Lignin phenol formaldehyde (LPF) resols were produced using depolymerized lignin fractions at various levels of phenol substitution (50 to 70 wt %). To produce monomeric-rich (BCD-oil) and oligomeric (BCD-oligomers) bio-based phenolic compounds, softwood kraft lignin was base-catalysed degraded. These base-catalysed depolymerized (BCD) building blocks were further used to substitute phenol in the synthesis of phenolic resins and were characterized in detail . The adhesive properties were compared to a phenol formaldehyde (PF) reference resin and a LPF with untreated kraft lignin. The resins synthesized with the two depolymerized lignin types differ significantly from each other with increasing phenol substitution. While with LPF-BCD-oligomers the viscosity increases and the bonding strength is not effected by increasing lignin content in the resin, a reduction of these properties could be observed with LPF-BCD-oil. Furthermore, LPF-BCD-oil showed similar curing behaviour and ultimate strength as the reference LPF. Adhesive bonds made using LPF-BCD-oligomers exhibited similar strength to those made using PF. Compared to the reference resins, it has been demonstrated that modified renewable lignin based phenolic components can be an equally performing alternative to phenol even for high degrees of substitution of 70%. Synthetic resins such as formaldehyde-based polycondensation adhesive systems are of great importance for the production of wood-based materials. Because of global environmental concerns and a possible scarcity of fossil resources, it is worthwhile to reduce the consumption of synthetic adhesives. During the last decade, a clear increase in research activity to improve the share of adhesives from bio-resources is evident . This tr6 t/a generated worldwide [3 t/a of kraft lignin are available on the market [Lignin—present in all vascular terrestrial plants—is a complex, amorphous polymer with phenylpropane units linked together by carbon–carbon or ether bonds. It is the second major compound in wood following cellulose and represents 30% of all non-fossil organic carbon on Earth . Technicorldwide . The domDue to a similar chemical structure, the potential of lignin and in particular technical lignins are attractive biopolymers to replace phenol in the production of PF resins has been an intensively researched topic in material sciences since the 1970s . In numeIn contrast to the great number of articles dealing with lignin as phenol substitute published so far, the record of industrial use for wood adhesives is rather poor. Nevertheless, there are some well documented cases of full-scale production experiments based on lignin proportion up to 75% for adhesives  but dueOne of the drawbacks of lignin compared to phenol is its reduced reactivity. Due to the complex chemical structure of lignin, various modification methods—such as methylolation, phenolation or demethoxylation—have already been investigated with the aim of improving the reactivity of lignin towards formaldehyde, with moderate success ,17. FurtAnother promising method to increase the applicability of lignin to generate value added products is the conversion of lignin into smaller fragments through depolymerisation (cracking). The primary purpose of lignin depolymerisation is to convert the complex lignin compound into small molecules and, moreover, increase the chemical reactivity of the degradation products ,20. ThesThe goal of this study is to describe the influence of base-catalysed depolymerized kraft lignin (BCD) on the resin properties of lignin-based phenolic resins. The prepared adhesives, containing up to 70% monomeric-rich (BCD-oil) and oligomeric (BCD-oligomers) bio-based phenolic compounds as phenol substituent, were further characterized in detail and compared to PF reference resin and LPF with untreated kraft lignin in performance.For the base catalysed depolymerisation, Metsä Fibre Oy  kindly provided precipitated pine kraft lignin, which was generated using carbon dioxide from the black liquor of a pulp mill . The usePhenol (≥99.5%) and sodium hydroxide  were purchased from Carl Roth GmbH & Co. KG . Industrial formaldehyde (54.5%), which contained 0.55% methanol and <100 ppm formic acid, was provided by Prefere resins Austria GmbH  and was stored at 60 °C.3 and were selected by focusing on homogenous and straight fibre alignment.For mechanical testing beech veneer strips with a thickness of 0.58 mm and beech boards with a thickness of 10 mm were acquired at J. u. A. Frischeis GmbH . Both veneers and boards had an average density of 591 kg/mLignin fractions were generated by base-catalysed depolymerisation (BCD) of purified softwood kraft lignin. For this purpose, the BCD feed solution was prepared by dissolving 7.50 wt % lignin in 3.75 wt % sodium hydroxide solution. Overall 40 kg lignin were processed in a continuous high-pressure pilot plant with a throughput of 1.4 kg dissolved lignin/h, a pressure of 250 bar and a reactor temperature of 320 °C. The average residence time of the alkaline lignin solution was approx. 10 min. Rößiger et al.  describeThe process leads to the hydrolysis of the aryl-aryl ether-bonds and aryl-methyl ether-bonds of the high molecular weight lignin polymers, the formation of low molecular weight phenolic mono-, di- and oligomers and degradation by-products . A detailed description of the proposed mechanisms is outlined in Rößiger et al. .The generated phenolic compounds were then further separated and purified into a monomer-rich base-catalysed lignin oil (BCD-oil) and an oligomer-rich base-catalysed lignin (BCD-oligomers). The summary of separation and purification process flow including the two products is shown in The purified BCD-oligomer filter-cake and the BCD-oil extract were gravimetrically characterized. The yield of the obtained lignin fractions was calculated as the dry mass percentage of the total.Ash content was determined according to ISO-1762 . 2 g of The specific structural changes in molecular weights were determined by gel permeation chromatography (GPC) using a high-performance liquid chromatography (HPLC) series 1260 from Agilent Technologies , equipped with GPC columns (AppliChrom ABOA DMSO-Phil-P Pore 250 & 350) and a refractive index detector.Furthermore, the concentration of individual phenolic, monomeric compounds was quantified by gas chromatography-mass spectrometry (GC-MS) (Agilent GC 7890A and Agilent MSD 5975C). The GC-MS was equipped with a HP-MS5 capillary column . As internal calibration standard, 1-methylnaphalene was used.The reference PF resin and untreated LPF reference resin with 50% unfractionated lignin were synthesized according to a recipe by Prefere Resin Oy , using the procedure described by Solt et al. .For the synthesis of LPF with BCD-oligomers, lignin powder was slowly dispersed in 50% NaOH-solution and mixed until a uniform slurry was achieved. Depending on the desired amount of phenol substitution , the proportion of lignin in the slurry varies and thus its solid content and slurry viscosity. In the case of BCD-oil, this pre-step was not necessary as the material is already in a liquid state and could be used without further treatment. The LPF resins, both with BCD-oligomers and with BCD-oil, were further produced according to the recipe described in Solt et al. . BecauseSolid content of the resins was determined according to EN-827 (2006) standard . TherefoThe pH value of each adhesive was measured according to ISO-8975 (1989) standard . For thiViscosity of the adhesives was obtained in accordance with ISO-3219 (1993) standard  standardFree formaldehyde content was determined based on ISO 11402 (2005) standard . TherefoFree phenol in the phenolic adhesives were determined on an ICS 5000 HPLC system  by Prefere resins Austria GmbH . The device was equipped with a UV-detector (271 nm) and Chromolith RP-18e 100 × 4.6 mm column. As external standard phenol was used. The measured value of free phenol had to be below the limit of 0.1 ± 0.03 wt %.The time required to reach the B-stage of phenolic resin curing (B-time) was measured according to German DIN-8987 (2006) standard  using se2. After press times from 30 s to 20 min  the tensile shear strength of the sample was tested with a testing speed of 60 mm/min. Exact method can be found in Solt et al. [To evaluate the development of the bonding strength as a function of hot pressing time a self-constructed hot-press was mounted in a Zwick/Roell Z100 universal testing machine . This specific setup was selected in order to allow testing according to ASTM-D7998-15  as origit et al. .2. For hot pressing (120 °C at 0.8 N/mm2), the pressing time of 20 min was derived from the ascertained curing time results of the automated curing evaluation system. This press time consists of 15 min until almost all systems reached final curing including 5 min through heating time. Samples were cut and treated with two different approaches according to the standard (A1 (dry): 20 °C, 65% rh for 7 days; A2 (wet): additionally stored in water at 20 °C for 4 days and tested in wet condition). The tensile shear strength of all adhesives was determined using the maximum recorded force divided by the overlap area of the joint.Tensile shear strength determination of the adhesives was carried out according to EN-302-1 (2004)  and the Mn/Mw = 1350/9850 g/mol). The isolated BCD-oligomers possessed a molar mass of 2250 g/mol and furthermore a polydispersity index (PDI) of 3.0. BCD-oil consists of low molecular weights compounds and had the narrowest molar mass distribution (PDI of 2.0) with Mw of 300 g/mol. Taking coniferyl alcohol with a molecular weight of 180 g/mol as a representative example for a lignin monomer, BCD-oligomers can be classified as oligomer and BCD-oil on average as dimer.Mw: 124 g/mol) and 27.6 wt % catechol (Mw: 110 g/mol). The residual 65.4 wt % of BCD-oil contains unknown or non-volatile mono-, di- and oligomeric phenolic compounds. The general objective of BCD is the reduction of complexities of the natural lignin molecule and increasing its chemical reactivity. These changes in structure can be qualified by analysing the molecular mass distribution. BCD oligomers were obtained as a brown powder. BCD oil is a dark brown liquid with a viscosity of 750 mPa·s.Quantitative GC-MS analysis of phenolic monomers in BCD-oil yields a content of 7.0 wt % guaiacol  of all LPF resins as well as of the reference resin were in the range between 0.3% and 0.9%. Compared to the standard PF-resole, all LPF-BCD-oligomers resins reached slightly higher values. For LPF-BCD-oligomers 70%, it was not possible to dissolve and further measure the HCHO amount. Furthermore, it can be seen that LPF with BCD-oil shows a significant increase in free formaldehyde with increasing phenol substitution and thus did not reach the selected target value of (≤0.5 wt %) above a substitution degree of 50%. Apparently, the components of BCD-oil have less reactive sites than phenol, resulting in remnant formaldehyde. The amount of free formaldehyde could be reduced by increasing condensation time, which was not done for reasons of comparability. Free phenol contents show again that beside of LPF-BCD-oil, the other resins reached the selected target values below 0.1 wt %.The B-Time was evaluated at 140 °C. Up to 60% phenol substitution, the BCD oligomers provide resins that are equally reactive as unmodified PF and are more reactive than LPF resins comprising uncracked lignin at a 50% substitution level. At 70% phenol substitution, the resin shows a slower gellation than PF but still similar to the reference LPF. The B-time of LPF-BCD-oil modified resins was in all cases shorter than the B-time of the reference PF resins, albeit at higher levels of free formaldehyde. Since the curing of phenolic resins correlates to the amount of free formaldehyde , this coMeasuring the development of the tensile shear strength during hot pressing is a method to investigate the curing properties of binders in the presence of adherents. The development of bonding strength is strongly influenced by a number of key factors such as pressing temperature, pressure and time but also by chemical properties of the adhesive and the adherend materials . Figure 2 with 100% wood failure) was reached with the LPF-BCD-oligomers resins at all three different phenol replacement levels. Furthermore, between the substitution levels no explicit difference can be found in the speed of strength development and the maximum measured tensile shear strength. Even at 70% substitution, a similar performance could be obtained, even though this resin showed a longer B-time. Thus, the BCD-oligomers seem to be reactive oligomers that can be perfectly bonded into the polymer network. For the LPF-BCD-oils a decelerated bonding strength development compared to PF reference was found. The bond strength developed slower as the lignin amount was increased. Especially in case of LPF-BCD-oil 70%, the curve shows a level-off in strength after 10 min pressing time at a value of 4.9 N/mm2, representing therefore a 25% lower result compared to the PF reference. Likewise, Ghorbani et al. [Using BCD-oligomers , a simil2 for thin bond lines. Interestingly, PF as well as the reference LPF resins bonded samples did not pass the standard tensile shear strength for A2  treated phenolic resins (6 N/mm2), whereas the LPFs with BCD-oligomers and BCD-oil (expect LPF-BCD-oil 70%) did. The lap joint bonded with PF reference resin has an average strength of 14 N/mm2 with a high wood failure (median: 90%) and after water storage a strength reduction of 60% with no wood failure. It should be noted that phenolic resins are typically applied where moisture and water-resistant bonding are needed and therefore commercial produced PF resins usually perform well under wet conditions. The weaker performance after treatment A2 could be attributed to the laboratory-scale production but also to the relatively low binder amount (200 g/m2). At a solid content of 43.6%, the adhesive joint achieves a solid resin content of 87 g/m2, which additionally complicates a homogeneous adhesive distribution. For the case of kraft lignin used in the form of a precipitated lignin without any further treatment in phenolic resins, the tensile shear strength—both wet and dry—is clearly below the PF resoles. As expected from the results of curing strength development, also final strength of LPF-BCD-oligomers show similar tensile shear strength as PF-reference. An apparent trend of increasing strength with increasing lignin content for LPF-BCD-oligomers could be observed, which, however can be neglected due to almost 100% wood failure for all degrees of substitution. As this strength value indicates the strength of the wood substrate, the value is not appropriate for differentiation between adhesive performance [2) but higher wet and dry strength level than LPF-reference. This observation for depolymerized kraft lignin is in line with the conclusion of Ma et al. [formance . LPF-BCDa et al. . They in median: 0% and afa et al. ,16,35. TKraft lignin was cracked into two fractions using a base-catalysed depolymerisation process to gain monomer-rich and oligomeric lignin units. These cracked lignins were further used to produce lignin-based phenolic resins with 50–70 wt % substitution of phenol by lignin and compared with untreated kraft lignin based phenolic resins and phenol formaldehyde reference resins. The results gained demonstrated that base-catalysed depolymerisation lignin oligomers (BCD-oligomers) can successfully substitute phenol in corresponding resins at high substitution levels of up to 70% without biasing final dry or wet tensile shear strength. BCD-oligomers showed consistently better performance compared to BCD-oil. Unique to this approach is that even for the high degree of phenol substitution the speed of bond strength development was fully comparable to the PF reference at 120 °C hot pressing temperature. Only the high viscosity of the LPF-BCD oligomer resin poses a challenge for industrial applications, which was not in focus of the present research. By further optimization, cracked lignins have the potential of achieving high performing resin meeting all industrial needs.


Hematoma after arterial cannulation is not so very rare but tends to occur during cannulation and immediately after decannulation. We report a case of delayed re-bleeding after the removal of a radial arterial cannula.A 74-year-old woman underwent revision total arthroplasty under general anesthesia with epidural anesthesia. She had no comorbidities and regularly took loxoprofen sodium hydrate. Examinations before the operation had no abnormal findings. Her right radial artery was punctured and cannulated uneventfully with a 22-gauge BD Insyte™ IV Catheter . The amount of bleeding was 320 mL, and she received 160 mL of autologous fresh-frozen plasma. The arterial cannula was immediately removed at the end of anesthesia. An anesthesiologist achieved hemostasis at the punctured site with 5 min of compression by hand. She exited the operation room with a hemostasis device  over the puncture site. The device was removed 2 h after the operation, when her bleeding was confirmed to have stopped. She was observed every 2 h without re-bleeding at the puncture site, and her systolic blood pressure was kept under 120 mmHg. A nurse noted bleeding from the puncture site 13 h after the operation. She compressed the site by hand and called a duty doctor, who advised watchful waiting. They confirmed the stop of bleeding and noticed slightly edema and internal hemorrhaging around the puncture site. The next day, internal hemorrhaging and edema from the fingers to the middle of the upper arm were noted (Fig. Indwelling arterial catheters is generally a safe procedure with few serious complications , 2. The The radial arterial puncture site is usually manually compressed at the time of decannulation. However, basic questions such as how long and strongly compression should be performed remain unclear.We should check for hemostasis frequently in cooperation with the ward staff.


Dishevelled (DVL) is an essential component of the Wnt signaling cascades. Function of DVL is controlled by phosphorylation but the molecular details are missing. DVL3 contains 131 serines and threonines whose phosphorylation generates complex barcodes underlying diverse DVL3 functions. In order to dissect the role of DVL phosphorylation we analyzed the phosphorylation of human DVL3 induced by previously reported  and newly identified  DVL kinases.2 enrichment of phosphorylated peptides followed by liquid chromatography tandem mass spectrometry on immunoprecipitates from HEK293T cells was used to identify and quantify phosphorylation of DVL3 protein induced by 8 kinases. Functional characterization was performed by in-cell analysis of phospho-mimicking/non-phosphorylatable DVL3 mutants and supported by FRET assays and NMR spectroscopy.Shotgun proteomics including TiOWe used quantitative mass spectrometry and calculated site occupancies and quantified phosphorylation of > 80 residues. Functional validation demonstrated the importance of CK1ε-induced phosphorylation of S268 and S311 for Wnt-3a-induced β-catenin activation. S630–643 cluster phosphorylation by CK1, NEK2 or TTBK2 is essential for even subcellular distribution of DVL3 when induced by CK1 and TTBK2 but not by NEK2. Further investigation showed that NEK2 utilizes a different mechanism to promote even localization of DVL3. NEK2 triggered phosphorylation of PDZ domain at S263 and S280 prevents binding of DVL C-terminus to PDZ and promotes an open conformation of DVL3 that is more prone to even subcellular localization.We identify unique phosphorylation barcodes associated with DVL function. Our data provide an example of functional synergy between phosphorylation in structured domains and unstructured IDRs that together dictate the biological outcome.Video Abtract. Wnt signaling pathway has been linked to an etiology of multiple developmental defects, inherited diseases and many types of cancer . Wnt patDrosophila, DVL1–3 in human). DVL proteins consist of three structured domains: N-terminally located DIX domain, centrally located PDZ domain and C-terminally located DEP domain. The individual domains are linked by intrinsically disordered regions followed by approximately 200 amino acid long, also intrinsically disordered, C-terminal tail.All known Wnt-induced pathways are transduced through Frizzled transmembrane receptors  and intrThere is a general agreement based on genetic experiments that DVL plays a crucial role as a signaling hub in both Wnt/β-catenin and non-canonical Wnt pathways . In addiDespite the well documented role of DVL in the Wnt signaling and the growing evidence for its participation in additional cellular processes, the molecular mechanisms that regulate DVL action in the cell are almost unknown. However, it is believed that post-translational modifications (PTM), particularly phosphorylation, represent a key component of such regulatory mechanisms. DVL proteins are very rich in serine (S) and threonine (T); for example, DVL3 contains 131 S/T residues, which can be potentially phosphorylated. The best described consequence of the activation of both Wnt/β-catenin as well as non-canonical Wnt pathway is phosphorylation of DVL by the Wnt-induced Casein kinase 1 ɛ (CK1ɛ) –14. In aIn this study we analyzed and compared the phosphorylation of DVL3 induced by eight S/T kinases. We have selected DVL3 as a candidate because its absence shows the strong phenotypes in vivo . We haveHEK293T, HEK293 T-REx and DVL1/2/3-null HEK293 T-REx cells were propagated in DMEM, 10% FCS, 2 mM L-glutamine, 50 units/ml penicillin, 50 units/ml streptomycin. CK1ε inhibitor PF-670462 was used at 10 μM. Cells were seeded on appropriate culture dishes  and the next day were transfected using polyethylenimine (PEI) in a stoichiometry of 3 μl PEI (0.1% w/v in MQ water) per 1 μg of DNA. Cells were harvested for immunoblotting or immunocytofluorescence 24 h after transfection, for immunoprecipitation after 48 h. The following plasmids have been published previously: FLAG-DVL3 , CK1ε 2, Myc-NEKFor the luciferase reporter assay, cells were transfected with 0.1 μg of Super8X TopFlash construct, 0.1 μg of pRLTKluc (Renilla) luciferase construct and other plasmids as indicated  24 h after seeding. Two ml of ice-cold lysis buffer supplemented with protease inhibitors , phosphatase inhibitors , 0.1 mM DTT, and 10 mM N-ethylmaleimide (Sigma E3876) was used for lysis of one 15 cm dish. Lysate was collected after 20 min of lysis on 4 °C and was cleared by centrifugation at 18000×g for 20 min. Three μg of anti-FLAG M2  antibody were used per sample. Samples were incubated with the antibody for 40 min, then 25 μl of G protein-Sepharose beads  equilibrated in the lysis buffer were added to each sample. Samples were incubated on the carousel overnight, washed 6 times with lysis buffer and finally, 40 μl of 2× Laemmli buffer was added and samples were subsequently boiled.The samples were loaded to 8% SDS-PAGE gels. Electrophoresis was preformed through the stacking gel at 100 V and through the separating gel at 150 with PageRuler Prestained Protein Ladder  as a marker. Gels were then process for mass spectrometry or the proteins were transferred to the polyvinylidene difluoride membrane  by Western Blotting (WB). Subsequently, the membrane was blocked in 5% defatted milk or 3% BSA for 1 h with shaking. After the blocking step, the membrane was incubated with corresponding primary and secondary antibodies. Proteins were visualized by ECL . The signal was detected using Vilber FUSION-SL system. Western blot was quantified using ImageJ software.http://www.moravian-biotech.com).The antibodies used were: anti-FLAG M2  for WB and IP, anti-CK1ɛ , anti-GFP , HA11 (Covance #MMS-101R), VSV (Sigma-Aldrich #V 5507), c-Myc , α-tubulin , anti-DVL3  and anti-β-actin . Following phospho-specific antibodies have been published previously – pS280-DVL3 (pS280) [N = 3) were analyzed and scored according to their phenotype into two categories (punctae/even). The antibodies used were as follows: anti-FLAG M2 , anti-DVL3 , anti-CK1ɛ , HA11 (Covance #MMS-101R), VSV (Sigma-Aldrich #V 5507), c-Myc  and anti-GFP , CAP350 [Cells were seeded onto glass coverslips and next day transfected according to the scheme. 24 h post transfection medium was removed, cells were washed by PBS and fixed by 4% PFA for 10 min or ice-cold MetOH  for 20 min. Cells were transfected 16–18 h later using Effectene (Qiagen), according to the manufacturer’s instructions. DMEM was replaced 6 h later and the analysis was done 24 h after transfection.FlAsH labeling of the DVL3 FlAsH III sensor was performed as previously described . Shortly. Fluorescence intensities data were acquired using Clampex software (Axon Instruments). During measurements, cells were maintained in imaging buffer; 5 mM of 2,3-dimercapto-1-propanol  was added to the cells approximately 40 s after the recording started. Recovery of ECFP fluorescence was monitored over time and FRET efficiency was calculated as described [To determine the FRET efficiency of the DVL3 FlAsH III sensor, coverslips with the cells were mounted using an Attofluor holder (Molecular Probes) and placed on a Zeiss inverted microscope (Axiovert200), equipped with 63x oil objective lens and a dual-emission photometric system (Till Photonics). Cells were excited at 436 ± 10 nm using a frequency of 10 Hz with 40 ms illumination time out of a total of 100 ms. Emission of ECFP (480 ± 20 nm) and FlAsH (535 ± 15 nm), and the FRET ratio (FlAsH/ECFP) were monitored simultaneously. Fluorescence signals were detected by photodiodes and digitalized using an analogue-digital converter The mutagenesis reactions were performed using the QuikChange II XL Site-Directed Mutagenesis Kit following the manufacturer’s instructions . All mutations described in this study were verified by Sanger sequencing. Following primers were used:For transfection HEK293 T-REx (WT) and DVL1/2/3-null HEK293 T-REx cells (D1/2/3 TKO HEK293 T-REx) cells were seeded at 200000 cell/well in 24 well plate density. Next day, the cells were transfected using polyethylenimine (PEI) in concentration 1 μg/ml and pH 7.4 and the PEI ratio 6 μl of PEI/ 1 μg DNA. Mixture of transfected plasmids  luciferase construct per well, the total amount of DNA was equalized by pcDNA3.1 to 400 ng DNA/well) and PEI was diluted separately in plain DMEM . After 6 h, medium containing transfection mix was removed and exchanged by complete DMEM medium. In all experiments cells were treated by 1 μM porcupine inhibitor LGK974  to reduce the autocrine secretion of all Wnt ligands and recombinant human R-spondin1 250 ng/ml . For stimulation of the cells, Wnt3a recombinant protein was used for 14 h in concentration 80 ng/ml . Control stimulations were done with 0.1% BSA in PBS. Samples were then analyzed by TopFlash reporter assay and WB.6-tag, a lipoyl domain tag and a TEV cleavage site. All proteins were expressed in BL21-DE3(RIL) cells. For NMR studies, cells were grown in minimal medium (M9) supplemented by 15NH4Cl (1 g/l) and/or 13C6 glucose (2 g/l) and induced with 0.5 mM IPTG for 24 h at 16 °C. Proteins were purified as described before [Human DVL2 PDZ domain (aa 265–361) and its phosphomimicking mutant (S281E + S298E) were cloned into pET vector with N-terminal His1H/13C/15N TCI cryogenic probe head with z-axis gradients. Chemical shift assignments of the phosphomimicking PDZ mutant (S281E + S298E) were obtained automatically using 4D-CHAINS technology [1H-15N HSQC spectra using 100 μM of 15N-labeled protein (wild type or phosphomimicking mutant) and increasing amounts of DVL_C (stock concentration of 800 μM).NMR experiments were carried out at CEITEC Josef Dadok National NMR Centre on a 700 MHz Bruker Avance III spectrometers equipped with chnology  as descrchnology . NMR titImmunoprecipitates were separated on SDS-PAGE gel electrophoresis, fixed with acetic acid in methanol and stained with Coomassie brilliant blue for 1 h. Corresponding 1D bands were excised. After destaining, the proteins in gel pieces were incubated with 10 mM DTT at 56 °C for 45 min. After removal of DTT excess samples were incubated with 55 mM IAA at room temperature in darkness for 30 min, then alkylation solution was removed and gel pieces were hydrated for 45 min at 4 °C in digestion solution . The trypsin digestion proceeded for 2 h at 37 °C on Thermomixer . Subsequently, the tryptic digests were cleaved by chymotrypsin  for 2 h at 37 °C. Digested peptides were extracted from gels using 50% ACN solution with 2.5% formic acid (FA) and concentrated in speedVac concentrator (Eppendorf). The aliquot (1/10) of concentrated sample was transferred to LC-MS vial with already added polyethylene glycol  was used for phosphopeptide analysis. Sample was diluted with acidified acetonitrile solution . Phosphopeptides were enriched using Pierce Magnetic Titanium Dioxide Phosphopeptide Enrichment Kit  according to manufacturer protocol and eluted into LC-MS vial with already added PEG . Eluates were concentrated under vacuum and then dissolved in water and 0.6 μl of 5% FA to get 12 μl of peptide solution before LC-MS/MS analysis.LC-MS/MS analyses of peptide mixture were done using RSLCnano system connected to Orbitrap Elite hybrid spectrometer (Thermo Fisher Scientific) with ABIRD  and Digital PicoView 550 (New Objective) ion source (tip rinsing by 50% acetonitrile with 0.1% formic acid) installed. Prior to LC separation, peptide samples were online concentrated and desalted using trapping column (100 μm × 30 mm) filled with 3.5 μm X-Bridge BEH 130 C18 sorbent (Waters). After washing of trapping column with 0.1% FA, the peptides were eluted (flow 300 nl/min) from the trapping column onto Acclaim Pepmap100 C18 column  by 65 min long gradient. Mobile phase A (0.1% FA in water) and mobile phase B (0.1% FA in 80% acetonitrile) were used in both cases. The gradient elution started at 1% of mobile phase B and increased from 1 to 56% during the first 50 min (30% in the 35th and 56% in 50th min), then increased linearly to 80% of mobile phase B in the next 5 min and remained at this state for the next 10 min. Equilibration of the trapping column and the column was done prior to sample injection to sample loop. The analytical column outlet was directly connected to the Digital PicoView 550 ion source.6 ions, one microscan and maximum injection time of 200 ms. High resolution  HCD MS/MS spectra were acquired with a target value of 50,000. Normalized collision energy was 32% for HCD spectra. The maximum injection time for MS/MS was 500 ms. Dynamic exclusion was enabled for 45 s after one MS/MS spectra acquisition and early expiration was disabled. The isolation window for MS/MS fragmentation was set to 2 m/z.MS data were acquired in a data-dependent strategy selecting up to top 10 precursors based on precursor abundance in the survey scan (350–2000 m/z). The resolution of the survey scan was 60,000 (400 m/z) with a target value of 1 × 10http://www.thegpm.org/crap/). Mass tolerance for peptides and MS/MS fragments were 7 ppm and 0.03 Da, respectively. Oxidation of methionine, deamidation  and phosphorylation  as optional modification, carbamidomethylation of C as fixed modification, TrypChymo enzyme specifity and three enzyme miss cleavages were set for all searches. The phosphoRS (version 3.1) feature was used for preliminary phosphorylation localization. Final localization of all phosphorylations  was performed by manual evaluation of the fragmentation spectra of the individual phosphopeptides. Based on the presence of individual fragments in the peptide sequence, it was decided whether the localization was accurate or not.The analysis of the mass spectrometric RAW data was carried out using the Proteome Discoverer software  with in-house Mascot  search engine utilization. MS/MS ion searches were done against in-house database containing expected protein of interest with additional sequences from cRAP database . Normalization of the data was performed using the set of phosphopeptide standards  and by non-phosphorylated peptides identified in direct analyses.All quantitative data (peptide intensities) were processed by two approaches see Fig. a. In theData of absolute peptide intensities were log-transformed (log10) because of their log-normal distribution. Log-transformed data of each replicate were standardized to control (kinase subtracted from “no kinase” control) and averaged from 3 replicates. Cluster analysis was applied both for kinases and for phosphosites; Ward’s minimum variance method based on the Lance-Williams recurrence and Euclidean distance were used. Circular plot accompanied by heat map was used for visualization of complex relation among phosphosites and kinases. All analyses were performed using R software.One-way ANOVA and Tukey Post tests were calculated by GraphPad Prism (GraphPad Software Inc.).Our previous work identified interactions between DVL3, CEP164, and TTBK2 kinase , 39. To Mobility-shift of DVL2 and DVL3 induced by TTBK2 Fig. a can be The best-defined role of DVL is the positive regulation of the Wnt/β-catenin pathway. In order to address if TTBK2 modulates this DVL function we analyzed the ability of TTBK2 to promote or to inhibit DVL-induced TCF/LEF-dependent luciferase reporter (TopFlash) in HEK293 cells. Interestingly, TTBK2 did inhibit efficiently DVL1- and DVL2-induced TopFlash activation in a kinase activity-dependent manner. Because DVL3 alone induces TopFlash very poorly . All samples were subsequently analyzed by liquid chromatography coupled to mass spectrometry (LC-MS/MS).Following validation of our experimental system Fig.  we perfoPeptides with accurately characterized phosphorylated site(s) were identified alongside with peptides of ambiguous phospholocalization, as it is common to proteomic studies. Therefore, in the accurate position focused analysis (pipeline #2) only a subgroup of peptides with clearly identified phosphorylated positions  were included. In the overall quantitative analysis (pipeline #3) all phosphorylated peptides were processed despite the uncertainty of phosphoresidues and as such were presented as “phosphorylated clusters”. For the detailed information on the quantification in these two pipelines see Materials and Methods.Combination of the above-mentioned approaches allowed us to cover more than 95% of the DVL3 sequence across all experiments  covering individual phosphosites or clusters to total intensity (phosphorylated peptide(s) + corresponding non-phosphorylated peptide(s)). Phosphorylated sites/clusters with > 5% site occupancy in one sample are plotted in Fig. We observed an increase in the phosphorylated site occupancy (above 5%) after overexpression of at least one protein kinase in all but one  cases F. The not2. This approach, commonly utilized for detailed screening of protein phosphorylation, allows detection of less abundant phosphopeptides. Phospho-enrichment data were processed in two ways  function by kinases and phosphatases.In this study we have identified TTBK2 and Aurora A as novel DVL kinases. For TTBK2 we propose a possible negative role in Wnt/β-catenin signaling. TTBK2 is found to basal bodies or distal appendages, respectively. Indeed, DVL has been associated with centrosome and basal bodies , 10, 53.The parallel application of several pipelines for sample preparation and data analysis, in particular the newly introduced site occupancy analysis, demonstrated the potential to detect phosphorylation events that are biologically relevant. This approach identified S268 as signature site for CK1ε, S280 as a signature site for NEK2 and S633–S643 cluster as a signature for CK1ε, NEK2 and TTBK2. All these phosphorylation changes have been validated by additional, more demanding proteomic pipelines and functional analysis.The data collected by various approaches shown in Figs. It is behind the scope of this study to test the functional consequences of all phosphorylation events. However, several interesting candidates for further analysis have been identified. For example, a striking phosphorylation pattern has been observed for two highly conserved regions between DIX and DEP domains – corresponding to S112–S140 and S188–S197. For the second cluster we were able to confirm the proteomic data by a phosphorylation-specific antibody targeted against pS192-DVL3. These sites showed high level of basal phosphorylation in the absence of any exogenously co-expressed kinase. Interestingly, expression of CK1ε (and to a lesser extent TTBK2 and PLK1) dramatically reduced phosphorylation of these motifs. This suggests that there is a so far unidentified endogenous kinase that very efficiently phosphorylates these regions, and (ii) that the binding and/or phosphorylation by CK1ε and TTBK2 interferes with this process or perhaps promotes removal of these phospho-moieties by activation of specific phosphatase(s). So far, only protein phosphatase 2A has been shown to have a positive function in the Wnt/β-catenin signaling upstream of DVL , 56, whiOur functional analysis of PTMs that control subcellular localization of DVL3 showed that phosphorylation in the domains (namely PDZ) and in the IDRs can lead to the same outcome. CK1ε, NEK2 and TTBK2, were the only kinases that could trigger the even distribution of DVL3 in the cytoplasm, in line with earlier reports , 12, 13 In summary, our study identified unique DVL3 phosphorylation barcodes associated with individual DVL kinases and DVL functions. Our data represent an important reference point and a toolbox for further analysis of DVL as exemplified by the functional analysis of the phosphorylation in the PDZ domain. From the more general point of view, our data pinpoint the importance of functional synergy between phosphorylation in the structured domains and in the unstructured IDRs that together dictate the biological outcome.Additional file 1: Figure S1. SDS-PAGE gels from three independent experiments. FLAG-DVL3 was overexpressed in HEK293, with or without the studied kinase, immunoprecipitated using the anti-FLAG antibody, separated on SDS-PAGE and stained with Coomassie Brilliant Blue. In red boxes are parts that were cut out.Additional file 2: Figure S2. Phosphorylated clusters map of DVL3. All identified phosphorylated clusters obtained from the pipeline #3 are visualized as a heatmap. Mean absolute intensities of phosphorylation clusters in the control  are expressed in the shades of blue. Numbers indicate decadic logarithm of the mean of 6 control samples. Nine columns represent heat map of relative change of phosphorylated peptide intensities  obtained for individual kinases (relative to control). Numbers in the heat map fields  indicate the number of experimental replicates with the positive identification of the given phosphorylated site.Additional file 3: Figure S3. Cluster analysis of the individual phosphorylated sites/clusters and individual kinases. Mean phosphorylated peptide intensities  standardized to control are used in the circular plot. Peptides expressed more intensively compared to control are in the shades of green, whereas less expressed peptides are in red. Dendrograms both for the kinases and the phosphosites are shown.Additional file 4: Figure S4. NMR comparison of DVL2 PDZ wild-type and phosphorylation-mimicking mutant S281E + S298E. A. 1H, 15 N HSQC spectra of PDZ wild-type (blue) and phosphorylation-mimicking mutant S281E + S298E (red). All amide frequencies have been assigned unambiguously using 4D spectra. The e cross-peaks of wild-type and corresponding glutamic acids cross-peaks of the mutant are marked. B. Weighted chemical shift differences between PDZ wild-type and S281E + S298E mutant versus the protein sequence.Additional file 5: Table S1. Intensities of the individual phosphorylated sites (raw data). Mean phosphorylated peptide intensities  obtained for individual sample from direct (pipeline #1) and enriched analysis processed (pipeline #2) are expressed in the shades of blue. ND corresponds to not detected signals or signals with intensity below 1 × 106.Additional file 6: Table S2. Sequence coverage of DVL3 in the individual experiments. Sequence coverage of DVL3 obtained for individual kinases in each replicate, in individual sample  and throughout the experiment.


Results from in silico study indicates that caffeine has comparatively good affinity with collagenase , elastase  and tyrosinase  and formed the stable protein-ligand complex as validated by molecular dynamics simulation . Moreover, in vitro data showed that caffeine (1000 µg/mL) has statistically significant maximum inhibition activity of 41.86, 36.44 and 13.72% for collagenase, elastase and tyrosinase, respectively.Skin ageing results from enhanced activation of intracellular enzymes such as collagenases, elastases and tyrosinase, stimulated by intrinsic ageing and photoageing factors. Recently, caffeine-based cosmetics are introduced that demonstrates to slow down skin photoageing process. However, no attempts have been done so for to understand caffeine functional inhibitory activity against photoageing related enzymes. Hence, this study established the caffeine molecular interaction and inhibition activity profiles against respective enzymes using  Clostridium histolyticum collagenase, porcine pancreatic elastase and Agaricus bisporus tyrosinase, respectively was investigated. The molecular interaction analysis was further evaluated using molecular dynamic simulation studies to decipher the solidity of respective enzyme-caffeine docked complex, and finally, caffeine inhibition activity for the selected enzymes along was calculated by in vitro approach as shown in Therefore, for the present study, we attempt to elucidate the functional inhibitory potential of caffeine as therapeutic agent against photoageing of skin by inhibiting the MMPs (collagenase and elastase) and tyrosinase activity. Initially, we employed 2.2.1.Clostridium histolyticum collagenase, porcine pancreatic elastase, Agaricus bisporus tyrosinase, 4-phenylazo benzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg substrate, N-succinyl-(Ala)3-p-nitroanilide and tyrosinase were procured from Sigma-Aldrich CO., St. Louis, MO, USA. All the other chemicals used in this study were of analytical grade.Analytical grade caffeine along with selected chemicals i.e. epigallocatechin gallate (EGCG), ursolic acid, arbutin, 2.2.https://pubchem.ncbi.nlm.nih.gov)http://www.swissadme.ch/)%20)http://www.rcsb.org/pdb/home/home.do)Three-dimensional (3D) structure of test compound i.e. caffeine (CID 2519) and reference compounds i.e. EGCG (CID 65064), ursolic acid (CID 64945) and arbutin (CID 440936) were downloaded from PubChem database ,The active site prediction in the selected receptors was conducted using CASTp 3.0 web server  simulations as conducted earlier2.6.The enzyme inhibition assay was performed for both the test (caffeine) and reference compounds i.e. epigallocatechin gallate for collagenase, ursolic acid for elastaseg for 5 min and absorbance of the collected supernatant was measured at 320 nm using spectrophotometer . The enzyme reaction mixture with buffer only was considered as negative control and subjected to similar experimental conditions as mentioned above. Finally, the percentage inhibition of collagenase was derived using the Collagenase inhibition assay was performed with modification as reported earlier in reaction buffer of 0.1 M tris-HCl (pH 7.8), 150 mM sodium chloride and 10 mM calcium chlorideAlso, the elastase inhibition potential of caffeine was investigated in tris-HCl buffer solution with modified method as described earlierMeanwhile, inhibitory effect of caffeine or arbutin on tyrosinase activity was evaluated using modified method as described earliert-test with statistical significance at p ≤ 0.05. Moreover, graph plotted between the various concentrations and percentage enzyme inhibition was used to calculate the putative IC50 values for each inhibitor against respective enzyme in Origin9 software .All the enzyme inhibition reactions were carried out in triplicates and inhibition activity was expressed as mean ± standard deviation. Further, statistical analysis for the obtained results was conducted using Microsoft excel package for Windows . Significant differences between the caffeine treatment against selected enzymes were determined by 3.3.1.Table S1). Whilst, crystal structure of collagenase (in silico studies. The structure of collagenase (Chain A) contained 671 residues folded to form 30 helix loops and 11 beta sheets  are essentially required to conduct the molecular docking between ligand and receptor. Therefore, 3 D structures of caffeine or reference compounds and respected enzyme crystal structure were downloaded from protein data bank (PDB). The caffeine which is classified as methylxanthine alkaloid  was anallagenase , elastaslagenase  and tyrolagenase  resolveda sheets  while ela sheets . Also, ta sheets .3.2.Table S2).Molecular docking has been reported as effective method to predict the impact of biomolecules in skin photoageing regulationS1(a)). However, EGCG was marked with four strong hydrogen bonds of length 2.05, 3.06, 1.59 and 1.73 Å at TRY496, GLU498, GLU524 and GLU555 residue, respectively. Also, single pi-pi stacking interaction at TRP539 residue and participation of HIS523, HIS527 and THR551 residues in the polar interactions was recorded in collagenase-EGCG complex. Additionally, hydrophobic and negative interaction was recorded for LEU495, TRY496, ILE497, PRO499, TRP539, TYR607 and GLU498, GLU524, GLU555, ASP554 residues, respectively in collagenase-EGCG complex (S2(a)). The function of occupied residues by EGCG i.e. GLU498 ; HIS523, HIS527 and GLU555 ; and GLU524 (active site) was also elucidated for the collagenaseMolecular docking analysis for the caffeine and EGCG with collagenase revealed a highest binding affinity of −4.66 and −6.16 kcal/mol, respectively. Moreover, caffeine was found to form signal moderate hydrogen bond (3.14 Å) and polar interaction with GLU498 and HIS527 residues, respectively, and identified as metal binding sites for calcium and zinc cations in the enzymesS1(b)). Whilst, ursolic acid depicted two strong hydrogen bonds at SER96 (1.65 Å) and ASN192 (2.71 Å) along with significant polar  and hydrophobic interactions  in the elastase-ursolic acid complex. All the interacting residues with EGCG were identified as disulfide binding site in the elastase crystal structure except SER96, ASN97 and LEU99 residues (S2(b)).Also, docked complex of elastase-caffeine showed highest docking energy of −3.36 kcal/mol while −4.23 kcal/mol in the elastase-ursolic acid complex. Besides, two moderate hydrogen bonds at GLY216 and SER195 residues of length 3.03 and 2.99 Å, respectively, as well as polar interactions by HIP57 , ASN192 and SER195 residues was observed in the elastase-caffeine docked complex. However, all these residues were reported for disulfide bond formation except HIS57 and SER195 residue that belongs to the conserved catalytic triadS1(c)). It was reported that HIS259 and HIS263 residues acted as site for copper binding via tele nitrogen except HIS263 residue which also played role for substrate binding site in the enzymeS2(c)).Meanwhile, docked complex of caffeine with tyrosinase showed maximum −2.86 kcal/mol binding energy and no significant hydrogen bond was observed. Whilst, HIS61, HIS85, HIS244, HIS259 ASN260, HIS263, SER282 and PHE90 VAL248, PHE264, MET280, VAL283, ALA286, PHE292 residues showed polar and hydrophobic interactions, respectively except GLU256 residue (negative interaction) in the tyrosinase-caffeine complex . Based on molecular interaction analysis for caffeine against reference compounds for the respective enzymes, it was concluded that caffeine has comparatively weak interactions and occupied less residues in the active region of model enzyme. Hence, only respective enzyme-caffeine complexes were further evaluated for their stability using MD simulation.Also, caffeine was aligned with reference compounds in the active site of respective model enzymes to demonstrate their docked configuration ). Such vibrations were suggested due to large number of 671 residues in the collagenase ). Moreover, RMSD for elastase ) and tyrosinase ) also depicted stable configuration except an intense deviation around 6 ns followed by stabilization. Whilst, RMSF calculations generated for elastase ) and tyrosinase ) exhibited acceptable convolutions in the respective atoms of protein and ligand. Additionally, atoms in the caffeine complexed with respected enzymes also depicted the acceptable and stable RMSF fluctuations during the simulation interval . These observations predicted that caffeine has attained a relatively stable complex system with the respective proteins as predicted during 10 ns simulation interval.Molecular dynamics (MD) simulations, in contrast to a static description of protein-ligand interactions, explicated water treatment and intrinsic flexibility of the receptor, and apprehended the dynamic nature of protein-ligand interactions. These advantages make MD an important computational tool for understanding the physical basis of the structure and function of biological macromolecules by investigating atomic-level interactions. In this study, changes in conformation and stability of predicted receptor-ligand complexes were investigated through 10 ns MD simulation. The trajectories obtained from 10 ns simulation for collagenase, elastase and tyrosinase complexed with caffeine were analyzed using the simulation interaction diagram option of the Desmond program. Following, four properties, where first three i.e. (a) RMSD, (b) RMSF, (c) Secondary structure changes, were analyzed for the selected protein-ligand complex to ensure that there are no abnormal structural changes over the simulation time while (d) protein-ligand contact, which is the most important feature for receptor-ligand complex, was studied to analyze the presence and absence of specific atomic-level interactions of ligand with binding site residueslagenase  and suggS1(a)). These results suggested the significant blockage of GLU498 residues contributed by caffeine-induced the inhibition of collagenase enzyme, and meanwhile, PRO476, TYR506, ARG508, THR509 and GLN512 residues significantly participated in the stability of protein receptor complex.Protein-ligand contact profiles for caffeine complex with collagenase, elastase and tyrosinase were also accessed from the simulation trajectories as shown in S1(b)). It was concluded that caffeine inhibits the enzyme activity by disturbing the active site as well as tertiary structure of elastase enzyme responsible for stable conformation.Meanwhile, elastase-caffeine interaction map predicted during the simulation showed participation of HIS57, LEU99, ASP102, THR146, ASN147, SER189, SER190, CYS191, ASN192, GLY193, ASP194, SER195, GLY196, VAL213, SER214, PHE215, GLY216, SER217, LEU218, GLY219, CYS220, TYR221, SER226 and VAL227 residues in hydrogen bonding, hydrophobic, ionic and water bridging . HoweverS1(c)), indicates comparatively less distortion in the enzyme structure. These observations suggested the caffeine as weak inhibitor against tyrosinase enzyme. Furthermore, number of residues contacts and their density i.e. darker shade of orange indicates more than one contact on that frame for the selected enzymes was also studied .Moreover, participation of various residues i.e. HIS61, GLY62, LEU63, TYR65, ASN81, CYC83, HIS85, HIS244, VAL248, GLU256, HIS259, ASN260, PHE264, ARG268, GLY281, VAL283, PRO284, GLU322 and ALA323 in hydrogen bonding, hydrophobic, ionic and water bridging interactions was observed for the tyrosinase-caffeine complex during the simulation . Interes3.4.In vitro enzyme inhibition assays are one of the therapeutic approach used by pharmaceutical and cosmetic industries to accesses the activity of bioactive compounds,Tables S4 and S5). It was observed that enzyme inhibition activity of all the three enzymes decreases with an increase in dilution of caffeine . Results show that caffeine has highest 41.86% and lowest 2.86% inhibition activity at 1000 and 10 µg/mL, respectively against the collagenase ). Also, caffeine displayed significant elastase inhibition of 36.44 and 3.42% at 1000 and 10 µg/mL dilution, respectively ). Furthermore, only higher concentration (1000 µg/mL) was marked with statistically significant 13.72% tyrosinase inhibition activity for the caffeine  for the inhibition activity induced by caffeine against collagenase, elastase and tyrosinase showed 0.9944, 0.9993 and 0.988 values, respectively, indicates the concentration-dependent inhibition of the respective enzymes by the various dilutions of caffeine. Moreover, reference compounds i.e. EGCG, ursolic acid and arbutin with R2 values of 0.9706, 0.9758 and 0.9685, respectively also exhibited the concentration-dependent inhibition against respective enzymes . Hence, these results suggested that the inhibition of enzyme by caffeine and reference compounds was directly proportional to the intensity of predicted intermolecular interactions in the respective protein-ligand complexes . It was recorded that caffeine exhibited IC50 values of 1749.7, 3023.2 and 168455 µg/mL against collagenase, elastase and tyrosinase, respectively while reference compounds i.e. EGCG, ursolic acid and arbutin showed 51.51, 238.36 and 1473.9 µg/mL against collagenase, elastase and tyrosinase, respectively. As reported in the literature, collagenase from Clostridium histolyticum and porcine pancreas elastase are topologically very similar to human collagen and neutrophil elastase and has been used for phenolic compounds to predict as respective inhibitors,,Agaricus bisporus is frequently used as an enzymatic in vitro model for developing the skin whitening substances targeting human tyrosinasein vitro assays performed with the collagenase, elastase, tyrosinase and respected synthetic peptides are reliable tool to get first indications of the capability of caffeine to relatively prevent the extracellular matrix degradation as well as pigmentation that coherently results into skin ageing.Also, coefficient of determination (Romplexes . Further4.in silico and in vitro approach and our results revealed that caffeine comparatively strongly inhibited the collagenase followed by elastase and week inhibition activity was recorded for the tyrosinase in concentration-dependent manner. Furthermore, in vitro experimental results showed correlation with predicted molecular interaction profiles from in silico analysis for the respective enzyme-caffeine complex. Thereof, due to the high drug-likeness and high skin permeability of caffeine as predicted from the SwissADME, we purpose that caffeine can be used for inhibition of collagenase, elastase and tyrosinase for cosmetic purposes comparatively at non-lethal doses but respective effects will be eventually visible after long usage.Caffeine has been demonstrated to act as UV protective agent for the skin but inhibitory activity of caffeine against collagenase, elastase and tyrosinase, purposed as major protein responsible for skin ageing induced by UV-light, is not yet studied. We conducted the research in this direction using


Sediment cores from Kløverbladvatna, a threshold lake in Wahlenbergfjorden, Nordaustlandet, Svalbard were used to reconstruct Holocene glacier fluctuations. Meltwater from Etonbreen spills over a threshold to the lake, only when the glacier is significantly larger than at present. Lithological logging, loss-on-ignition, ITRAX scanning and radiocarbon dating of the cores show that Kløverbladvatna became isolated from Wahlenbergfjorden c. 5.4 cal. kyr BP due to glacioisostatic rebound. During the Late Holocene, laminated clayey gyttja from lacustrine organic production and surface runoff from the catchment accumulated in the lake. The lacustrine sedimentary record suggests that meltwater only spilled over the threshold at the peak of the surge of Etonbreen in AD 1938. Hence, we suggest that this was the largest extent of Etonbreen in the (mid-late) Holocene. In Palanderbukta, a tributary fjord to Wahlenbergfjorden, raised beaches were surveyed and organic material collected to determine the age of the beaches and reconstruct postglacial relative sea level change. The age of the postglacial raised beaches ranges from 10.7 cal. kyr BP at 50 m a.s.l. to 3.13 cal. kyr BP at 2 m a.s.l. The reconstructed postglacial relative sea level curve adds valuable spatial and chronological data to the relative sea level record of Nordaustlandet. This is not compatible with postglacial isostatic rebound evidence reconstructed from raised beaches around the Svalbard archipelago, which suggests a maximum LGM ice load over Kong Karls Land to the southeast of Nordaustlandet5. The location of LGM ice domes, timing, and implications for ice sheet dynamics is still debated7. Postglacial relative sea level changes on Nordaustlandet and eastern Spitsbergen are not well constrained in time and space. Blake8 reconstructed the relative sea level history of Lady Franklinfjorden in northwest Nordaustlandet, and suggested a Late Weichselian-Holocene marine limit of at least 50 m above high tide (a.h.t.). A study9 from Svartknausflya at the south coast of Nordaustlandet suggested a marine limit of more than 70 m a.h.t. Marine isolation ages from lakes on Nordaustlandet provide ancillary information about the postglacial relative sea level changes12.Nordaustlandet is a key locality for understanding the glacial history of Svalbard. Recent studies suggest that during the Last Glacial Maximum (LGM), ice domes in the Svalbard-Barents Sea Ice Sheet were centered over the southern opening of the Hinlopen Strait and Nordaustlandet2) and the smaller Vestfonna (2455 km2)14. The location between these two ice caps makes the inner part of Wahlenbergfjorden an important site for studying Holocene glacier variations. Flink et al.15 suggested that the Wahlenbergfjorden trough acted as an ice stream onset zone during the LGM, feeding westerly flowing ice into an ice stream in the Hinlopen Strait16. The inner part of the fjord was deglaciated prior to 11.3 cal. kyr BP17, and Etonbreen, a major surge-type outlet glacier of Austfonna, now terminates in the inner part of Wahlenbergfjorden. The last surge of Etonbreen was captured on oblique aerial photographs from 193818. To study the Holocene glacier activity, we obtained sediment cores from a proglacial threshold isolation basin, Kløverbladvatna, located at this part of the fjord. This new sediment sequence provides an opportunity to link the terrestrial geological record to the marine record17. Flink et al.15 found increased sedimentation rates in inner Wahlenbergfjorden during the late Holocene, which may suggest surge activity of Etonbreen, or at least greater glacial activity. At the sea floor, a multi-crested terminal moraine and stacked debris flow lobes suggest that Etonbreen has advanced several times in the late Holocene. Flink et al.15 showed that the last surge of Etonbreen in 1938 reached a position approximately 7 km further out than its present glacier margin. It is, however not known when or how many times Etonbreen advanced to this position (or positions further upglacier) prior to the 1938 surge. The Ice Rafted Debris (IRD) record from the middle part of Wahlenbergfjorden indicates increased IRD flux during the last c. 1000 years, which also suggests larger glacial activity than earlier17. Previous lake sediment studies from Nordaustlandet have focused on palaeoecological and palaeoclimatic reconstructions19, and have not been targeting proglacial threshold lakes that potentially contain glacial on/off signals22. Data highlighting Holocene glacier variations on Svalbard are important to understanding the observed recent ice loss and place it in a long-term perspective26. The aim of this study is (1) to reconstruct the late Holocene glacier fluctuations of Etonbreen, based on threshold lake sediment cores from Kløverbladvatna, and (2) to decipher the postglacial relative sea level changes in Palanderbukta, Wahlenbergfjorden, based on radiocarbon dating of raised beach ridges. Finally, we discuss our results and their implications for reconstructions of the relative sea level change, spatial pattern of glacioisostatic rebound, and palaeoglaciology following the last deglaciation of northeast Svalbard.At present, about 80% of Nordaustlandet is glaciated, mainly by the two ice caps Austfonna  is fed by surface runoff in a local catchment (c. 1.5 km2) at Oxfordhalvøya. An abandoned, dry channel indicates that the lake was previously fed by meltwater from Etonbreen  is located on Oxfordhalvøya in the innermost part of the 50 km long Wahlenbergfjorden at the western part of Nordaustlandet, Svalbard Fig. . The bed30. At the mouth of Palanderdalen, a gently sloping plain of raised beach ridges dominates the landscape. A meltwater river running northwards through the valley has eroded geological sections into the raised marine sediments. Currently the river is forming a large delta, prograding into Palanderbukta.Palanderbukta is a c. 20 km long tributary fjord south of Wahlenbergfjorden Fig. . The stuWe recovered one 76-cm-long surface core KLØV S2) with the gravity corer, and two piston cores (KLØV P2 and KLØV P1B)  and a paired bivalve shell of the marine species Nuculana pernula were observed in the facies. We interpret this facies as marine mud with IRD. The red-brown color suggests a sediment source from the Rijpbreen granitoid substratum of Etonbreen. However, we do not interpret the red color as an on/off glacial signal to the basin. Outsized clasts occur both in the olive-grey and the red-brown sediments of this facies, suggesting ice rafting during deposition in both cases.Figure 31. One sample from 77 cm depth of a paired bivalve shell of the marine species Nuculana pernula was radiocarbon dated, yielding a 2σ age range of 9768–9500 cal. yr BP  was observed and dated  record36. Radiocarbon age Ua-53789 is based on a sample immediately below the marine-lacustrine transition and yields a 2σ age interval of 5473–5313 cal. yr BP  suggest a major change in depositional environment. We interpret this as a dramatically increased inflow of glacial meltwater to the lake, comparable with evidence from other studies of threshold lake sedimentation37. There is currently no inflow of glacial meltwater to Kløverbladvatna, and we therefore interpret the upper 6 cm of laminated clayey gyttja as formed in the present-day environment. This similarity in the lithology also suggests that the laminated clayey gyttja facies is deposited in an environment similar to the present, i.e. surface runoff to the lake but no glacial meltwater inflow. Three samples of Salix polaris macrofossils were obtained from KLØV S2 for radiocarbon dating  and the deposition of the clay-silt unit in KLØV S2 (134 ± 36 14C yr BP)  the presence of outsized clasts within the marine phase and 2) the red-brown mud within the lacustrine phase. The marine muds in KLØV P1B and the lower part of KLØV P2 contain outsized clasts interpreted as IRD. This agrees with observations of outsized clasts throughout the Holocene from a marine sediment core dated to 11.3–0.2 cal. kyr BP and retrieved from the central part of Wahlenbergfjorden the red- P2 Figs . The dis17 and formation of the uppermost dated beach ridge in Palanderbukta at 10.7 cal. kyr BP. On a line, Palanderbukta is located exactly between Svartknausflya, southern Nordaustlandet (marine limit of 70 m a.h.t)9 and Lady Franklinfjorden in northwestern Nordaustlandet (marine limit of 50 m a.h.t.)8. Hence, a marine limit of 65 m a.s.l. in Palanderbukta  of uplift and Late Weichselian ice dome(s)6.We were not able to date the postglacial marine limit in Palanderbukta at 65 m a.s.l., but it most likely formed in the 600-yr long time window between the 11.3 cal. kyr BP deglaciation of Wahlenbergfjordenet al.25 showed that a nearby major outlet glacier, Gullfaksebreen, in De Geerbukta at the west side of the Hinlopen Strait advanced in the early Holocene, a period characterized by mild climate and peak insolation  in the marine part of the Kløverbladvatna sedimentary record indicates glacier and/or sea ice activity in Wahlenbergfjorden until the basin isolation. This supports recent modelling projections of some Svalbard glaciers surviving the Holocene thermal optimum. The presence of ice rafted debris in the sediment cores even suggests that glaciers could have been calving into Wahlenbergfjorden in the early Holocene. The geological sections in Palanderbukta reveal that the site was not glacially overridden after deposition of the Our reconstruction of the postglacial relative sea level history of Palanderbukta is consistent with data on Holocene relative sea level changes from elsewhere at Nordaustlandet, and from northeast Spitsbergen. The altitude of the postglacial marine limit increase along a transect from Lady Franklinfjorden to Palanderbukta and Svartknausflya supporting a larger center of ice mass located to the southeast of Nordaustlandet.53 and magnetic susceptibility (MS)54. The XRF and MS records were measured with 1 mm averages and 4 mm intervals, respectively. We use the Ti signal to illustrate the changes between organic and minerogenic sediment. This is commonly used to infer the amount of glacial meltwater input to proglacial lakes53. We normalized the Ti peak area (counts per second) with the incoherent (inc) and coherent (coh) Rh scatter peaks from the X-ray tube (Ti/(inc + coh)) to remove scattering from the instrument as suggested by Kylander et al.55. The ratio between Ca and Fe peak areas was used to detect the transition from marine to lacustrine sediments 33. Stratigraphic correlation between the cores was established with AnalySeries 2.0.4.256, using tie-point in the elemental data. Loss-on-ignition (LOI) was measured to determine the total organic content57. Samples of 2 cm3 were taken every 2 cm and dried at 110 °C for 24 hours. The dry samples were ignited at 550 °C during four hours to determine the amount of organic material (LOI). Alongside with the Ti/(inc + coh) signal, the LOI record illustrates transitions between organic and minerogenic sediment.The bathymetry of Kløverbladvatna was surveyed with a hand-held depth sounder  for point measurements, and cores were retrieved from the central, deepest part of the basin at a depth of 17.5 m. Coring was carried out from a small zodiac using a hand-held lightweight piston corer with 60 mm diameter coring tubes. The coring was performed through a hose in the floor of the zodiac, which was anchored in a stable position on the lake surface. Additionally, surface sediments were obtained with a hand-held lightweight gravity corer. The lithology and stratigraphy of the cores were visually inspected and logged immediately after splitting and cleaning the cores in the laboratory. The cores were analyzed using an ITRAX core scanner providing optical and radiographic line scan images along with records of element contents measured using X-ray fluorescence technique (XRF)58 using the IntCal13 and Marine13 calibration curves for the terrestrial and marine macrofossils, respectively. Since the Marine13 curve has a built-in global marine reservoir effect of −440 ± 52 years, a local ΔR value for the Spitsbergen area of 105 ± 24 years was applied59. The reported radiocarbon ages are given in calibrated years before present 60. Age-depth relationships for the core sequence were established with the Bayesian based code ‘Bacon’ v. 2.261 in ‘R’ v. 3.4.062.To establish the chronology of the sediment cores, we sampled macrofossils retrieved from residues of 0.5 mm sieving, identified and isolated using a binocular microscope. Radiocarbon ages were obtained through accelerator mass spectrometry (AMS) at the Ångström Laboratory, Uppsala University. All radiocarbon ages were calibrated in the online CALIB 7.1 software63. All elevations were extracted from the same ArcticDEM strip, and with the slope-induced error being negligible, the internal 2σ error from the ArcticDEM to the elevation measurements is 0.4 m. In Palanderbukta, the relief of each raised beach is typically c. 1 m, which also limits precision. Hence, a conservative estimate of this sampling uncertainty is 0.5 m. Error propagation yields 1.1 m (2σ), which is the error of the heights of the samples relative to each other. As this error shows the internal consistency of the relative sea level curve, we plot this error on Fig. 64. We assume that the area has been microtidal at least since the LGM66. The sampled organic material from the beach ridges was AMS radiocarbon dated at the Ångström Laboratory, Uppsala University. The internal, dense parts of whalebone samples were subsampled and submitted for dating. Ages were calibrated as described above. A relative sea level curve was constructed from the pairs of calibrated radiocarbon ages and raised beach altitudes68. In Svalbard, elevations of raised beaches reflect the interplay between postglacial eustatic sea level rise and glacioisostatic rebound following the deglaciation of the Svalbard-Barents Sea ice sheet69.We determined the location of driftwood, whalebones, and mollusk shells on raised beach landforms in Palanderbukta by hand-held GPS, using a mean value of three instruments. To obtain an accurate elevation above sea level, we extracted the elevation of the raised beach ridges from the ArcticDEM using the hand-held GPS coordinates. Average slopes in the surveyed area are <10%, keeping the slope-induced error on elevation to a minimum. The ArcticDEM has absolute errors of 2–3 m RMSE in the plane and c. 2 m in the vertical. Most of the error is due to biases, and when these are removed through co-registration by translations, the error is reduced to 0.2 m RMSESupplementary Information