Fluorous-modified surfaces have emerged as a powerful tool for the immobilization of fluorous-tagged biomolecules based on their specificity and the strength of fluorous-fluorous interactions. To fabricate a fluorous-based protein microarray, we designed two strategies for site-specific modification of proteins with a fluorous tag: attaching the fluorous tag to the C-termini of expressed proteins by native chemical ligation (NCL) or to the Fc domain of antibodies through boronic acid (BA)-diol interactions. The perfluoro-tagged proteins could be easily purified by fluorous-functionalized magnetic nanoparticles (MNPs) and immobilized on a fluorous chip with minimal non-specific adsorption. Importantly, proteins immobilized on the solid support through non-covalent fluorous-fluorous interactions were sufficiently stable to withstand continuous washing. We believe that this fluorous-fluorous immobilization strategy will be a highly valuable tool in protein microarray fabrication. However, a protein’s activity may be significantly influenced by its orientation on the solid support. Additionally, the non-specific adsorption of proteins from biological samples to the solid support may result in high background noise and consequently interfere with the accurate interpretation and analysis of results2. Therefore, fabricating a high-activity protein microarray with suppressed non-specific interactions is the most critical task in this field.One of the most powerful tools in proteomics is the protein microarray, which utilizes immobilized proteins on a solid support for high-throughput analysis of biochemical properties and biological activities4 , which could dramatically reduce the activity of the target protein. Hence, many chemists have developed oriented and orthogonal immobilization methods to help resolve the potential problems of protein activity loss; these methods include Diels-Alder ligation5, Staudinger ligation6, Cu(I)-catalyzed 1,2,3-triazole formation7, thiol-ene ligation8, enzymatic post-translational modification9, and cyanobenzothiazole (CBT) condensation10. In contrast, many oriented immobilization strategies utilize non-covalent interactions11. Popular examples include DNA-directed immobilization12, avidin-biotin interaction13, and affinity immobilization of His-tagged fusion proteins to a metal-chelate support15. However, such methods have potential drawbacks like the non-specific adsorption of non-target proteins in complex biological samples to the solid support, and insufficient strength of non-covalent interactions, which make the immobilized proteins susceptible to falling off after prolonged incubation under complex biological conditions (such as serum), or continuous vigorous washing. Nevertheless, to take advantage of the high specificity of noncovalent binding strategies, the immobilization surface must be able to resist the adsorption of other proteins that lack an affinity tag. Usually, during the glass preparation process, the glass surface is coated by poly(ethylene) glycol to minimize protein non-specific adsorption16. However, both the quality of the glass surface and also the strength of the affinity ligand can affect the immobilization results. Thus, generating a convenient, non-specific binding-resistant, and easily prepared glass surface for the oriented and noncovalent immobilization of proteins is highly challenging.The immobilization of proteins via covalent attachment has been thoroughly studied. However, the most common strategies for protein immobilization rely on random covalent bond formation19. Recently, Pohl and co-workers created a fluorous carbohydrate microarray utilizing highly specific fluorous affinity interactions between fluorous-tagged saccharides and fluorous-modified glass surfaces21. Spring et al.22 and Schreiber et al.23 also developed fluorous small molecule microarrays for analyzing small molecule-protein interactions. More recently, Willi Bannwarth and co-workers24 attached a tri-perfluorotag to the enzymes RNase H and rcHRP via random amide bond formation and then immobilized them on fluorous glass surfaces via fluorous-fluorous interactions. These fluorous-based microarrays have the following advantages: (i) high signal-to-noise ratios, (ii) low non-specific binding, (iii) low and uniform background fluorescence, and (iv) simple fabrication workflows.Fluorous-based substrates have emerged as an attractive candidate for biomolecule immobilization as they possess the unique characteristic of having specific and strong affinity to molecules containing a fluorous group, yet also being highly resistant to non-specific adsorption from non-fluorous molecules25. Accordingly, BA has been applied as a probe for recognizing various saccharides and glycoproteins in numerous applications26. Previously, we reported the use of BA-functionalized glass slides to fabricate oriented protein microarrays by targeting the glycan chain of Fc-fusion proteins27. Herein, we developed a straightforward protein microarray fabrication method utilizing a fluorous-based surface for the oriented and noncovalent immobilization of proteins on the surface through fluorous-fluorous interactions. Two types of fluorous probes were designed for the site-specific labeling of proteins with a fluorous tag: one involving the combination of expressed protein through ligation with their N-terminal cysteine residue and the other via boronate ester formation between a BA-probe and an antibody’s glycan moieties  are known to rapidly form reversible cyclic boronate esters with cis-diolsies Fig. . To makeies Fig.  or boronies Fig.  and then28 and was modified by NCL to incorporate a fluorous tag on the protein C-terminal end (Ftag-protein). To achieve a high ligation yield, the fluorous probe must have sufficient water solubility because the protein labeling yield is positively correlated with the probe’s hydrophilicity.As a proof of concept, eGFP was chosen as a target protein for a model study. eGFP was expressed using the IMPACT system (New England Biolabs)1–4, we incorporated a short-chain tri(ethylene glycol) linker within the probe, a common reagent generally used for increasing solubility in buffered-aqueous solutions. We also insert polar and charged amino acid residues including cysteic acid to further reduce the hydrophobicity of the fluorous-tagged probes. The preparation of probes 1–4 started from fluorous carboxylic acid 6, which was first coupled to N-Boc-2,2′-(ethylenedioxy)bis(ethylamine) (7) to provide compound 8 in 81% yield  generated an amine, which was reacted with 9  in the presence of 2.5% triisopropylsilane (TIPS) as a scavenger, which also affected cleavage of the Boc group in 10, producing probe 1 in essentially quantitative yield. To access BA-containing probe, Boc-protected cysteic acid linker (11) was incorporated on amine form of 8 to afford an intermediate 12 (68% over two steps), which was followed by Boc deprotecion and subsequent 1-ethyl-3-(3-dimethylaminorpopyl)carbodiimide hydrochloride (EDC-HCl)/1-hydroxybenzotriazole (HOBT)-mediated coupling with 3-carboxyphenylboronic acid to afford fluorous-tagged BA probe 2 in a modest 18% yield over two-steps. For the synthesis of arginine-containing probe, Fmoc-Arg(Pbf)-OH was first reacted with the amine generated from flourous tag 8, and extension of the synthesis of Fmoc-protected intermediate was then carried out using 20% piperidine in DMF as the Fmoc deprotecting agent followed by coupling to the carboxylic acid of the incoming amino acid Boc-Cys(Trt)-OH affording 13 . In a similar manner, compound 14 was synthesized in 69% yield starting from 8 and using Boc-Cys(Trt)-OH. Simultaneous cleavage  of the trityl ether, Pbf, and Boc protecting groups in 13 and in 14 straightforwardly produced the target probes 3, and 4, respectively. The fluorous-tagged small molecule 5 was used for purification of fluorous-modified proteins in this study -1-piperazineethanesulfonic acid (HEPES) buffer. At pH 8.0, the sulfonic acid group of cysteic acid acquires a negative charge, which can improve the water solubility of the fluorous probe.Herein, we tested multiple combinations of ethylene glycol, with the amino acids arginine and cysteic acid to find an ideal structure for improving the solubility of the fluorous probe Fig. . In the 1 was added to the column in the presence of an auxiliary thiol reagent (2-mercaptoethanolsulfonic acid) to allow on-column cleavage and subsequent covalent labeling of the protein with the fluorous probe via NCL  water compatible, the nanoparticle surface was incorporated with ethylene glycol, which is also able to suppress non-specific protein adsorption. The Ftag@MNPs were synthesized by modifying our previously reported method31. A fluorous functionality was introduced onto the particle surface through a sol-gel process using tetraethyl orthosilicate (TEOS) followed by the addition of 2-[methoxy(polyethyleneoxy)propyl]trimethoxysilane (mPEG) and triethoxysilane (Ftag-(OEt)3) at various volume ratios—3:1, 4:1, 5:1, 6:1, 8:1, and 10:1—to give Ftag@MNPs, as illustrated in Fig. tag@MNPs increased as the amount of mPEG increased, and the non-specific adsorption decreased as the amount of (Ftag-(OEt)3) increased 3 volume ratio of 6:1, the Ftag@MNPs become soluble in buffer. It should be noted that the poor solubility of the Ftag@MNPs can also be improved by the addition of detergent (discussed in the next paragraph). Therefore, to maintain the solubility of the MNPs while simultaneously maximizing the amount of available fluorous tags on the MNPs, Ftag@MNPs with a mPEG/Ftag-(OEt)3 volume ratio of 5:1 were chosen for use in target protein purification.The labeling of a protein with a specific functional group is important for studying its interactions with small molecules, DNA, and other proteins. Thus, developing a tool to facilitate the isolation of such modified proteins is imperative because the labeling efficiency is often less than 100%. Magnetic nanoparticles (MNPs) are a well-developed platform for biochemical researchtag@MNPs, MBP was chosen as a model protein and modified with the fluorous NCL probe (1) as described previously. We noted that after concentrating the fluorous tag-modified eGFP to a high concentration, the proteins tended to precipitate, possibly because of fluorous tag self-assembly in the aqueous solution (data not shown). Native polyacrylamide gel electrophoresis (PAGE) analysis was used to investigate the purification results  were captured by the Ftag@MNPs . Encouraged by these results, we designed two strategies for eluting Ftag-MBP from Ftag@MNPs by the addition of 1-mM (or 0.65%) compound 5 (to give Ftag-MBPF) or 0.1% triton X-100 (to give Ftag-MBPtriton) in the elution buffer ; this analysis gave a molecular weight (MW) of 43971 Da, in agreement with the calculated MW of 43905 Da  and anti-RCA120 pre-incubated with Ftag-BA (compound 2) and anti-RCA120 were spotted on a fluorous slide. The fluorescence image-based binding results  was dissolved in a mixed solution of TFA/CH2Cl2/triisopropylsilane (TIPS)/H2O . The mixture was stirred at room temperature for 30 minutes to deprotect the triphenylmethyl (Trt) and Boc groups. The solution was concentrated under reduced pressure. The resulting residue was washed by hexane (Hex) and EA to give probe 1, which was used in the next reaction without further purification . Rf = 0.4 (1:3 MeOH-DCM) 1H NMR  δ 4.85-4.78 , 4.11-4.03 , 3.63-3.60 , 3.59-3.53 , 3.47-3.35 , 3.30-3.24 , 3.15 , 3.05 , 2.61-2.45 ; 13C NMR  δ 173.0 (x2), 172.1, 168.3, 71.3 (x2), 70.5, 70.3, 56.1, 52.7, 52.5, 40.6, 40.5, 31.1, 27.8 , 27.5, 26.1; 19F NMR  δ −81.8 , −115.0–−115.3 , −122.0–−122.2 , −122.2–−122.5 , −123.1–−123.4 , −123.8–−124.1 , −126.6–−126.9 ; HRMS (ESI) m/z Calcd. for C23H28F17N4O8S2− [M-H]−: 875.1077 found 875.1074.Compound 12  in DCM (4 mL) in an ice bath. The reaction was stirred at room temperature for 2 h, and then, the solvent was removed under reduced pressure. A solution of the above residue, 3-carboxyphenylboronic acid , EDC , HOBt , and Et3N  in anhydrous DMF (5 mL) was stirred at room temperature for 12 h. The solvent was removed under vacuum. The crude product was purified by silica gel chromatography (30% MeOH in 1:1 EA-Hex) to give probe 2 in 18% yield over two steps (82.9 mg). Rf = 0.23 (20% MeOH in 1:1 EA-Hex); 1H NMR  δ 9.7 , 8.61 , 8.15 , 7.9 , 7.28-7.19 , 6.91 , 4.42-4.36 , 3.5 , 3.42-3.36 , 3.24-3.12 , 2.91-2.80 , 2.48-2.37 ; 13C NMR  δ 170.7, 169.4, 165.9, 157.3, 135.5, 129.3, 118.2, 117.6, 114.1, 69.5 (x2), 69.0, 68.8, 51.9, 51.3, 38.8, 38.6, 25.9 , 25.7; 19F NMR  δ −81.9 , −115.0–−115.3 , −122.0–−122.2 , −122.2–−122.5 , −123.1–−123.4 , −123.8–−124.1 , −126.6–−126.9 ; HRMS (ESI) m/z Calcd. for C27H29BF17N3O10S[M-H]−: 920.1322 found 920.1323.TFA (1 mL) was added to a solution of compound 13  was dissolved in a mixed solution containing TFA/CH2Cl2/TIS/H2O . The mixture was stirred at room temperature for 30 minutes to deprotect the Trt and Boc groups. The solution was concentrated under reduced pressure. The resulting residue was washed with Hex and EA to give probe 3, which was used in the next reaction without further purification . Rf = 0.5 (2:2:1 EA-MeOH-H2O) 1H NMR  δ 4.35 , 4.22 , 3.64-3.60 , 3.58-3.53 , 3.44-3.36 , 3.25-3.17 , 3.10-3.05 , 2.58-2.48 , 1.92-1.79 , 1.79-1.64 ; 13C NMR  δ 173.7, 173.0, 168.8, 158.7, 71.3, 71.3, 70.5, 70.4, 55.8, 55.0, 42.0, 40.5, 40.4, 30.1, 27.8 , 27.5, 26.5, 26.1; 19F NMR  δ −81.9 , −115.1–−115.4 , −122.1–−122.3 , −122.3–−122.6 , −123.1–−123.4 , −123.9–−124.2 , −126.7–−126.9 ; HRMS (ESI) m/z Calcd. for C26H36F17N7O5S [M + H]+: 882.2305 found 882.2301.Compound 14  was dissolved in a mixed solution containing TFA/CH2Cl2/TIS/H2O . The mixture was stirred at room temperature for 30 minutes to deprotect the Trt and Boc groups. The solution was concentrated under reduced pressure. The resulting residue was washed with Hex to give probe 4, which was used in the next reaction without further purification . Rf = 0.18 (1:9 MeOH-DCM); 1H NMR  δ 4.01 , 3.64-3.61 , 3.61-3.51 , 3.48-3.33 , 3.03 , 2.97 , 2.61-2.42 ; 13C NMR  δ 172.9, 168.5, 71.3, 71.2, 70.5, 70.3, 56.1, 40.5, 40.5, 27.8 , 27.5, 26.3; 19F NMR  δ −81.9 , −115.1–−115.4 , −122.0–−122.3 , −122.3–−122.6 , −123.1–−123.4 , −123.9–−124.2 , −126.7–−126.9 ; HRMS (ESI) m/z Calcd. for C20H25F17N3O4S [M + H]+: 726.1294 found 726.1299.Compound The perfluoro-tagged protein solution was prepared in a printing buffer  and dispensed using a robotic contact arrayer  fitted with Stealth Pins SMP3 (Arrayit Corp.) onto a fluorous-coated slide. The printing process was performed at 90% relative humidity, and the temperature was maintained below 26 °C. The printed slide was kept in a humidified chamber and maintained at 4 °C for 12 h. Before incubation, the slide was left at room temperature for an additional 2 h to enhance the fluorous-fluorous interactions. The slides were then blocked twice with 1% bovine serum albumin (BSA) solution in phosphate-buffered saline (PBS) for 5 min each and then washed twice with deionized water for 5 min each.® 4550 scanner with a Cy3 filter. The activity of fluorescent eGFP on the slide was directly measured with a NovaRay Microarray Scanner using a fluorescein isothiocyanate (FITC) filter. The fluorescence intensity of the spots was quantified using the ArrayVision software package (version 8.0) with correction for local background. The mean intensity of each spot was taken as a single data point analysis. Spot size is approx. 110 µm in diameter with a spot-to-spot variation (CV) of 10.1%.The functional protein microarray activities were characterized by using either a biotinylated antigen or a pair of antigens and the corresponding biotinylated secondary antibody and streptavidin-Cy3 in a sandwich detection format (see Supporting Information for details). All images were acquired using a VIDAR RevolutionSupporting information


AMP does not have that effect and HDX measurements for myristoylated and non-myristoylated AMPK in the presence of AMP are similar. These differences in dynamics may account for a reduced basal rate of phosphorylation of Thr172 in myristoylated AMPK in skeletal muscle where endogenous ATP concentrations are very high.The heterotrimeric AMP-activated protein kinase (AMPK), consisting of α, β and γ subunits, is a stress-sensing enzyme that is activated by phosphorylation of its activation loop in response to increases in cellular AMP. N-terminal myristoylation of the β-subunit has been shown to suppress Thr172 phosphorylation, keeping AMPK in an inactive state. Here we use amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) to investigate the structural and dynamic properties of the mammalian myristoylated and non-myristoylated inactivated AMPK (D139A) in the presence and absence of nucleotides. HDX MS data suggests that the myristoyl group binds near the first helix of the C-terminal lobe of the kinase domain similar to other kinases. Our data, however, also shows that ATP.Mg AMPK is an AMP/ATP-sensing enzyme that plays a crucial role in regulating cellular energy metabolism in eukaryotesMammalian AMPK is a heterotrimeric complex consisting of catalytic α- and regulatory β- and γ-subunits3456101156711121416172021222+/Calmodulin-dependent protein kinase β (CaMKKβ) kinases are two of the upstream kinases (AMPKKs) that catalyze the phosphorylation of AMPK at Thr172 of the α-subunit kinase domain, leading to an increase in AMPK activity by several hundred-fold2425262793031Liver kinase β1 (LKB1) and CaThe N-terminal myristoylation of the β-subunit has a binary function in AMPK regulation. Firstly, it localizes and suppresses activity, as the removal of the myristoyl group by mutation of Gly2 to Ala showed a wide distribution of AMPK into the cytoplasm and a four-fold increase in AMPK activity14141533In this study, we used amide hydrogen-deuterium exchange mass spectrometry (HDX MS) to map the myristoyl binding pocket as well as structural changes due to myristoylation. In addition, myristoylation-dependent differences in the peptide residues known to have significant roles in interactions with nucleotides were also investigated.Prior to carrying out the HDX experiments, sequencing of all pepsin digest fragment peptides of non-myristoylated and myristoylated AMPK was carried out as described in materials and methods. A good sequence coverage of 82%, 90% and 91% for α, β and γ subunits respectively of undeuterated non-myristoylated AMPK, and 78%, 86% and 91% for α, β and γ subunits of undeuterated myristoylated AMPK with a large number of overlapping fragments was obtained and analyzed under deuterium exchange conditions . For theConsidering the general trend of exchange, deuteration in the absence of ligands appears moderately faster for the majority of peptides for myristoylated AMPK compared to non-myristoylated AMPK . For theThe most significant difference between myristoylated and non-myristoylated AMPK for the α-subunit is observed for the peptide 122–131 of the kinase domain, which showed a substantial increase in HDX for myristoylated AMPK . This ped for CBS 1 is 2.5 μMd of 80 μM, would be 42% saturated. The most significant effects observed are within the γ-subunit: peptides within CBS3 and between CBS3 and CBS4 show the most substantial protection upon binding AMP and to a lesser extent those within CBS4. The increase in protection likely reflects AMP binding to site 3. Peptides within CBS1 show a small degree of increased protection whereas those in CBS2 demonstrate very little protection. However, peptides between CBS1 and CBS2 (108–115 and 116–123) show an increase in protection, and these would reflect binding of AMP to site 1. The marked difference in deuteration for site 3 despite its lower level of saturation with nucleotide perhaps suggests a greater change in dynamics at site 3 and is consistent with previous studiesWe next set out to monitor the effects of AMP on dynamics of non-myristoylated and myristoylated AMPK. The addition of AMP to both non-myristoylated and myristoylated AMPK has similar effects to each other and is consistent with previously reported resultsComparison of AMP binding to non-myristoylated and myristoylated AMPK highlights several features . It is cd of ATP.Mg2+ for site 1 and site 3 is 18 and 230 μM respectively, therefore we expect under our sample conditions site 1 to be 76% saturated and site 3 to be 21% saturated. The addition of ATP.Mg2+ to both non-myristoylated and myristoylated AMPK show broadly similar effects with respect to the γ-subunit as observed for AMP  and a connecting loop (278–283) to the AIS  the myristoylated β-subunit promotes AMPK association, phosphorylation and activation at cellular membranes, indicating a myristoyl-switch mechanism14153333 protein . As we h protein . In contted AMPK .2+. Firstly, the γ-subunit for both myristoylated and non-myristoylated show similar HDX patterns in the presence of ATP.Mg2+. The CBS3 and the connection between CBS3 and CBS4 show the most significant changes on addition of nucleotide for either form of the protein, and our observations are similar to those previously reportedThere are several interesting similarities and some notable differences between myristoylated and non-myristoylated AMPK in the presence of ATP.Mg2+ 2+ are likely to be physiologically relevant. A relative increase in AMP levels, and binding to AMPK, would lead to an increase in the flexibility of myristoylated AMPK allowing access to the phosphorylation site and mobility of the β-CBM to occur.In conclusion, comparison of the HDX of myristoylated and non-myristoylated kinase dead AMPK suggest a binding site for the myristoyl group similar to other myristoylated kinases. Myristoylation resulted in increased deuteration for the peptide 122 to 131 that marks this binding site, however in the presence of ATP.Mg6-α1(D141A)β1γ1 was expressed in E. coli strain Rosetta (DE3) using the pET DUET expression system (Novagen) as described previouslyHeterotrimeric human kinase dead AMPK His2O) without ligand (AMP or ATP.Mg2+) or with 0.6 μl of 2 mM ligand pre-dissolved in 17.4 μl deuterated buffer. The final concentrations of the protein and ligand in the exchange reactions were 3 and 60 μM, respectively. The labelling mixtures were incubated at room temperature and deuterium exchange was carried out for three time points , followed by quenching of the reaction, by lowering the pH to 2.5 via addition of 230 μl of pre-chilled 2% trifluoroacetic acid. Quenched samples were digested, desalted and separated online using a Waters ultra-performance liquid chromatography system based on a nanoACQUITY platform as described36−1. The resulting peptic peptides were trapped for 7 min on a 2.1 × 5 mm C18 peptide trap . The trapped peptides were eluted from the trap column onto a reversed phase Waters ACQUITY UPLC BEH C18 1.7 μm, 1 × 100 mm column held at 0 °C using an 8 to 40% gradient of acetonitrile in 0.1% formic acid at 40 μL.min−1 over 10 min. The eluate was directed into a Synapt ESI Q-Tof mass spectrometer . Mass spectra were acquired in MSE mode, a nonbiased, nonselective CID methodDeuterium exchange was initiated by diluting 2 μl of 30 μM protein, 10-fold with 18 μl deuterated buffer .Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Ornithodoros moubata was studied using differentiated MUTZ-3 cells as a model of immature dendritic cells of the host skin. Following internalization, cystatin OmC2 was initially found to inhibit the activity of several cysteine cathepsins, as indicated by the decreased rates of degradation of fluorogenic peptide substrates. To identify targets, affinity chromatography was used to isolate His-tagged cystatin OmC2 together with the bound proteins from MUTZ-3 cells. Cathepsins S and C were identified in these complexes by mass spectrometry and confirmed by immunoblotting. Furthermore, reduced increase in the surface expression of MHC II and CD86, which are associated with the maturation of dendritic cells, was observed. In contrast, human inhibitor cystatin C, which is normally expressed and secreted by dendritic cells, did not affect the expression of CD86. It is proposed that internalization of salivary cystatin OmC2 by the host dendritic cells targets cathepsins S and C, thereby affecting their maturation.To ensure successful feeding tick saliva contains a number of inhibitory proteins that interfere with the host immune response and help to create a permissive environment for pathogen transmission. Among the potential targets of the salivary cystatins are two host cysteine proteases, cathepsin S, which is essential for antigen- and invariant chain-processing, and cathepsin C , which plays a critical role in processing and activation of the granule serine proteases. Here, the effect of salivary cystatin OmC2 from  Borrelia causing relapsing fever in infected humans, are transmitted by the soft ticks of Ornithodoros genus , as well as it was able to reduce the DC-mediated proliferation of naive CD4+ T cells. In addition, the vaccination of mice with recombinant cystatin OmC2 decreased the success of tick feeding. Ticks that fed on the mice with the highest level of anti-cystatin OmC2 antibodies had the highest mortality  in the host skin may represent an additional mechanism utilized by ticks to modulate the immune response of their host. Cystatin OmC2 is naturally present in the saliva, salivary glands and gut of the soft tick via the TLR9 pathway  and dermal DC have been distinguished among DC in epidermis and dermis  was used for the prediction of possible O-glycosylation sites in cystatin OmC2.The baculovirus expression system was used to prepare the salivary cystatin OmC2 as a recombinant protein with an oligo His-tag added to its C-terminus in insect Sf9 cells  or 0.1 M acetate buffer  for 5 min. The active cathepsins were next incubated with tick cystatin OmC2 or with human cystatin C at a 1:1 molar ratio for 10 additional min at 37°C. IEF was then performed using the XCell SureLock Mini-Cell system (Invitrogen-Life Technologies). The proteins were separated on vertical pre-cast IEF gels (pH 3–7 and pH 3–10) from Invitrogen. IEF Markers 3–10 (SERVA Liquid Mix) were applied as protein standards. The proteins were fixed in 10% TCA for 30 min, followed by 1% TCA for 10 min. The changes in isoelectric points (pI) of the applied proteins (recombinant proteins and proteins from the cell lysates) were examined. Both recombinant tick cystatins; isolated from insect cells and from 3 (Fluka). Selected bands were excised and analyzed using mass spectrometry or the proteins were transferred to PVDF membranes. The proteins from the SDS-PAGE or IEF gels were electrotransferred to PVDF membranes and immunolabeled with rabbit anti-cathepsin S pAb  were separated on 12.5% pre-cast gels from Lonza using the Mini-PROTEAN 3 Cell system (Bio-Rad). Protein standards from Fermentas (PageRuler Prestained Protein Ladder) were applied. The separated proteins were stained with 0.1% PhastGel Blue R (Coomassie R 350 dye) from GE Healthcare or with 0.2% AgNO2O and 2 μl of a 2,5-dihydroxyacetophenone matrix solution. The mixture was applied to a matrix target plate, allowed to dry at room temperature, and analyzed.Selected bands were excised from the precast SDS-PAGE or IEF gels and prepared for mass spectrometric analysis as described . The cells were grown in α-MEM with 20% heat-inactivated FBS , 1% Glutamax (Life Technologies) and 40 ng/ml GM-CSF (CellGro) as previously described . Both cystatins weren't labeled with Alexa Fluor 488. The cells were pre-incubated at 4°C to prevent non-specific internalization of the fluorescently labeled primary antibodies: anti-CD86 (clone FUN-1) conjugated to CY-CHROME and anti-MHC II  conjugated to R-phycoerythrin (both from BD Biosciences-Pharmingen). A total of 5000 cells per sample were gated and evaluated for specific labeing using a FACSCalibur instrument (BD Biosciences) and BD CellQuest software (version 3.3). Differentiated MUTZ-3 cells treated with cystatin OmC2 and LPS were compared to non-treated differentiated MUTZ-3 cells.MUTZ-3 cells  were cytocentrifuged to glass slides coated with poly-L-lysine (Sigma Aldrich). The cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. The cells were then immunolabeled with rabbit anti-cathepsin S pAb, rabbit anti-human cystatin C pAb, sheep anti-cathepsin L  for 1 or 3 h. Internalization of cystatin OmC2 to acidic vesicles within the endocytic pathway of treated cells, i.e., to the late endosomes and lysosomes, was followed by using confocal microscopy and fluorescently labeled organelle markers. The LysoTracker Blue DND-22 (a marker for lysosomes), CellMask Orange plasma membrane stain, and CellLight Late Endosomes-RFP/Bac Mam 2.0 system (for the expression of Rab7a-RFP in late endosomes) were from Life Technologies-Molecular Probes. All commercial reagents were used according to the supplier's recommendations. Also, differentiated MUTZ-3 cells were pre-incubated for 30 min at 4°C to decrease their endocytic capacity. Then, cells were cultured with fluorescently labeled cystatin OmC2 at 4°C for 1 and 3 h. Cells cultured at 4°C were compared to the cells cultured at 37°C (and not pre-incubated at 4°C).6 cells/ml) were cultured in the presence of 2, 12, or 15 μM cystatin OmC2 for 1 or 3 h. The cells were pelleted, vigorously rinsed and resuspended in 0.1 M phosphate buffer with 1 mM EDTA (pH 6.0). The cells were sonicated on ice using a Branson Digital W-450 Sonifier . The non-soluble fraction was pelleted and removed by centrifugation at 16,000 g. The soluble fraction was aliquoted and stored at −80°C. The residual activity of the cysteine proteases in the treated cells was compared to the non-treated cells (control). In addition, lysates for analysis by mass spectrometry and Western blotting were prepared from cells that had been cultured in the presence of 12 μM non-labeled cystatin OmC2 (pull-down assay).Differentiated MUTZ-3 cells  were all compared to corresponding controls (non-treated cells). P-values of 0.05 or less were considered significant. Statistical analysis was performed using IBM SPSS Statistics 20.The residual endoprotease and exoprotease activities were measured using the fluorogenic substrates Z-Phe-Arg-AMC and H-Gly-Phe-AMC (Bachem) as described  was performed using immobilized-Ni affinity chromatography. Cell lysates were prepared from differentiated MUTZ-3 that had been cultured in the presence of 12 μM cystatin OmC2 for 1 or 3 h as described above. PBS was added to non-treated cells as a control. The lysates from the treated and non-treated cells were normalized to the same protein content  and incubated with Ni-precharged Sepharose CL-6B (Roche). After 30 min of incubation at 4°C and rigorous shaking (200 rpm), the beads with the bound proteins were rinsed, mixed with SDS-PAGE loading buffer with DTT and boiled. The samples were centrifuged at 16,000 g, and the soluble fractions were subjected to electrophoresis. The separated proteins were stained with Coomassie dye and analyzed using mass spectrometry. Alternatively, the separated proteins were blotted to nitrocellulose membranes and labeled with anti-cathepsin C or anti-cathepsin S antibodies.18 trapping column (Proxeon EASY-ColumnTM) and separated on a C18 PicoFritTM AQUASIL analytical column . The mobile phase A solvent (0.1% formic acid) was used for loading and a 60 min gradient consisting of 5–40% of mobile phase B  was used for peptide separation. The overall flow rate was 300 nl/min. The MS spectra were acquired in the Orbitrap analyser with a mass range 300–2,000 m/z and a resolution of 30,000. The MS/MS spectra were obtained by CID fragmentation  of the nine most intense precursor ions from the full MS scan. Dynamic exclusion was enabled with repeat count of 1 and 60 s exclusion time. The database search and quantification by spectral counting were performed using the MaxQuant proteomics software (version 2.0.18), with the embedded Andromeda search engine  compared to the fluorescence measured in fraction with the labeled cystatin (25900 a.u.)  of cystatin OmC2 that were observed in the IEF gels under non-denaturating conditions in the range of pH 3–7 Figure . Prior tThe endoprotease activity toward Z-Phe-Arg-AMC Figure  and the The possible formation of complexes of cystatin OmC2 with the selected lysosomal endoprotease cathepsin S, and of cystatin OmC2 with the exoprotease cathepsin C, was verified using IEF (pH 3–7) under native conditions. The observed changes in the pI's of cathepsin S and of cathepsin C, both pre-incubated with cystatin OmC2, were compared to those of the non-treated cathepsins Figure . The preThe bands representing cathepsin S (without cystatin OmC2) were observed at pI 7.4 or higher Figure . MultiplE. coli was also observed at pI 7.1  or cathepsin C (21 kDa) in the treated cells (lanes 2 and 4) compared to the cells grown in the culture medium without cystatin OmC2  from family C13 . Likely, the concentration used in in vitro studies is higher than the concentration of cystatin OmC2 in saliva. Then again, the saliva is injected directly into the tissue and the local concentration of its components may be quite high around the cells in that tissue. Though the tick may secrete only small amounts of cystatin OmC2, its inhibitory potency may be adequate to achieve inhibition of the targeted proteases.We don't have quantitative data on the concentration of cystatin OmC2 in the tick saliva. In the paper by Grunclová et al.  rough esInhibitory activity of cystatin OmC2 has been determined in enzymatic assays with the recombinant mammalian cathepsins  were increased in pull-down fractions from the cells with internalized cystatin OmC2, whereas only cathepsin S (an endoprotease) was increased when compared to non-treated cells. Neither cathepsin L nor other endoproteases were increased. Among the significantly increased proteases in pull-down fractions, we have focused on the two of them; on cathepsin S due to its known involvement in antigen processing and presentation in DC. Second, we have focused on cathepsin C, a dipeptidyl peptidase, often included in proteolytic cascades, but rarely studied in DC  could affect the processing of several other proteins . Interestingly, cathepsin C is highly expressed in monocyte-derived DC  and NF-κB pathway in macrophages that were infected with parasitic In summary, exogenous cystatin OmC2 entered the late endocytic compartments and acted effectively on cysteine proteases inside the MUTZ-3-derived immature human DC. Among these proteases, two cathepsins, S and C, which are involved in antigen processing and proteolytic cascades, were accessed. In addition, the expression of CD86, a costimulatory molecule that is associated with the maturation of DC, was diminished. We propose that the diminution of the activity of two key cysteine proteases contributes to the overall ability of tick saliva to affect the immune response of tick's host.Conceived the study, designed the experiments and wrote the paper: TZ. Performed the experiments: TZ, RV, and AS. Analyzed the data: TZ, RV, and MF. Contributed reagents/materials/analysis tools: TZ, RV, MF, JS, LG, and PK. Contributed insightful suggestions on the study: PK and BT. Supervised the project: TZ and BT.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Falco sparverius), in a fruit-growing region of Michigan. During the 2013–2016 study, we added a total of 23 new nest boxes in addition to 24 intact boxes installed previously; kestrels used up to 100% of our new boxes each season. We conducted temporally-replicated surveys along four roadside transects divided into 1.6 km × 500 m sites. We developed a multi-season occupancy model under a Bayesian framework and found that nest boxes had strong positive effects on first-year site occupancy, site colonization, and site persistence probabilities. The estimated number of occupied sites increased between 2013 and 2016, which correlated with the increase in number of sites with boxes. Kestrel detections decreased with survey date but were not affected by time of day or activity at the boxes themselves. These results indicate that nest boxes determined the presence of kestrels at our study sites and support the conclusion that the local kestrel population is likely limited by nest site availability. Furthermore, our results are highly relevant to the farmers on whose properties the boxes were installed, for we can conclude that installing a nest box in an orchard resulted in a high probability of kestrels occupying that orchard or the areas adjacent to it.Nest boxes for predators in agricultural regions are an easily implemented tool to improve local habitat quality with potential benefits for both conservation and agriculture. The potential for nest boxes to increase raptor populations in agricultural regions is of particular interest given their positions as top predators. This study examined the effects of cherry orchard nest boxes on the local breeding population of a declining species, the American Kestrel ( Upupa epops)[Coracias garrulus)[Falco tinnunculus)[Increases in anthropogenic land use, particularly agricultural expansion and intensification, pose a significant threat to many species and the functional diversity of species assemblages ,2. One fpa epops), Europeagarrulus), and Eurnunculus). Nest bonunculus)–17.Falco sparverius; hereafter “kestrels”) are of interest as predators in fruit-growing regions because their diet typically consists of insects, small mammals, and birds [2 [Raptors are potentially important predators in agroecosystems based on their positions as top predators . For exand birds  and can nd birds . Furthernd birds –22 that nd birds ,23. Whilnd birds . Thus, tbirds [2 , 25, no birds [2 .This study used surveys of kestrel presence/absence and multi-season occupancy modeling to determine whether nest boxes in a fruit-growing region of northwestern Michigan  increaseThis research was approved by the Institutional Animal Care and Use Committee at Michigan State University (AUF #01/13-014-00). Private landowners granted us permission to conduct this study on their properties.We conducted this study on the Leelanau and Old Mission Peninsulas of northwestern Michigan , a major cherry-growing region that also produces apples and wine grapes, among other crops . The regKestrels are present in the study area during the summer breeding season; the kestrel population in this region is considered entirely migratory ,29. PopuOur survey design consisted of 4 roadside transects (9.6 km– 12.8 km long) divided into 1.6 km survey sites . We placWe conducted temporally-replicated surveys in 2013–2016 during the kestrel breeding season. The surveys began each year between 9–21 June, after most of the known kestrel nests had hatched , so thatDuring a survey round, we surveyed a site on foot from south to north , waited for 5 min, and then surveyed the site again from north to south (return survey). Each survey round therefore yielded 2 surveys. These sequential time intervals may violate the assumption of independent and equal detection probabilities among surveys within the survey season , but beczi,t = 1 if at least one kestrel was present at site i in year t. The first year site occupancy probability ψi,1 determined the initial occurrence state at site i; the site colonization probability γi,t and site persistence probability φi,t determined the occurrence state in subsequent years.We investigated the effects of nest boxes and other factors (detailed below) on kestrel presence and detectability using multi-season occupancy modeling under a Bayesian framework . This moyi,k,t = 1 if a kestrel was detected at site i during survey k in year t. The occurrence state zi,t and the detection probability pi,k,t. determined whether a kestrel was detected at site i during survey k in year t.The multi-season occupancy model also included the following observation process:We included the 28 segment sites as spatial replicates within the 4 transects. We included the transect level to account for the potential non-independence associated with using groups of sites along 4 roads (site boxes). We chose this distance given that a typical kestrel home range is 0.5 km– 2.4 km in diameter [We predicted that kestrel presence would be more likely at sites with nest boxes. We therefore characterized each survey site based on whether it had nest boxes within 0.8 km of the transect  [neighbor box).We also considered the potential spatial dependence of sites along a transect. We included transect ID to account for the possibility that kestrel presence was more similar at sites within the same transect compared to sites from other transects (ransect) . In additransectj represented the random effect of transect j, and α1,2,4,5,7,8 represented the logit-linear coefficients for model covariates [We modeled the site occupancy, colonization, and persistence probabilities as follows:variates .date) to determine whether kestrels became more or less conspicuous as the survey season progressed [time).We first considered the potential effects of survey timing on kestrel detections. Kestrels exhibit conspicuous hunting behaviors, such as hover-hunting and use of elevated, exposed perches . We predogressed . We alsosurvey). We predicted that kestrels might avoid the survey area after the initial survey, thus decreasing the detection probability during return surveys.We also addressed a biological explanation underlying the potential temporal dependence between initial and return surveys of a site during a survey round. We investigated whether observer presence during an initial survey of a site influenced kestrel behavior and affected detection during the return survey (nest distance).Finally, kestrel activity at known nest sites can bias survey detections . We atteWe modeled the detection probability as follows:λ [We estimated model parameters using Markov Chain Monte Carlo (MCMC) methods. For each model we used uninformative priors and ran two chains for 30,000 iterations, discarding the first 20,000 runs as burn-in and thinning by 2. We ran all models using package “R2jags” in Program R (3.3.1) . We asseλ .Over the 4 years of this study, we detected kestrels at 22 of the 28 survey sites and during 133 out of 1176 total surveys. We detected kestrels at sites along all 4 transects; however, we observed kestrels along the western transect in 2016 only, after we had installed nest boxes during the fall of 2015 . The numsite box in all ecological process models had 95% CRIs that did not overlap zero, indicating that nest boxes were an important predictor of first-year site occupancy, site colonization, and site persistence  detections over the breeding season for unsuccessful breeders only, which the authors attributed to a decrease in conspicuous territorial behaviors after reproductive failure. Because kestrels using the new nest boxes during this study showed a 91% success rate, the decrease in detections observed during this study is more likely due to another source of behavioral change rather than reproductive failure. An alternative explanation is that kestrels are less conspicuous later in the breeding season due to lower hunting activity, as we predicted. Prey consumption requirements for breeding kestrels increase when the eggs hatch because the adults must provision their young [Julian date had a negative effect on kestrel detectability; kestrel detections decreased between June and August. Previous research  has showir young , and whiir young . Thus, air young . Withouttime suggests that kestrels are equally conspicuous in the morning and afternoon. This conclusion is supported by previous observations that kestrels hunt throughout the day without apparent peaks in activity [survey suggests that observer presence during the initial survey did not affect kestrel detectability during the return survey, thus any potential temporal dependence between surveys is not the result of a behavioral response to the observer. Finally, site distance did not have an important effect, which indicates that detections were not biased towards kestrel activity at boxes with active nesting attempts within 250 m of the transect. This conclusion is further supported by the fact that we made no kestrel sightings at a nest box. The results from the detectability model indicate that kestrel researchers have flexibility in the timing of their surveys during the day and the placement of survey sites with regard to nest boxes.None of the remaining covariates had important effects on kestrel detectability. The lack of an effect of activity . The lacAlthough kestrels are conspicuous open-habitat raptors, the occupancy model estimated our detection probabilities as <0.4 . These dOur results support the conclusion that nest boxes determined the presence of kestrels at our study sites; however, our study design and modeling framework do no allow us to conclude that the breeding kestrel population has increased as a direct result of nest box installation. It is possible that the installation of nest boxes in the survey sites drew in breeding kestrels that had used other territories previously; the true nature and extent of nest site-limitation cannot be determined without also monitoring other sources of cavities throughout the region . NeverthOur survey design also included logistical constraints; we maximized the spatial and temporal replicates needed for occupancy modeling at the possible expense of introducing unresolved spatial and temporal dependence between sites and surveys. However, we included additional variables in our ecological and observation models to attempt to identify and explain possible biological sources for any dependence. We found that adjacent sites were dependent in that sites neighboring those with nest boxes were more likely to be colonized by kestrels than sites neighboring those without boxes. We also did not find an important difference in detections among initial and return surveys, which suggests that observer presence during the initial survey did not affect kestrel detectability during the return survey; however, the pairs of surveys could still be dependent in other ways. Nevertheless, we do not expect that temporal dependence significantly biased our detectability estimates because we observed all possible combinations of detection and nondetection for pairs of initial and return surveys .Tyto alba) and their diets [Although our results do not directly correspond to an increase in the breeding kestrel population, our results indicate that kestrels in this fruit-growing region are more likely to choose breeding territories with nest boxes. Given the high reproductive success observed at nest boxes in this region , we therir diets ; ongoingIn addition, our territory-level results are highly relevant to the farmers and landowners on whose properties the boxes were installed, for we can conclude that installing a nest box in an orchard resulted in a high probability of kestrels occupying that orchard or the areas adjacent to it. Our results could encourage additional farmers to install and maintain nest boxes in fruit-growing regions where agricultural practices create open hunting habitat for kestrels that are therefore limited in these habitats primarily by nest site availability. Ongoing work aims to further inform box installation practices by exploring the relationship between the number of boxes installed at a site and kestrel presence in a fruit-growing region with lower nest box occupancy rates than observed in this region. Two major issues with nest box programs that can potentially be detrimental to kestrel populations are placement of boxes in low quality habitat and the installation of boxes that are not monitored or maintained . Thus, iS1 File(TXT)Click here for additional data file.S2 File(R)Click here for additional data file.


Rising temperatures in the Arctic cause accelerated mass loss from the Greenland Ice Sheet and reduced sea ice cover. Tidewater outlet glaciers represent direct connections between glaciers and the ocean where melt rates at the ice-ocean interface are influenced by ocean temperature and circulation. However, few measurements exist near outlet glaciers from the northern coast towards the Arctic Ocean that has remained nearly permanently ice covered. Here we present hydrographic measurements along the terminus of a major retreating tidewater outlet glacier from Flade Isblink Ice Cap. We show that the region is characterized by a relatively large change of the seasonal freshwater content, corresponding to ~2 m of freshwater, and that solar heating during the short open water period results in surface layer temperatures above 1 °C. Observations of temperature and salinity supported that the outlet glacier is a floating ice shelf with near-glacial subsurface temperatures at the freezing point. Melting from the surface layer significantly influenced the ice foot morphology of the glacier terminus. Hence, melting of the tidewater outlet glacier was found to be critically dependent on the retreat of sea ice adjacent to the terminus and the duration of open water. Of particular interest is the more than 200 tidewater outlet glaciers around Greenland3 because their direct contact with coastal water masses constitute a link between ocean heat transport, sea ice and melt of glacial ice4. However, coastal water masses around Greenland vary considerably between the east and west coast5 and also along the climatic gradient from southern to northern Greenland .The Greenland Ice Sheet mass loss has accelerated7 and western9 coasts of Greenland where they provide energy for subsurface melt11. Tidewater outlet glaciers at high polar latitudes have been studied at the floating ice tongue of Nioghalvfjerdsfjorden (79°N) and melting of glacier ice has been explained by heat from deeper and warmer  recirculated Atlantic water12. Studies of the floating ice shelf in the Peterman Fjord  also showed that subsurface melting could account for basal melt of the ice shelf13. Other studies of polar tidewater outlet glaciers include studies at Svalbard (~79°N) where the influence from ocean heat transport was manifested by a strong correlation between calving activity and subsurface ocean temperatures14. These studies elucidate the important role of sub-surface ocean heat transport for melting glacial ice.Relatively warm water masses have been shown to be in contact with tidewater outlet glaciers along the eastern15. Ice mélange and a protecting fast ice cover in front of glacier termini have been shown to influence the stability of the floating ice shelf in Nioghalvfjerdsfjorden on decadal to millenial time scales16 and recent speed-up and mass loss of both Nioghalvfjerdsfjorden and Zachariae Isstrøm occurred in periods with increased air temperature and low concentrations of sea ice and further enhanced by entry of warm subsurface-temperatures17. Retreats of the major outlet glacier at Jakobshavn Isbræ have also been related to a weakening of the ice mélange in front of the terminus19. The Ellesmere Island ice shelves, facing the Arctic Ocean, have decreased significantly during the last century20 and, recently, a large fraction of the remaining shelves calved during summer 200821. Calving events of the Ellesmere Island ice shelves have also been associated with longer periods of open water and loose pack ice along shelf margins22. Such conditions increase surface melt, wave erosion and removal of a resisting pressure on calving ice from surrounding land-fast ice and, thereby, promote fast disintegration of the glacier tongue23. Thus, these observations indicate the important role of sea ice cover and ocean heat transport in front of the glaciers.However, other processes also affect terminus stability. For instance, an analysis of satellite images showed that changes in atmospheric conditions only had a minor influence on terminus position of five large outlet glaciers and, therefore, it was hypothesized that other processes such as ice thickness, subglacial topography and fjord bathymetry may regulate the retreat or advance of these glaciers24. Thus, coastal and shelf water masses in the western Fram Strait are generally transported southward from the Wandel Sea . In addition, we analyze water masses near the terminus and argue that the terminus is a floating ice shelf. The observed ice foot morphology at the terminus is related to large melt rates in the surface layer, and we show that the short open water period has a major influence on the heat budget near the outlet. Finally, our results are used to explain the observed decadal variation of the outlet glacier.In this study, we present hydrographic measurements from the northeastern coast of Greenland at a tidewater outlet glacier from the largest peripheral ice cap in GreenlandThe fjord complex in northeastern Greenland associated with Independence fjord  showed that the glacier front continued vertically towards the bottom with no signs of an ice foot. Together with visual observations of ‘fresh’ glacier ice above sea level these observations indicated that calving had occurred recently at this location. We witnessed several occasions of calving. The bottom depth was about 60 m along this transect Fig. . TransecWater masses near the terminus were analyzed by combining all deep stations collected close to the terminus in the period 10–25 August Fig. . SurfaceThe bottom water temperatures at a depth of 44 m along the terminus decreased from −1.68 °C at the MYI edge to the coldest temperatures at transect III of −1.74 °C Fig. , i.e. a in situ freezing temperature, was observed during a deep profile (bottom depth 70 m) taken ~50 m from the glacier terminus on transect III. Low temperatures were also observed between 45–57 m of the water column, where in situ temperatures were within 0.005 °C of the freezing point and where super-cooling of up to 0.002 °C was observed in the layer between 48 and 50 m. Note that the super-cooling was within the instrument uncertainty range of ±0.005 °C. However, such low temperatures strongly indicated that subsurface freezing had occurred at the glacier.A very low temperature of −1.807 °C , i.e. 0.014 °C above in situ freezing temperature of −1.86 °C  showed that the highest salinity along the terminus was found at this deepest location and temperature was well above the  °C Fig. . High sa °C Fig. . These o28 and with only latent heat exchange and freshwater dilution considered, indicated a cooling of ~0.3–0.5 °C of the bottom water mass at transect II and III. The near-glacial profile at transect III showed a layer between 35 and 60 m with in situ freezing temperatures located above a warmer and more saline bottom water mass  was located at station NS northwest of the terminus at the MYI edge and both T and S were significantly higher than observed at the terminus Fig. , definedass Fig. . A visitContinuous measurements from the CTD-mooring deployed at 13.5 m, St. M, Fig.  from May2 and is likely formed after the Holocene climatic optimum29. The main north flowing outlet of the ice cap is a ~25 km broad outlet located east of VRS  could not be explained by mixing with nearby relatively low-saline water masses along or in front of the terminus. No sufficiently deep connections were observed west off the terminus to explain a deep transport from station NS to the bottom water at transect III Fig. . This su °C Fig. , could aThe presence of relatively warm bottom water (>−1.4 °C compared to freezing temperatures below −1.7 °C) near FIIC showed that coastal water masses may cause significant bottom melt below the ice shelf. A comparison between bottom water masses and observations from station YK175 located on the continental slope off FIIC  on the seafloor further towards the east.Based on these observations we suggest that intrusions of coastal water masses below a ~45 m thick ice shelf result in ice melting and cooling of water below the ice shelf to the freezing point of seawater. Subsurface freezing temperatures indicate freezing, and possibly refreeze of melted ice, below the glacier. This cold-water phenomenon, observed at glaciers around Antarctica35, and this shows that a significant amount of meltwater is mixed into the surface layer in this region.Surface layer temperature and salinity were analyzed in the upper 20 m of the water column from locations off VRS, at station NS near the MYI and at transect III at the inner part of the NW-outlet glacier Table . The are−2. The average incoming solar insulation from 1–25 August was 164 W m−2 corresponding to a total solar heat flux of 333 MJ m−2 during the open water period . Measurements of light attenuation off VRS showed that the euphotic zone (i.e. 1% light level) was only ~6 m deep, in accordance with the observed shallow summer thermocline. Thus, solar heating during the short open water period in August in front of FIIC can explain the observed change in heat content between early spring and late summer. The incoming solar heat flux on top of the glacier during this period of ~106 MJ m−2  is comparable to the increased energy content in the surface layer, and corresponds to the energy required for melting 0.4 m m−2 of ice.In order to evaluate glacier melt in the surface layer we assess the energy budget. Our measurements showed that surface water temperature in the upper 10 m increased by more than 2.5 °C in the open water areas near the glacier compared to early spring conditions (Table 36 (Methods), was 0.5 m month−1. Including the influence from wave erosion, calculated from wind speed and direction38, increased this value by an order of magnitude to 0.7 m day−1. The surface temperature increased from near freezing temperature during the open water period and, therefore, these melt rates should be reduced by about 50% (e.g. assuming a linear temperature increase of the surface layer). Thus, melt from the surface layer resulted in ~10 m month−1 during the short open water period in August.The direct melt from a surface layer with an average temperature of 0.7 °C  while the gradual increase in July could be related to open water areas north of the MYI cover as evident from MODIS satellite imagery from 22 June  and this showed that the temperature near the glacier, at ~13 m depth, was significantly colder than at the mooring. This could be explained by heat loss for melting of glacier ice. The melt rates calculated from the CTD-mooring, therefore, represent an upper bound of melting below the upper halocline. Thus, subsurface melting at this depth level is relatively small when compared to melt from the surface layer in the open water period. Surface heating in August increased surface water temperature to above 2 °C in front of the glacier corresponding to a surface melt rate in the upper 5 m of ~0.8 m month−1, whereas melt rates calculated at station NS corresponded to relatively low values of 0.1 m month−1 in the upper few meters (due to the MYI cover) and ~0.4 m month−1 at ~5 m depth  in the upper 10 m  was compared with temperatures at station NS (−1.2 °C) and at transect III (−1.5 °C) at the corresponding density level  between 1950 and 1964 show that the area was covered by sea ice throughout the year42. Higgins30 noted that extensive open water leads were visible close to the terminus on aerial photographs (from plane) in 1978. The presence of open water has been visually observed around Station Nord every summer in recent years.The climatological annual averaged sea ice extent in the Arctic Ocean has decreased by 14% between the period 1963 and 1998 and by 27% since 1950 considering the minimum sea ice extent in 2012Our results show that the duration of open water increases heat content in the surface layer significantly and transport of warm surface water may, therefore, impact the NW-outlet glacier. Melting of the disintegrated ice tongue in front of the NW-outlet during the last decades and the subsequent replacement of the ice tongue with MYI can, therefore, be explained by transport of warm surface water from open water areas Fig. . The rol−1), width (~10 km) and depth (~40 m) of the glacier tongue yield ~7.4 PJ yr−1. The ocean heat transport in the surface layer (containing 103 MJ m−2) towards the terminus with a length of ~10 km and assuming an ocean current of 1 cm s−1 corresponds to an energy flux of 0.9 PJ day−1. These values are rough estimates based on our current knowledge of the ice flow and ocean currents estimated from the relatively low salinity and observed tidal motion of ice floes. A transport towards the glacier would cause melt along the terminus and in the wide cracks into the glacier. Some of the heat will also be used for melting sea ice and convergence in front of the glacier would redirect currents along the terminus and, therefore, only a portion of the heat flux calculated above would be available for melting glacier ice. However, transport of warm surface water towards the glacier during a few weeks would equal the energy needed for melting the annual ice flow from the NW-outlet and is comparable to the energy flux from solar insulation in August (i.e. ~106 MJ m−2) above a corresponding area (i.e. 100 km2) of ~10 PJ. Thus, heating of the ice free surface layer is a main component of the energy budget around the NW-outlet.Relating the melt rates, inferred from the 2015 observations, with ice dynamics reveal that annual ice flow can easily be balanced by short periods of open waters. Estimating the heat required for melting the annual ice flux of the glacier tongue from the NW-outlet based on the ice flow . Water mass properties at the bottom of the terminus and ice surface elevation data supported that the NW-outlet has a floating ice shelf. Bottom water at the terminus was super-cooled or very near the freezing point, providing a strong indication that net freezing took place at the glacier ice-ocean interface. During the short (~1 month) open water period the increased heat content of the surface layer was estimated to account for up to ~10 m of ice melt per month. The presence of open water in front of the terminus and the associated solar heating of the surface layer were found to be a major contribution to the annual melt. Reduction in sea ice cover in front of tidewater outlet glaciers along the north coast of Greenland could, therefore, result in a significant increase of ocean heat transport towards tidewater outlet glaciers.The field campaign was carried out from two inflatable boats (Zodiac) equipped with a winch and a SeaBird Electronics SBE19plus SEACAT profiler measuring conductivity, temperature and depth (CTD) and a PAR Licor sensor. The CTD-mooring off VRS  was a SeaBird-Electronics SBE37SMP Microcat in a frame deployed on the bottom (sensors ~0.2 m above bottom) at 13.5 m depth  were mounted at the rear of the two zodiacs and about 10 cm below water level to continuously record bottom depth. Map of northeastern Greenland  was calculated as:p are the density and heat capacity for seawater, respectively. The temperature difference between in situ summer temperature (T) and early spring temperature, determined as ΔT = (T − Tspring), correspond to the change in heat content during the summer season. The heat content is integrated in the upper 10 m and z the vertical coordinate.The seasonal change in heat content  was calculated by integrating the difference of m, m yr−1) at the ice-ocean interface was calculated according to Neshyba and Josberger36 as follows:The meltwater rate  was calculated from the formula provided by White et al.37:a and b are constants , zr is the roughness height of the ice surface (~1 cm) and Hw and Tw are significant wave height and period, respectively. Significant wave height and period were calculated from half hourly measurements of wind speed assuming a fetch of 2 km, corresponding to the estimated open water area in front of the glacier. Only wind forcing from directions between 150 and 330 degrees, corresponding to wind directions towards the glacier, were considered. Significant wave height and period were calculated according to Kahma and Calkoen38.Melt from wave erosion (VSupplementary Information


In addition, docking simulation results demonstrated that ASN37, SER35, SER36, TRP76, and ARG128 residues of BACE1 interacted with biochanin A. Moreover, the binding energy of biochanin A was negative , indicating that it might potentiate a strong binding between the compound and the allosteric site of BACE1, resulting in further effective BACE1 inhibition. The present novel findings raise the possibility that biochanin A may be used as a preventative, developed into a therapeutic agent for AD, or both.Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the enzyme involved in the abnormal production of the amyloidogenic peptide Aβ, one of the major causes of histological hallmarks of Alzheimer’s disease (AD). Thus, BACE1 represents a key target protein in the development of new potential target for the prevention and treatment of AD. In this study, in vitro anti-AD activity of biochanin A, a dietary isoflavone found in legumes and most notably red clover, were evaluated via human recombinant BACE1 inhibition assay, as well as enzyme kinetic and molecular docking predictions. Enzyme-based assays revealed that biochanin A exhibited a non-competitive inhibitory effect on BACE1 with an IC Alzheimer’s disease (AD) is a neurodegenerative disorder presenting with symptoms such as memory loss and disruption in judgment, reasoning, and emotional stability. The predominant pathological hallmarks of the disease are amyloid plaques, i.e., extracellular deposits of polymerized amyloid-β peptides (Aβ), and intracellular aggregates of misfolded tau protein ,2. Much β-Secretase and γ-secretase have been identified as the two key proteases responsible for the processing of the membrane-bound APP to the 40/42 residue Aβ . β-APP c25–35-induced PC12 cell injury and apoptosis via preventing mitochondrial dysfunction  and a Linwever-Burk plot [a plot of 1/enzyme velocity (1/V) versus 1/substrate concentration (1/[S])]. The modes of inhibition were determined by both methods, whereas the inhibitory constant . Marvin was used for drawing, displaying, and characterizing the chemical structures . Potentt-test and Duncan’s multiple range tests using Statistical Analysis System (SAS) version 9.3.8 .All experiments were performed in triplicate. Data of each experiments were expressed as mean ± SE. Significant differences were analyzed by Student’s p < 0.05 and p < 0.001), with an IC50 of 28 μM.We investigated whether biochanin A has BACE1 inhibitory activity by an in vitro BACE1 activity assay. BACE1 activity was found to decrease in a dose-dependent manner to 69.54% ± 3.62%, 43.89% ± 0.52%, 30.31% ± 1.97%, and 25.31% ± 1.02% by biochanin A at 12.5, 25, 50, and 100 μM, respectively . Statistx-axis implies –Ki when 1/V = 0. As shown in x-intercept, indicating non-competitive inhibition, with Ki values of 43 μM, suggesting that they might bind to either a β-secretase sub-site or to another regulatory site. It implies that biochanin A reduces the activity of the BACE1 and binds equally well to the enzyme whether or not it has already bound the substrate.In an attempt to explain the mode of enzymatic inhibition of biochanin A, kinetic analyses were performed using different concentrations of the substrate  and an inhibitor. Concentrations of biochanin A  were also selected for this study. The reaction mixture consisted of the same, aforementioned BACE1 assay method. In non-competitive inhibition, the The molecular docking models of biochanin A (green color) is illustrated in The molecular culprit in AD is the Aβ, which is derived through sequential cleavage by the two proteases β- and γ-secretase. In contrast, TACE cleaves APP within the Aβ domain, thus prevents Aβ generation. In addition, the closest structural relatives to BACE 1 are the aspartyl proteases trypsin, chymotrypsin, and elastase. To determine the enzyme specificity and selectivity, the inhibitory property of the biochanin A against TACE and other serine proteases such as chymotrypsin, trypsin, and elastase was investigated . BiochanIn this study, the impact of biochanin A on BACE1 inhibition was investigated. The current failure rate of finding BACE1 inhibitors has driven research interests towards the search for alternative small molecules with therapeutic potential for reducing risk or slowing progression of dementia associated with AD. Given the brain localization of the target, inhibitors are required to be of low molecular weight and should have reduced susceptibility to P-glycoprotein (Pgp)-mediated efflux transport at the blood–brain barrier (BBB) . To effeBiochanin A was proved to possess anti-angiogenic and neuroprotective properties. Administration of 50 mg/kg biochanin A significantly reduced tumor size and edema in the brains of male Fisher rats . In addiIn vitro and in vivo studies have shown that biochanin A is metabolized to genistein by a demethylation pathway . After dBioavailability of biochanin A is still controversial. Moon et al. (2006) suggested that the bioavailability of biochanin A was only 2.6% after a 5 mg/kg dose and 1.2% after a 50 mg/kg dose of biochanin A, which may be due to extensive first-pass metabolism by phase I oxidative metabolism and phase II glucuronidation and/or sulfation in the intestine as well as in the liver . HoweverOur findings indicated that biochanin A effectively inhibited the activity of BACE1 in a dose-dependent and non-competitive manner. Furthermore, our results provided important mechanistic insights into the binding mechanism of this compound to BACE1. Taken together, this study reveals that the biochanin A contained herein would clearly play a beneficial role in the development of therapeutic and preventive agents of AD and suggest potential guidelines for designing BACE1 selective inhibitors.


Abnormal tau metabolism followed by formation of tau deposits causes a number of neurodegenerative diseases called tauopathies including Alzheimer’s disease. Hyperphosphorylation of tau protein precedes tau aggregation and is a topic of interest for the development of pharmacological interventions to prevent pathology progression at early stages. The development of a mathematical model of multisite phosphorylation of tau would be helpful for searching for the targets of pharmacological interventions and candidates for biomarkers of pathology progression. In the present study, we for the first time developed a model of multisite phosphorylation of tau protein and elucidated the relative contribution of kinases to phosphorylation of distinct sites. The model describes phosphorylation of tau or PKA-prephosphorylated tau by GSK3β and CDK5 and dephosphorylation by PP2A, accurately reproducing the data for short-term kinetics of tau (de)phosphorylation. Our results suggest that kinase inhibition may more specifically prevent tau hyperphosphorylation, e.g., on PHF sites, which are key biomarkers of pathological changes in Alzheimer’s disease. The main features of our model are partial phosphorylation of tau residues and merging of random and sequential mechanisms of multisite phosphorylation within the framework of the probability-based approach assuming independent phosphorylation events. Microtubule-associated protein tau (MAPT) stabilizes microtubules regulating cell architecture and cargo transport . As an iGlycogen synthase kinase 3 beta (GSK3β) is a proline-directed protein kinase generally considered one of the key players in tau phosphorylation. This kinase phosphorylates mainly (S/T)P motifs and specifically recognizes the (S/T)XXX(pS/pT) consensus motif whereby the first priming phosphorylation event is performed by GSK3β itself or another kinase. Priming  may enhance or silence the catalytic efficacy of the kinase. Prephosphorylation by CDK5 increases subsequent phosphorylation catalyzed by GSK3β but not vice versa, i.e., no significant stimulation of phosphorylation is observed when phosphorylation by GSK3β is followed by phosphorylation by CDK5 ; this prKinase p38γ, another candidate for pharmacological intervention, phosphorylates tau in vitro at four sites  in a short-term reaction and hyperphosphorylates tau during long-term assays. T205 is a primary p38γ site in tau, and phosphorylation of T205 alleviates amyloid β toxicity in a mouse model of AD [The tau phosphorylation state depends strongly on phosphatases. PP2A is the most abundant phosphatase in the human brain . PP2A acPharmacological interventions in tauopathies and targeted tau hyperphosphorylation have been proposed. Several GSK3β inhibitors such as tideglusib, lithium, valproic acid, and AZD-1080 do not show improvements in cognitive outcomes in clinical studies . A blockQuantitative analysis of the contribution of kinases to the phosphorylation state of each residue should be helpful for searching for proper targets of pharmacological interventions and candidates for biomarkers of pathology progression or treatment efficacy. Ten tau sites and kinases incorporated into the model are presented in Several mathematical models describing different outputs of a variety of molecular mechanisms involved in the processing of phosphorylation sites have been developed . These an phosphorylable sites can exist in 2n phosphorylation microstates, which may have different functional characteristics. Explicit mathematical description of such states by usual kinetic methods and differential equations is impossible due to combinatorial complexity. Various approaches have been developed to describe such complex systems, e.g., rule-based approaches [Compared to phosphorylation of a single residue, multisite phosphorylation increases the possibilities for regulating protein function considerably. A protein with proaches . These aproaches , 17. In proaches  or the cproaches  for kineOur main goal was to develop for the first time the quantitative model of multisite (de)phosphorylation of tau protein and to elucidate the relative contribution of kinases and phosphatases to the phosphorylation of distinct sites.A short description of the probability-based approach for description of multisite protein phosphorylation is presented below. For a detailed description, see n phosphorylable sites. The concentration of the protein in a prefixed state (phosphorylated or unphosphorylated ith site) is a macrostate S(ai) where ai = 1 or 0. It is defined as a sum of microstates in which each site except ai can be in a phosphorylated (ai = 1) or unphosphorylated state (ai = 0):Let us consider a protein with The reduction of such a high-dimensional phosphorylation microstate space to a smaller number of functional states may be performed by a probabilistic method .By definition, the probability to find the protein in a particular microstate equals to the proportion of the protein in this microstate:If we assume independent phosphorylation of distinct sites, then microstate probability equals the product of probabilities of the corresponding macrostates:S(ai = 0) = Ri are substrates for kinases, and macrostates S(ai = 1) = Pi are products of phosphorylation (see formulas below).Therefore, the theory of probability, when applied to multisite protein phosphorylation, links the concentration of the protein in any microstate to the concentration of distinct (un)phosphorylated sites that is measured experimentally via formulas  and 3)..3). MacrThis approach enabled the description of multisite phosphorylation by a random or sequential mechanism  avoidingExisting published theoretical approaches to multisite protein phosphorylation describe a situation when all phosphorylation sites are fully available for a kinase . The appTau residues that undergo phosphorylation and are taken into account in the model were chosen based on available in vitro experimental data . PhosphoThere are “closed” and “open” states of the site for a kinase. Because experimental phosphorylation kinetic curves for some residues and total tau did not reach a plateau, we can assume that very long-term kinetics make tau residues fully available for kinases. “Closed” states of residues exist, and they are assumed to be in a very slow equilibrium with an “open” state. Hence, during a short-term phosphorylation reaction (when time is not sufficient to observe opening of a closed residue), only some tau molecules undergo phosphorylation at a specific site, depending on the proportion of initially opened sites, as confirmed by in vitro studies  and by ii describing a residue’s extent of openness) or to an increase in kinase catalytic efficiency (ki).The reactions of phosphorylation of protein sites are independent. Residues of tau protein are phosphorylated independently from each other  but compete for binding to a kinase. Priming (phosphorylation of S214) may influence subsequent phosphorylation of other sites (positive or negative priming), potentially via the conformational change that leads to opening of the closed sites  were chosen arbitrarily so that T205 phosphorylation would be dependent strongly on p38γ activity because APP23 mice with depletion of p38γ show a lack of T205 phosphorylation [Only protein phosphatase 2A (PP2A) is taken into account in this model. PP2A dephosphorylates all residues.M equal for all residues.Each residue in the open state is considered a substrate, and its phosphorylation follows standard Michaelis–Menten kinetics with substrate competition (assumption 3) and KPartial residue phosphorylation is described by means of the initial state of the system:αi is a probability that residue i is in an open state at the initial moment of the in vitro experiment used for calibration; then N is stoichiometry of tau phosphorylation when the system is close to the “steady-state”. For long-term simulations, we assume tau conformational transitions leading to complete opening of the sites, i.e., all α parameters are equal to 1.0.Here, The fitting is performed in the DBSolve Optimum software, v.36 . The objhttps://automeris.io/WebPlotDigitizer/index.html).Data extraction from figures , 20 was f (between arbitrary units and concentration) and parameters corresponding to phosphorylation  were determined. Then, phosphorylation level for each residue was calculated as described in the experimental procedure [ki for phosphatase) at the second stage.The calibration of model parameters was carried out in two main stages. At the first step, proportionality coefficient rocedure  and servThe majority of experimental data used for construction and calibration of the model were measured for full-length tau protein. Data for model calibration included kinetics of tau phosphorylation  and dephki and αi were determined for each dataset; proportionality coefficient f was shared among all four datasets. Michaelis constants for GSK3β and CDK5 were fixed at 1 μM estimated from experimental data [Four datasets were selected for fitting of the phosphorylation parameters: the kinetics of the phosphorylation of unphosphorylated tau or PKA-prephosphorylated tau by GSK3β or CDK5. Individual estimates of tal data , 23.f, but the initial value for its fitting was estimated via the following reasoning  to the total measured level of tau phosphorylation (tau ⋅ N), assuming that 10 sites cover all phosphorylation stoichiometry :There are no available experimental data to evaluate parameter ning see . Becausef were obtained for the dataset on tau phosphorylation by CDK5 without prephosphorylation, and it was chosen as a unique initial value for subsequent simultaneous fitting to all datasets including data for total tau phosphorylation stoichiometry.A maximum of four calculated parameters αi were subdivided into groups according to similar values after preliminary fitting and were assumed to have a unique value inside the group in order to set the reasonable limits for parameters and behavior of the simulated curves. This approach also allowed for reducing the number of parameters and improved computational efficiency. The same reduction was applied to the number of parameters ki. At the last step, ki and αi (which remained independent) were refitted. Thus, 36 of 42 α parameters were placed into 11 groups based on similarity of the preliminarily fitted values  were calculated after calibration of phosphorylation parameters (for GSK3β and CDK5). Experimental data were available only for six residues  for which catalytic constants were identified. For the remaining five residues  catalytic constants were equal to the averaged value of the six identified constants. The Michaelis constant was fixed at 11.6 μM taken from a literature source .These intervals were estimated for all the parameters fitted to the experimental datasets by the linearization method . The estOptimization of MLE (maximum likelihood estimator) in DBSolve Optimum v.36 .dbs.Estimation of confidence intervals based on the covariance matrix using R package For some simulations confidence bands were calculated via the Monte-Carlo approach.M and catalytic constants), 105 random sets of parameters were generated from a uniform distribution. Limits for parameter generation were as follows: a) from zeros to doubled values obtained from fitting for catalytic constants related to GSK3β, CDK5, and PP2A; b) from zero to 1.0 for the level of enzymes; c) from zero to 100 for Michaelis constants and p38γ-related catalytic constants. For each set of parameters, endpoints  were calculated, and then for each pair (endpoint–parameter), Pearson’s correlation coefficients were computed and presented as a heatmap.For selected parameters  depends on PKA. Data revealed that PKA-prephosphorylation influences the final level of residue phosphorylation (determined by αi) and the total stoichiometry that cannot be attributed to catalytic constants.The proportion of opened residues (ki) for specific residues are also PKA-dependent.Kinases’ catalytic constants  for distinct sites (A) and its contributions to total tau stoichiometry (B). For six residues  without PKA-prephosphorylation, the”openness” for CDK5 is higher than that for GSK3β and vice versa for the three residues . The pseudoresidue as a conglomeration of all other potentially phosphorylated residues is considered more”open” for GSK3β than CDK5 regardless of tau pretreatment. This situation makes tau protein a more preferable substrate for GSK3β than for CDK5, and this notion is supported by a greater number of GSK3β-phosphorylable sites [f and parameters related to the pseudoresidue  of GSK3β differs from that of CDK5 by up to three orders of magnitude . For kinase p38γ, catalytic constants were not fitted and were chosen for sensitivity analysis so that only T205 phosphorylation would be dependent strongly on p38γ activity in contrast to the other three sites.The slow dephosphorylation kinetics (S404 or S199) were described worse than fast kinetics . This ouPseudoglobal sensitivity analyses were conducted to explore 1) how the uncertainty in the output of a mathematical model can be apportioned to the different sources of uncertainty in its inputs; 2) how sensitive the different tau states are toward potential disturbances in the system. Phosphorylation states of residues and total tau phosphorylation stoichiometry were chosen as endpoints.ki) of GSK3β is much higher , which is the shared feature of published models , hamperiαi and ki. One of the possible solutions (implemented in our model) is to reveal a similarity between the fitted values of parameters, equate them, and use shared values for such groups. Thus, grouping of similar parameters can help to overcome uncertainty when researchers use biological data with unknown or large measurement inaccuracy or in case of a lack of data. We estimated the model parameters by means of in vitro data on the phosphorylation of tau or PKA-prephosphorylated tau by GSK3β and CDK5 and determined confidence intervals for them. Overall, several groups of α-parameters have emerged phosphorylation reported in refs. , 20. Becrged see  with nonrged see . AlthougIt is well known that hyperphosphorylation of tau is associated with tau aggregation , but notOur pseudoglobal sensitivity analysis can be considered an analysis of sensitivity of specific tau states to potential therapeutic interventions (kinase inhibition or phosphatase activation).Phosphorylation of tau at S396 is significantly reduced by a GSK3β knockdown, whereas phosphorylation of T181 not reduced by suppression of GSK3β in SH-SY5Y cells . In manyVarious kinds of cross-talk between kinases and phosphatases subtly regulating tau phosphorylation have been investigated , 38. PP2αi and ki. Besides, it is impossible to evaluate the contribution of GSK3β autophosphorylation to kinetic constants of tau phosphorylation; therefore, determination of the GSK3β phosphorylation state during the reaction should provide some helpful information. Biochemical approaches, such as immunoblotting with phosphospecific antibodies, are routinely used for monitoring of (previously identified) phosphorylation sites. Mathematical description requires quantitative information that can hardly be obtained by immunoblotting. There are some limitations of the antibody-based approach for monitoring of phosphorylation events. First, there is the issue of availability of specific antibodies for distinct sites that can be potentially phosphorylated. Second, resolution is low due to spatial closeness of phosphorylation sites. Typically, antibody epitopes are 4–12 amino acid residues long [Experimental data on the dynamics of key phosphorylation events are essential for the development of accurate quantitative and mechanistic models of multisite phosphorylation and priming. On the other hand, experimental data often are not very amenable to calibration of model parameters and require making some assumptions. Model predictions may help to improve experimental design. For example, it would be better to increase the time resolution of phosphorylation kinetics, especially at the beginning of the reaction, and to continue the reaction until a plateau is reach. This strategy would have confirmed the transient peak during S404 phosphorylation by GSK3β  and alloues long , suggestues long , 35.In the present study, a mathematical mechanism-based model of multisite (de)phosphorylation of tau protein was developed. The main features of the model are partial phosphorylation of tau residues, and merging random and sequential mechanisms of multisite phosphorylation within the framework of the probability-based approach assuming independent phosphorylation events. This approach illustrates how analysis of in vitro kinetics allows for the choice of better candidates and for a greater understanding of relations between therapeutic targets and biomarkers. The model describes phosphorylation of tau or PKA-prephosphorylated tau by GSK3β and CDK5 and dephosphorylation by PP2A, thereby accurately reproducing the data on short-term kinetics of tau (de)phosphorylation. Our results suggest that kinase inhibition may prevent tau hyperphosphorylation more specifically, e.g., on PHF sites, which are the key biomarkers of AD pathology. The model may serve as a submodel for subsequent development of a systems pharmacological model of tau pathology for research into biologically relevant challenges.S1 Appendix(DOCX)Click here for additional data file.S1 File(XML)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.