Sequencing of mutant HE genes revealed 25 amino acid substitutions at 16 positions in three antigenic sites: A-1, A-2, and A-3, and a newly designated Y-1 site. In the 3D structure, the A-1 site was widely located around the receptor-binding site, the A-2 site was near the receptor-destroying enzyme site, and the Y-1 site was located in the loop on the topside of HE. The hemagglutination inhibition reactions of the MAbs with influenza C viruses, circulating between 1947 and 2016, were consistent with the antigenic-site amino acid changes. We also found some amino acid variations in the antigenic site of recently circulating strains with antigenic changes, suggesting that viruses that have the potential to alter antigenicity continue to circulate in humans.We mapped the hemagglutinin-esterase (HE) antigenic epitopes of the influenza C virus on the three-dimensional (3D) structure of the HE glycoprotein using 246 escape mutants that were selected by a panel of nine anti-HE monoclonal antibodies (MAbs), including seven of the C/Ann Arbor/1/50 virus and two of the C/Yamagata/15/2004 virus. The frequency of variant selection in the presence of anti-HE MAbs was very low, with frequencies ranging from 10 Orthomyxoviridae family of enveloped and segmented negative-sense RNA viruses, together with the influenza A virus and influenza B virus. The fourth segment of the influenza C viral genome encodes the hemagglutinin-esterase (HE) glycoprotein, which is present in virions as a homotrimer. The HE monomer, which contains 641 amino acids excluding a signal peptide, is composed of two subunits that are cleaved by a host protease. HE1 (432 amino acids) is the globular region of the HE protein, and HE2 (209 amino acids) is the stalk region ) of purified egg-grown C/Yamagata/15/2004 virus and an equal volume of incomplete Freund adjuvant (DIFCO) three times at intervals of 10–11 days. After 3–5 days of final boosting, the mice were anesthetized and their splenocytes were extracted and fused with myeloma X63-Ag8.653 cells. The hybridoma cells binding to the C/Yamagata/15/2004 whole virus were selected by enzyme-linked immunosorbent assay (ELISA) and then screened using the hemagglutination inhibition (HI) test. The hybridomas producing antibodies with HI activity were cloned twice at terminal dilution and then injected intraperitoneally into pristane-primed BALB/c mice. The resulting ascitic fluids were collected and used as a source of antibodies. The MAbs isotypes were determined using the Ouchterlony double-diffusion method. The animal experiments were carried out with approval from the committee for animal experiments of Yamagata University .To select escape mutants in this study, we used eight MAbs  of the HE of C/Ann Arbor/1/50, and two MAbs (YA3 and YA5) of the HE of C/Yamagata/15/2004. The eight MAbs against the C/Ann Arbor/1/50 strain were prepared in our previous studies ,21, and Anti-C/Ann Arbor/1/50 (C/Taylor lineage) chicken antiserum and anti-C/Yamagata/10/89 (C/Yamagata lineage) chicken antiserum were used as previously described. 5 PFU/mL) viral antigen in 0.05 M carbonate buffer (pH 9.6) and held for 2 h at 4 °C. After blocking with 1% BSA in PBS, the plates were washed four times with PBS containing 0.05% Tween 20 (PBS-T), and 50 μL of two-fold serially diluted MAbs were added to each well. After incubation overnight at 4 °C, the plates were washed four times with PBS-T and incubated for 1 h at room temperature with peroxidase-labeled goat anti-mouse IgG . After washing the plates, antibodies were detected by using the Peroxidase Substrate Kit . Optical densities (ODs) were read at 415 nm. ELISA titers are expressed as the reciprocal of the highest antibody dilution with an OD greater than 0.20.Antibody titers of two MAbs (YA3 and YA5) were determined by ELISA using purified egg-grown C/Yamagata/15/2004 viruses as antigens. Ninety-six-well microplates were coated with 50 μL of 50 μg/mL . Synthesis of HE gene cDNA and subsequent polymerase chain reaction (PCR) were carried out in a 25-μL reaction mixture containing 2.5 μL RNA, 0.5μL 20 μM forward primer FluCHEKN8 , and 0.5The sequences of 8 strains isolated in Yamagata in 2016 (DDBJ/GenBank accession numbers LC385860 to LC385867), together with 107 sequences previously reported , were anAmino acid positions were plotted on the 3D structure of HE molecules using the PyMOL Molecular Graphics System, version 1.8.6.1 . The crystal structure of HE was obtained from the Protein Data Bank (PDB ID 1FLC).−4.62 to 10−7.58  ,16,17 or 10−7.58 . D37-res 10−9.25 . Consequ 10−9.25  and C/Ya 10−9.25 , respectThe reactivity of each MAb with the panel of escape mutants was examined using the HI tests . Based oSingle amino acid changes at positions 173, 192, or 269 only affected reactivity with MAb J9, U9, or J14, respectively. Furthermore, a single mutation at positions 125, 172, and 175 selected by MAb J9 affected the reactions of the mutants with MAbs J9 and Q5 or J14, whereas amino acid substitutions at positions 193 and 235 decreased the reactivities of MAbs U9, Q5, and/or J14 with the mutants.Escape mutants, each possessing a single mutation at positions 65, 68, 351, and 353, were selected by MAb K16, and these mutations affected the reactivity with K16. Moreover, K16 showed decreased reactivity with the other mutants carrying an E193K and a D269N change, which were selected by MAbs belonging to the A-1 site.A single mutation at positions 164, 198, 201, and 203 affected reactivity with U1 and U2. Interestingly, U1 and U2 showed decreased reactivity with L164P or L201R, but reacted with L164S or L201I.Three escape mutants, each possessing a single amino acid change at position 172, 190, or 193, were selected by MAbs YA3 and/or YA5 . The reaThe reactivity of chicken antisera against C/Ann Arbor/1/50  or C/YamO-acetylesterase active site in the 3D structure  instead of position 175. Thus, glycosylation at position 173 appears to influence the reactivity with J14. MAb Q5 recognized the amino acid substitutions in two distantly located epitopes: position 172 (epitope J9) and positions 193 and 235 (epitope Q5). MAb J14 also recognized amino acid substitutions located on epitope J9 (positions 125 and 175) and epitope J14 (positions 235 and 269). Residue 175 is an oligosaccharide attachment site that is used for glycosylation , and theTo investigate the possibility of antigenic drift among the six genetic lineages, genetic analyses were performed for influenza C viruses, including 107 strains previously reported between 1947 and 2014 ,9 and eiThe course of amino acid changes in the antigenic sites for each lineage are shown in There were only three strains belonging to the C/Taylor lineage: C/Taylor/1233/47, C/Ann Arbor/1/50, and C/Paris/1/67. C/Paris/1/67 showed decreased reactivity with U9 and Q5 compared with C/Taylor/1233/47 , possiblC/Mississippi/80 lineage strains were sporadically isolated in Japan in two periods: between 1982 and 1985 and between 1992 and 2004  5,9,11,,28,29,30We previously reported that C/Aichi lineage strains disappeared from the world after 1992 . We. We12]. Viruses belonging to the C/Sao Paulo lineage dominantly circulated in Japan between 2005 and 2016  9,12]. ,12. 9,12We analyzed the antigenic structure of HEs of C/Ann Arbor/1/50 and C/Yamagata/15/2004 using the respective anti-HE MAbs and their escape mutants. By neutralizing three parent viruses, we were able to obtain more mutants carrying different mutations of the C/Ann Arbor/1/50 strain compared to our previous study . This stA novel finding was that the A-1 site is widely located surrounding the receptor-binding site. In particular, epitope J9, which is composed of residues 125, 172, 173, and 175, was located near the residues forming hydrogen bonds with the HE receptor, which included Y127, T170, G172, Y227, and R292, as reported by Rosenthal et al. . As showBefore the crystal structure of HE protein was reported, we predicted that residues 172 to 192 would be located close to residue 269 on the HE protein because site A-1 epitopes, recognized by four MAbs , overlapped each other ,16. HoweEpitope U9 in the A-1 site, which is composed of residues 192, 193, and 235, is located far from epitope J9 because no overlapping reactivity was observed between MAbs J9 and U9 . In the O-acetylesterase active site  [In this study, we found that epitope K16 in the A-2 site, which is composed of residues 65, 68, 351, and 353, is near the 9-nd H355) . Althougnd H355) , may be nd H355) . Moreovend H355) . ResidueUnlike the A-1 and A-2 sites, epitopes U1 and U2 in the A-3 site are far from the biological active site, such as receptor-binding sites or receptor-destroying sites. In the present study, the variants with K198E comprised 49% (29 of 59) of all variants with mutations in the A-3 site . An aminA new antigenic site, Y-1, is located in the loop on the topside of the HE protein as well as epitope U9 in the A-1 site. Epitopes YA3 and YA5, which consist of residues at positions 192 to 195 and 198, overlap with epitope U9 in the A-1 site and possibly with epitope Q5 or J14. Interestingly, two escape mutants selected by MAbs YA3 and YA5 possessed deletions at positions 192 to 195 and position 198, respectively, rather than a single amino acid change. Surprisingly, this region may not be essential for viral propagation. Among the natural isolates, a loss of amino acids in the HE protein rarely occurs and only viruses belonging to the C/Mississippi lineage possess a deletion at position 194 . TherefoThe antigenicity of influenza C virus is highly stable, as indicated by the amino acid sequence identity of more than 93% obtained for the HE1 protein of natural isolates during a 68-year period . However−4.62 to 10−7.58 for the C/Ann Arbor/1/50 virus (−7.11 to 10−9.25 for the C/Yamagata/15/2004 virus (−2.97 to 10−4.84 for the A(H1N1)pdm virus [The mutations in the antigenic sites of the natural isolates affected the reactivities of the corresponding MAbs in the HI test . There i50 virus  and 10−704 virus , which wdm virus . TherefoIn the present study, we evaluated the epitopes of nine MAbs on the 3D structure of the HE protein. The data presented here are useful as a basis for future studies of the immunological and epidemiological characteristics of influenza C virus. Continued surveillance and antigenic analysis using the panel of MAbs characterized here would help determine the succeeding replacement of antigenic lineages or the possible emergence of a novel lineage of influenza C virus.


N-glycosylation pathway. In various tumour cell lines, the distribution of N-linked sugars on the cell surface is modified and correlates with the progression of tumour metastasis. GMII therefore is a possible molecular target for anticancer agents. Here, we describe the identification of a non-competitive GMII inhibitor using computer-aided drug design methods including identification of a possible allosteric binding site, pharmacophore search and virtual screening.Golgi α-mannosidase II (GMII) is a glycoside hydrolase playing a crucial role in the  N-glycan processing. Its inhibition has been linked to an induced tumour repression  represents the inhibitor concentration. For the KM value determination, substrate concentrations ranging from 0.4 to 8.0 mM for dGMII and from 0.008 to 1.6 mM for JBM were used. For the IC50 determination of swainsonine, concentrations ranging from 6 to 120 nM were applied. For the IC50 determination of compound 18, the substrate concentrations ranged from 10 to 1000 μM. In order to define the mode of inhibition, the determination of the IC50 values was carried out using different substrate concentrations ranging from 0.1 to 3 mM, since in case of non-competitive inhibition the IC50 value should be independent from the substrate concentration [50 values.ICowing Eq :y=ymax−ntration . The proThe protein structure of dGMII was analyzed to detect pockets that could be targeted by allosteric inhibitors. A total of 60 cavities were identified in the protein structure (PDB code: 1HWW ). After A comparison of the amino acid composition revealed some differences .The known catalytic site is compact, hydrophilic and predominantly negatively charged in the region where potential ligands are deeply buried . The potAmino acids such as D204, D340, D92 or D472 may accordingly interact with positively charged binding partners. The GMII is a metallo enzyme and contains a zinc ion in its active site. Noticeably, as with all zinc sites, the metal ion is enclosed by a shell of hydrophilic groups which is surrounded from a larger shell of hydrophobic groups . On the The druggability of the potential allosteric binding site identified was evaluated using the DoGSite Scorer considering the following properties: pocket volume, surface area, depth, druggability score and the apolar amino acid ratio .With a druggability score of 0.83, the DoGSite Scorer predicted a high druggability for the potential allosteric site, since scores > 0.5 indicate a druggable binding site . FurtherIn the absence of a known ligand for this potential allosteric binding pocket, the pharmacophore model was built based on its amino acid composition. Here, a special focus was set on charged amino acids buried inside the potential binding pocket to facilitate favourable electrostatic interactions between ligands and protein. The pharmacophore model included an excluded volume covering all receptor atoms to prevent a steric clash upon binding. Additionally, six other pharmacophore features were included in the model .Two pharmacophore features were included to achieve interactions with E459. On the one hand, a hydrogen-bond donor or a cationic functionality was required close to the glutamate. On the other hand, the counterpart of this interaction, the carboxylic acid function of E459, was included as a hydrogen-bond acceptor. Further, a hydrophobic moiety was required close to the side chains of L965 and F1016. Another pharmacophore feature was located between E459 and R462 to either act as hydrogen-bond donor to E459 or as acceptor for R462. An additional feature determined an area of a hydrogen bond acceptor that may interact with the protonated side chain of K967 and the last feature represented the position of an anionic and hydrogen bond-accepting group presumably interacting with R462. In addition to the excluded volume area, the features accounting for the interaction of proposed ligands with E459 via either hydrogen bonding or electrostatic interactions and for the interaction network to E459 and/or R462, respectively, were determined as being mandatory. Ligand conformations could only pass the pharmacophore search if six out of seven features (with excluded volume) were matched.Several million conformations of the > 600,000 molecules passing the first filter step for molecular properties were calculated. However, only the conformation with the lowest rmsd-value between the matching atom positions and the pharmacophore query points was kept in the results table. Altogether, 41,815 molecules passed this second filter step. Subsequently, these molecules were subjected to docking into the potential allosteric binding site.For 36,607 compounds, a docking pose was obtained. Compounds for subsequent biological evaluation were selected according to the following criteria:total docking score (ΔG) predicted to be ≤ -25 kJ/molpredicted ligand efficiency (LE) as defined by Eq  ≥ 1 kJ/mvisual inspection of the predicted pose including an accurate analysis of the postulated protein-ligand interactions: hydrophobic parts should not be solvent-exposed and strong polar ligand groups were required to interact with at least one binding site residue via electrostatic and/or ion interactions in particularly with E459 und R462.-1×HAC-1 were finally purchased. In total, 27 out of the 41,815 compounds were selected for testing  and the benzothiophene 20 (ca. 41% inhibition at 300 μM).Two out of the 27 compounds exhibited inhibition > 40% at 300 μM, namely the bis-pyridyl derivative 18 , .In the predicted binding mode of compound 18 a bidentate hydrogen bond between the carbonyl oxygen of the amide bond and the protonated side chain of R462 is postulated . The dGMII protein Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 FigDrosophila proteins exported to the culture medium (A). Detection of the α-mannosidase II in the culture medium by western blot (B). Lines 1 to 5 show the expression of the α-mannosidase II in not transfected Drosophila cultures (1), Not induced, co-transfected cultures (2), transfected cultures with 5 (3), 10 (4) and 15 μM CdCl2 (5). α-mannosidase II was purified by incubating the culture medium with Affi-Gel Blue resin to eliminate the BSA from fetal bovine serum and subsequently by immobilized metal affinity chromatography (IMAC). Protein Molecular Weight Marker  was used to identify the position of α-mannosidase II (121.54 kDa).SDS-PAGE of (TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.


The mathematical model of the polymer plasticization in the reciprocating screw injection moulding machine is presented in this paper. Methods of calculation of the most important flow characteristics, such as the solid bed profile, the pressure and temperature profiles, the mass flow rate, the power demand, the screw torque and the energy consumption were analysed. According to the mathematical model, a computer program was developed. Based on the computer program, simulation studies of the injection moulding process were conducted. Thereafter, the experimental studies, evaluating the theoretical model from the accuracy and usefulness point of view, were carried out. Important output quantities, such as the temperature and pressure profiles, the power demand by the screw, the torque on the screw and the screw rotation time were measured. The studies were performed on a specially made research office. The simulation results were compared with the experimental data measured for the most popular polymers and different operating parameters of the injection machine. The experimental studies have indicated the need to introduce some corrections to the mathematical model. Several modifications have been made to the model, mainly related to the methods of stress determining in the polymer layer. Finally, the output characteristics of the plasticization process in the injection moulding are now correctly determined by the model with an average error less than 10%. One of the main factors that minimizes the production costs is the optimal choice of the processing equipment and processing conditions. For a long time, the optimization of geometry of plasticizing systems and forming tools in the injection moulding process have used the experience of designers and manufacturers. Recently, the theoretical approach has been having the increasing importance. It relies on using mathematical models for the plasticization process on the basis of the law of mass, momentum and energy conservation and the characteristics of a material. The models join the output characteristics of the plasticization process, such as pressure and temperature distribution, throughput, power demand and so forth, with the geometry of the plasticizing system, the adjustable process parameters and the material data, allowing thereby the optimization of the equipment design .The theoretical approach to the plasticization process through the creation of computer-based simulation models is widely used mainly in the case of the extrusion process. Many models can be found in the literature. They describe, in less or more complex way, the plasticization of polymers in single-screw extruders. These models commonly use similar principles but differ in detailed assumptions. Thus, the extruder is divided into three main functional zones—the zone of solid conveying, the transient zone and the zone of melting and melt conveying. The description of the solid conveying zone is based on the dry friction mechanism represented usually by the classic approach of Darnell and Moll  with subThe existing models of the polymer melting in the screw systems can be divided into two categories, that is, the models of contiguous solid melting (CSM) and the models of dispersed solid melting (DSM). The Tadmor (CSM) model  and its The DSM melting mechanism is characteristic for the starve-fed extrusion regime, which is more relevant of the twin-screw extrusion. The starve-fed conditions in single-screw extruders can be obtained with a controlled feeding, because for typical processes the flood feeding described by the CSM models prevails. Although the starve-fed in the single-screw extruders has been known for a long time ,20, its In the last two decades, there has also been observed a significant development in the field of modelling of the plasticization process in the twin-screw extruders, both in the co-rotating systems ,25,26,27Despite the significant development of the extrusion simulation programs, only very few simulation models describe the plasticization process in the injection moulding. The main cause is a much more complex dynamic of the plasticization process resulting from the cyclical nature of the injection moulding. It involves the existence of coupled static and dynamic melting phases (stationary and rotating screw) accompanied by an axial screw movement with an adjustable stroke. The model approach to melting mechanism in the injection moulding has been the subject of a few works ,32,33,34It follows from the foregoing literature data that the creation of an adequate and comprehensive simulation model of the plasticization process in the injection moulding has not been fully completed yet. It confirms that there exist at least several commercial software programs, such as EXTRUD, SSD, REX, SSEM and NEXTRUCAD for the extrusion process but there is probably one computer program—PSI available for the analysis of the plasticization process in the injection moulding.The purpose of this article is to present the comprehensive model of the polymer plasticization process in the injection moulding. The previous version of this model and the simplified model verification (limited only to comparison of the screw rotation time) was presented in ,45. NextThe model description is comprised of three parts. The first one consists of definitions and main assumptions. Then, the key segments of its construction are presented. The last part of the description discusses the calculation procedure.D, width W (constant on the whole screw length), lead S and flight width e. The channel depth H in feed and metering zones are constant and equal to fH and mH, respectively. The channel depth in a compression (transition) zone changes linearly from fH to mH. The additional geometric parameters of the model are flight clearance Sδ (thickness of the slit between the top of screw flight and the inner barrel surface) and the lengths of feed, compression and metering zones. The helix angle ϕ depicted in D and S. It is also possible to analyse flows in two or more parallel aligned channels. The above geometric parameters are principally the same as those used in various extrusion models.The plasticizing unit shown in In order to create a mathematical model of the plasticization process during the injection moulding, several general assumptions were made.(a)The injection moulding is a quasi-steady process, that is, the process characteristics such as the pressure, temperature, solid polymer and melt profiles change with time during a single injection cycle but they are constant for the same moments of time in different cycles.(b)The plasticization during the injection moulding in contrast to the extrusion contains two different and strictly coupled melting phases–static melting at stationary screw and dynamic melting at the rotating screw and its axial backward movement.(c)For the assumed screw geometry and adjustable operating parameters  the screw retreat is generally determined by the equality of the calculated pressure in the front of the screw and the pre-set back pressure.(d)The most important process characteristics correspond to the moment of time just before the end of the screw rotation, that is, to the maximal filling degree of the screw channel with the mixture of solid polymer and melt.(e)Description of various phenomena connected with the plasticization process, for example, the solid polymer and melt transportation, the static/dynamic melting mechanism, non-isothermal flow conditions and so forth, requires additional detailed assumptions discussed below.They are the following:U . The first smaller loop calculates the coupled (time dependent) solid bed profiles after static melting and dynamic melting according to the assumption (b). It uses the assumption that the profile after the static melting is the initial profile for the dynamic melting and inversely. The third loop calculates the time dependent melt temperature changes during the dynamic melting assuming that the initial melt temperature after the static melting is approximately constant and equal to the barrel temperature. Other calculation details are discussed below.It can be seen that the algorithm comprises three loops. The largest loop is connected with the pressure calculations according to the assumption (c). This makes it possible to calculate the screw backward movement velocity a feed port and a solid conveying zone;a transient (delay) zone;a melting and melt conveying zone.Firstly, the existence of three dynamical zones in the plasticizing system was assumed:Furthermore, the flat (rectangular) screw channel model was assumed.-at the end of screw rotation (the beginning of static melting);-at the beginning of screw rotation (the beginning of dynamic melting).The starting point for the model is the model of steady-state extrusion that is similar to the classical extrusion model of Tadmor and Klein . Howeverτ—the extra stress tensor, d—the rate-of-strain tensor, dII—the second invariant of d-tensor, 0T—the reference temperature , k0, n, a—rheological parameters: k0—the consistency coefficient, n—the power-law exponent, a—the temperature coefficient.Moreover, it was assumed that the melt behaviour can be described by the power-law of the form: U of rotating and withdrawing screw should be additionally taken into account. The generalized mechanism of solid conveying with axial screw motion is shown in It was assumed that the dynamic equilibrium in the solid conveying zone is established fast enough. Hence, its operating characteristics can be adequately described by means of relations, that are valid for the steady-state conditions . HoweverH—the channel height, W—the average channel width, szV—the solid bed velocity along the screw channel (in z-direction), sρ—the density of solid polymer, D—the outer screw diameter, U—the axial screw velocity, mρ—the average density of polymer melt; The szV velocity is determined from Assuming the flow continuity, the mass flow can be calculated both from the solid bed velocity and from the screw withdraw velocity asszV and U can be calculated and this makes possible to calculate the solid conveying angle θ from (4). If this angle is known, the pressure profile in the solid conveying zone can be determined using the force and torque balance [k—the parameter determined from the balance of forces and moments of force acting on the material layer of elementary thickness dz [z-direction.If the mass flow  balance ,44. A gep0 in the feed port region can be calculated according to the simple formula proposed in [ρ0—the bulk density, g—the gravitational acceleration.The initial pressure posed in :(5)p0=ρ0Se in the solid conveying zone as the sum of power dissipated at the barrel, screw root, screw flights and the power used to increase the pressure in the solid bed:l) is the pressure in l-location; l is the location on the screw channel length (in z-direction) measured by a number of computation steps from the beginning of the screw; bf, sf—the barrel and screw friction factors of solid polymer, respectively, jV—the velocity of solid bed transport relative to the barrel, determined from For the given pressure profile, it is possible to determine the power demand SM in the solid conveying zone was determined in the classical way as the product of the dry friction force and the lever arm:The torque From the presented relations, it follows that the main difference in description of solid conveying zone action during extrusion and injection moulding is the existence of the non-zero retraction velocity of the screw.wδ [wδ over the zone length. These changes depend on the rate of dynamic melting, that can be calculated from [U [The transient zone in the model starts at a point, where melt layer appears at the solid bed surface. It was adopted, that this is the place of the screw channel which corresponds with the beginning of the barrel heating zone at a given moment of time. The end of the transient zone in the screw channel corresponds with the point, where the melt film thickness reaches a critical value δw . In contδw  it was ated from , assumin from [U .t, s and m mean: the transient zone, the solid conveying zone and the melting zone, respectively, x—the weight factor, changing from 0 to 1 on the length of the transient zone.The calculations of the pressure changes in the transient zone base on the assumption that the pressure gradient in this zone can be determined as a weighted average of the pressure gradient at the end of the solid conveying zone and the pressure gradient at the beginning of the melting zone:This semi empirical approach that provides a smooth pressure profile at the zone boundaries was introduced, because there is no exact method of pressure calculation in the case, if the flow is determined by both dry and viscous friction.Te in the transient zone was defined as the sum of the power dissipated in the thin melt film at the barrel surface, on the root surface and the flight surfaces, as well as the power desired to increase the pressure. It can be calculated for one computation step from the equation:jτ—the shear stress in the polymer melt layer in the screw channel, mδ—the melt layer thickness in the screw channel in the transient zone, changing linearly from 0 to critical value wδ: mδ = w xδ (x—the weight factor), eτ—the shear stress in the polymer melt layer in the slit between the top of the screw flight and the barrel surface, e—the width of the screw flight; jτ and eτ quantities are defined according to the power law as follows:k0, a, n—the parameters of the power law equation, sδ—the slit thickness, bT—the average barrel temperature in the heated zone, mT—the melting (flow) temperature of polymer, The power demand p (1 − x)) and viscous (τ·x) friction. The last term of the Equation (9) refers to the power dissipated in the thin polymer layer existing between the top of the screw flight and the inner barrel surface as a result of the leakage flow. The determination of the polymer temperature in the slit bases on an energy equation, which assumes neglecting of the convection and the conductivity along and across the slit for the generalized Newtonian liquid and takes the form:The first term in Equation (9) assumes the occurrence of the polymer-barrel friction as a weighted average of dry  takes the form:Due to the high intensity and the short shear time as well as the partial compensation of thermal effects at the surface of the barrel and the flight it is assumed that the flow is approximately adiabatic. Thus, the Equation (12) assumes the simplified form:, the shear time it in the slit is equal to the transition time from one to the other edge of the slit with the width e and the helix angle ϕ in the direction and the velocity determined by the vector Analysing the displacement of the polymer particle in the slit see , the shemc—the average specific heat of the polymer melt.The Equation (14) with the viscosity expressed by the Formula (15), integrated with the initial condition TM in the transient zone was determined as:τ is the shear stress defined as:The torque In conclusion, it should be noted that the transient zone could not be omitted from the model calculations, as it is in the case of some other available models. This is due to the experimental studies that indicated this zone is necessary for obtaining the correct values of the output characteristics. It is also important that the weighted average of dry friction and the viscous friction at the polymer-barrel contact should be maintained in the transient zone. Adopting viscous friction only results in significant overestimation of the pressure, power and torque characteristics.The melting process during the injection moulding is more complicated in comparison with the extrusion, mainly due to the existence of the static melting phase (for the stationary screw). Moreover, the phase of the dynamic melting must additionally take into account the axial screw motion. Both phases are coupled. The final conditions for one of them are the initial conditions for the second one.Defining of the function of the solid bed width distribution in time and space  is the basis for the plasticization model with the three-zone-screw. It can be described with the general relation:l  in the Equation (20) is expressed in the number of computation steps.The spatial coordinate It was assumed that three areas of different behaviour of the polymer material, shown in A—the ratio of the cross-sectional area of the channel occupied by the solid bed to the total cross-sectional area of the channel (see H(l)—the relative height of screw channel, determined as H(l) = H/fH, changing from 1  to mH/fH  on the screw channel length.Due to the variable channel height along the screw it is convenient to express the solid bed profile as the functionnnel see , H(l)—thA(l) function in the two basic moments of time, is necessary to describe of the melting process in the injection moulding: just after the start of the screw rotation (beginning of dynamic melting): rt is the recovery time defined as:SN is the screw stroke (expressed in number of coils).just after the finish of the screw rotation (beginning of static melting): Knowing the The static melting begins after the stopping the rotational screw motion. The solid polymer is molten in a certain time interval, which is approximately equal to the cooling time and then the screw is shifted forward on the distance of the screw stroke. The polymer is molten now in the time approximately equal to the hold time. The method of calculating the solid bed profile after the static melting process is given in .Dynamic melting starts at the moment of the beginning of the screw rotation. The calculation of the solid bed profile after the screw rotation period was done using the theory of dynamic extrusion . The metuL defined by Equation (23) describes the solid bed displacement due to the rotary-backward motion of the screw and it is expressed in the computation steps. The solid bed evolution during the screw rotation can be followed by changing uL from 0 to the end value given by the Equation (23).The quantity eA characteristic for the extrusion process [iA profile after static melting can be determined. It is the initial value for the new profile of the solid bed fA calculated for dynamic melting. The iteration is repeated until A profiles (after static and dynamic melting) will be established. It could be shown that the time required for stabilization of fA and iA is not shorter than the passage time of the first polymer portion over the whole channel length. It corresponds to a certain value of iN iteration cycles [Equilibrium values of A after the static and dynamic melting can be calculated using the iteration method. The steady-state profile  process  taking a process  .The total power demand in melting zone MM in the melting zone was determined as a sum:xzτ and eτ are defined respectively as The torque U and the pressure profile, where the pressure value at the screw end is equal to the known back pressure (operating parameter). Both quantities are strictly coupled and their determination closes the computation cycle. Hereafter, that makes it possible to calculate the most important process characteristics such as the plasticization rate, the power requirement, the screw torque, the average melt temperature and the specific energy consumption. The choice of the proper backward velocity U for a given back pressure was done with the iteration method using a special control algorithm. It increases or decreases the U value depending on the calculated pressure on the screw end and the assumed back pressure, until both pressures become equal with a desired accuracy. The algorithm adopts the following form:One of the most fundamental questions in the model is the determination of the screw retraction velocity In summary, some earlier concepts of the modelling of the plasticization process in the injection moulding have been taken into account. Due to the partial similarity between the extrusion and the reciprocating injection moulding, some solutions from the existing extrusion models have also been applied. However, many other issues are the original solutions: the generalization of the static and dynamic melting process for the three-zone screw, the description of reciprocating screw motion with the adjustable stroke, the description of two-dimensional, non-isothermal and non-Newtonian (power-law) melt flow in the channel, the solution for the time dependent mass balance during the dynamic melting and the description of the instant melt temperature changes ,48,49.The full operation algorithm of a computer program based on the model shown above is presented in For evaluation of prediction accuracy and possible improvements, the simulation model required a full experimental verification based on the comparison of its output characteristics mentioned above with the experimental characteristics of the real plasticizing unit. For this purpose, the measuring system as the special equipment connected with the conventional injection moulding machine was designed and built ,50. Selethe injection moulding machine, Battenfeld Plus 350/70,four pressure/temperature sensors , range 0–150 MPa, 0–300 °C, OE: ±0.5% FS),the torque—measuring device , range 0–250 Nm, OE: ±1% FS),the inductive sensor for the screw rotational velocity measurements ,the screw linear displacement sensor , range 0–150 mm, LE: ±0.08%),the control cabinet with the touch screen.The test office for the measurements of the output parameters of the plasticization process during the injection moulding consists of the suitably instrumented injection moulding machine linked to the collecting and processing data module and the computer for imaging and saving of the collected data. The test office shown in the back pressure (changed in range of 4–24 MPa),the screw velocity ,the dwell time (changed in range of 8–50 s)—approximately equal to the cooling time of the product in the mould,the average barrel temperature in the heated section.The results presented below are related to the studies involving five typical thermoplastic polymers characterized in The study by changing only one of the parameters listed in The study by changing simultaneously two of the adjustable parameters listed in The studies on the plasticization process in injection moulding were carried out as two independent series of experiments:Because of the periodical (unsteady) character of the injection moulding process, all the results refer to the moment just before the end of the screw rotation. This moment corresponds to the maximum filling degree of screw channel with solid polymer and it is critical from the main plasticization characteristics point of view. It is of a fundamental importance for such quantities as the pressure and temperature profiles along the screw length, the power demand by the screw and the screw torque.The pressure and temperature profiles for the variable input parameters: the back pressure, the screw rotational velocity, the dwell time and the barrel temperature are presented only for both extreme values of these parameters . The results for the intermediate values of the input parameters are not shown to keep the readability of the pictures, because these characteristics commonly change linearly with respect to the characteristics for extreme values.Because there is a lot of results and they are very similar for different polymers in aspect of the shape of curves and the differences for the relevant theoretical and experimental characteristics, it was decided to present the output characteristics of one polymer for each variable process parameter. However, all studied polymers will be discussed for each analysed parameter. In order to standardize the charts, the characteristics obtained from the model are shown as thick lines without markers, while the experimental profiles represent the markers indicating the measurement points.The first part of the studies was related to change of one input process parameter, measurements of output characteristics and comparison with the characteristics determined by the model.The comparison of the theoretical and experimental data for the pressure and temperature profiles of the molten polymer shows a good agreement. The model slightly underestimates the pressure values and overestimates the temperature values by a few degrees for all polymers. These differences are the greater, the greater is the MFI of the polymer. We can also see a very small effect of the back pressure on the power demanded to the screw during its reciprocating motion, the process efficiency and the torque on the screw. The model predicts the values of these characteristics with a good accuracy. The model overestimates insignificantly the values of the power, the yield and the torque for the other polymers. The compliance with the model is also good and differences do not exceed a maximum of 20%.The variable parameter of the rotational velocity of the screw brings the greatest dynamics of changes in output characteristics of the plasticization process of polymers during the injection moulding. An expected increase in the maximum pressure of the polymer with increasing screw rotational velocity is visible in Differences in the temperature determining by the model are the greater, the greater is the rotational velocity of the screw. The differences do not exceed a few degrees for low screw velocity and they approach up to 20 °C for high screw velocity. This is probably caused by the overestimation of the effect of the viscous friction on the temperature increase of the polymer melt and it is related to the applied rheological model. However, an inaccurate measurement of the polymer temperature in the barrel can also be important. These issues will be discussed later.The dwell time parameter means exactly the downtime of the screw in the rear position and quantitatively is practically equal to the product cooling time in the injection mould. The dwell time does not significantly affect any output characteristics of the plasticization process except the process yield, as shown in We observe significant differences in the experimental and model pressure profiles in As the temperature of the barrel increases, the power supplied to the screw decreases. However, the total power demanded to the plasticizing system of the injection moulding machine increases, which is related to the increase in the power demand by the barrel heaters .The second part of the studies was related to the change of two input process parameters. At the same time, the rotational velocity of the screw and the back pressure in the tests were changed. These parameters are presented in In (a)the pressure profile of the polymer in the screw channel,(b)the temperature profile of the polymer in the screw channel,(c)the profile of the solid polymer bed width in the screw channel (not verified),(d)the power demanded by the screw in the plasticizing system,(e)the torque on the screw,(f)the yield of the plasticization process,(g)the recovery time (or the injection cycle time),(h)the SEC (the specific energy consumption).The mathematical model of the plasticization process in the injection moulding predicts values of the following characteristics:The pressure of molten polymers in the screw channel is determined substantially correctly. The results of the comparison of the theoretical and experimental characteristics for all tested polymers indicate that the mean differences do not exceed 20%. Mostly, the model profiles cover with the experimental points very well. It is worth noting that the largest deviations from the real values are observed for very low rotational velocity of the screw and very low and high barrel temperature, that is, for the process conditions, which are very rarely used in practice. The model better predicts the behaviour of molten polymers in the conditions similar to those most commonly used in injection processing, that is, quite high rotational velocity and average, typical values of processing temperatures.the Carreau-Yasuda model, slightly modified by the authors ,δ, m—the parameters of the equation, η0—the lower Newtonian viscosity.the author’s rheological model based on the free volume theory: In order to better predict polymer pressure values, several problems will be analysed. The first problem to study in the near future is using a different rheological model. Two rheological models will be analysed:The second factor, which will be analysed for improving the compliance of the pressure characteristics is the analysis of methods of polymer-metal friction coefficients determining. The values of the dynamic friction coefficients of the polymers used in this study were taken as the mean values from the literature and they were equal 0.1–0.2 for PP and PE-HD to 0.4–0.5 for PS and PE-LD. The pressure value of the solid polymer in the screw channel (which determines the pressure of the polymer melt) in the model significantly depends on friction coefficients values. It is known from the literature that the values of friction coefficients are variable and depends not only on the temperature but also on the pressure and the rotational velocity of the screw. In order to analyse this problem more closely, the apparatus for dynamic friction coefficients measuring for different polymers in variable temperatures, pressures (load) and a variable velocity of the movable wall will be constructed in the near future. This will allow to adapt the corrected values of the friction coefficients to the model. It is supposed to improve the compatibility of the model results with the experience.Another problematic issue that appeared during the experimental research is the model of barrel feeding with the solid polymer during the injection process. In the previous studies authors assumed that in accordance with the known Tadmor model of the plasticization process in extrusion , a polymThe comparison of the temperature characteristics of molten polymers generally indicates that the model overestimates the real temperature values by approx. 2–15 °C. The greater are the differences between the theoretical and experimental profiles, the more viscous is the polymer. These differences increase as the rotational velocity of the screw increases. They decrease with the increase of the screw downtime as well as the barrel temperature increase and they are practically independent of the variable back pressure.The temperature profiles determined experimentally are practically unchangeable for the different back pressures. The analysis of the model indicates that it overestimates the amount of the heat generated in the material due to the viscous friction. Moreover, large increases in the polymer temperature above the barrel temperature are not observed in the real plasticizing system of the injection moulding machine. Regardless of the polymer type and the process parameters, this increase always equals about 2–6 °C. This is thought-provoking because it seems that the extreme values of some operating parameters  should affect the temperature of the molten polymer in a way. Meanwhile, for a given polymer the temperature of the melt is practically constant in the same points of the barrel, regardless of the value of the screw velocity or the back pressure. Due to the above-mentioned facts, doubts relating to the method for measuring the temperature of the molten polymer in the screw channel appeared.The polymer temperature measured by the pT sensor refers to the temperature of the polymer melt layer at the inner barrel wall. The model determines average temperature of the polymer melt in the entire cross section of the screw channel. This can be a source of differences, especially for a low fluidity of the material, when the viscous friction increases. It was decided to measure the temperature of molten polymers inside the screw channel in the future. We plan to place the tip of a thin thermocouple in the molten material in the screw channel through the hole in the barrel. The barrel already has 16 holes made for the pT sensors. After proper processing of locking bolts placed in the barrel holes, the thermocouple will be placed in the molten polymer on the depth of several mm into the screw channel, just after the end of the screw rotation. We also plan to carry out studies on a temperature of the solid polymer in the initial part of the barrel in the same way. It will verify whether temperature of the polymer melt inside the screw channel is comparable or different from the temperature of the polymer boundary layer. It will also enable to develop a mathematical model for determining the polymer temperature in the solid bed.The methodology of the power demand and the torque determining has been corrected in the model in a relation to the works presented earlier ,44. As aThe yield of the process is very well determined by the model and the differences in theoretical and experimental values do not exceed a maximum of 10%. The model overestimates this parameter slightly in each case. This characteristic is closely connected with the next parameter determined by the mathematical model that is, with recovery time. This parameter is underestimated by the model by approx. 20%, that is, by about 1 s. It is caused by the method used to determine the recovery time by the measuring system. However, in practice, the value of the full injection cycle time is more important than the recovery time. The model also determines the cycle time. Thus, a typical recovery time error of 1 s/5 s = 20% means that the average error of the cycle time equals 1 s/20–30 s = 3–5%. It is therefore relatively small.The model verification procedure should provide sensitivity analysis in addition to compliance tests. The sensitivity analysis involves examining changes in the output (variable) values of the model when the input parameters of the model change. It is required from a good model that small changes in the input parameters cause small changes in its output values only.When an explicit algebraic equation describes the relationship between the independent variables and the dependent variable, the sensitivity analysis is easy to perform. In this case, the sensitivity coefficient SC, for a particular independent variable can be calculated from the partial derivative of the dependent variable with respect to the independent variable :(32)SCiyIn simplification, it can be assumed that the sensitivity function (32) indicates how many percent will change the function value if the specified variable increases by 1%.The matrix of results in the form of a table is presented in the temperature of the barrel,the temperature of polymer melting,the polymer-barrel friction coefficient,the power-law exponent.The model is sensitive to four parameters:n). On the one hand, this fact requires an accurate determination of the power-law equation parameters for each polymer used for the modelling process. On the other hand, it indicates the potential benefit of using another equation that describes the viscosity of molten polymers. However, as previously described, the applied power-law equation describes the molten polymer behaviour very well. It is indicated by the correct results of the model work. In addition, there is no guarantee that the model will be less sensitive to the parameters of another rheological model.We can see the high sensitivity of the model to one of the parameters of the rheological equation  occur in the determination of the pressure and temperature characteristics. It is worth noting, however, that the knowledge of the pT profiles is scientific and cognitive in nature. On the other hand, the results such as the power demand, the torque, the process yield and the SEC are of a practical importance. Here the model shows the results more compatible with the experience.The model uses three groups of input parameters: geometrical parameters of the plasticizing system (mainly the screw), working parameters of the injection moulding machine and material parameters of the polymer being processed. In the experimental verification of the model, the second and third parameter groups are changed. All tests were conducted using one injection moulding machine and one screw. It is worth performing similar studies for a larger injection moulding machine as well as at least two different screws with the different length of feed, compression and metering zones to generalize or detail the results presented in this work.


Contents: Danshen is a popular herb employed to treat cardiovascular and cerebrovascular diseases worldwide. Danshen–drug interaction has not been well studied.Objective: The inhibitory effects of four major tanshinones  on UDP-glucuronosyltransferases (UGTs) isoforms were determined to better understand the mechanism of danshen–prescription drugs interaction.Materials and methods:In vitro recombinant UGTs-catalyzed 4-methylumbelliferone (4-MU) glucuronidation reaction was employed. Tanshinones (100 μM) was used to perform the initial screening of inhibition capability. High-performance liquid chromatography (HPLC) was used to separate 4-MU and its glucuronide. In vitro–in vivo extrapolation (IV–IVE) was employed to predict in vivo inhibition situation.Results: Cryptotanshinone inhibited UGT1A7 and UGT1A9 with IC50 values of 1.91 ± 0.27 and 0.27 ± 0.03 μM, respectively. Dihydrotanshinone I inhibited UGT1A9-catalyzed 4-MU glucuronidation reaction with the IC50 value of 0.72 ± 0.04 μM. The inhibition of cryptotanshinone towards UGT1A7 and UGT1A9 was best fit to competitive inhibition type, and UGT1A9 was non-competitively inhibited by dihydrotanshinone I. Using in vitro inhibition kinetic parameters (iK) and in vivo maximum plasma concentration (Cmax) of cryptotanshinone and dihydrotanshinone I, the change of area-under-the-concentration–time curve (AUC) was predicted to be 0.4–4.2%, 3.7–56.3%, and 0.6–6.4% induced by cryptotanshinone and dihydrotanshinone inhibition towards UGT1A7 and UGT1A9, respectively.Discussion and conclusion: The inhibitory effects of tanshinones towards important UGT isoforms were evaluated in the present study, which provide helpful information for exploring the mechanism of danshen–clinical drugs interaction. Salvia miltiorrhiza Bunge, has been widely utilized to treat cardiovascular and cerebrovascular diseases all over the world, including China, Japan, the United States of America (USA), and European countries , important phase II enzymes, are responsible for catalyzing the glucuronidation reaction by adding the glucuronosyl group of UDP-glucuronic acid (UDPGA) as a cofactor  is the NAD(P)H:quinone oxidoreductase 1 (NQO1)-mediated quinone reduction and subsequent glucuronidation, and UGT1A9 plays an important role in this process  were performed to predict possible danshen-drug interactions due to the inhibition of UGT isoforms by tanshinones.Tanshinone I, tanshinone IIA, cryptotanshinone, and dihydrotanshinone I (purity ≥98%) were purchased from Aladdin Corp. . 4-Methylumbelliferone (4-MU), 4-methylumbelliferone-β-D-glucuronide (4-MUG), Tris-HCl, 7-hydroxycoumarin, and uridine 5′-diphosphoglucuronic acid (UDPGA)  were purchased from Sigma-Aldrich . Recombinant human UGT supersomes  expressed in baculovirus-infected insect cells were obtained from BD Gentest Corp. . All other reagents were of HPLC grade or of the highest grade commercially available.2, 50 mM Tris-HCl buffer (pH = 7.4), and 4-MU in the absence or presence of different concentrations of danshen's components. The concentrations of 4-MU were as follows: 110 μM for UGT1A1, 1200 μM for UGT1A3, 110 μM for UGT1A6, 15 μM for UGT1A7, 750 μM for UGT1A8, 30 μM for UGT1A9, 80 μM for UGT1A10, 350 μM for UGT2B7, and 250 μM for UGT2B15. The compounds were dissolved in DMSO, and the final concentration of DMSO was 0.5% (v/v). After 5 min pre-incubation at 37 °C, the UDPGA was added in the mixture to initiate the reaction. The incubation time was 120 min for UGT1A1, UGT1A10, UGT2B7, and UGT2B15, 75 min for UGT1A3, 30 min for UGT1A6, UGT1A7, UGT1A8, and UGT1A9. The reactions were quenched by adding 100 μL acetonitrile with 7-hydroxycoumarin (100 μM) as an internal standard. The mixture was centrifuged at 20,000 × g for 10 min and an aliquot of supernatant was transferred to an auto-injector vial for HPLC analysis. The HPLC system  contained a SCL-10A system controller, two LC-10AT pumps, a SIL-10A auto injector, a SPD-10AVP UV detector. Chromatographic separation was carried out using a C18 column  at a flow rate of 1 mL/min and UV detector at 316 nm. The mobile phase consisted of acetonitrile (A) and H2O containing 0.5% (v/v) formic acid (B). The following gradient condition was used: 0–15 min, 95–40% B; 15–20 min, 10% B; 20–30 min, 95% B. The calculation curve was generated by peak area ratio  over the concentration range of 4-MUG 0.1–100 mM. The curve was linear over this concentration range, with r2 value >0.99. The limits of detection and quantification were determined at signal-to-noise ratios of 3 and 10, respectively. The accuracy and the precision of the back-calculated values for each concentration were less than 5%.The probe substrate for all the UGT isoforms is 4-MU which is a non-selective substrate of UGTs. The incubation and analysis conditions were performed as previously described  was determined as previously described  were determined utilizing various concentrations of 4-MU in the presence of different concentrations of components. Dixon and Lineweaver–Burk plots were adapted to determine the inhibition type, and the second plot of slopes from Lineweaver–Burk plot versus compound concentrations was utilized to calculate the iK value.The half-inhibition concentration , and the value was calculated to be 3.1 μM. The Dixon plot  of cryptotanshinone and dihydrotanshinone I after administration of a mixture of danshen components for rats was used to predict the in vivo danshen–drugs interaction. The Cmax was reported to be 42.85 ng/mL (0.14 μM) and 11.29 ng/mL (0.04 μM) for cryptotanshinone and dihydrotanshinone I, respectively. Besides in vivo concentrations of danshen components, the alteration of AUC for co-administered drugs also depends on the contribution of UGT isoforms (mf) towards the metabolism of drugs. So, the relationship between AUC's alteration and mf is described in In the present study, the maximum plasma concentration –drug interaction or drug-induced metabolic disorders of endogenous substances  were demonstrated to be good substrates of UGT1A7   should be considered to be another important factor influencing the final in vivo herb–drug interaction results. Previous studies have that the initial oral dose of Schisandra lignan extract (SLE) leads to an apparent inhibition towards CYP3A; however, its long-term consumption exhibits significant induction of CYP3A . Further in vivo experiments will be performed to evaluate the net effects of inhibition and induction of drug-metabolizing enzymes by danshen's components.The explanation of these in vitro data to extrapolate in vivo situation, more in vivo experiments should be carried out in the future.In conclusion, the inhibitory effects of tanshinones towards important UGT isoforms were evaluated in the present study, which provide helpful information for exploring the mechanism of danshen–clinical drugs interaction. Given that the complication when using these


A novel organic-inorganic hybrid containing allyl benzoxazine and polyhedral oligomeric silsesquioxane (POSS) was synthesized by the thiol-ene (click) reaction. The benzoxazine (BOZ)-containing POSS (SPOSS-BOZ) copolymerized with benzoxazine/epoxy resin was used to prepare composites of SPOSS-PBZ-E nanocomposites(NPs). The polymerization behavior was monitored by FTIR and non-isothermal differential scanning calorimetry (DSC), which showed that the composites had completely cured with multiple polymerization mechanisms according to the oxazine ring-opening and epoxy resin (EP) polymerization. The thermal properties of the organic–inorganic polybenzoxazine (PBZ) nanocomposites were analyzed by DSC and thermogravimetric analysis (TGA). Furthermore, the X-ray diffraction analysis and the scanning electron microscopy (SEM) micrographs of the SPOSS-PBZ-E nanocomposites indicated that SPOSS was chemically incorporated into the hybrid nanocomposites in the size range of 80–200 nm. The flame retardancy of the benzoxazine epoxy resin composites was investigated by limiting oxygen index (LOI), UL 94 vertical burn test, and cone calorimeter tests. When the amount of SPOSS reached 10% or more, the vertical burning rating of the curing system arrived at V-1, and when the SPOSS-BOZ content reached 20 wt %, the thermal stability and flame retardancy of the material were both improved. Moreover, in the cone calorimeter testing, the addition of SPOSS-BOZ hindered the decomposition of the composites and led to a reduction in the peak heat release rate (pHRR), the average heat release rate (aHRR), and the total heat release (THR) values by about 20%, 25%, and 25%, respectively. The morphologies of the chars were also studied by SEM and energy dispersive X-ray spectroscopy (EDX), and the flame-retardant mechanism of POSS was mainly a condensed-phase flame retardant. The ceramic layer was formed by the enrichment of silicon on the char surface. When there are enough POSS nanoparticles, it can effectively protect the combustion of internal polymers.  Benzoxazine has a stable aromatic ring structure and can be cross-linked under certain conditions to form a structure such as a phenolic resin. . During Polyhedral oligomeric silsesquioxane (POSS) is a novel organic–inorganic hybrid filler with a cage structure, which is mainly composed of three-dimensional cage-like structures connected by Si–O–Si bonds and is very stable. The incorporation of POSS into benzoxazine monomers can improve the thermal, mechanical ,14,15,163-POSS) and 3,4-dihydro-3-(prop-2-ynyl)-2H-benzoxazine (P-pa) by a click reaction. However, the preparation of octa-azido functionalized POSS is too complicated. In previous studies, no researchers used thiol-based POSS and allyl benzoxazine to obtain POSS-BOZ nanocomposites nor did they study their effects on the thermal stability and flame retardancy of BOZ/E composites.It has been increasingly recognized that “good” reactions for making POSS-related materials including the criteria of efficiency, versatility, and selectivity; therefore, “click” reactions are ideal tools . Li et aTherefore, in this paper, an organic–inorganic hybrid benzoxazine was prepared by the thiol-ene (click) reaction between benzoxazine and sulfur-containing POSS. The nanocomposites were blended with benzoxazine containing allyl group (BOZ)/epoxy composites and cured. The effect of POSS on the thermal stability and flame retardancy of the material was studied.Allylamine was obtained from the Chengdu Huaxia Chemical Reagent Co. Ltd. , >95%. Paraformaldehyde was obtained from the Tianjin Fuchen Chemical Reagent Factory . Phenol was obtained from the Beijing Chemical Plant . The SPOSS (TH-1550) was obtained from Hybrid Plastics Inc. , >99%. DMPA was purchased from Aladdin Biochemical Technology Co. Ltd. , >99%. Tetrahydrofuran was obtained from Beijing Tongguang Fine Chemical Company . Epoxy resin was purchased from E-44, Beijing Tongguang Fine Chemical Company .2 atmosphere. Then, phenol  was added into the reaction system. The mixture was stirred at 80 °C for 2 h. Subsequently, the solution was cooled to room temperature, and washed using 1 mol/L NaOH solution and distilled water several times. Then, the organic layer was dried over anhydrous Na2SO4. The solution was dried under vacuum to afford a light-yellow liquid. Yield: 56 g, 80%; 1H NMR : δ 6.80–7.13 , 5.92 , 5.27–5.18 , 4.88 , 4.00 , 3.40 . FTIR (cm−1): 3077 cm−1 (stretching of =C–H), 1843–1560 cm−1 (skeleton vibration of benzene ring), 1646 cm−1 (stretching of C=C), 914 cm−1 (symmetric stretching of C-O-C), 746 cm−1 (out-of-plane bending vibrations of olefinic =C–H). HRMS (MALDI-TOF): m/z 175 .The synthesis route of BOZ is shown in Benzoxazine POSS was synthesized using the procedure presented in Composites (SPOSS-PBZ-E) containing polybenzoxazine, POSS, and epoxy resin were prepared by the procedure in 2.UV-curing: A HWUV0133X three-dimensional UV curing box  was used for the experiment. The wavelength of the ultraviolet light was 300–400 nm, the lamp power was 400W, and the radiation intensity was 100 mW/cm1H-NMR spectra were recorded on a Bruker Avance 600 NMR spectrometer  operated in the Fourier transform mode. CDCl3 was used as the solvent, and the solution was measured with tetramethyl silane (TMS) as an internal reference.Nuclear magnetic resonance (NMR) spectroscopy: −1.Fourier-transform infrared (FTIR) spectroscopy: FTIR spectra were recorded on a NICOLET 6700 IR spectrometer . The spectra were collected at 32 scans with a spectral resolution of 4 cmMass spectrometry: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was performed using a Bruker BIFLEX III device  equipped with a pulsed nitrogen laser . Samples were measured in positive ion modes with usually 50 spectra accumulated. For the MALDI mass spectrum, we used a-Cyano-4-hydroxycinnamic acid matrix and the salts were a mixture of NaCl and KCl. X-ray diffraction (XRD). The samples were scanned at a speed of 8 °C/min at ambient temperature using an X-ray diffractometer  at a generator voltage of 35 kV and a current of 25 mA. The data were collected from 2° to 40° intervals.Thermogravimetric analysis (TGA) was performed with a NETZSCH 209 F1 thermal analyzer  at a heating rate of 10 °C/min under nitrogen and air atmosphere, respectively. The temperature ranged from 40 to 800 °C.Differential scanning calorimetry (DSC) curves of the EP composites were measured using a Netzsch 204 F1 differential scanning calorimeter  with a pressure cell. Samples (5–10 mg) were tested at a heating rate of 10 °C/min and results from the second heating were in the range of 35–300 °C. The dynamic mechanical(DMA) properties of the cured blends were performed using a METTLER TOLEDO SDTA861 instrument  with a sample dimension of 5 × 5 × 3 mm in a controlled strain tension mode and a temperature ramp rate of 4 °C/min from −70 to 200 °C at a frequency of 1.0 Hz.The limiting oxygen index (LOI) was obtained using the standard ASTM D 2863 procedure, which involves measuring the minimum oxygen concentration required to support candle-like combustion of plastics. An oxygen index instrument  was used on barrel-shaped samples with the dimensions of 100 × 6.5 × 3 mmVertical burning tests were performed using the UL 94 standard on samples with the dimensions of 125 × 13 × 3.2 mm with the CZF-5A horizontal vertical burning tester by Jiangning Analytical Instrument Factory . In this test, the burning grade of a material was classified as V-0, V-1, V-2, or NR (no rating), depending on its behavior (dripping and burning time).Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) images were taken with a Hitachi SU8020  with a 20 kV accelerating voltage. The samples were coated with thin layers of gold to make the surface conductive. 2, according to the ISO 5660 protocol, using a Fire Testing Technology apparatus  with a truncated cone-shaped radiator. The specimen (100 × 100 × 3 mm) was measured horizontally without any grids. Typical results from the cone calorimeter were reproducible within ±10%, and the reported parameters were the average of three measurements.Cone calorimeter measurements were performed at an incident radiant flux of 50 kW/m1H NMR . Moreover, the molecular quality of P-ala was obtained in Different synthesis methods can be used to prepare benzoxazine monomers. Agag and Takeichi prepared monomers with a solventless method ; However1H NMR . The res−1 disappeared after SPOSS was modified by benzoxazine. The chemical structure of SPOSS-BOZ was confirmed by 1H NMR , 0.93 ppm (Peak a), and 1.88 ppm were caused by the seven isobutyl hydrocarbon substituents of the POSS. Next, we magnified the spectra of the allyl moiety  has been prepared using different methods ,33. Howey 1H NMR . The resl moiety B. FurtheTi, Tp, and Tf were 144 °C, 210 °C, and 240 °C, respectively, and the resin system started to react in the range of 144 to 210 °C. Combined with the relevant data [−1 and 916 cm−1 decreased due to the allyl and the characteristic peaks of the oxazine ring respectively, which indicated that the ring-opening reaction and polymerization of the oxazine ring had taken place. Meanwhile, the peak at 3203 cm−1 was due to the phenolic hydroxyl group that occurred at 160 °C/1 h. With curing proceeding, the peak at 1224 cm−1 vanished because the epoxy group decreased, whereas the peak at 1012 cm−1 increased due to the ether bond. The reaction between the phenolic hydroxyl group and epoxy group was found. Therefore, these results proved that the copolymer had been completely polymerized [The thermally activated polymerization reaction of the copolymers was also studied by non-isothermal DSC, as shown in ant data , the curTGA in the nitrogen and air atmospheres, derivative thermograms, and tan δ curves of the nanocomposites are shown in Tg) of the nanocomposites was slightly lower when compared with the BOZ:E matrix. As the POSS cage structure consisting of Si atoms and O atoms was bulky, this hindered the formation of the benzoxazine/epoxy resin cross-linked network to a certain extent and reduced the crosslink density [2 yield that was produced by the thermal degradation of POSS. The char yield in the air atmosphere was far less than in N2. As can be seen from the thermograms, not only in the N2 but also in the air atmosphere was the weight loss curve of the BOZ:E matrix not significantly altered by the presence of SPOSS. Similar weight loss traces were found from the TGA profiles. The TGA clearly showed that the 5% mass loss temperature (T5%) of SPOSS-PBZ-E nanocomposites was slightly lower than the BOZ:E matrix except for the modified composites with a 20 wt % addition of SPOSS-BOZ in the N2 atmosphere Therefore, the decrease in T5% and Tg was consistent. At a later period of the degradation process, the degradation rate slowed down with the increased POSS content and the Tpeak of all the SPOSS-PBZ-E hybrid nanocomposites almost remained unchanged with respect to that of the BOZ:E matrix. The steric hindrance and interaction with the polymer chains caused by the incorporation of the POSS cages may result in a limit to the moving of polymer chains during the thermal degradation and lead to a small increase in thermal stability. Moreover, the thermal stability of the inorganic component (POSS) was very good and, during combustion, the POSS units could form a ceramic superficial layer because of the low surface energy of the siloxane structure of POSS [2 and 2.8% in air, respectively. From the DMA experimental data, the glass transition temperature , and cotton ignited by flaming particles or drops. The composites of PBZ-E without SPOSS-BOZ and 5% SPOSS-BOZ burned readily and had no rating; however, even with an incorporation of 10% of SPOSS, the burning behavior of blends altered and there was no dripping. The UL 94 V-1 rating was obtained and the total afterflame time (t1 + t2) was reduced in comparison to the 5% POSS-BOZ-E. During combustion, the SPOSS-BOZ nanocomposites formed a dense SiO2 layer that could prevent the penetration of combustion. When the POSS content cannot reach this level, an effective SiO2 layer will not be formed during combustion. The flame retardancies of the SPOSS-BOZ-E composites are presented in Cone calorimetry is used as a more promising technique to compare and evaluate the fire performances of polymeric materials. The fire performances of the composites were compared using time to ignition (TTI), peak heat release rate (pHRR), average HRR (aHRR), total heat release (THR), total smoke production (TSP), and total smoke release (TSR). These parameters are reported in TTI is used to determine the influence of flame retardant on ignitability. As shown in 2, and the pHRR values of the 5% SPOSS-PBZ-E, 10% SPOSS-PBZ-E, 15% SPOSS-PBZ-E, and 20% SPOSS-PBZ-E were 957, 962, 942, and 769 kW/m2, respectively. It is clear that SPOSS-BOZ apparently hinders the decomposition of the composites and improves the flame retardancy. The addition of 20 wt % SPOSS-BOZ caused reductions in the pHRR, aHRR, and THR values by about 33%, 27%, and 25%, respectively  was synthesized by the thiol-ene (click) reaction. The curing process of SPOSS/polybenzoxazine/epoxy has multiple polymerization mechanisms according to the oxazine ring-opening and epoxy resin (EP) polymerization, which was characterized by FTIR, and showed that the phenolic hydroxyl group was formed after the ring opening of the oxazine ring, which promotes the ring-opening polymerization of epoxy. The X-ray diffraction analysis and the SEM micrographs of the SPOSS-PBZ-E nanocomposites indicated that SPOSS was chemically incorporated into the hybrid nanocomposites in the size range of 80–200 nm. The thermal properties and the char yield of the nanocomposites increased, and the highest maximum rate of weight loss decreased with respect to the addition of the SPOSS-BOZ organic–inorganic hybrid. At the same time, the flame retardancy of nanocomposites was improved when compared with the pure benzoxazine/epoxy blend. When the amount of SPOSS reached 10 wt % or more, the UL 94 vertical burning rating reached V-1, and when the SPOSS content reached 20 wt %, both the thermal stability and the flame retardancy of the curing system were improved. In the cone calorimeter testing, the addition of SPOSS-BOZ hindered the decomposition of the composites and the addition of 20 wt % SPOSS-BOZ caused reductions in the pHRR, aHRR, and THR values of approximately 33%, 27%, and 25%, respectively. Then, the morphologies of the chars were also studied by SEM and EDX, which indicated that during the combustion process of the nanocomposites, the POSS nanoparticles accumulated on the surface and formed a ceramic layer in the exterior char residue.


Contrary to some modeled reconstructions, our findings indicate an asymmetric LIS topography with higher ice on Keewatin Dome (~3200 m) and thinner ice in the prairies along the Plains divide (1700–2100 m) during LGM. The resiliency of icy permafrost to past warm intervals preserved relics of the LIS; these ice-marginal landscapes, now poised for thaw, should uncover more valuable clues about the conditions of the last major ice sheet on Earth.The Laurentide Ice Sheet (LIS) occupied a large part of North-America during the late Pleistocene. Determining the proper surface geometry and elevation of the LIS is of central importance to estimate global changes in sea-level and atmospheric circulation patterns during the late Pleistocene and Holocene. Despite largely disappearing from the landscape during the late Holocene, LIS remnants are found in the Penny and Barnes ice caps on Baffin Island (Canada) and ongoing permafrost degradation has been exposing relics of the LIS buried along its northern margin since the late Pleistocene. Here, we use the δ The models predict largely conflicting ice configurations, from a single dome centered near Hudson Bay7 to an ice sheet with multiple ice accumulation centers8, with significant differences in ice elevations (in the order of ±1000–2000 m in some places). These different LIS configurations translate into large uncertainties in modeling late Wisconsinan climate9 and melt-water contribution8. A reason for the large uncertainties in LIS topography is that the glacio-isostatic models are being calibrated to relative sea level curves and paleo lake shorelines  and subsequent models are adjusted as more of these data are becoming available12. However, to improve our assessment of the impacts of the LIS to global changes in climate and sea-level during late Pleistocene and Holocene , determination of its surface geometry and elevation from other methods is required, especially from actual remnants of the LIS.The maximum and recessional limits of the LIS have been defined using empirical data18O-elevation relation. This approach was used to reconstruct Holocene changes in ice surface elevation for the Greenland Ice Sheet and Agassiz Ice Cap17, with the findings used to infer an increased contribution from Greenland Ice Sheet to sea level change17. For the former North American late Wisconsinan ice sheets, this approach is challenged by the fact that the ice had largely disappeared from the landscape by 3–4 ka BP; nonetheless, remnants of late Wisconsinan LIS ice are still found in Penny and Barnes ice19 , with ice sourced during the LGM further up the flow-line and connecting it to the higher Keewatin Dome (3200 m)23. These elevations were derived from a glacial model using ideal ice rheology that is insensitive to unknowns parameters, the spatial extent of grounded LIS ice, yield shear stresses and the ice frozen to the bed of Hudson Bay at LGM2; no independent assessment of the reconstructed elevations using the δ18O-elevation relation was made.In glaciated places with poor known boundary conditions , an alternative approach for the reconstruction of elevation of glaciers is to use the δ24. Such sites have been discovered near the northern maximum limit of the LIS, including: (1) Peel Plateau, NWT, Canada; (2) Richards Island, NWT; (3) north-west Victoria Island, NU, Canada; (4) south-west Bylot Island, NU, Canada . The sites extend across the northern margin of the LIS which allows us to determine the elevation at key sectors: Foxe and Keewatin domes, Mc’Clintock and Plains ice divides. Our findings are compared to the various modeled LIS configurations during the late Wisconsinan, including Fisher-1985, Tarasov-2012, ICE5G, ICE6G, NAICE and an alternative version of the Fisher-1985 LIS elevation model2.In addition to the LIS remnants in Penny and Barnes ice caps, climate warming in the Canadian Arctic has been increasing thermokarst activity and buried relics of the LIS are being exposed along headwall of thaw slumps and other natural exposurescluding:  Peel Pla18O records of Penny and Barnes ice caps have been correlated with other ice cores in Arctic Canada and Greenland and cover most of the Wisconsinan glaciation19. Near the bottom of Penny Ice Cap, late Pleistocene-age ice has average δ18O of −31.2 ± 1.2‰  with the overlying Holocene-age ice having average δ18O of −23.7 ± 0.6‰. On Barnes Ice Cap, late Pleistocene-age LIS ice is exposed along the western margin in a distinctive bubble-rich white band that spreads over 200 m22. This band of white ice has average δ18O of −34.3 ± 2.4‰ , much lower than the average of −22.6 ± 1.7‰ in the Holocene-age blue ice18. At Barnes and Penny ice caps, the shift in δ18O between modern and LGM ice (δ18O[modern-LGM] = 12–15‰) greatly exceeds that expected due to changing air temperatures only 27.The four buried LIS sites consist of ice that was stranded or buried shortly after the LIS reached its maximum extent at ~21–17 ka BP28 and within the Tutsieta Lake Phase recessional limit dated to 15 k cal yr BP29. The ice sheet had receded from the region by 13.5 k cal yr BP based on 14C ages obtained from remains of steppe bison near Tsigehtchic30. In this region, a major ice lobe flowed north-northwest along the Mackenzie Valley with the Mackenzie Through and Bear Lake ice streams likely bringing LIS ice from the more southerly Plains ice divide14.On the Peel Plateau Fig. , buried −1). The white ice also has the lowest δ18O values , whereas the other three ice types have slightly higher δ18O values, ranging from −31.2 to −27.3‰  clear ice with mm-size spherical gas inclusions; (2) sub-vertically banded clear ice and fine sediments; (3) bubble-poor blue ice; (4) white ice rich in spherical gas inclusions, similar to the bubble-rich white ice band on Barnes Ice Cap. The four ice types were characterized by a Ca-Na-Cl geochemical facies  with low cation contents. Together with the stratigraphic context, the ice was inferred by ref33 to be buried LIS ice .Along the shore of YaYa Lake, massive ice was identified by radar and borehole drilling beneath 7-m of glaciofluvial sand34. Former ice margins suggest that the north-west sector of Victoria Island represented the maximum extent of the LIS8; however, a revision of the extension of the ice based on 14C of marine molluscs suggest that the LIS extended across Banks Island35, such that northern Victoria Island and the Jesse Moraines along the east coast of Banks Island represent a recessional phase of the LIS36. The LIS likely occupied north-west Victoria Island near 21–18 ka BP and the ice had receded by 14–12 ka BP36. On the Prince Albert Peninsula, the LIS originated from the Mc’Clintock ice divide and was likely transported by the Mc’Clintock Channel and possibly the Collinson Inlet ice streams14.On the Prince Albert Peninsula on north-west Victoria Island, late Wisconsinan tills are cored by buried LIS ice34 identified buried LIS ice beneath 1-m thick till extending 60–450 m laterally. The englacial ice, which contained a small amount of dispersed sediments, had a sub-vertical banding with alternating bubble-rich and bubble-free ice and its δ18O averaged −30.1 ± 1.4‰  with ice flowing northward from the Lancaster Sound ice stream40. Radiocarbon age of shells in nearby marine sediments indicate the region was deglaciated near 9860 ± 140 14C BP41.Buried LIS ice was discovered in the Qarlikturvik Valley on southwest Bylot Island37. The ice had a clear to whitish appearance, similar to the late Pleistocene-age white ice band on Barnes Ice Cap. The buried ice contained many small spherical and coalescing gas inclusions. The ice was dominated by Ca2+, Na+, Mg2+ cations and their concentration was low and within the range of ice sampled from the nearby C-93 glacier . Further, the ~4‰ difference between the δ18O record of the LIS remnants cannot solely be explained by regional temperatures or major moisture source differences because the δ18O composition of modern precipitation is strongly zonal and varies by less than 1‰ along latitudes of 67–72°; late Pleistocene δ18O precipitation records from 154 empirical proxy data showed a similar zonal distribution43. However, like Penny and Barnes ice caps20, the difference in Δ18O[modern-late Wisconsinan] for the buried LIS sites is higher than expected for their latitudes  to a deep ice core at the head of its flow line near the center of the ice sheet (Pakitsoq site and GISP2 ice core)26; such ice transport and preservation of δ18O record was also incorporated in 3D isotope stratigraphy models44 . The modern δ18O-elevation slope cannot be directly transferred to different climate periods, but empirical proxy data suggest that the δ18O-elevation relation during the late Pleistocene changed by −0.05‰43, providing a late Pleistocene δ18O-elevation relation of −0.67 ± 0.03‰ per 100 m. The increase in slope value during the colder late Pleistocene is similar to that reported from modern-day Antarctica . Applying the late Pleistocene δ18O-elevation correction and accounting for the 5–7‰ shift in δ18O of precipitation during the late Wisconsinan along the latitudes of our sites  allows us to define the LIS surface elevation in their source area during the late Wisconsinan  suggest surface ice elevations of 1900–2000 m and 2500 ± 100 m. For Barnes Ice Cap, the late Wisconsinan and LGM δ18O values are much lower , suggesting surface ice elevations for most of the last glacial period of 2200–2400 m and near 3200 ± 100 m during the LGM , the ice likely originated at similar elevation to most of the last glacial ice preserved in nearby Barnes Ice Cap (2150–2350 m). The ice at Barnes and Penny ice caps and on south-west Bylot Island would have been sourced from Foxe Dome for much of the late Wisconsinan, hundreds of km from their modern location. The very low values near −40‰ on Barnes Ice Cap and near −36‰ on Penny Ice Cap would suggest ice sourced at LGM at higher elevation and connecting it to the 3200 ± 100 m Keewatin Dome23  were measured from foraminifer shells in the Gulf of Mexico and linked to LIS meltwater sourced from the Keewatin Dome draining through the Mississippi River watershed51. On north-west Victoria Island, the buried LIS ice (δ18O = −30.1 ± 1.4‰) that flowed from the Mc’Clintock ice divide likely originated from ice sourced at surface elevations of 1500–1900 m. Finally, the buried LIS ice on the Peel Plateau (δ18O = −31.2 ± 0.6‰) and Richards Island (δ18O = −32.4 ± 1.1‰) suggests ice sourced from the Plains ice divide at surface elevations of 1700–2100 m. The Peel Plateau and Mackenzie Delta region have its highest divide at the Plains divide because the transition between the hard-frozen beds of the Canadian Shield and the deforming soft-beds of the prairies translates into a very sharp change in flowline direction 27, we performed a frequency distribution analysis of the late Pleistocene age δ18O records of GISP2, Penny and Barnes ice caps . Additionally, the variance in δ18O for the late Wisconsinan ice was assessed for the GISP2 and Penny δ18O records and standard deviations of 1.1 to 1.4‰ were calculated. As such, random sampling of buried late Wisconsinan ice does not have δ18O values that would deviate greatly from LGM-age ice . Increasing the uncertainties in δ18O to 3‰ would translate into an elevation error of 500 m.The δaps Fig. ; the lik18O records of six LIS remnants sites indicate an asymmetric LIS during the LGM, with ice surface elevations near 2200–2400 m on Foxe Dome, 3200 ± 100 m on the Keewatin Dome and 1700–2100 m along the Plains ice divide  with the mapped glacial limits in the south-eastern Mackenzie Mountains where the LIS reached maximum elevation of ~1550 m52 (descending in elevation to 750 m in the Richardson Mountains)28; (2) surface exposure age near 21 kyr for elevations >1400 m in the western Mackenzie Mountains53; and (3) reported δ18O values from other buried glacier ices and segregated ices in northwestern Canada, which are all greater than −34‰ (mainly in the −32 to −28‰ range)54. The ICE5G, ICE6G and NAICE models generally predicted too much ice in the Keewatin Dome and/or Plains ice divide. Given some uncertainties in our approach and in geophysical models, we highlight differences in the order of 1000 m between these various paleo-elevation reconstructions at LGM. For example, the ICE5G model55 has similar ice elevations for the Foxe Dome and along the Mc’Clintock and Plains ice divides, but it has a much thicker Keewatin Dome (~4000 m). Conversely, the ICE6G model6 has similar ice elevations for the Keewatin and Foxe domes, but increased ice elevation along the Plains ice divide (2800–3200 m). The NAICE model4 also has much higher ice elevation along the Plains ice divide (~2500–3000 m).Together, the paleo-elevation reconstruction derived from the δide Fig. , and the18O records from LIS remnants in glaciers and permafrost to derive surface elevation of the LIS provides valuable constraints to modeling efforts. Our δ18O-derived paleo-elevations have decent fit with the Fisher-1985 glacial model and the Tarasov-2012 glacio-geophysical model and supports the estimated LIS ice volume at LGM of ~21.1 × 106 km3 . Some of the latest modeled glacio-geophysical LIS elevation reconstruction are generally found to be over-estimating its surface elevation near the LGM on the Keewatin Dome and along the Plains ice divide, although these may also relate to uncertainties in our ice-age estimates and the dynamic nature of the LIS. There are probably other remnants near former ice-marginal extents of past ice sheets in North-America and Siberia that contain information that would help reconstruct their interior geometry and global-scale impacts. Given that the ongoing climate warming and associated thermokarst is exposing new sites that potentially contain LIS relics in permafrost24, these should be found and sampled before they melt away as they contain key information about the conditions prevailing for the last major ice sheet on Earth .The concept of using δ28.Distinguishing between buried glacial ice and other massive ice types in permafrost is usually based on cryostratigraphy and combines detailed physical, geochemical and isotopic measurements of the ice and may also include analyses of occluded gases, while its chronology usually relies on the age of the surrounding sedimentsThe buried ice on the Peel Plateau and Bylot Island were sampled in summer 2016 and 2012, respectively. At both sites, a cryostratigraphic approach was used to describe the exposed ice and the overlying sediments. Samples of ice were collected using either an ice pick or a portable core-drill. All samples were shipped and stored in our laboratories until analyses.18O/16O) and hydrogen (D/H) were determined using a Los Gatos Research high-precision liquid water analyzer coupled to a CTC LC-PAL autosampler. The results are presented using the δ-notation (δ18O and δD), where δ represents the parts per thousand differences for 18O/16O or D/H in a sample with respect to Vienna Standard Mean Ocean Water (VSMOW). Analytical reproducibility for δ18O and δD was ±0.3‰ and ±1‰, respectively.The stable isotope ratios of oxygen  at the University of Ottawa. Solutes are expressed in mg L17; (2) the δ18O-elevation relation adjusted for late Pleistocene conditions43; and (3) the concept of an ice sheet flow-line model that predicts that the margins of ice sheets contain ice that originate from their local accumulation area at much higher elevation56.The approach used to derive the surface elevation of the LIS during the late Wisonsinan for sites along its northern margin combines: (1) the stable isotope-based paleo-altimetry method used to reconstruct Holocene changes in surface elevation of the Greenland Ice Sheet and Agassiz Ice Cap56. The latter was observed on Greenland Ice Sheet where the δ18O record of ice sampled along a horizontal transect from the western edge (Pakitsoq site) was remarkably similar to that of the GISP2 δ18O ice core record situated near the center of its accumulation area due to ice resurfacing in the ablation zone26. The ice-flow on Greenland Ice Sheet was successfully modeled using a 3D transport model fitted with δ18O data from ice cores44. In fact, both Pakitsoq and Barnes Ice Cap exposed late Pleistocene-age ice near their margins over a horizontal distances of ~200–500 m; with Holocene-age ice above it . The steeper slope in Arctic region is attributed to colder-drier conditions and near adiabatic lapse rate with little mixing of air masses as they gain elevation; this is particularly the case on large ice sheets. In the lower latitudes, vertical advective mixing is much stronger and the mixing of air masses reduces the δ18O-elevation slope.The δ18O of precipitation during the late Wisconsinan is largely affected by changes in air temperature, site elevation, seasonal distribution in amount of precipitation and atmospheric circulation, the latter two relate to changes in moisture source. The effect of a change in zonally-averaged air temperature during the late Wisconsinan, including a shift in the position of sea-ice front, was associated with a latitudinal-dependent shift of 3–10‰ in δ18O of precipitation, with the higher latitudes experiencing a greater shift21. This trend was also observed in 154 late Pleistocene δ18O proxy records43. The modern δ18O-elevation slope cannot be directly transferred to other and different climate periods, such as the cooler late Pleistocene. However, based on empirical data, ref.43 suggested that the global δ18O-elevation relation during the late Pleistocene changed by −0.05‰ per 100 m. We therefore use a late Pleistocene δ18O-elevation relation of −0.67 ± 0.03‰ per 100 m for Arctic regions. This late Pleistocene δ18O-elevation slope is nearly identical to that observed in the colder Antarctica . Changes in seasonal distribution in amount precipitation and atmospheric circulation can be assessed from the D-excess parameter, which can provide clues into changes in distance to moisture source 57. Examining the D-excess record of Penny Ice Cap  and north-western  sectors of the LIS have more uncertainty. The D-excess values of the buried white ice on the Peel Plateau are within the range of that on Penny and Barnes ice caps and also of that calculated in modern-day precipitation at nearby sites 58. Therefore, if there was a change in distance to moisture source, it is not reflected in the D-excess values. However, to truly assess source distribution changes, one would have to do a trajectory tracing of ancient climates over the proposed ice sheet geometries. The biggest unknown relates to the age of the sampled buried ices. We ascribed to the four buried LIS ice a near LGM-age (29–19 ka BP)27 based on the existing chronologies of the timing of advance and retreat of the LIS in these areas. The LIS reached its maximum extent at ~21k-17 ka BP8 and had receded from these regions by 10–14 ka BP1. This timing of retreat of the LIS at the sites provides a minimum age constraints for the sampled ice with the actual age at each site being dependent on the ice flow rate in the ice sheet. The uncertainty in the age of the sampled buried LIS sites is the biggest uncertainty when comparing reconstructed paleo-elevations derived from the δ18O-elevation relation to those from various glaciological and geophysical models that have reconstruction for specific times because the LIS topography is evolving over time.The δCap Fig.  shows va2. It uses a simple plastic ice model that is insensitive to unknown parameters and uses as inputs the margins of the ice sheet, present-day topography and an assumed yield shear stress. References60 showed that elevation estimates using this model are good to 6% on present-day Greenland, with ridges and domes having smaller errors. No assumptions are made in advance about ice divides and accumulation centers. At the time of its publication in 1985, some uncertainty still existed about northern and northeastern LIS margins, thus ref.2 presented the LIS surface elevation for the minimum-concept of grounded ice margins. The maximum-concept of ice margins was produced at that time also and is shown in Fig. 6 km3). Note that the Cordilleran and Innuitian ice sheets have not been included in these reconstructions so their volumes are not included.The reconstruction of late Wisconsinan LIS surface elevation at 18 ka is from ref.Supplementary information