases, a physical understanding of the spatial and temporal variation of COF anisotropy is missing8. Anisotropy may explain some of the major discrepancies between modelled and observed surface velocities in highly dynamic areas such as ice streams10 since the commonly used isotropic flow law is no longer valid when the COF is anisotropic. The resulting mechanical anisotropy potentially introduces errors of unknown magnitude in modelled strain rates and, thus, bias in basal sliding velocities obtained by inversion methods7.Most state-of-the-art large-scale ice-flow models either ignore the mechanical anisotropy of ice entirely13. Accordingly, it is crucial to accurately reproduce ice stream dynamics in prognostic ice-sheet models to reduce uncertainties in estimates of future ice-sheet evolution and sea-level rise. In regions of fast flow (> 10 ma−1) with lateral compression and along-flow extension through flow acceleration, the development of strong, spatially variable COFs is expected and might facilitate streaming ice14. A number of ice-flow models capable of simulating COF evolution or its effects on ice deformation15 as well as combining these two mechanisms17 were already developed more than a decade ago. However, both the application of these models to ice streams and spatially extensive in-situ observations of the COF in these dynamic regions remain rare.Ice streams are the primary source of dynamic ice mass loss and are hence of great importance in the stability of ice sheets18. These sites are unrepresentative of the COF in more dynamic areas, such as ice streams and their immediate surrounding where the strain history is more complex.Direct observations of COFs in ice cores are limited to point measurements and by the unknown orientation of the core. Furthermore, most deep ice cores are drilled at ice domes or at ice divides, where the ice tends to flow slowly, and the COF is primarily a result of uniaxial or confined vertical compression22. Single ice crystals show dielectric anisotropy due to their uniaxial birefringent property, i.e. the relative dielectric permittivity is larger in the direction of their c-axis than in the direction of their basal plane. The COF of polycrystalline ice determines its bulk relative dielectric permittivity and affects the speed of electromagnetic wave propagation. Horizontal anisotropy modulates RES signals24 such that the propagation speed of electromagnetic waves depends on their polarisation relative to the preferred orientation of the c-axes and techniques have recently been developed to derive information on the horizontal component of the COF from RES28. In areas where a horizontally anisotropic COF prevails, two different effects are observed in radargrams that can be related to the strength of horizontal anisotropy: (1) Interference of wave components travelling at different speeds leads to nodes of maximum/minimum radar return power23 termed here as birefringence-induced beat signature  surveys are widely used in glaciology for mapping ice thicknesses, basal properties, and internal stratigraphy with electromagnetic wavessotropy:  Interfersotropy:  Interfer28 have used similar methods in isolation to infer the horizontal COF anisotropy of ice streams from radar data. The application of such methods on a large spatial scale and the comparison between different radar methods, COF-evolution modelling, and ice-core data allows us to obtain a robust understanding of the COF distribution and to constrain mechanical anisotropy by calculating directional flow-enhancement factors. Our results show a spatially variable horizontal COF anisotropy that considerably affects the ice stiffness in the NEGIS onset depending on the deformation mechanism and its direction relative to the COF principal axes.In this study, we use the characteristic radar signals induced by the COF to determine the distribution of the horizontal anisotropy at the onset of the Northeast Greenland ice stream (NEGIS), the largest ice stream in Greenland. Using an extensive data set of radar measurements with various radar systems and a combination of independent methods, we determine the distribution of anisotropy in the horizontal plane. Previous studies2 and encompasses the fast-flowing central part as well as the shear margins of the NEGIS onset  deep ice-core site, as well as polarimetric phase-sensitive radio-echo-sounding (pRES) measurements at six distinct locations  retrieved at the southeastern shear margin  is generally high in the ice-stream centre in the upstream part of the NEGIS, suggesting the development of a COF with a pronounced horizontal anisotropy. Downstream of EGRIP, the horizontal anisotropy disappears within a distance of roughly 60 km, particularly evident in the results from the travel-time analysis . Both methods, in that sense, provide apparent horizontal anisotropies that are lower-bound estimates and complement each other.The differences in the strength of horizontal anisotropy between the travel-time and beat-signature analyses can be explained by the polarisation angles under which each method performs best: The strongest effect of horizontal anisotropy from travel-time differences is obtained for waves polarised parallel to the COF’s principal axes, while the obtained horizontal anisotropy seems weaker for misaligned wave polarisations, and no effect is observable for angles of 45Additional differences between panels a–c in Fig. 34 coupled with a COF-evolution model accounting for lattice rotation16 assuming a steady-state ice-stream geometry. The values of Δλ vary between 0–0.9, with 95% of values in the range 0–0.67  imply a horizontal single maximum, intermediate values (Δλ ≈ 0.3–0.6) generally indicate a vertical girdle, and low values provide little information . COFs are constrained to be universally isotropic close to the surface, and are generally vertical single maxima near the bed, though in some regions, the basal COF is partway between a vertical girdle and vertical single maximum.The COF evolution was modelled along flow tubes with the full-Stokes ice-flow model Elmer/Ice.67 Fig. d. The ve35.Horizontal shear causes rotation of the horizontal eigenvectors away from the flow direction , initialised with a vertical girdle of the type observed in the EGRIP ice core31, experiencing the deformation along a downstream flow line. In contrast to Elmer/Ice, Specfab solely simulates the COF development as a response to accumulated strain without being coupled to an ice-flow or temperature model. The COF evolution obtained by this simpler simulation shows a gradual decrease of the horizontal anisotropy, as the COF transitions from a vertical girdle to a vertical single maximum roughly 50 km downstream of EGRIP as a consequence of flow-transverse extension and decreased along-flow acceleration  and the radar-derived horizontal anisotropy, in particular with the travel-time method applied on pRES and airborne crosspoint data (Δλ ≈ 0.4–0.55), agrees well with a vertical girdle observed over large depths in the EGRIP ice core (Δλ ≈ 0.6)31. The results of our radar-based analyses and the Specfab COF evolution modelling consistently indicate a decreasing horizontal anisotropy in the downstream part of the NEGIS. The Elmer/Ice-modelled horizontal anisotropy (Δλ ≈ 0.48) is much smaller than observed at the southeastern shear margin in the S5 shallow core (Δλ ≈ 0.85), while in the northwestern shear margin, the simulations indicate the development of a horizontal single maximum comparable to the observations in the S5 core. Due to the lack of signal return power across large depths of the shear margins, the COF distribution within the shear margins cannot be confirmed independently by radar-based analyses. Outside the NEGIS, the pRES measurement indicates a decreased horizontal anisotropy compared to inside the ice stream, which agrees with the results from the beat-signature analyses and the Elmer/Ice model at the corresponding location.Near the EGRIP drill site, the Elmer/Ice-modelled , corresponding to a distance of around 50 km  based on the model- and radar-derived horizontal anisotropy. The enhancement factors are defined as the longitudinal and shear strain rates with respect to the principal COF axes, divided by the expected strain-rate magnitude assuming isotropic ice. An enhancement factor of one is, by definition, identical to the viscosity of isotropic ice under otherwise identical conditions (e.g. temperature and impurity content). Enhancement factors below or above one imply a decrease or increase in susceptibility towards the corresponding deformation and strain orientation, respectively.The local deformation rate of glacier ice depends on its effective viscosity, which is predominantly controlled by temperature and the COF, while other factors such as grain size distribution, impurities, and water content play a notable but minor role40. While the former can partially be estimated from surface velocities, the latter cannot be inferred from direct radar-based observations on large spatial scales, so is limited to model results in our analysis. The enhancement factors presented here are therefore calculated assuming the strain-rate tensor is oriented in alignment with the COF principal frame. In this sense, our method estimates the eigenenhancements, understood as the maximal effect that COF anisotropy can have on flow under favourable conditions where the strain-rate tensor is aligned with the COF (i.e. an upper-bound estimate). While this is likely to be true for both the ice-stream interior and outside, considerable strain–COF misalignment is expected and modelled in the shear margins  derived from the radar data  compared to further upstream (Exx ≈ 0.02). Enhancement factors based on modelling results  outside the ice stream, with radar-derived enhancement factors ranging from 0.3–0.7 . Unsurprisingly, Elmer/Ice shows the strongest shear enhancements in the shear margins, indicating that they are soft for horizontal shear deformation. The modelled shear-margin softening in Fig. Exy ≈ 1.7–2.3) than in the southeastern margin (Exy ≈ 1.1–1.7) because of the higher residence time in the shear zone of the modelled flow lines. The enhancement factor calculated from the S5 core , as suggested by the calculated enhancement factors inside the ice stream, cannot realistically be achieved through temperature, as it would require the ice stream to be ~15–30 ∘C colder than the surrounding ice under the assumption of cold-ice conditions. However, shear-margin softening of a factor of two, as suggested by Elmer/Ice and the S5 core, could also be obtained by a temperature anomaly of 6.6 ∘C across the shear margins. This estimate is in line with the results of a recent study43 using a 3D-thermomechanical model, which showed that temperature anomalies of up to 6 ∘C are plausible in the NEGIS shear margins. Hence, COF and temperature might be equally important for shear margin softening.To illustrate the significance and magnitude of the flow-enhancement for deformation, we compare it to the influence that temperature would have on viscosity by calculating equivalent temperature anomalies for isotropic ice  lead to a further COF transformation into a vertical girdle, although in a weaker form than upstream.The influence of the COF on the effective viscosity depends on the deformation type and direction Fig. . We find48, and in particular in ice streams with low basal friction49. Calving and similar perturbations at the glacier front or at the bed, propagate upstream through geometrical changes in the flow field or through direct transmission of membrane stresses . A previous study50 found that disturbances on decadal or subdecadal forcing periods propagate upstream through processes dominated by pure shear and that the decay length of the perturbations is longer for harder ice. A simple calculation of the characteristic time for viscous response , describing the density distribution of c-axes orientations: its eigenvectors, ai, and the corresponding eigenvalues, λi, describe the orientation and length of the three principal axes56. By convention, the three eigenvalues are defined as λ1≤λ2≤λ3 and λ1 + λ2 + λ3 = 1. We derived the horizontal COF anisotropy (Δλ) from ground-based and airborne radar profiles, as well as from polarimetric pRES measurements at distinct locations, assuming that one of the principal eigenvectors is vertically oriented. These results are complemented and validated by COF evolution models, as well as observations from the EGRIP and S5 deep and shallow ice cores, respectively. Flow-enhancement factors were calculated from the estimated COF. Details on each method, validation and uncertainties are presented and further discussed in the The orientation of ice crystals is commonly represented as a second-order orientation tensor, 29. The survey was conducted with a Multichannel Coherent Radar Depth Sounder (MCoRDS 5) mounted on the Polar 6 BT-67 aeroplane57. The radar system consists of eight antenna elements, each functioning as a transmitter and a receiver using a transmit–receive switch. The antennas were oriented such that the E-field polarisation is parallel to the flight direction (HH polarisation), and the transmitting wave beam was pointed towards the nadir. The profiles were recorded using linear frequency-modulated chirps in the frequency band of 180–210 MHz at a pulse repetition frequency of 10 kHz, and the received signals were sampled at a frequency of 1.6 GHz. The aeroplane flew at an approximate altitude of 360 m above ground and a velocity of 260 km h−1. Processing was performed with the post-processing CReSIS Toolbox58 and includes pulse compression using a Tukey time–domain weighting and filtering with a Hanning window. We refer to ref. 29 for further details.We used an extensive set of airborne radar sounding data (EGRIP-NOR-2018 survey) which was recorded in 2018 over the onset region of NEGIS and its shear margins59.The airborne survey was complemented with a dedicated ground-based UHF radar profile 3. The sample surfaces were carefully polished with a microtome knife in the EGRIP trench at –18 ∘C. After one hour of controlled sublimation, c-axes were measured with an automated COF analyser by Russel-Head Instruments (FA G50). The data were background corrected before processing, and the COF was derived via digital image processing. Here, we used the COF measurements to validate our geophysical observations and modelling results. The COF information from the EGRIP ice core stems from depths of 1830, 2024, and 2087 m, as published in ref. 31, and shows a vertical girdle with a superimposed horizontal single maximum. Three samples at a depth of 68 m in the S5 shallow core show a strong horizontal single maximum COF.Observational COF data were retrieved from two ice cores: the deep EGRIP ice core and a close-by shallow core (around 14 km from EGRIP) in the southeastern shear margin of NEGIS called S5. The preparation and measurement of the samples from both ice cores follow the same procedures: All samples were analysed from thin sections that were cut vertically to the ice-core axis and have dimensions of about 90 × 70 × 0.3 mmex) and extraordinary (ey) components when travelling through polycrystalline ice with bulk anisotropic dielectric properties. Varying wave speeds for waves polarised in different directions result in a two-way travel-time difference Δt when reflected off the same reflector at depth and received at the surface. The value of Δt at a certain depth depends on the strength of the horizontal anisotropy. At points where two radar waves are polarised orthogonal to each other , the travel time of reflections can be exploited to derive the directional depth-average dielectric permittivities and the degree of horizontal anisotropy. For the airborne data, we manually picked the arrival times of internal reflections in crossing radar profiles. The depth of these reflectors varied from 450 to 3000 m and was determined with a relative permittivity profile derived from Dielectric Profiling (DEP) measurements on the EGRIP ice core. The depth-averaged apparent horizontal anisotropy was calculated from the slopes of a linear regression through the picked arrival times with depth . Additionally, we developed an automated method that was applied to the entire airborne data set. Due to the automatisation, the resulting horizontal anisotropy of the beat-signature analysis is a depth average, representing approximately the top 1700 m of the ice sheet. As for the travel-time difference analysis, the beat signature is only visible for certain antenna polarisations in relation to the COF principal axes. Consequently, The inability of these methods to detect horizontal anisotropy at particular polarizations does not prove the absence thereof. Further details on the manual and automatic methods can be found in the Supplementary Information Section Here, we manually picked the beat signatures in selected airborne and ground-based radargrams to determine the horizontal COF anisotropy  near the EGRIP core site. The flow tubes are wide enough to average out some of the effects of small-scale topography that can detrimentally affect flow line models65, but overlap with each other at some points . While the full stress state is included by the parameterised half dimension, the model is not fully three-dimensional. As a result, the across-flow resolution is very coarse (effectively 2.5 km), and the horizontal shear stresses must be imposed. This requires smoothing of remotely sensed velocity products to obtain reasonable strain rates, which may degrade sharp gradients in the COF. While the model assumes isotropic ice at the surface, ice cores often show anisotropic COFs near the surface69. Despite these limitations, the model matches the pattern of COF inferred from radar and measured by ice cores well enough (i.e. within the uncertainties of the methods) to provide a reasonable estimate of the COF between data points. Further details of the model and our application in the NEGIS can be found in ref. 14 and Section Though this is arguably the most advanced coupled model of COF evolution and flow, it still has a number of shortcomings, particularly for dynamic areas. First, the COF evolution considers only lattice rotation; a number of studies suggest a role for dynamic recrystallisation in the evolution of COFs in warm ice, and even in colder ice when stress is high , and not stress or temperature.Due to the loss of numerical stability associated with high ice-flow velocities, the domain modelled with Elmer/Ice only extends 40 km downstream of EGRIP. To explore the COF evolution in the downstream part of NEGIS, we used the spectral COF model for polycrystalline materials ‘Specfab’ by ref. 31 and let it travel downstream along a flow line obtained from the surface velocities by ref. 64. The Lagrangian COF update of the parcel for incremental steps of 1 year is given by strain-rate and spin tensors derived from the surface velocities in the corresponding flow line segment. The end of the simulation corresponds to a distance of ~120 km from EGRIP and an advection time of 2000 years. Further details of the model and our application can be found in Section 30.We considered an ice parcel which is statistically representative of the COF seeded at EGRIP with an initial COF similar to the average girdle-type measured in the ice core30. While our radar-based COF analyses only allow inferring the difference in horizontal eigenvalues (Δλ), the required full COF estimate can be obtained by applying a few assumptions about the expected COF types inside/outside the ice stream and in the vicinity of the shear margin. These assumptions  that would be required to obtain ice softening/hardening equivalent to that suggested by the COF. Thereby, ΔT is derived from the formula of temperature-dependent enhancement factors assuming isotropic ice and setting the enhancement factors to the values obtained from COF effects. For further details, see Supplementary Information, Sections Besides the COF, the temperature is another major factor controlling the ice viscosity. For comparison, we used the calculated enhancement factors to estimate the temperature difference (ΔSupplementary InformationPeer Review File