We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with μ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME.Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells’ limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the μ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. μ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2’s cargo affinity   •AP2 μ2T156 phosphorylation starts early at CCP inception and increases as CCP grows•Phosphorylation favors an AP2 conformational change and also triggers NECAP binding•NECAP PHear simultaneously binds P-AP2 and membrane-remodeling proteins on opposite faces•Disrupting the P-AP2:NECAP:membrane-remodeling protein network reduces CME rates Wrobel et al. show that phosphorylation of the mammalian endocytic AP2 adaptor causes a conformational change that allows it to efficiently bind the protein NECAP. This, in turn, recruits membrane-remodeling proteins into clathrin-coated pits, which drive their formation toward final scission from the parent membrane. The levels of a vast array of transmembrane proteins of widely differing functions must be tightly but dynamically regulated. This is achieved by controlling the rate of each protein’s internalization, which occurs mainly by clathrin-mediated endocytosis (CME) and balancing it with its rates of delivery to the surface after biosynthesis, transport down the endocytic system, and recycling back to the cell surface. CME is mediated by clathrin-coated vesicles (CCVs) that matured from clathrin-coated pits (CCPs). The most abundant proteins in endocytic CCVs are clathrin itself, which forms the outer polyhedral scaffold, and the clathrin adaptors AP2 and clathrin assembly lymphoid myeloid leukemia protein (CALM), which couple the clathrin layer to the inner membrane. Lower levels of other clathrin adaptors and CME accessory proteins are also found in the central layer . Other p2 by AP2 aided by Fcho1/2 and Eps15/R that can be stabilized by clathrin recruitment, followed by expansion of a clathrin-coated structure through recruitment of more cargo-bound clathrin adaptors and clathrin, and finally coat and membrane deformation. Similar CCVs can also be formed from the periphery of large, flat pre-existing clathrin “plaques” in an analogous manner  A is esselic form , resultilic form . Thus, clic form .in vivo combined with biophysical studies from our laboratories suggested that phosphorylation was required for cargo capture via a phosphorylation-induced conformational change that facilitates cargo and membrane binding. However, this hypothesis is based largely on in vitro studies, and it conflicts with aspects of temporal ordering in CCV formation. For instance, phosphorylation of μ2T156 by a Numb-associated kinase (NAK) family member, either α-adaptin associated kinase (AAK1) or potentially BIKE (aka BMP2K) (AP2 is subject to multiple phosphorylation events . The besa BMP2K) , is thoua BMP2K) . Consequa BMP2K) .To unravel the functional significance of μ2 phosphorylation, we applied an array of complementary approaches: quantitative immunofluorescence was used to localize phosphorylated AP2 to CCPs; X-ray crystallography to determine a new open conformation adopted by phosphorylated AP2 (termed P-AP2 from now on); surface plasmon resonance (SPR) to measure the affinity of AP2 and P-AP2 to endocytic signals; biochemical and mass-spectrometry-based analyses to identify and characterize the phosphorylation-dependent binding of a new μ2 interaction partner, NECAP; and structural NMR analysis to characterize this interaction at the molecular level. Finally, highly specific NAK kinase inhibitors were used in combination with high temporal resolution, multi-color, live-cell imaging to show that AP2 phosphorylation is required for efficient NECAP recruitment to CCPs and the maturation of productive CCVs.2 binding site, showed markedly reduced levels of μ2 phosphorylation   is a recently evolved potent (low nM IC50) inhibitor of the μ2-phosphorylating NAK family members in human AAK1 and BIKE, which binds in their structurally similar ATP-binding pockets . We usedNAK−) . Use of  of T156 C and S1DNAK− cells  internalization: its rate decreased by approximately 30% in both inhibitor-treated cells and also in μ2K− cells B and S1DK− cells F, indicaUsing quantitative immunofluorescence microscopy, we first analyzed global distribution of total AP2 and P-AP2 in CCPs and calculated the proportion of CCPs that contain AP2 and P-AP2. We plotted these values as a function of the respective EGFP-CLCa intensities in the detections C. In conThe correlation analysis of paired fluorescence intensity values between P-AP2, AP2, and EGFP-CLCa reveals that the quantity of P-AP2 and EGFP-CLCa and the quantity of P-AP2 and total AP2 are positively correlated in CCPs. In comparison, the correlation between the quantity of AP2 and EGFP-CLCa is even higher D. These Previous studies used several different experimental systems to test the role of AP2 phosphorylation in cargo recruitment to CCPs and yielded conflicting results  to investigate which stage of CCP assembly fails in the absence of phosphorylation and compared CCP assembly rates in inhibitor-treated cells and control cells. Inhibition of μ2T156 phosphorylation resulted in a ∼10-fold increase in the density of diffraction-limited clathrin-coated structures that failed to pinch off during the 10-min video acquisition time (persistent structures) G and 1H.In sum, these data indicate that μ2T156 phosphorylation affects CCP maturation and the rate of CME but not likely due to alterations in the efficiency of cargo sequestration.2 analog IP6 by molecular replacement using the closed structure (PDB 2VGL) as a search model (Recombinant T156-phosphorylated AP2 core (Pcore) with >95% stoichiometrically phosphorylated T156 was produced as in ch model . The str2XA7) and subsequently Cμ2 from the same structure  is that Cμ2 is in a different orientation, rotated by ∼90° and translated by ∼9Å with respect to N-μ2 such that it packs against residues 300–400 of β2, burying a solvent accessible surface area of 640Å2 (The structure is clearly in a new active and open conformation that we term open+ A: the boof 640Å2 A–S3F. Thof 640Å2 . The losThe μ2 linker in the open form (2XA7) is mostly ordered with residues 142–157 adopting a helix. T156 is buried and so inaccessible to a kinase and hence should not be phosphorylatable. Further, a phosphate at this position would also likely cause steric clashes with β2 and Nμ2, which would disfavor an open conformation for Pcore and explain our inability to crystallize Pcore in the open conformation. In the new open+ form, there is no traceable density for the residues 134–158 including the phosphorylated threonine C despiteCrystals with similar morphology and space group dimension to those of open+ Pcore were also obtained of core, but none diffracted beyond an ∼6-Å resolution. Molecular replacement clearly showed that the core in this crystal form was in the open+ conformation : this wa2 binding  and 30- to 100-fold (slow) stronger than the fast and slow phases seen when cargo is not present. However, there is only a modest (∼3-fold) increases in YxxΦ-containing liposome-binding affinity on core phosphorylation, likely resulting from phosphorylation stabilizing the open+-cargo-binding competent core conformation. There are no obvious differences upon AP2 phosphorylation in binding the dileucine cargo. In support of this, using fluorescence anisotropy, we show that μ2T156 phosphorylation causes no obvious change in the affinity of AP2 for YxxΦ cargo peptides, when AP2 is stimulated by the addition of polyanionic heparin to mimic the presence of a PtdIns4,5P2-enriched membrane -tagged core and Pcore coupled to sepharose beads and mass spectrometry for protein identification. Any protein with binding to GST and/or core was rigorously filtered from the data. In the remaining list, NECAP1 and NECAP2 were identified as the two top hits that bound Pcore only . These d elegans , publishC. elegans NECAP orthologs. NECAPs are known to bind other endocytic proteins containing FxDxΦ-like motifs, such as amphiphysin or Bin1 and CALM with KDs in a range of hundreds of μM as determined in NMR titrations  C–S4E showork and ). Using work and E.D ∼55 μM GQIGWRR) and NECAP1 PHear domain demonstrated that the interaction possessed a KD ∼55 μM F and S4Epeptides F, contropeptides F, indicapeptides D. This dD interaction between the AP2 α-appendage and a WxxF motif at the C terminus of mammalian NECAP-1 has been characterized  or LP inhibitor (lower panel).LP inhibitor treatment reduced the number of CCPs that recruit NECAP1-mRuby2 to 31% of all CCPs and the overall levels of NECAP1-mRuby2 declined by ∼50% D–5F. TheIn conclusion, the quantity of NECAP1 in CCPs is positively correlated with the level of P-AP2. NECAPs are recruited in increasing amounts over time to the majority of CCPs throughout their life in a manner dependent on μ2T156 phosphorylation. Quantification of the immunofluorescence images E and S5Fde novo determining the structures of the PHear domain and a complex between the PHear domain and the μ2 linker peptide SQITSQV(pT)GQIGWRR using multidimensional, heteronuclear NMR (We probed the molecular details of the interaction by lear NMR . The NEClear NMR A (Ritterlear NMR . Comparilear NMR A–S6D. Thlear NMR B–6D and lear NMR E. The silear NMR  using HMlear NMR J. The re9-fold) in the pull-down with R102A and the double mutant R90A/R102A. We note that AP2 capture was affected significantly, not only by the R90A mutation but also by the R102A mutation. This may be due to AP2 being at the hub of a large interaction network involving proteins binding via FxDxF motifs at the R102 site: in line with this, the levels of AP2 detected on pull-down with the double R90A/R102A mutant, decreased to the limits of detection.Structure-based mutations, which did not affect the domain’s fold as determined by circular dichroism and physical properties (data not shown), confirmed the mode of binding: mutation of R90A, R113D, and S89D and a double mutation of A33S/L38S abolished binding of the phosphorylated μ2 peptide, while mutation of S88A and R32D reduced binding . In contNECAP1-mRuby2 WT or R90A were expressed at near endogenous levels in a background of siRNA-mediated depletion of endogenous NECAPs G and S6HTo quantitatively assess the changes in the AP2 interactome upon its phosphorylation, we used Stable Isotope Labeling with Amino acids in Cells (SILAC)-based proteomics combined with native co-immunoprecipitation of AP2. Since the NECAP PHear domain binds P-AP2 and can simultaneously bind endocytic accessory proteins via FXDXΦ motifs, this interaction survey should also identify binding partner(s) of NECAP. RPE cells were treated with LP inhibitor, while control cells were treated with just DMSO. NECAP1 and NECAP2, as expected but also the PX-BAR protein SNX9, underwent significant and reproducible depletion upon the inhibition of μ2T156 phosphorylation A. ITC meWe tested the hypothesis that SNX9 recruitment and activation in CCPs may be dependent on the presence of NECAPs and their association with P-AP2 using TIRFM B. SNX9 rCells were treated with control non-targeting siRNA (upper panel) or depleted simultaneously for NECAP1 and NECAP2 (lower panel).To confirm these data in other cell lines, we used NECAP1 1–133 as bait in “pull-down” experiments with cytosolic extracts of three non-neuronal cell lines . Identification of the bound proteins by mass spectrometry  revealedde novo by a pioneer complex of AP2 assisted by Fcho1/2 and Eps15/R .Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact Dr Z Kadlecova  were used as a parental cell line. It was obtained from ATCC and cultured in DMEM medium  supplemented with 10% FBS (Thermo Fisher Scientific). All cells were cultured at 37°C under 5% CO2. Cell lines were routinely tested for absence of mycoplasma contamination with MycoAlert Mycoplasma Detection Kit (Lonza). HepG2 and HeLa S3 cells used for preparation of cytosolic extracts were also obtained from the ATCC and were cultured under the same conditions.6β2t+μ2myc, pMWHis6β2t+μ2myc+AAK1(1-325), pMWαtGST+σ2, pMWαGST+σ2 were as described in  and (26-350) were made by cloning the appropriate length fragments of AAK1 into the NcoI and XhoI of pET28b.6-NECAP1 (1-275), pET28bHis6-NECAP1 (1-133) were both created by cloning appropriate length fragments of NECAP1 into pET28b using NdeI and NotI sites. pGEX4T3NECAP1(1-275), pGEX4T3NECAP1(1-133) and pGEX4T2NECAP133-275 were created by cloning appropriate fragments of NECAP1 into pGEX4T3 using EcoRI and NotI sites. Point mutant versions A33S, S88A, S89D, R90A, R90D, R95A, R102A, R113A NECAP1(1-133) were generated using standard pcr-based mutagenesis in pGEX4T3NECAP1(1-133).The following constructs were created for bacterial expression of various forms of NECAP1. pET28bHisTo make a construct for expressing GST alone, termed pETHis6-GST, GST was cloned using XbaI and EcoRI sites into pETM30.All constructs were verified by sequencing.Phosphorylated and non-phosphorylated AP2 heterotetramers were expressed and purified as previously described . Phospho6 tag and this protein was additionally purified using NiNTA agarose to remove non-full-length material.NECAP1 GST-fusion proteins were expressed in E. coli BL21DE3 pLysS at 22°C for 16 hours. Cells were lysed and insoluble material removed by centrifugation. Proteins were bound to GST sepharose in bufferA  and washed extensively with ∼100 volumes of bufferA and the proteins cleaved from GST by overnight incubation at 22°C with thrombin. Cleavage was halted by the addition of 0.1 mM AEBSF and the free proteins collected and concentrated to c. 20 mg/ml and further purified by S200 gel filtration in appropriate buffers. Full length NECAP1 was appended at its C-terminus with a His1 2 1 with one molecule in the asymmetric unit cell of dimensions 121Å, 121Å, 258Å; 90, 90, 120 and diffracted at best to 2.6 Å at the Diamond Light Source beam line IO4-1. The data were integrated with Mosflm and scaled and merged in Aimless. The structure was solved using Phaser with the closed core structure (PDB ID 2VGL) as a search model and refined using alternating cycles of Refmac and manual rebuilding in Coot to the final R and Rfree of 0.230 and 0.259 respectively , 200 mM NaCl, and 10 mM DTT. The crystals were mounted and cryo-protected in a solution containing: 25% glycerol, 20% PEG 1000; 100 mM Na+/K+ phosphate buffer (pH 7.2), 200 mM NaCl, and 10 mM DTT, prior to flash cooling by plunging them into liquid nitrogen. The crystals were of the space group P 3ectively . When ov1 21 2 with unit cell dimensions of 182 Å, 182 Å, 211 Å; 90, 90, 90.Crystals of AP2 Pcore and core in the open+ state were grown by hanging drop vapour diffusion from a solution containing 12 mg/ml AP2 core or Pcore, 1mg/ml TGN38 YxxΦ-based cargo peptide DYQRLN, and 5 mg/ml functional variant dileucine cargo RM(P)SEIKRLLSE from CD4 against a reservoir containing 7.5% PVA, 9% 1-propanol, 90 mM Hepes pH 7.4, and 100 mM guanidine hydrochloride over a period of two days. Drops were set up with a 2:1 protein/precipitant ratio. The crystals were cryo-protected with 8% PVA, 12% 1-propanol, 90 mM Hepes pH 7.4, 100 mM guanidine hydrochloride, 20% glycerol, 1 mg/mL TGN38 peptide, and 5 mg/mL CD4 peptide before being plunged into liquid nitrogen. Data were collected at the Diamond Light Source beam lines IO4 and IO4-1, integrated in Mosflm and were of the space group P42XA7) as the search model. In the end, an “optimal” data selection was chosen by comparing the data from the five crystals with the calculated intensities from the initial model, as a function of image number: data from two crystals and from the later parts of two others were rejected as having too much radiation damage, leaving data from three crystals. Using this data set, the best molecular replacement solution was found using the bowl  from the open form and the Cμ2 domain from the same structure as independent search models (as opposed to the bowls and Cμ2 structure of other AP2 complexes). This final solution still exhibited very weak electron density for the first 80 to 100 amino acids of the N-terminal part of the β2 trunk although fully present as shown by complete rebinding of an aliquot of the crystallised Pcore to NiNTA resin through the His6 tag on the very N-terminus of β2 (data not shown). Various approaches were employed in an effort to locate these missing residues but all failed. Extensive refinement was carried out on the structure using Refmac. The initial refinement was attempted using rigid bodies with a hope that it would reveal the density for the N-terminal part of the β2 solenoid. As it did not, the β2 solenoid up to the residue 87 was removed, as the electron density around this region remained very weak. Prosmart was used to generate restraints for low-resolution refinement. Finally rounds of TLS refinement  from the Pcore open+ form. The resulting solution showed clear electron density corresponding to the Cμ2 domain at the new position observed only in the open+ conformation. The validated full open+ core structure was then refined in Refmac to R=0.259 and Rfree=0.285. Crystallographic statistics of all three structures  are listed in All programs used in crystallographic structure determinations were launched from and are part of CCP4 suite .15NH4Cl (for single-labelled samples) or 13C6 glucose vitamins, trace elements, antibiotics, and 15NH4Cl (for double-labelled samples). Following purification by gel filtration, samples were exchanged into 75mM sodium acetate pH 6.5 using a PD10 column  in accordance with the manufacturers instructions for the ‘gravity’ method.Samples for NMR were expressed in M9 minimal media supplemented with natural isotopic abundance glucose, vitamins, trace elements, antibiotics, and 15N- or 15N,13C-labelled protein solutions in 70 mM 2H3 acetate buffer pH 7.0 in 95:5 H2O:2H2O or 99% 2H2O. In addition to the native NECAP residues 1-133, the sequence studied here carried a non-native N-terminal sequence GSPNS (here numbered -4-0). NMR samples of the complex comprised 0.3-0.4mM equimolar mixtures of 15N- or 15N,13C-labelled NECAP 1-133 and natural abundance μ2 149-163 in 70 mM 2H3 acetate buffer pH 7.0 in 95:5 H2O:2H2O or 99% 2H2O. NMR data were acquired using Bruker DRX600, AV-1 600, AV-3 800 spectrometers, each equipped with a cryogenically cooled triple resonance (1H/15N/13C) 5mm probe. Experiments were conducted at 25°C unless otherwise stated, and 1H chemical shifts were calibrated using sodium 3,3,3-trimethylsilylpropionate (TSP) as an external 1H reference; 15N and 13C chemical shifts were indirectly referenced to the 1H shifts using the ratio of gyromagnetic ratios. An essentially complete set of resonance assignments was made using the experiments described below.NMR samples of NECAP 1-133 comprised 0.3-0.6mM 2O samples of free protein , the following datasets were acquired: 2D datasets: [15N-1H] HSQC; [13C-1H] HSQC ; constant-time [13C-1H] HSQC . 3D datasets: CBCANH; CBCA(CO)NH; HNCA; HBHA(CO)NH; [1H-13C-1H] HCCH-COSY; [13C-13C-1H] HCCH-TOCSY; 15N NOESY-HSQC (tm = 150 ms); 13C NOESY-HSQC .For H2O samples of complex containing 1:1 15N,13C-labelled NECAP 1-133 and natural abundance μ2 149-163, the following datasets were acquired: 2D datasets: [15N-1H] HSQC; [13C-1H] HSQC ; [1H-1H] filtered NOESY (tm = 150ms) with filter elements set to reject protons coupled to 13C or 15N in F1 and to accept protons coupled to 13C or 15N in F2, so as to detect intermolecular cross-peaks; [1H-1H] filtered NOESY  and [1H-1H] filtered TOCSY (tm = 40 ms and 80 ms), all with filter elements set to reject protons coupled to 13C or 15N both in F1 and in F2, so as to detect intramolecular cross-peaks within μ2149-163. 3D datasets: HBHA(CO)NH; HNCACB; 15N NOESY-HSQC (tm = 150 ms); 13C NOESY-HSQC .For H2H2O samples of complex containing 1:1 15N,13C-labelled NECAP 1-133 and natural abundance μ2146-163, the following datasets were acquired: 2D datasets: [13C-1H] HSQC , constant-time [13C-1H] HSQC , [1H-1H] filtered NOESY (tm = 150ms) with filter elements set to reject protons coupled to 13C or 15N in F1 and to accept protons coupled to 13C or 15N in F2, so as to detect intermolecular cross-peaks. 3D datasets: [1H-13C-1H] HCCH-COSY; [13C-13C-1H] HCCH-TOCSY; 13C NOESY-HSQC ; 13C filtered NOESY-HSQC , with filter elements set to reject protons coupled to 13C or 15N in F1, so as to detect intermolecular cross-peaks.For 13C experiments, a period of 15N decoupling equal in length to the acquisition period was applied at the beginning of the interscan delay, and for 15N experiments an equivalent period of 13C decoupling was similarly applied.All of the NOESY datasets used for structure calculations (see below) were acquired using pulse sequences modified to ensure equal RF heating in each case, e.g. for 15N-1H] HSQC spectra at 25°C during addition of natural abundance μ2146-163 to 15N-labelled NECAP 1-133. Chemical shift perturbations were calculated according to Δδ = √((Δδ(1H))2 + (Δδ (15N) / 5.0)2).Backbone amide chemical shift perturbation analyses were performed by recording   as described in . Resulti2.The liposomes were synthesized by rehydration of a desired lipid film in 0.3 M sucrose followed by sedimentation, resuspension in 20 mM HEPES, pH 7.4, and extrusion through a 100 nm membrane. The “basic” liposomes contained 80% pig-brain-derived phosphatidylcholine and 20% phosphatidylethanolamine. Complex liposomes were prepared by substituting PC with 2% of a lipid-linked peptide and/or 4% of a phosphoinositide PIPAll SPR experiments were run on BIAcore T200 machine  with 10 mM Tris pH 8.7, 250 mM NaCl, 1 mM DTT used as a running buffer. Liposomes were deposited on a surface of an L1 biosensor to generate a “membrane mimic”. In order to do so, the system was equilibrated in the running buffer and the surface of the L1 sensor was primed with three 60 sec injections of 20 mM CHAPS. This was followed by sequential injections of the liposomes  aiming to reach the target of 7000 RU in each flow cell. Subsequently, the loosely bound liposomes were removed by two pulse injections of 50 mM sodium hydroxide for 5 s each. The entire procedure was run at 10 μl/min. The affinities of the AP2 cores and Pcores to the immobilised liposomes were then measured at a flow rate of 30 μl/min. The proteins were injected at concentrations ranging from 50 nM to 50 μM for 90 sec (association) followed by a buffer flow for 510 sec (dissociation). The liposome surfaces were then regenerated by a 10 s pulse injection of 50 mM NaOH. In all analyses, the background signal from the protein binding to the basic liposomes  measured at flow cell 1 was subtracted from the binding to the complex liposome species.The kinetic analysis of Pcore and core binding to the liposomes was then carried out. None of the systems studied gave single exponential kinetics. The association and dissociation phases were therefore routinely analysed using a double exponential function, which returned reaction amplitudes for the fast and slow components of each phase. Reaction amplitudes for the fast and slow components of the dissociation phases were generally more reproducible than those for the association phases. These data were therefore used to obtain apparent equilibrium dissociation constants associated with the fast and slow components by fitting the change in amplitude with protein concentration using a simple 1:1 binding model. The results of the analysis are shown in D of the reaction, its stoichiometry (n), and their corresponding SEM values  were titrated in with 20 injections of 2.43 μl each separated by 2.5 minutes. For ITC with WT PHear and Pcore, the proteins were gel filtered into 100 mM TRIS pH 8, 250 mM NaCl, 0.25 mM TCEP and experiments were carried out at 10°C with between 0.05 and 0.1 mM Pcore in the cell and with PHear domain between 2 and 3 mM being titrated in with 20 injections of 2.43 μl each separated by 2.5 minutes. A relevant syringe-solution-into-buffer blank was subtracted from all data and for constructs, which displayed measurable binding, a minimum of three independent runs that showed clear saturation of binding were used to calculate the mean Klues see . Analysi°C with a Clariostar plate reader (BMG Labtech) using a 482/530 nm fluorescence polarisation module. Where used, polymeric heparin  was added to ∼500 μM concentration, and NECAP PHear domain was added to 200 μM. Mean fluorescence anisotropy values (3 or more measurements) were plotted against protein concentration and the curves fitted to a single-site binding model:A peptide encoding the TGN38 YxxΦ motif (sequence ASDYQRL) and modified at its N-terminus with fluorescein (Sigma-Genosys) was used in all binding experiments at a concentration of 50 nM. Various quantities of recombinant AP2 core or isolated C-μ2 were premixed with the fluorescent peptide and incubated for 10 minutes to allow the mixture to come to equilibrium. Protein / peptide mixtures were prepared in 10 mM Tris pH 8.7, 250 mM NaCl (for AP2 core / phosphorylated AP2 core) or 20 mM HEPES pH 7.4, 500 mM NaCl (for C-μ2). Fluorescence anisotropy measurements were carried out at 25f + (Fb - Ff).[L]/(Kd + [L])where F is the measured anisotropy in a mixture of free (f) and bound (b) fluorescent peptide with anisotropy of Ff and Fb respectively, [L] is the pseudo-first order concentration of ‘‘ligand’’  and Kd is the equilibrium dissociation constant. Curves were fitted with ProFit software using the Levenberg-Marquardt algorithm. F = FIn addition to C-μ2, and P-AP2 core pre-incubated with heparin or with a mixture of heparin and NECAP PHear domain, binding titrations were performed with AP2 core in the absence of heparin, resulting in only a slow linear increase in anisotropy as previously reported , indicatInteraction partners of various recombinant fusion proteins were identified after incubation brain or tissue-culture cell lysates. For brain lysates, a single frozen mouse brain was thawed in 1 ml of buffer-T , cut to small pieces and slowly homogenized with a glass homogenizer. The final brain lysate was collected as the supernatant of three consecutive centrifugations . Lysates of tissue-cultured cells were prepared from 15 confluent dishes (15cm ø). Cells were washed with PBS, scraped off the dishes and collected by centrifugation for 5min/500xg. The derived pellets were resuspended in 5x Vol buffer-T, incubated for 20min at 4°C and the final lysates were gained as the supernatants of two consecutive centrifugations .The following proteins were used as baits: GST (negative control), GST-Core, GST-Pcore, GST-NECAP1 PHear (residues 1-133) and mutants of thereof . Equal amounts (∼ 10 μg) of GSH-bead immobilized bait proteins were incubated with 1-3 mg of tissue or cell lysates in a total volume of 1 ml buffer-T and incubated 1h/4°C. Afterwards, beads with bound proteins were collected by centrifugation for 5min / 500xg. Lysates were removed and beads were processed for mass spectrometry-based identification of bound proteins after washing 2x with buffer-T and 2x buffer without detergent.To test binding of full-length NECAP1 and NECAP1 PHear (1-133) to core and Pcore, they were expressed as N-terminally GST-tagged fusion proteins and immobilized to GSH-beads. An equivalent of 6μg was incubated with 20μg of core or Pcore for 15min at RT in buffer T. Subsequently, the beads were collected by centrifugation and washed 1x with buffer T and 1x with buffer T without detergent. Bead-bound proteins were resolved by SDS-PAGE, followed by western blotting and detection of the AP2 μ-subunit.Binding of full-length NECAP1 and NECAP1 PHear (1-133) to various AP2 species was also tested in buffer B  following the same protocol but with proteins detected with Coomassie stain. In such experiments NECAP1 constructs were used as baits and AP2 core, AP2 Pcore, AP2 core or Pcore in presence of 0.5 mM or 5 mM CSFFEDNFPE peptide , AP2 α-FLcore, and AP2 α-FLPcore were preys.(122-171) and GST-μ2(149-163), NECAP were expressed and isolated as N-terminally 6His-tagged fusion proteins and immobilized to NiNTA beads. Details of incubation and washing were as above and binding was detected after SDS-PAGE and Coomassie staining.To assay the binding to phospho- / non-phospho versions of GST-μ2Beads from pulldown experiments with bound proteins were resuspended in 25μl 50 mM Tris pH 7.5, 2M Urea, 1 mM DTT, 150 ng Trypsin and incubate for 30 min at RT. Then, 50 μl of 50 mM Tris pH 7.5, 2M Urea, 50 mM Chloracetamid were added followed by centrifugation (3 min / 500xg). This supernatant and a second supernatant derived from an additional washing with Chloracetamid containing buffer were combined and incubated overnight at RT. Digestion was then terminated by addition of 1μl TFA. The derived tryptic peptides were loaded onto activated stage tips (Sigma) and washed with 0.1% TFA and 80% acetonitrile, 0.1% TFA.For LC/MS analysis, samples were analyzed on a Q-Exactive Plus mass spectrometer (Thermo Scientific) coupled to an EASY nLC 1000 UPLC (Thermo Scientific). Peptides were loaded with 0.1% formic acid in water (solvent A) onto a self-packed Poroshell column  and separated at a flow rate of 250 nL/min using the following gradient: 3 - 5% solvent B (0.1% formic acid in 80 % acetonitrile) within 1 min, 5-30% solvent B within 40 min, 30-50% solvent B within 8 min, followed by washing with 95 % solvent B for 10 min. The mass spectrometer was operated in data-dependent acquisition mode. The MS1 survey scan was acquired from 300-1750 m/z at a resolution of 70.000. The top 10 most abundant peptides were isolated within a 1.8 Th window and subjected to higher energy collisional dissociation fragmentation at a normalized collision energy of 27%. The automatic gain control target was set to 5e5 charges, allowing a maximum injection time of 110 ms. Product ions were detected in the Orbitrap at a resolution of 35.000. Precursors were dynamically excluded for 20 s.All mass spectrometric raw data were processed with Maxquant (version 1.5.3.8) using default parameters. Briefly, MS2 spectra were searched against a FASTA database constructed from reviewed and unreviewed human sequences extracted from Uniprot (downloaded at: Sept 2018). A list of common contaminants was included in the searches. False discovery rates on protein and PSM level were estimated by the target-decoy approach to 1% (Protein FDR) and 1% (PSM FDR) respectively. The minimal peptide length was set to 7 amino acids and carbamidomethylation at cysteine residues was considered as a fixed modification. Oxidation (M) and Acetyl (Protein N-term) were included as variable modifications. The match-between runs option was enabled.After mass spectrometry-based identification of proteins captured in a pulldown experiment, we first eliminated any hit (identified by the presence of one or more peptides) that was captured by GST, which served as a negative bait control. In a second step we deleted any low abundance protein (identified by the presence of two or less peptides) from the list, as we wished to focus only on the most prominent binding partners. For the identification of proteins binding specifically to Pcore, we eliminated any protein hit (more than a single peptide present) that bound to GST and/or Core. The remaining lists of proteins were ranked from the top downwards based on the iBAQ values of any hit. Results are summarised in NAK− and μ2T156D sequences were generated using standard site-directed mutagenesis in the μ2 cDNA inserted in retroviral bicistronic IRES-BFP vector PMIB6 previously described , N-terminally fused with mRuby2-Flag-GGGS and flanked by Not1 and Sal1 were custom synthesized by IDT. Standard restriction cloning techniques were used for insertion into retroviral vector PMIB6.The cDNA of SNX9, with a sequence corresponding to NM_016224.4, was a gift of Folma Buss, CIMR. The SNX9 construct was made using Gibson Assembly Master Mix  to introduce mRuby2-Flag-GGGS at the N-terminus together with NotI and SalI restriction sites. The cDNA was inserted into PMIB6 using standard restriction cloning techniques.Retrovirus particles were generated by co-transfecting HEK 293T cells in the presence of polybrene (Merck) with 3 plasmids coding for Gag-Pol, VSV-G and the retroviral expression constructs in PMIB6. FACS was used to sort population of cells expressing fluorescent marker as described below.NAK- cell line was tested with three different anti-μ2pT156 antibodies: rabbit monoclonal ab109397 (Abcam), rabbit monoclonal D4F3 , rabbit polyclonal #3843 .After retrovirus infection, cohorts of cells expressing various levels of mutant μ2 subunits were sorted by FACS based on BFP intensity. The levels of expression of μ2 subunit within each stable cohort was validated by western blotting using the anti- μ2-antibody . The cell population with the expression levels closest to the endogenous μ2-adaptin was selected for further experiments. The absence of phosphorylation in μ2Cohorts of RPE cells expressing several different levels of NECAP1, NECAP2 or SNX9 were sorted by FACS based on mRuby2 fluorescent intensity. Anti-NECAP antibody  or anti-SNX9 antibody  was used to detect expression levels closest to the endogenous protein by western blotting.siRNA mediated depletion of endogeneous μ2, NECAP1 or NECAP2 was carried out with 3 rounds of siRNA transfection at 12, 36, and 60h after plating. The experiments were performed on the fifth day. The target sequences for siRNAs were located at 3’UTR of the genes. The sequences were as following: NECAP1: GCTTAAAAGGCCAGCGTCT, NECAP2: GTAAATTGGCACCGTGTCA. Separate set of control experiments were carried out with NECAP siRNA targeting ORF for NECAP1: CGACCGAGTTGGAGTACGA and NECAP2: GTGGAGAGTGTGACGGATT. μ2 siRNA sequence was CCAAAGGCCAGUAAUGGAU. Targeting siRNA together with non- targeting control sequence was acquired from Dharmacon ON-TARGETplus Human siRNA series.5 cells. Cells were fixed with methanol at -20°C for 45s and incubated with 2% BSA for 30 min at 4°C. Cells were either incubated for 3h at 10μM of LP or in vehicle control (0.1% (v/v) DMSO) prior to the fixation. Cells were incubated with a rabbit monoclonal anti- μ2pT156 antibody  or anti-AP2α antibody AP6 to stain for total AP2 at 4°C overnight. Cells were washed three times with PBS and further incubated with appropriate AlexaFluor labeled secondary antibodies (Life Technologies) for 1h. After three washes with PBS the cover slips were mounted in stainless-steel chamber and imaged in PBS on Zeiss Elyra microscope in TIRF mode at room temperature.Immunofluorescence of μ2pT156-AP2-CLCegfp, μ2pT156-CLCegfp-NECAP and AP2-CLCegfp-NECAP were prepared to decipher the quantity and distribution relationship between the stated components. Parental cell lines expressing CLCegfp alone or in combination with NECAP1-mRuby2 were grown overnight in 6-well plates on a glass cover slips at a quantity of 1x10To assess the dependency of cargo colocalization to CCPs on μ2T156 phosphorylation, cells were pretreated with 10μM of LP or in vehicle control (0.1% (v/v) DMSO) for 3h. Cells were chilled at 4°C and pre-chilled solution of mouse monoclonal anti-Tfr antibody OKT9 (Bio-X-cell) containing also LP was added to the wells at a final concentration 4μg/mL. Cells were incubated at 4°C for 30min, fixed and permeabilized using PFA and 0.1% saponin for 20 min and processed for TIRFM as described above.NAK− cells or cells exposed to NAK inhibitors were imaged under identical conditions, and post-acquisition image processing and analysis was also identical for all conditions to allow for direct comparison. The spatial relationship and the degree of colocalization between signals in dual-colour or triple-colour immunofluorescence images were analyzed using Matlab . The aim of the analysis was to characterize the recruitment of the CCP components  in relation to AP2 phosphorylation. To that end we compared the results of the analysis for immunofluorescence images acquired with control cells and cells treated with NAK inhibitors or μ2NAK− cells. We adapted CMEanalysis software   for the PCCs capture only linear relations of the paired intensity values . To haveDetections in master channel were divided into bins based on their fluorescence intensity. Non-overlapping intensity intervals of bins were estimated in such a way that the bins contain approximately the same number of detected spots. The proportion of spots in each bin that were above the given significance level in the slave channel was plotted as a function of the bin intensity. This function thus depicts the impact of the appearance of the signal in slave channel on the amount of fluorescence intensity in the master channel.NAK− cells, or cells exposed to NAK inhibitors were acquired under identical illumination and acquisition conditions. Quantitative analysis to distinguish bona fide CCPs and to measure CCP initiation rates, lifetime distributions, and density of static structures was performed using CMEAnalysis software  and mounted on inverted microscope equipped with the Definite Focus System. Time-lapse image sequences from different cells were acquired at a frame rate of 0.5 frame/s using a sCMOS camera with 6.45 × 6.45 μm2 pixels (Photometrics). Similarly, near simultaneous 2-channel (488nm/561nm TIRF) videos were acquired at 0.5 frame/s. Cells in control experiments, μ2software . On minisoftware  that is NECAP-mRuby2 averaged intensity values for individual cohorts  were smoothed by Gaussian kernel (standard deviation 1.2) and a maximum was found with subsample precision using cubic interpolation. SNX9-mRuby2 maximum recruitment rate was computed in the same way, as a maximum of intensity differences . Averaged intensity values were calculated from videos acquired with minimum 10 cells in 2 separate experiments using SNX9-mRuby2 cells or NECAP1-mRuby2 RPE cells.4 cells/well. Plates were incubated with 4 μg/ml of OKT-9 at 37°C for the indicated time points. Cells were then immediately cooled down (4°C) to arrest internalization. Following a washing step to remove unbound ligand , the remaining surface-bound ligand was removed from the cells by an acid wash step . Cells were washed with PBS and then fixed in 4% PFA in PBS for 20 min and further permeabilized with 0.1% Triton X-100/PBS for 10 min. Internalized OKT-9 was assessed using a goat anti-mouse HRP-conjugated antibody (Life Technologies), further developed with OPD , and the reaction was stopped by using 2M H2SO4. The absorbance was read at 490 nm with BMG CLARIOstar Microplate Reader.Transferrin receptor (Tfn) internalization at 37°C experiments were performed using anti-TfnR mAb . Cell lines were grown overnight in 96-well plates at a density of 2.0 × 10NAK- cells, NECAP1-WT or NECAP1-R90A were carried out after siRNA mediated knockdown of endogenous protein (see above). Results of individual independent experiments (n=3) were pooled, averaged and presented as mean±SD, n=3. The statistical significance was analyzed by two-tailed, unpaired t-test.Internalization efficiency measured upon acute treatment with LP inhibitor was carried out in the presence of 10μM LP with cells pre-incubated with LP for 3h. Internalization experiments measured in μ2g for 10 min. An equal amount of each protein lysate was incubated with anti-AP2α antibody AP6 conjugated to protein G-Sepharose beads overnight at 4°C. The immune complexes were analyzed by Western blot with rabbit anti-AP2- μ2 ), anti-AP2α , Anti AP2-μ2 pT156 D4F3 , SNX9 , anti-NECAP 1/2 .For immunoprecipitation experiments, cells were grown to 80% confluency in 15cm dishes. NAK kinase inhibitor 1, LP-935509 and NAK kinase inhibitor 2 (Cas No1093222-27-5) were reconstituted in DMSO as a stock solution at 10mM and used at 1/1000. Cells were incubated in fresh medium containing 10uM solution of either inhibitor or 0.1 v/v% DMSO for 3h. Media was removed from the dishes and cells were washed three times with ice cold PBS. Cells were scraped and then solubilized in 0.1% NP40 lysis buffer using 10x passage through a 25G needle, insoluble debris was removed by centrifugation of the lysates at 10,000 × RPE cells were grown in custom-made DMEM  supplemented with 10% dialyzed fetal calf serum (Invitrogen). l-arginine  and l-lysine  were added to the “light” media, while and l-arginine 13C/15N and l-lysine 13C/15N (Cambridge Isotope Laboratory) to the “heavy” media at the same concentrations. The amino acid concentrations are based on the formula for normal DMEM (Invitrogen). Once prepared, the SILAC media was mixed and filtered through a 0.22-μm filter (Millipore) and stored at 4°C. RPE cells were passaged in SILAC media for at least 2 weeks (or 5 cells doublings) before harvesting to ensure complete incorporation of isotopic amino acids. Cells were harvested for heavy amino acid incorporation check and immunoprecipitation. Immunoprecipitation was carried out as described above using RPE cell lysate prepared from three 15cm dishes for each condition. 100uL of G-Sepharose beads conjugated to 500 ug of anti-AP2α antibody AP6 were used for each immunoprecipitation. After incubation at 4°C O/N beads were rapidly washed with lysis buffer at 4°C three times. We followed manufacturer's instructions for Trypsin-LysC mix digestion (Promega). Beads from both conditions were combined and incubated with aqueous solution of dithiothreitol at a final concentration of 5 mM at room temperature for 1h. Alkylation was carried out with with freshly thawed aqueous solution of iodoacetamide at a final concentration of 5 mM. The immunoprecipitated proteins were digested in 200 μL of buffer containing 4M urea, 50 mM Tris-HCl pH 7.5 and 5 μg/mL Trypsin and LysC mix at 37°C overnight. After the initial digestion, the samples were centrifuged for 5 min at 14 000 rpm and the supernatant was collected in a clean tube separate from the beads. Beads were washed twice in 100 μL of urea buffer and the supernatants were pooled. Digestion with fresh enzyme mix continued at 37°C for additional 12h at 2M urea buffer. In the next step samples were treated with trifluoroacetic acid (TFA) to stop digestion. We followed previously published protocol  to desalPeptides were eluted from SDB-RPS tips with 30 μl of 5 % (w/v) ammonium hydroxide in 80% acetonitrile (ACN) and dried a speed vac.All samples were analyzed on a Q-Exactive Plus (Thermo Scientific) mass spectrometer that was coupled to an EASY nLC 1000 UPLC (Thermo Scientific). After resuspension in 10 μl 5% formic acid, 2% ACN the peptides were loaded with 8 μl solvent A (0.1% formic acid in water) onto an in-house packed analytical column . Peptides were separated at a constant flow rate of 250 nL/min using the following gradient: 3 - 5% solvent B (0.1% formic acid in 80 % ACN) within 1 min, 5-30% solvent B within 40 min, 30-50% solvent B within 8 min, followed by washing with 95 % solvent B for 10 min. The mass spectrometer was operated in data-dependent acquisition mode.The MS1 survey scan was acquired from 300-1750 m/z at a resolution of 70.000. The top 10 most abundant peptides were isolated within a 1.8 Th window and subjected to HCD fragmentation at normalized collision energy of 27%. The AGC target was set to 5e5 charges, allowing a maximum injection time of 110 ms. Product ions were detected in the Orbitrap at a resolution of 35.000. Precursors were dynamically excluded for 20 s.All mass spectrometric raw data were processed with Maxquant (version 1.5.3.8) using default parameters. Briefly, MS2 spectra were searched against a fasta database constructed from reviewed and unreviewed human sequences extracted from Uniprot (downloaded at: 16.6.2017). The Maxquant default list of common contaminants was included in the searches. False discovery rates on protein and PSM level were estimated by the target-decoy approach to 1% (Protein FDR) and 1% (PSM FDR) respectively. Trypsin was chosen as the digestion enzyme with a maximum of 2 missed cleavages. The minimal peptide length was set to 7 amino acids and carbamidomethylation at cysteine residues was considered as a fixed modification. Oxidation (M) and Acetyl (Protein N-term) were included as variable modifications. The match-between runs option was enabled. The LFQ algorithm implemented in Maxquant was used for label free quantification. For SILAC quantification (Arg10 and Lys8) multiplicity was set to 2 and the re-quantify option was enabled. Statistical analyses, data transformation, and filtering were performed in Perseus 1.6.1.1 and results were visualized using the same software. Identifications were filtered by removing reverse hits, matches based on modified peptides only, and common contaminants (‘standard filtering’).Quantification analysis for the all the data presented in this manuscript have already been detailed in the The data sets generated during this study are available as follows:6QH5 closed Pcore, 6QH6 open+ core, 6QH7 open+ Pcore and NECAP PHear+μ2phosphopeptide 6RH6 and PHear 6RH5.Structure coordinates have been deposited on the PDB with the accession codes Mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD013468.Live cell imaging datasets and immunofluorescence data sets are available from corresponding author ZK on request.


Hand, foot and mouth disease (HFMD) is endemic among population of young children in Thailand. The disease is mostly caused by enterovirus 71 (EV71) and coxsackievirus A16 (CA16).This study conducted serosurveillance for neutralizing (NT) antibodies to EV71 subgenotypes B5 and C4a, and to CA16 subgenotypes B1a and B1b, in 579 subjects of various ages using a microneutralization assay in human rhabdomyosarcoma (RD) cells. These test viruses were the major circulating subgenotypes associated with HFMD in Thailand during the study period.We found that the levels of seropositivity against all 4 study viruses were lowest in the age group of 6–11 months, i.e., 5.5% had antibody to both EV71 subgenotypes, while 14.5% and 16.4% had antibody to CA16 subgenotypes B1a and B1b, respectively. The percentages of subjects with antibodies to these 4 viruses gradually increased with age, but were still less than 50% in children younger than 3 years. These laboratory data were consistent with the epidemiological data collected by the Ministry of Public Health which showed repeatedly that the highest number of HFMD cases was in children aged 1 year. Analyses of amino acid sequences of the test viruses showed 97% identity between the two subgenotypes of EV71, and 99% between the two subgenotypes of CA16. Nevertheless, the levels of seropositivity and antibody titer against the two subgenotypes of EV71 and of CA16 were not significantly different.This study clearly demonstrated NT antibody activity across EV71-B5 and EV71-C4a subgenotypes, and also across CA16-B1a and CA16-B1b subgenotypes. Moreover, there were no significant differences by gender in the seropositive rates and antibody levels to any of the 4 virus subgenotypes.The online version of this article (10.1186/s12985-018-1074-8) contains supplementary material, which is available to authorized users. Hand, foot and mouth disease (HFMD) is common in children below the age of 5. The disease is characterized by acute fever with vesicular rashes or blisters on the palms, soles, and oral mucosa. HFMD can be caused by any of several serotypes of human enteroviruses, most commonly enterovirus 71 (EV71) and coxsackievirus A16 (CA16). The co-circulation of EV71 and CA16 in the same season has been observed in many countries . CA16-asHFMD was recognized as an emerging disease after an EV71-associated outbreak in Malaysia in 1997 which involved approximately 2628 cases with 29 deaths . In 1998Picornaviridae, Genus Enterovirus, species Enterovirus A. An average genome of picornavirus is about 7500 nucleotides long and encodes for a polyprotein which is cleaved into 4 functional structural proteins: VP1, VP2, VP3 and VP4 of the viral capsid. VP1 is immunodominant and functions as the principal neutralizing (NT) domain. VP2 and VP3 also induce NT antibodies, but VP4 does not [EV71 and CA16 serotypes belong to Family does not . As NT adoes not , 27. Gendoes not , 27. Simdoes not . These dSeveral epidemiological studies demonstrated NT antibody to only one subgenotype of EV71 and/or CA16 , 29–32, This study was approved by two Ethical Committees: the Siriraj Institutional Review Board, Faculty of Medicine Siriraj Hospital, Mahidol University and the Ministry of Public Health Review Board, Nakhon Ratchasima Province. Adult subjects or parents of the child subjects signed the consent form for participation in the study.Nakhon Ratchasima Province is 259 km from Bangkok. It is the major gateway to the northeast of Thailand. In 2013, this province had a population of 2.6 million, with the second highest population next to Bangkok. Based on data from the Bureau of Epidemiology, MOPH, Thailand, this province had the highest incidence of HFMD cases among the northeast provinces, and ranked third in incidence for all of Thailand. .A total of 579 serum samples were collected from subjects who lived in Nakhon Ratchasima Province in 2013. These subjects ranged in age from < 1 to 60 years old. Samples from children under 5 were the leftover sera from non-HFMD cases after routine investigations for disease diagnosis/treatment in hospitals located in various districts of the province. Sera from those of age older than 5 years were collected from healthy subjects in several communities. The test sera were aliquoted and stored at − 20 °C until used.The test viruses used in MN assays for NT antibodies were: two strains of EV71, SiICRC10/TH/2011 subgenotype B5 (EV71-B5/2011) and SiICRC01/TH/2014 subgenotype C4a (EV71-C4a/2014); two strains of CA16, SiICRC06/TH/2011 subgenotype B1a (CA16-B1a/2011) and SiICRC01/TH/2012 subgenotype B1b (CA16-B1b/2012). These viruses were isolated from clinical specimens from HFMD patients in Vero cells  and propagated in RD cells (derived from human rhabdomyosarcoma). Both Vero and RD cell lines were grown in Eagle’s minimum essential medium (EMEM)  supplemented with 10% fetal bovine serum (FBS) (Gibco); infected RD cell monolayers were maintained in EMEM supplemented with 2% FBS. The complete VP1 regions of these virus isolates were sequenced and phylogenetically analyzed for genotypic and subgenotypic identification. The virus titers were determined in RD cells and expressed as 50% tissue culture infectious dose (TCID50) according to the Reed-Muench method . The tesThe CPE-based MN assay was conducted in 96-well micro-culture plates. Each test serum was heat inactivated at 56 °C for 30 min and serially two-fold diluted with maintenance medium starting from a dilution of 1:10 to 1:1280. The assay was performed by mixing 60 μl of the diluted serum with 60 μl of the test virus suspension at a concentration of 200 TCID50 and incubated at 37 °C for 2 h. Then, a volume of 100 μl of the virus-serum mixture was transferred into a well of RD cell monolayer and further incubated at 37 °C for 5–6 days. In order to verify the amount of virus inoculum at the concentration of 100 TCID50, the virus back-titration at doses of 0.1, 1, 10, and 100 TCID50 were included in every assay plate, together with positive control with known antibody titer and cell culture controls. Test reactions were run in duplicate. The inoculated cell monolayers were observed for appearance of CPE indicating viral infection. The antibody titer was defined as the reciprocal of the highest serum dilution that protected 50% of the inoculated cell monolayers from the virus infection, i.e., the last dilution of NT antibody-positive serum which exhibited ≤2+ degrees of CPE. Test sera with NT antibody titers of ≥10 were considered seropositive. To calculate the geometric mean titer (GMT), sera with NT antibody titers of < 10 were assigned a value of 5 and sera with NT antibody titers of ≥1280 were assigned a value of 1280.Total RNAs were extracted from the virus suspensions using the QIAamp Viral RNA . The extracted RNAs were used as targets for amplification of VP1 to VP4 regions by reverse transcription-polymerase chain reaction (RT-PCR) using the One Step RT-PCR kit (QIAGEN). Briefly, a total 50 μl volume for RT-PCR consisted of 10 μl of RNA template in master mix containing 1X RT-PCR buffer, 2 μl of enzyme mix, 2 μl of dNTP mix, 5 units of RNase inhibitor , and 400 nM of primers. The reaction was carried out in a thermal cycler . The PCR cycling program consisted of one cycle at 50 °C for 30 min and one cycle at 95 °C for 15 min, followed by 35 cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 60 s and a final extension step cycle at 72 °C for 10 min. The amplified product was electrophoresed in 1.5% agarose gel in TAE buffer and purified using a QIAEX II gel extraction kit (QIAGEN). The primer sets, shown in an Additional file According to a report from the Bureau of Epidemiology (BOE), Department of Disease Control, MOPH, from 2007 to 2017, HFMD occurred commonly in children less than 5 years of age  nor antibody levels  between the B5 and C4a subgenotypes. The seropositive rates of NT antibody to CA16 were slightly higher than those to EV71. Percentages of subjects with NT antibody to CA16-B1a or CA16-B1b were greater than 90% in the age-groups of 7–11 years and older  and antibody levels  between the B1a and B1b subgenotypes. Our study clearly demonstrated the presence of NT antibody activity to the EV71-B5 and EV71-C4a subgenotypes, and also to the CA16-B1a and CA16-B1b subgenotypes. After merging the data sets, the seroprevalence of EV71 and CA16 NT antibodies in various age-groups, irrespective of subgenotypes, is shown in Fig. The EV71 seropositive rates rose to peaks is the age-group 12–17 years (90.2% for antibody to EV71-B5 and 85.2% to EV71-C4a), and then declined Fig.  and b. Tder Fig.  and b. TP > 0.05) nor their GMTs  to any of the 4 test viruses . There were no statistically significant gender-based differences in the number of seropositive subjects Additional file 2:Figure S1. Age-related HFMD cases in Thailand during 2007–2017. A summary graph of the data reported by the Bureau of Epidemiology, MOPH, Thailand. (PDF 242 kb)Additional file 3:Figure S2. Seropositivity of NT antibodies against EV71 (A) and CA16 (B) by gender. (PDF 393 kb)Additional file 4:Figure S3. VP1-VP4 amino acid alignment of EV71. (PDF 183 kb)Additional file 5:Figure S4. VP1-VP4 amino acid alignment of CA16. (PDF 250 kb)


Curcuma longa essential oil (CLLEO) extracted from leaves, and its major constituent α-phellandrene have proven to be a novel biopesticide candidate against third instar larvae (L3) of Cochliomyia macellaria. In this way, groups of 20 L3 were placed on filter paper impregnated with different concentrations of CLLEO, from 0.31 to 2.86 μL/cm2 and α-phellandrene, from 0.29 to 1.47 μL/cm2. The extracts were solubilized in ethanol. Data shown in this article is related to the research article “Can an overlooked by-product from turmeric industry be effective for myiasis control?” Chaaban et al., 2019. Data on L3 toxicity was observed after 6 and 24h of contact with both extracts, as well as a marked reduction of L3 movement, color changes in the cuticle and progressive darkening in their body. Major cuticle damage and L3 mortality were reported.Morphological biomarkers can be used to establish a diagnosis of fly larvae structural damage and toxicity to target cells by biopesticide candidates. Insecticide activity of natural compounds such as  Moreover, progressive darkening in L3 body, marked reduction of larval movement, color changes in L3 cuticle and death were observed after treatment .The data of this paper involves the experimental analysis regarding the cuticular damage of the natural compounds Cochliomyia macellaria. a) Control group (ethanol) 6 h after exposure. b) Control group (ethanol) 24 h after exposure. Observe the normal size and movement and regular cuticle color. c) L3 treated with 1.59 μL/cm2 of Curcuma longa leaves essential oil (CLLEO) 6 h after exposure. Observe the change in the color of L3 body, decrease motility and some dead larvae. d) L3 treated with 1.59 μL/cm2 of CLLEO 24 h after exposure. Note the reduction of movements and progressive larval darkening.1Video 1Third instar larvae (L3) of Cochliomyia macellaria. a) Control group (ethanol) 6 h after exposure. b) Control group (ethanol) 24 h after exposure. Observe the normal size and movement and regular cuticle color. c) L3 treated with 1.47 μL/cm2 of α-phellandrene 6 h after exposure. Observe the change in the color of L3 body, decrease motility and some dead larvae. d) L3 treated with 1.47 μL/cm2 of α-phellandrene 24 h after exposure. Note the progressive darkening of the body of L3 and a marked reduction of movements.2Video 2Third instar larvae (L3) of The following is/are the supplementary data related to this article:22.1Curcuma longa (leaves) used in this work were cultivated at the Medicinal Plants Unit of the Catarinense Federal Institute (IFC), located at 26° 23′ 33.6691″ S and 48° 44′ 18.3336″ W. The location is at 10.6 m above the sea level in the city of Araquari, Santa Catarina, South of Brazil. The plant cultivation, essential oil extraction and chemical characterization were carried as described in the companion paper α-Phellandrene (CAS: 99-83-2) was acquired commercially  and certified as having ≥99% purity.2.2. The toxicity of CLLEO and α-phellandrene over L3 of C. macellaria was performed using groups of 20 L3, which were placed on filter paper that were impregnated with a range of concentrations of CLLEO (0.31–2.86 μL/cm2) and α-phellandrene (0.29–1.47 μL/cm2). L3 were put into glass vials containing filter papers (12.56 cm2) impregnated with 0.2 mL of EO solution, that were solubilized in ethanol using the protocol described by Chaaban et al. (2017) Data of the establishment of stock colonies, insects’ identification, mass reproduction, and the protocol for the biological tests were performed as described in the companion paper [1]2 of CLLEO and 1.47 μL/cm2 of α-phellandrene, both solubilized in ethanol. The data on these alterations can be observed in the companion paper Damages were measured by macroscopic biomarker changes and microscopic lesions using histological sections and scanning electron microscopy in L3 treated with 1.59 μL/cm


Objectives: The aim of this study was to investigate the proliferative and protective effects of striatisporolide A (SA) obtained from the rhizomes of Athyrium multidentatum (Doell.) Ching on human umbilical vein endothelial cells (HUVECs). Methods: Cell viability was measured by the MTT method. Cell apoptosis was determined by flow cytometry. Intracellular ROS was measured by the 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe. Results: The viability rate in cells treated with 100 µM SA alone was increased to 128.72% ± 0.19% and showed a significant difference compared with the control group (p < 0.05). Meanwhile, SA augmented the cell viabilities in H2O2-treated HUVECs, and the cell viability was enhanced to 56.94% ± 0.13% (p < 0.01) when pre-incubated with 50 µM SA. The cell apoptosis rates were reduced to 2.17% ± 0.20% (p < 0.05) and 3.1% ± 0.34% (p < 0.01), respectively, after treatment with SA alone or SA/H2O2. SA inhibited the overproduction of reactive oxygen species (ROS) in HUVECs induced by H2O2 and the fluorescent intensity was abated to 9.47 ± 0.61 after pre-incubated with 100 μM SA. Conclusions: The biological activities of SA were explored for the first time. Our results stated that SA exhibited significant cytoproliferative and minor cytoprotective effects on HUVECs. We presume that the mechanisms of the proliferation and protection actions of SA involve interference with the generation of ROS and the cell apoptosis. These findings provide a new perspective on the biological potential of butenolides. Caesalpinia bonduc, could inhibit MCF-7 breast cancer cell line cells with IC50 values of 12.8 and 6.1 μM [50 value of less than 30 μM [C. islandica (Iceland moss) had substantial antitrypanosomal activity against T. b. brucei [.Natural products have been attracting continuous attention due to their unique structures and activities. Butenolides, a class of unsaturated lactones with a four-carbon 25H)/23H) furanone heterocyclic ring skeleton, are usually found in fungi, bacteria and gorgonians. Their saturated analogues serve as signaling molecules in bacteria to amplify spore formation in streptomycetes or induce metabolite production . Some buH furanonan 30 μM . The γ-aH/23H) fuPenicillium striatisporum and Penicillium janthinellum [4-dihydroxylation and dehydrative cyclization pathway [50 value of 36.5 μM [Striatisporolide A (SA)  is a simhinellum ,8. Inspi pathway . A recenAthyrium multidentatum (Doell.) Ching is a terrestrial fern in the family Athyriaceae, used as an largely unexplored folk medicine that has been used for thousands of years for conditions such as high blood pressure, anxiety and arthritis [Athyrium multidentatum (Doell.) Ching. In order to explore the antiaging potential of SA, the in vitro cell viability, cell apoptosis and ROS levels were investigated employing HUVECs as a model system. rthritis . In our p < 0.05) at the concentration of 100 µM, which was much stronger than resveratrol. However, a higher dose of SA (150 µM) led to decreased cell viability (99.76% ± 0.2%). This meant that HUVECs displayed the best viability when exposed to 100 µM SA. In addition, SA showed protective effects on HUVECs against H2O2-induced oxidative injury . After pre-incubation with 50 µM SA, the cell viability was enhanced to 56.94% ± 0.13% and showed a significant difference compared with the control group (p < 0.01). However, there was no dose-effect relationship between SA and cell viabilities in H2O2-treated cells. These results indicated that SA was more powerful at boosting cell proliferation than protecting the cells against H2O2-induced oxidative damage. Chemical molecules with proliferative and protective properties are beneficial for repairing and regenerating damaged tissues. Natural products with proliferation activity are very popular for their low toxicity and minor side effects. Heteropolysaccharide from the fruiting bodies of Lactarius deliciosus Gray could significantly promoted B cell and RAW264.7 cell proliferation at 5 μg/mL with proliferation rates of 124.73% and 138.01%, respectively [Terminalia bellirica extract with gallic acid as a major component was found to possess cytoprotective activity and could significantly boost human mesenchyamal stromal cell (hMSC) proliferation and cell attachment [The proliferative and protective effects of SA on HUVECs are displayed in e injury B. In comectively . A Termitachment . Resverase of SA 0 µM led 2O2, which was much higher than that in the control group (8.57% ± 0.56%). As expected, SA greatly inhibited the apoptosis in both H2O2-induced and non-induced cells (2O2. Differences were significant (p < 0.05) compared with the control group and to the H2O2 group (p < 0.01). SA showed stronger anti-apoptosis effects than resveratrol in the experiments. Apoptosis is a genetically controlled and evolutionarily conserved form of cell death that contributes to the normal embryonic development and the maintenance of tissue homeostasis in the adult organism [2O2. Apoptosis is desired in cancer treatment. However, excess apoptosis can lead to neurodegenerative diseases, hematologic diseases and tissue damage, which can be found in myocardial ischemia/ischemia reperfusion injury and HIV infection. Anti-apoptotic drugs, such as nerve growth factor and antioxidant, have been proved to be effective for the prevention and amelioration of these pathological conditions. Protocatechuic acid has been reported to be effective in preventing neurodegenerative diseases such as Parkinson’s disease in PC12 cell model, by virtue of its prominent antioxidative, anti-cytotoxic and anti-apoptotic activities [Cells were treated with 100 µM SA in the apoptosis assays for its marked proliferation promoting effect on HUVECs at this concentration. As presented in ed cells H,G. The organism . It occuorganism . SA redutivities ,19. Taki2O2. 2O2 groups. However, there was no distinct dose-response relationship between SA and ROS levels at concentrations from 50 to 150 μM. After pre-incubated with 100 μM of SA, the fluorescent intensity in the cells was abated to 9.47 ± 0.61, which was much less than the control group (22.85 ± 0.43). SA remarkably crippled the ROS levels in HUVECs. Oxidative stress has been linked with the pathogenesis of several degenerative diseases including aging, atherosclerosis, cardiovascular diseases, neurodegenerative diseases and cancer [2−•), hydroxyl radical (•OH), hydrogen peroxide (H2O2), singlet oxygen (1O2), and nitric oxide (NO•) are highly reactive species and their uncontrolled generation induces oxidative modification of cellular macromolecules, which can ultimately lead to apoptotic or necrotic cell death [2O2-induced oxidative stress via suppression of the accumulation of intracellular ROS, which involved the activation of the Nrf2/HO-1 pathway [C. elegans from oxidative damage potentially by modulating genes involved in the insulin/insulin-like growth factor (IGF) signaling (IIS) pathway and the oxidoreductase system [The overproduction of the intracellular ROS in HUVECs was induced by Hd cancer . ROS in ll death ,21. In te system . In viewAthyrium multidentatum (Doll.) Ching rhizome, which was collected in Changbai Mountain area of China in 2012 and identified by Professor Chongmei Xu . Fetal bovine serum (FBS) was purchased from Sijiqing Biological Engineering Materials Co., Ltd . Hyclone RPMI 1640 medium, phosphate buffered saline (PBS), penicillin-streptomycin, MTT cell proliferation and cytotoxicity assay kit, Annexin V-FITC/PI apoptosis detection kit and dimethyl sulphoxide (DMSO) were from Solarbio® Science & Technology . Reactive oxygen species assay kit was purchased from Beyotime® Biotechnology . Resveratrol was from Sinopharm Chemical Reagent Co., Ltd .SA was obtained from Athyrium multidentatum (Doll.) Ching was finely chipped and soaked in methanol at room temperature for 20 days. The resultant extract was suspended in distilled water and partitioned successively with petroleum ether and ethyl acetate. The ethyl acetate extract was further eluted and purified by silica gel column chromatography (100–200 mesh) eluting with petroleum ether and ethyl acetate. SA was obtained from the fraction that eluted with 4:1 (v:v) petroleum ether and ethyl acetate. SA redissolved in DMSO was normalized to a final concentration of 0.2% DMSO in culture medium when applied to the cells.SA was prepared as described in our previous study . In brie2 at 37 °C. The culture medium was changed every other day and all the operations were processed under sterile conditions. Cells of passage 3–5 were used in all experiments. The HUVECs were obtained from Institute of Plastic Surgery, Weifang Medical University . Cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated FBS and 1% antibiotics in a water-saturated atmosphere of 5% CO2O2-induced oxidative damage [3 cells per well in a 96 well plate. Following stabilisation for 12 h, the cells were treated with 100 µL different concentrations of SA (50–300 µM) or resveratrol (20 µM). Meanwhile, the control group (CG) was added 100 µL of 1640 growth medium instead of medicine. The group without cells was designed as vacuity control group. After 48 h of incubation, cells were washed two times with PBS, then 90 µL fresh 1640 growth medium and 10 µL MTT reagent (5 mg/mL) were added sequentially for the formation of formazan. Four hours later, 110 µL of DMSO was added and gently shaken for 10 min to dissolve the formazan crystals. Finally, the absorbance was read at 570 nm using a microplate reader . The best non-toxic sample concentration (over 100% cell viability) in the cell proliferation was selected for cell apoptosis evaluation assay. The MTT assay was conducted to evaluate the cell proliferation and protection effects of SA against He damage . In the 2O2-induced oxdative injury was performed as a modified method of Guan et al. [4 cells/mL and inoculated into a 96-well cell culture plate (100 μL per well), then cultured at 37 °C for 12 h. Afterwards, cells were treated with SA (50–300 µM) or resveratrol (20 µM) for 48 h before co-incubated with 300 µM H2O2 solution for 40 min. At the end of treatment, cells were washed once with PBS and the cell viability was measured as described previously. Cell proliferation and protective effects by the samples were expressed as the percentage of cell viability, and calculated according to the following formula:The cytoprotective effect of SA against Hn et al. . Briefly6 cells (per well) were harvested by centrifugation at 900 rpm for 3 min, successively washed once with cold PBS and binding buffer, then resuspended with 500 µL binding buffer. 5 µL Annexin V-FITC was added into the cells (100 µL) and reacted in the dark for 10 min. Afterwards, 5 µL PI was added and incubated for another 5 min. Finally, 300 µL PBS was added and the cell apoptosis was analyzed on a flow cytometer . Apoptosis in HUVECs was analyzed by flow cytometry with the Annexin V-FITC/PI apoptosis kit according to the manufacturer's protocols. Cells were exposed to SA (100 µM) or resveratrol (10 µM) for 48 h, then washed twice with PBS. About 1 × 102O2-treated HUVECs was determined by the same method. Briefly, after treatment with SA (100 µM) or resveratrol (10 µM) for 48 h, cells were added 500 µL H2O2  solution and endured for 40 min, then washed twice with PBS. After stained with Annexin V-FITC and PI, the cells were applied to the analysis of apoptosis. Apoptosis in H5 cells (per well) were plated into a 6-well plate and allowed to stabilize for 12 h, then treated with SA (50–150 µM) or resveratrol (10 µM) for 48 h. At the end of treatment, 0.5 mL H2O2 solution  was added to induce the overproduction of ROS. 40 min later, the cells were washed with PBS and collected by centrifugation at 900 rpm for 3 min, then incubated in the dark with 10 µM DCFH-DA prepared in serum-free media (1:1000). After incubation for 20 min at 37 °C, the cells were washed three times with FBS-free 1640 medium and transferred to a 96-well black plate. Cellular fluorescence was measured using a Fluoroskan Ascent fluorometer (Thermo Scientific). Reading was taken at excitation and emission wavelengths of 485 and 538 nm, respectively. Results were expressed directly as fluorescent intensity.The intracellular ROS levels were measured according to a modified method of Yang et al. . About 7p < 0.05.Data were presented as means ± SD from three independent experiments and evaluated using the statistical software of Microsoft Excel 2003 coupled with a two-tailed paired t-test. Statistical significance was considered significant if The results clearly demonstrated that SA exhibited excellent proliferative and slight protective effects on HUVECs through a combinination of anti-apoptosis and ROS inhibition actions. The current findings give support for the potential application of SA in the prevention of degenerative diseases and the development of protective ingredients against oxidative damage.


Although supramalleolar osteotomy is the main joint-preserving method for the treatment of varus ankle osteoarthritis, it tends to be ineffective when ankle osteoarthritis presents in combination with an excessive talar tilt angle. The purpose of this study was to present a new surgical technique, supramalleolar osteotomy combined with lateral ligament reconstruction and talofibular immobilization, for the treatment of varus ankle osteoarthritis with an excessive talus tilt angle and to evaluate the clinical and radiological results.From January 2013 to October 2016, a total of 17 patients with 17 cases of varus ankle arthritis with excessive talar tilt angles (larger than 7.3°) underwent surgical treatment using our new technique. The American Orthopaedic Foot and Ankle Society (AOFAS) clinical ankle-hindfoot scale and a visual analogue scale (VAS) were used to evaluate ankle function and pain before surgery and at the last follow-up. The medial distal tibial angle (MDTA), anterior distal tibial angle (ADTA), talar tilt angle (TTA), and hindfoot moment arm values (HMAVs) were evaluated on weight-bearing radiographs acquired preoperatively and at the last follow-up.p < 0.001), and the VAS score decreased from 4.9 ± 0.4 to 1.1 ± 0.2 (p < 0.001). The MDTA, TTA, and HMAV changed from 80.9° ± 0.4° to 90.1° ± 0.4°, 11.7° ± 0.6° to 1.4° ± 0.3°, and 12.6 mm ± 0.8 mm to 4.2 mm ± 0.6 mm, respectively (each p < 0.001). The ADTA showed no obvious change (p = 0.370). The staging of 11 cases (65%) improved. Intramuscular vein thrombosis of the lower limbs occurred in 1 patient 1 week after surgery, and superficial infection occurred in 1 patient.The AOFAS score improved significantly from 45.8 ± 2.1 before surgery to 84.8 ± 1.8 after surgery (Supramalleolar osteotomy combined with lateral ligament reconstruction and talofibular immobilization can correct the load of the weight-bearing ankle and effectively improve the ankle function. As the talar tilt angle can be significantly improved after surgery, this technique can be used for the treatment of varus ankle osteoarthritis with an excessive TTA. Ankle osteoarthritis (OA) can be manifested as varus, valgus, or neutral alignment, in which varus accounts for approximately 52% of the cases , 2. AbnoA deformation apex of varus ankle arthritis  may peak at the distal tibia, tibia-talus joint, subtalar joint, or at the calcaneus. As the main joint-preserving surgery, supramalleolar osteotomy can adjust the alignment of the tibia and talus and normalize the stress distribution on the ankle joint surface, thus preventing degeneration of the joint; this technique is increasingly widely used –8. AlthoChronic lateral instability of the ankle (CLIA) can cause varus tilt of the talus and is considered to be the most common cause of varus ankle OA , 5. In tAfter Institutional Review Board approval (No. 2018CTEC-SL-008), we retrospectively reviewed our data from January 2013 to October 2016. The inclusion criteria were as follows: (1) stage 2 or stage 3 adult ankle OA according to Tanaka ankle arthritis staging , with clThere were a total of 17 patients, including 13 men and 4 women with a mean age of 56.5 years (50 to 65). Fifteen patients had a history of severe ankle sprains, 5 had a history of diabetes, and 5 had a history of smoking. There were 2 patients in stage 2, 11 patients in stage 3a, and 4 patients in stage 3b.Clinical evaluations were conducted using the American Orthopaedic Foot and Ankle Society (AOFAS) clinical ankle-hindfoot scale  and a viPreoperatively and at the last follow-up, weight-bearing radiographs in the anteroposterior view, lateral view, and hindfoot alignment view were applied for the following measurements Fig. : 1) MDT MDT1];  and his assistants. Under epidural or general anesthesia, the patient was placed in a supine position with a tourniquet placed on the proximal end of the leg. An anteromedial and an anterolateral incision were made to expose the ankle joint.The osteophytes were debrided, and the anterior talofibular ligament (ATFL) and calcaneofibular ligament (CFL) were explored. Bone marrow stimulation was performed by cartilage drilling, if necessary . The oblAfter fixation of the distal tibia, the talus was everted to observe the medial space of the ankle, and the deltoid ligament was released if the medial gutter was narrow. Once the varus and supination deformities of the talus could be corrected passively, and the heel alignment returned to neutral, the modified Brostrom procedure  was perfThe wound drainage tube was removed approximately 48 h after surgery. The ankle was kept in a neutral position with a below-knee plaster cast for 6 weeks. Follow-up was conducted at 6 weeks, 3 months, 6 months, 1 year, and 2 years after surgery. The Kirschner wires for fixation of the lateral malleolus and talus were removed under local anesthesia 6 weeks after surgery, and partial weight-bearing movements were permitted, as tolerated. Full weight-bearing was allowed when complete bone healing was confirmed by radiography approximately 3 to 6 months after surgery.The AOFAS ankle-hindfoot scale was employed to assess the functional results, and a VAS score was employed to assess pain. Radiological evaluations included measurements of the MDTA, TTA, ADTA, and HMAVs. The matching of the tibia talus and the stage of the ankle joint were observed.t tests. Differences were considered statistically significant at p ≤ 0.05.All data were analyzed using SPSS version 21.0 . Continuous variables including the AOFAS score, VAS score, MDTA, ADTA, TTA, and HMAV were presented as the mean ± standard deviation and were analyzed using paired p < 0.001), and the VAS score decreased from 4.9 ± 0.4  to 1.1 ± 0.2  (p < 0.001).For the group as a whole, the mean follow-up time was 32.2 months . At the last follow-up, the AOFAS score improved significantly from 45.8 ± 2.1  preoperatively to 84.8 ± 1.8  (p < 0.001). The ADTA changed from 82.1° ± 0.3°  to 82.5° ± 0.3° , and there was no statistical significance (p = 0.370)  to 90.1° ± 0.4° , 11.7° ± 0.6°  to 1.4° ± 0.3° , and 12.6 mm ± 0.8 mm  to 4.2 mm ± 0.6 mm , respectively . The arthritis staging improved in 11 cases (65%); 2 cases improved from stage 2 to stage 1, 7 cases from stage 3a to stage 2, and 2 cases from stage 3b to stage 3a. No change was observed in 6 cases, and there were no aggravated cases  to narrow the mortise and reduce the TTA . HoweverThe need for reconstruction of the lateral malleolar ligaments after distal tibial osteotomy is controversial. Lee et al.  and MannSome limitations of the present study must be acknowledged. A small number of cases were included in our study, and there was no control group to confirm the positive effect of the 6-week postoperative tibia-talus fixation by Kirschner wires. In addition, the mean follow-up time was 32.2 months, but the range was large (24–40 months), which may have an impact on the efficacy results. Moreover, this study has not successfully analyzed the individual factors  that might be perhaps predictive of success or failure as a result of the sample size. Large-scale and long-term follow-up studies are needed to provide more convincing clinical data for the application of this technique.Supramalleolar osteotomy combined with lateral ligament reconstruction and talofibular immobilization can effectively improve the load of the ankle in the treatment of varus ankle arthritis. As the talar tilt angle can be significantly improved and effectively maintained, this technique could be an alternative method for the treatment of varus ankle arthritis with an excessive TTA.


N-butylpyridinium hexafluorophosphate, [BPy]PF6), as solid solvent and phenanthroline (PT) as an extractant was developed to determine micro levels of Fe2+ in tea by PT spectrophotometry. TC-SLE was carried out in two continuous steps: Fe2+ can be completely extracted by PT-[BPy]PF6 or back-extracted at 80 °C and the two phases were separated automatically by cooling to room temperature. Fe2+, after back-extraction, needs 2 mol/L HNO3 as stripping agent and the whole process was determined by PT spectrophotometry at room temperature. The extracted species was neutral Fe(PT)mCl2 (m = 1) according to slope analysis in the Fe2+-[BPy]PF6-PT TC-SLE system. The calibration curve was Y = 0.20856X − 0.000775 (correlation coefficient = 0.99991). The linear calibration range was 0.10–4.50 μg/mL and the limit of detection for Fe2+ is 7.0 × 10−2 μg/mL. In this method, the contents of Fe2+ in Tieguanyin tea were determined with RSDs (n = 5) 3.05% and recoveries in range of 90.6%–108.6%.At the present paper, an analytical method based on temperature controlled solid-liquid extraction (TC-SLE) utilizing a synthesized ionic liquid, ( In the human body, iron is a constituent of hemoglobin, myoglobin, the cytochrome system and a variety of enzymes in the blood. Absence of iron could lead to hypoferric anemia and hyperlipemia PF6) that are solids at room temperature are employed as extraction solvents when the temperature is higher than their melting points. Water insoluble complexes are readily extracted into the molten organic phases and distribution equilibrium is rapidly achieved at the higher temperature. Phase separation can be easily obtained by cooling the extraction system to room temperature. Some metals generate metal chelates with organic extractants at elevated temperature  as an extractant and [BPy]PF6 as a “green” solvent.Temperature controlled solid-liquid extraction (TC-SLE) is considered a branch of extraction chemistry ,20. In t chelate ,22), so 6, the extraction efficiency is not influenced by the temperature. TC-SLE can be achieved within 10 min. As a result, in the experiment a 10 min extraction is performed at 80 °C. The highest extraction efficiency was obtained with 0.5 g~3.0 g of [BPy]PF6-PT when the other conditions were kept unchanged. Thus 1 g of [BPy]PF6-PT was used in the experiment.Different factors affect the TC-SLE process. It is very important to optimize them to obtain the best recovery. When the extraction temperature is higher than the melting point of [BPy]PF2+ extraction into [BPy]PF6 with or without PT as a function of the pH value of the aqueous phase is plotted in The percentage of Fe2+ extraction efficiency is less than 50% at pH 1.50~9.50 when there is no PT in [BPy]PF6, and extraction efficiency of Fe2+ is higher than 99.6% at pH 5.00~7.00 when there is 8.1 × 10−3 mol·L−1 PT in [BPy]PF6. These results indicate that the optimal pH value was 5.50 for Fe2+ extraction at this TC-SLE system. The Fe2+ extracted into the solid [BPy]PF6-PT phase can be quantitatively back-extracted into 2 mol·L−1 HNO3 solution at 80 °C. Extraction of Na+ or K+ into [BPy]PF6-PT was negligible.It shows that the Fe−4~1.08 × 10−2 mol·L−1 on Fe2+ TC-SLE behavior. When the PT concentration was 8.04 × 10−3 mol·L−1, the extraction efficiency of Fe2+ reached 100% and remained constant at pH = 5.50. Consequently 8.10 × 10−3 mol·L−1 of PT in [BPy]PF6 was selected for determination of Fe2+ in tea.2+  and [Cl−] are the equilibrium concentration of Fe2+ and Cl− in the aqueous phase and [Cl−] is regarded as constant as a result of the large amount of Cl− in the TC-SLE system. [PT]IL and [Fe(PT)mCl2]IL are the equilibrium concentrations of PT and Fe(PT)mCl2 in the IL phase at 80 °C, and m, the composition ratio of PT to Fe2+ in the extractive compound.Corresponding When Equation (3) is expressed in logarithmic format, it can be rewritten as: D is linear with a slope of m along with concentration of PT in IL phase when the temperature and pH are fixed and the concentration of Cl− is kept constant in the TC-SLE system. As shown in ca. 1. The result indicates that Fe2+ is extracted into the ionic liquid phase in neutral Fe(PT)mCl2 (m = 1). Thus the extraction mechanism can be expressed by Equation (5): Obviously, log 2+ using HNO3 as stripping agent in the TC-SLE system is better. The results are shown in 2+ stripping percentage was obtained using 2 mol·L−1 HNO3 as stripping agent. Therefore 2 mol·L−1 of HNO3 was selected as stripping agent in the TC-SLE system for the determination of Fe2+ in tea.The experiments show that the effect of back-extraction for Fe2+, the [BPy]PF6-PT was washed three times with distilled and deionized water and could be used again in this TC-SLE system. The results indicate that only 8% of [BPy]PF6-PT was washed away after triplicate extraction of Fe2+ because of the solubility of PT PF6 at 80 °C. Stock solution of 1.0 × 10−2 mol·L−1 Fe2+ was prepared using GR NH4Fe(SO4)2·12H2O by a usual method. Other chemicals used were of analytical or guaranteed reagent-grade and used without further purification. Distilled water was used throughout unless otherwise specified. An Avatar 360 FT-IR  was used to record the IR spectrum. A V-1800 spectrophotometer  was used for the determination of Fe2+. A pHS-3C meter  was employed to monitor the pH values at room temperature. A WRS-1B digital melting point apparatus  was used to determine the melting point of [BPy]PF6. TC-SLE was carried out using the device described in our previous report [PT (0.015%) was freshly prepared prior to use. [BPy]PFs report . In the 6 are shown in The two steps of the synthesis and the structure of [BPy]PF6  is the bending asymmetric vibration peak of -CH3; 1173.46 cm−1 is the in-plane bending vibration peak of the ring C-H bonds; 782.57 cm−1 is the C=C bending vibration peak of the pyridine ring; 685.18 cm−1 is the bending (deformation) vibration peak of C-H on the ring and 833.86 cm−1 and 556.80 cm−1 are characteristic absorption peaks of PF6−.The infrared spectrum (IR) of [BPy]PF6  showed t6 was determined to be 1.2501 g·mL−1 with specific gravity bottle method [6 is 73.8 °C~75.5 °C.The density of [BPy]PFe method . The mel6 with PT (1 g) and a solution containing the desired amount of Fe2+ and buffer (10 mL) were placed in the glass extraction vessel described in PF6-PT phase was determined after back-extraction at 80 °C for 10 min in 10 mL of 2 mol·L−1 HNO3. The extraction percentage (%E) of Fe2+ was calculated by the ratio of the extracted amount of Fe2+ to the initial amount of Fe2+ in the aqueous phase. The distribution ratio (D) was estimated by the ratio of total concentration of Fe2+ in the solid phase to that in the aqueous phase. In the experiments, the ionic strength was maintained at 0.1 with sodium chloride.[BPy]PFribed in . The mixotometry . The con−1 HCl. After that the wetted tea powders were burned in a muffle furnace at 500 °C for 4 h then the wet tea powders were carbonized in an electric cooker at low temperature, and then the porcelain crucible was taken out and cooled to room temperature. 1:1 HCl was added into the porcelain crucible to dissolve the residue, the solution was filtered and the pH was adjusted to 7 or so and then transferred to a 100 mL volumetric flask to stand after adjusting to a constant volume.Commercial Tieguanyin tea  was accurately weighed, then powdered after drying at 75–80 °C. The tea powder were put in a porcelain crucible and soaked into 0.2 mol·L2+ is performed. Furthermore the determination of Fe2+ at tea can be achieved based on a fixed pH at room temperature. The unique properties of the IL allow a highly efficient and selective separation and determination of iron. Solid ILs can be recycled. Small amounts of water contained in [BPy]PF6 as determined by IR, may have contributed to the wide range of recoveries observed (90.6%–108.6%). The proposed TC-SLE with IL provides an alternative route for the separation and determination of iron in different matices.A novel TC-SLE system with an eco-friendly IL as a solid extraction solvent has been developed for the separation and determination of iron. Its extraction behavior is different from that of a liquid-liquid system . The IL


Even cycled at a current density of 5 A·g−1 for 650 cycles, the Cel-CS electrode maintained a specific capacity of 938 mA·h·g−1, which illustrates the potential application prospects of the Cel-CS electrode in microelectronic devices and systems.A novel integrated electrode structure was designed and synthesized by direct electrodepositing of Cu–Sn alloy anode materials on the Celgard 2400 separator (Cel-CS electrode). The integrated structure of the Cel-CS electrode not only greatly simplifies the battery fabrication process and increases the energy density of the whole electrode, but also buffers the mechanical stress caused by volume expansion of Cu–Sn alloy active material; thus, effectively preventing active material falling off from the substrate and improving the cycle stability of the electrode. The Cel-CS electrode exhibits excellent cycle performance and superior rate performance. A capacity of 728 mA·h·g With the development of the electronics industry and micro-machining technology, microelectronic devices have been well-researched with applications in many areas, such as pacemakers, miniature transmitters, sensors, hearing aids, defibrillators, etc. The trends for these devices are towards miniaturization and integration on a millimeter scale ,2. Clear−1 (Li22Sn5), low platform potential (0.3–0.6 V) and low cost, Sn-based anode electrodes have been researched widely as one of the most promising anode electrodes for microscale lithium ion batteries (LIBs) [−1 after 600 cycles at 500 mA·g−1. Zhang et al. [6Sn5/Cu composite by one-step dealloying of Al10Cu3Sn, which showed a capacity of 326 mA·h·g−1 after cycling at 100 mA·g−1 for 50 cycles. A Sn–Fe@C network synthesized by Shi et al. [−1 after 100 cycles at 100 mA·g−1. Though all these methods improve the cycle performance of Sn-based anodes to an extent, integration compatibility is still the main issue to consider.Owing to the high theoretical capacity of 992 mA·h·gs (LIBs) ,13,14,15s (LIBs) ,17,18,19s (LIBs)  improvedg et al.  prepared−1 LiSn5, low xMn3−xO4 array micro-/nanostructures grown on stainless steel by a solvothermal route and a subsequent annealing treatment as promising integrated binder-free electrodes for MLIBs. Guo et al. [2ZnSnS4 thin film with radio frequency magnetron sputtering and applied it in microscale thin-film LIBs. In our previous work [At present, the integrated electrode is the main method used to improve integration compatibility, which saves time and cost in the mixing and coating production process. Lou et al.  preparedo et al.  fabricatous work , multi-s−1 can be achieved after 250 cycles at the current density of 100 mA·g−1. Even cycled at the current density of 5 A·g−1 for 650 cycles, the Cel-CS electrode could still show a specific capacity of 938 mA·h·g−1, which illustrated the potential application prospects of Cel-CS electrode in microelectronic devices and systems.In this work, a novel integrated structure electrode was designed and synthesized by directly electrodepositing Cu–Sn alloy anode materials on the Celgard 2400 separator (Cel-CS electrode). Firstly, directly electrodepositing Cu–Sn alloy anode materials on the separator can greatly simplify the fabrication process and improve the integration compatibility of MLIBs. Secondly, by abandoning the traditional Cu current collector, the Cel-CS electrode not only increases the energy density of the whole electrode, but also buffers the mechanical stress caused by the volume expansion of Cu–Sn alloy active material, thus effectively preventing active material from falling off from the substrate and improving the cycle stability. The Cel-CS electrode exhibits excellent cycle performance and superior rate performance. A capacity of 728 mA·h·g4P2O7·3H2O, 6.2 g SnCl2·2H2O, 4.7 g CuCl2·2H2O and 5 mL electroplating refiner  in deionized water. The integrated electrode was obtained by keeping the voltage constant at 7 V for 300 s. The novel integrated electrode which composited anode material with the Celgard 2400 separator (Cel-CS electrode) was obtained. As a contract, the Cu–Sn alloy layer composite with the Cu collector (Cu-CS electrode) was also prepared in the same electroplating conditions. The loading of the Cu–Sn alloy layer was approximately 1.7 mg·cm−2.The separator used in this work is commercial Celgard 2400 . A conductive nano-carbon layer (about 5 nm) needs to be sprayed on the separator in advance by magnetron sputtering. The Cu–Sn alloy layer growth on the pretreated separator under appropriate electro-deposition conditions. The composite electrodes were prepared with DC current deposition by constant voltage electro-deposition. The electro-deposition process and parameters have been discussed in our previous work . ElectroThe phase structure of the Sn–Cu alloy anode was determined by X-ray diffraction (XRD) on a D8 Advance diffractometer . The morphologies and microstructures of the Sn–Cu alloy anode before and after cycling were characterized by using a field emission scanning electron microscope  equipped with an energy dispersive spectros-copy (EDS). The XPS was performed by a Perkin-Elmer PHI 5000C ESCA system  with Al Kα radiation operated at 250 W.6 in ethylene carbonate/dimethyl carbonate (EC/DMC = 1:1 by volume) as the electrolyte. Galvanostatic charge/discharge profiles and rate capability between 0.01 and 3.0 V (vs. Li+/Li) at various current densities were measured on a LAND Cell test system . Cyclic voltammograms (CV) and electrochemical impedance spectroscopy (EIS) were carried out on the CHI760E . CV tests were performed between 0.01 and 2.5 V (vs. Li+/Li) at a scan rate of 0.2 mV·s−1. The EIS data were tested between frequencies of 0.01 Hz and 100 kHz at a signal amplitude of 5 mV.Electrochemical performances of the Cel-CS electrode were measured using CR2032 coin-type half cells. The prepared Sn–Cu alloy electrode was cut into discs (12 mm in diameter) as work electrodes. Li foil was used as the counter and reference electrode, and 1.0 M LiPF6.5Sn5 , JCPDS card NO. 47-1575) phase, which was the main component of the electroplating Sn–Cu alloy.6.5Sn5. The surface element state of the Cel-CS electrode was measured by XPS. The high-resolution Sn 3d profiles  at 0.2 mV·s−1 are illustrated in 6Sn5 into Li2CuSn-type phase, and the alloying process involved the lithiation of Sn to finally yield Li4.4Sn from Li2CuSn, respectively [4.4Sn to Li2CuSn and Cu6Sn5. In the subsequent cycles, the two cathodic peaks change to board slope at 0.5 and 0.1 V, which can be attributed to the structure transformation and recombination [−1 between 0.01 V and 3.0 V (vs. Li+/Li). Consistent with the CV plots, during the first discharge step, a small voltage plateau at about 1.4 V was indicative of the formation of SEI film. The two obvious slope platforms located at 0.6 V and 0.1 V are associated with the alloying process of Cu6Sn5 to Li2CuSn and Li2CuSn to Cu6Sn5, respectively [Cyclic voltammograms (CVs) of the Cel-CS electrodes between 0.01 and 2.5 V . The initial discharge and charge capacities of Cel-CS electrode are 942 and 803 mA·h·g−1 with a coulombic efficiency of 85%. According to reports [2O during the first discharge process. The capacity of the Cel-CS electrode appeared stable at about 728 mA·h·g−1 at 250 cycles, which is close to the performance of Cu-CS electrode with the Cu current collector. Due to the Cu–Sn alloy layer of the Cel-CS electrode being directly electroplated on the separator without a current collector, the energy density of the Cel-CS electrode was much higher than that of the Cu-CS electrode. −1, the Cel-CS electrodes exhibited capacities of 582 mA·h·g−1; even cycled at a current density of 20 A·g−1, a capacity of 264.7 mA·h·g−1 and a capacity retention of 45.5% could still be achieved, while for the Cu-CS electrode only 201.2 mA·h·g−1 (30.1%) was kept. reports ,34,35,36−1 [+ diffusion, respectively. Before cycling, profiting by the smaller particle size and tighter structure, the Cel-CS electrode demonstrates the similar semicircle diameter and larger slope, implying a closed charge transfer resistances and faster Li+ mobility during the cycling process. After 250 cycles, the Rct and W of the Cel-CS showed an increasing trend to a smaller extent, while that of Cu-CS showed an obvious increase, implying better structure stability of the Cel-CS electrode. To illustrate the superior high rate performance of the Cel-CS electrode, the cycle curves at 5 A·g−1 were performed in −1, which is much higher than previously reported [To further analyze the dynamic behavior of the Cel-CS and Cu-CS electrodes, electrochemical impedance spectroscopy (EIS) measurement c was per−1 . All thereported ,22,32,38reported ,39,40,41+ diffusion path, providing faster kinetics and resulting in better rate performance.−1 can be achieved after 250 cycles at the current density of 200 mA·g−1. Even cycled at a current density of 6 A·g−1 for 650 cycles, the Cel-CS electrode still showed a specific capacity of 938 mA·h·g−1, which illustrated the potential application prospects of the Cel-CS electrode, with higher energy density and power density, on MLIBs.In summary, a novel integrated structure electrode (Cel-CS electrode) was designed and synthesized by electrodepositing alloy anode materials on the separator. The integrated structure of the Cel-CS electrode not only greatly simplifies the battery fabrication process and increases the energy density of the whole electrode, but also buffers the volume expansion and enhances the cycle stability. The Cel-CS electrode exhibits excellent cycle performance and superior rate performance. A capacity of 728 mA·h·g