Elevation of mTOR activity in injured neurons has been proposed as the primary underlying mechanism. Here we demonstrate that PTEN−/− also abrogates the inhibitory activity of GSK3 on collapsin response mediator protein 2 (CRMP2) in retinal ganglion cell (RGC) axons. Moreover, maintenance of GSK3 activity in Gsk3S/A knockin mice significantly compromised PTEN−/−-mediated optic nerve regeneration as well as the activity of CRMP2, and to a lesser extent, mTOR. These GSK3S/A mediated negative effects on regeneration were rescued by viral expression of constitutively active CRMP2T/A, despite decreased mTOR activation. Gsk3S/A knockin or CRMP2 inhibition also decreased PTEN−/− mediated neurite growth of RGCs in culture and disinhibition towards CNS myelin. Thus, the GSK3/CRMP2 pathway is essential for PTEN−/− mediated axon regeneration. These new mechanistic insights may help to find novel strategies to promote axon regeneration.Knockout of phosphatase and tensin homolog (PTEN Loss of the PTEN protein, an event that activates PI3K/AKT signaling, in neurons enables axon regeneration. Now Leibinger et al show in retinal ganglion cells and during optic nerve regeneration in mice, that this is mediated by inactivation of the AKT substrate GSK3, which in turn releases axonal CRMP2 from GSK3-mediatated inhibition. Therefore, CNS injuries are often associated with an irreversible functional loss, such as blindness after optic nerve injury or paralysis and sensation loss after spinal cord damage. However, research over the last two decades has developed several approaches to improve the intrinsic ability of adult CNS neurons to regrow axons. For instance, genetic or shRNA-mediated neuronal depletion of phosphatase and tensin homolog (PTEN), which activates the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway in respective neurons, enables axon regeneration over considerable distances into the injured optic nerve or spinal cord8. In the optic nerve, these effects can be mostly mimicked by expression of constitutively active AKT10. Active AKT modulates several downstream regulators by phosphorylation, such as tuberous sclerosis complex (TSC) or glycogen synthase kinase 3 (GSK3)14. Regarding Pten knockout (PTEN−/−)-mediated axon regeneration, the PI3K/AKT/TSC signaling pathway, which activates the mammalian target of rapamycin (mTOR) has been made accountable for the strong neuroprotective as well as axon regeneration promoting effects. Consistently, treatment with the (mTOR) inhibitor rapamycin partially compromises the beneficial effect induced by PTEN−/− on neuroprotection as well as regeneration4. However, targeting effectors directly downstream or even regulators upstream of mTOR, such as S6 kinase1 (S6K1), E4 binding protein (E4BP) or Ras homolog enriched in brain (Rheb), were able to induce neuroprotection, but failed to mimic the entire PTEN−/− effect on axon regeneration18. Interestingly, a recent study even proposed an adverse effect of S6K1 activity for spinal cord axon regeneration19. These reports suggest that the regenerative effects of PTEN−/− do not solely depend on mTOR, but also other downstream effectors of AKT.Mature neurons of the central nervous system (CNS) cannot normally regenerate injured axons due to an inhibitory environment of axonal growth cones and an insufficient intrinsic growth ability21. IS activates the JAK/STAT3 pathway but has little effect on the PI3K/AKT pathway26. Genetic or shRNA-mediated conditional knockdown of GSK3β (GSK3β−/−) markedly promotes IS-induced optic nerve regeneration by reducing inhibitory phosphorylation of axonal collapsin response mediator protein 2 (CRMP2) in RGCs, demonstrating that GSK3/CRMP2 signaling is highly relevant for optic nerve regeneration and most likely also other parts of the CNS27.Another approach to promote optic nerve regeneration is the stimulation of RGCs by cytokines of the interleukin 6 (IL-6) superfamily, which are released from activated retinal glial cells after an inflammatory stimulation (IS) induced by lens injury−/−-mediated axon regeneration. We found that PTEN−/− releases CRMP2 from GSK3-dependent inhibition in RGC axons. Moreover, PTEN−/− mediated optic nerve regeneration was significantly reduced by inhibitory CRMP2 phosphorylation in Gsk3S/A knockin mice, but rescued by viral expression of constitutively active CRMP2T/A, indicating its essential role in this context. As the number of publications using PTEN depletion mediated neuronal regeneration is still increasing28, these mechanistic findings are highly relevant for data interpretation and may facilitate new regenerative strategies mitigating the high cancerogenic risk of PTEN−/− or inhibition in the future.As GSK3 is a substrate of AKT, the current study tested the hypothesis that the GSK3/CRMP2 pathway could be an essential part of PTEN27. As neuronal Pten knockout (PTEN−/−) triggers AKT activation much stronger than ONC  or respective control virus (AAV-GFP) (PTEN+/+). Some animals were subjected to additional ONC 3 weeks after viral application. Consistent with previous data27, retinae of uninjured PTEN+/+ mice showed only a few pGSK3α- and pGSK3β-positive RGCs in retinal cross-sections, while ONC significantly increased numbers and intensities of positively stained RGCs when evaluated 5 days after surgery  causes moderate AKT activation and subsequent inhibitory phosphorylation of GSK3α  and GSK3β  in RGCs of adult miceONC Fig. , we firsery Fig. . PTEN−/−ect Fig. . The absies Fig. . Westernged Fig. . AKT phoone Fig.  and neitone Fig. , therebyols Fig. . Thus, bPten knockout-stimulated GSK3 inhibition was mTOR activity-dependent  or wild-types and received intravitreal injections of either AAV-GFP (PTEN+/+) or AAV-Cre (PTEN−/−). Three weeks after that, all animals were subjected to ONC. The resulting regenerative states of RGCs were evaluated either by quantification of spontaneous neurite growth in cultures that were prepared 5 days after ONC   Fig. . PTEN−/−ONC Fig. , demonst31. We, therefore, explored the possibility of whether GSK3β inhibition (phosphorylation) may indirectly contribute to the effect of PTEN−/− on mTOR activity  to determine the mTOR activity in RGCs15. Consistent with previous reports32, co-staining against βIII-tubulin showed ~15% of pS6-positive RGCs in wt retinae and an increase in the number and intensity of pS6-positive RGCs (~40%) as well as increased RGC soma size in PTEN−/− mice which was previously described as a consequence of S6K1 activity downstream of mTOR15  double knockin did so ity Fig. . To thisR15 Fig. . While O−/− Fig. . InteresGCs Fig. . Consistpes Fig. . HoweverGsk3α and/or Gsk3β knockouts have the opposite effect. To this end, we generated Gsk3βf/f/Ptenf/f mice, which along with Gsk3αf/f, Gsk3βf/f, Gsk3(α/β)f/f, and Ptenf/f animals received intravitreal injections of either AAV-Cre or AAV-GFP resulting in either PTEN−/−, GSK3α−/−, GSK3β−/−, GSK3(α/β)−/−, GSK3β−/−/PTEN−/−, or GSK3β+/+/PTEN+/+ (wt) RGCs. Half of the animals were additionally subjected to ONC and their retinae collected after 5 days. Consistent with previous reports32, ONC reduced mTOR activity in wt animals  by GSK3β  levels in proximal segments of optic nerves (either crushed or uninjured) from wt, PTEN−/−, and the different PTEN−/−/GSK3S/A genotypes  as described previously27. Western blot analyses of nerve lysates showed similar levels of total CRMP2 protein in all groups  compared with respective PTEN+/+ animals , which is resistant to inhibitory phosphorylation by GSK3β in RGCs of wt mice33. These mice remained either untreated or were subjected to ONC. The percentage of pS6-positive cells in CRMP2T/A expressing RGCs  in PTEN−/−-mediated axon regeneration. To this end, we intravitreally injected either AAV-Cre or AAV-GFP in PTENf/f mice and performed ONC 3 weeks after that. After another 5 days, retinae were used for culture preparation and the regenerative state of RGCs was again assessed by spontaneous neurite growth on a growth permissive (PDL) or inhibitory (CNS myelin) substrate. Cells were cultured in the presence of the CRMP2 inhibitor (R)-lacosamide35 or the mTOR inhibitor rapamycin27 either alone or in combination. Rapamycin-treated groups received pretreatment in vivo 12 days before culturing, while others were treated with the vehicle. PTEN+/+ (wt) RGCs showed moderate spontaneous neurite growth within 2 days in culture   showed similarly reduced neurite growth as LCM treated PTEN−/− cultures on PDL and myelin  Fig. . In contng) Fig. . Intereslin Fig. . MoreoveT/A) could rescue the inhibitory effect of GSK3S/A on PTEN−/−-mediated axon regeneration in vivo  and >90% (Cre) of RGCs, respectively, with a nearly 100% overlap of CRMP2T/A and Cre-co-expression after 3 weeks . Consistent with the hypothesis that the release of CRMP2 from GSK3-mediated inhibition is significantly involved in the mechanism, viral overexpression of constitutively active CRMP2T/A did not additionally enhance PTEN−/−-induced optic nerve regeneration  and even more effectively total GSK3(α/β)−/− in PTEN+/+ mice showed the opposite effect. This GSK3-dependent modulation of S6 phosphorylation was entirely blocked by rapamycin, indicating that it must have been regulated upstream of mTOR. Although modulation of mTOR activity by GSK3 has not yet been described in neurons, in non-neuronal cells active GSK3 was shown to phosphorylate and activate TSC2, which subsequently leads to Rheb and mTOR inhibition31. Whether a similar mechanism underlies the effects in RGCs (neurons) needs to be investigated in the future. On the other hand, rapamycin treatment did not affect PTEN−/−-mediated phosphorylation of GSK3α or GSK3β, thereby excluding a potential feedback loop of S6K1 on both isoforms in RGCs as previously shown in mouse embryonic fibroblasts40. Although the underlying mechanism awaits further investigation, the identified regulatory effect of GSK3 on mTOR activity in RGCs contradicts a previous study, suggesting that GSK3β−/− does not affect pS6 levels in RGCs and that both substrates act entirely independently from each other10. The differing findings of both studies could be because Guo et al. quantified pS6 staining intensities of RGCs on selected retinal sections, whereas we quantified RGCs in entire retinal whole-mounts. In addition, we used Western blot analysis to verify our findings and contrary to Guo et al., our quantification method in retinal whole-mounts showed a reduction of pS6 levels in RGCs upon ONC, which is consistent with the existing literature32. Therefore, full PTEN−/−-mediated activation of mTOR occurs to some extent indirectly via its inhibitory effect on GSK3 and both AKT targets cannot be regarded as independent from each other. In fact, the incomplete mTOR activation in PTEN−/−/GSK3S/A mice may also be the reason why the overexpression of CRMP2T/A only approximately rescued these animals from reduced optic nerve regeneration compared with PTEN−/− alone −/− induced higher pS6 levels, soma sizes and were neuroprotective suggests that mTOR only above a certain activity level affects neuroprotection and soma size. Consistently, only the double knockin Gsk3(α/β)S/A, but not the single Gsk3βS/A knockin compromised PTEN−/− dependent soma size, which was previously described as a consequence of S6k1 activity downstream of mTOR15. Also, the higher pS6 levels and soma sizes observed after PTEN−/− compared with GSK3(α/β)−/− correlated with stronger neuroprotection and may be due to the additional mTOR activation via the PI3K/AKT/Rheb axis.Knockout of GSK3α did not measurably alter mTOR activity in non-injured RGCs but prevented its inactivation upon axotomy. As GSK3α−/−, the release of active CRMP2 appears to be essential for axon regeneration. In support of this idea, TSC1−/− or expression of constitutively active Rheb or S6K1 reportedly mimicked PTEN−/−-mediated survival of axotomized RGCs, but fail to promote substantial optic nerve regeneration17. Consistently, we found that rapamycin, although completely preventing S6 phosphorylation, did not reduce PTEN−/−-induced neurite growth of RGCs cultured on a growth permissive substrate and had only a small effect on disinhibition toward CNS myelin  and was not further affected by additional rapamycin. Moreover, the inhibitory effect of LCM in PTEN−/− was similarly strong as observed in Pten−/−/Gsk3S/A knockin mice where lacosamide did not further compromise neurite elongation of RGCs from these animals. Most importantly, expression of constitutively active CRMP2T/A almost entirely rescued impaired optic nerve regeneration in PTEN−/−/GSK3βS/A animals despite reduced mTOR activity , RAS or extracellular signal-regulated kinase (ERK)42. Whether co-expression of constitutively active CRMP2 and mTOR activation, for example by constitutively active Rheb, can fully mimic the effect of PTEN needs to be addressed in the future. Nevertheless, activation of downstream targets without affecting PTEN itself is, from a clinical point of view, more feasible, because PTEN inhibition is highly cancerogenic43. Therefore, the finding of a role of the GSK3/CRMP2 pathway in PTEN−/−-mediated axon growth may open new therapeutic strategies for CNS repair.Whether inhibition of the remaining mTOR activity reduces the regenerative outcome in the presence of active CRMP2 was not addressed in the current study. However, as stated above, it was previously demonstrated that rapamycin does not reduce GSK3βSer9Ala, GSK3αSer21Ala, and GSK3αSer21Ala/βSer9Ala double knockin mice  and respective wt C57BL/6,129/Ola mice29 were provided by Prof. Dr. Dario Alessi  and crossbred with PTENf/f mice  provided by Prof. Dr. Raab . GSK3βf/f and GSK3αf/f mice  were provided by Prof. Dr. Woodgett 27. For all experiments, RGC-specific Pten or Gsk3 knockout was induced by intravitreal injection of AAV-Cre into the respective floxed mice f/f) 3 weeks before optic nerve injury or tissue collection as described previously with transduction rates of >90%. All mice were housed under the same conditions for at least 10 days before experiments and generally maintained on a 12-h light/dark cycle with ad libitum access to food and water. All experimental procedures were performed in compliance with ethical regulations and approved by the local animal care committee (LANUV Recklinghausen) and conducted in compliance with federal and state guidelines for animal experiments in Germany.Male and female mice (2–3 months old) were used for all experiments: GSK3β514 (CRMP2T/A) was overexpressed upon intravitreal injection of AAV-CRMP2T/A. In all cases, respective control animals were intravitreally injected with AAV-GFP. For surgery, animals were anesthetized by intraperitoneal injections of ketamine (120 mg/kg) and xylazine (16 mg/kg). The left optic nerve was intraorbitally crushed 1 mm behind the eyeball for 10 s using jeweler’s forceps , as described previously22. Two days before tissue isolation, regenerating axons were labeled by intravitreal injection of 2 µl Alexa Flour 594- or Alexa Flour 488-conjugated cholera toxin β subunit .Depletion of PTEN, GSK3α and/or GSK3β was induced by intravitreal injection of 2 µl AAV-Cre into the, respectively, floxed mice 3 weeks before surgery. CRMP2 lacking the phosphorylation site at T32.The specific mTOR inhibitor rapamycin  or the vehicle  was injected intraperitoneally starting 1 week before surgery. Rapamycin was dissolved at 20 mg/ml in ethanol and diluted in 5% Tween 80, 5% polyethylene glycol 400 in PBS before each experiment, and administered every 2 days3VO4, 1% Triton X-100, 0.1% SDS) with protease inhibitors  and phosphatase inhibitors  using 5 sonication pulses at 40% power . Lysates were cleared by centrifugation in an Eppendorf tabletop centrifuge at 4150 × g for 10 min at 4 °C. Proteins were separated by sodium-dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), using Mini TGX gels  according to standard protocols and transferred to nitrocellulose membranes . Blots were blocked in 5% dried milk in phosphate-buffered saline with 0.05% Tween-20 (PBS-T)  and incubated with antibodies against βIII-tubulin , S9-phosphorylated GSK3β  S21-phosphorylated GSK3α , total GSK3α/β , ribosomal protein S6 , T514-phosphorylated CRMP2 , total CRMP2 , or T308-phosphorylated AKT  at 4 °C overnight. All antibodies were diluted in PBS-T containing 5% bovine serum albumin . Anti-rabbit or anti-mouse immunoglobulin G (IgG) conjugated to horseradish peroxidase  were used as secondary antibodies. Phospho-GSK3α and CRMP2 primary antibodies were detected using conformation-specific horseradish peroxidase conjugated anti-rabbit (IgG) secondary antibody . Antigen-antibody complexes were visualized using an enhanced chemiluminescence substrate (Bio-Rad) on a FluorChem E detection system . Western blots were repeated at least three times to verify results. Band intensities were quantified relative to respective loading controls using ImageJ software.For protein lysate preparation, mice were killed, and retinae or optic nerve segments proximal to the lesion site were isolated and dissected. Respective tissues were homogenized in lysis buffer . Eyes with or without attached optic nerves were removed from connective tissue, post-fixed overnight in 4% PFA solution at 4 °C and subsequently transferred to 30% sucrose for at least 4 h. The tissue was then embedded in KP-cryo compound  and longitudinal sections (14 µm) were cut on a cryostat, thaw-mounted onto charged glass slides  and stored at −20 °C until further use.Retinal whole-mounts were prepared, fixed in 4% PFA for 30 min at room temperature and incubated in PBS containing 2% Triton X-100 for 1 h to improve antibody penetration. RGCs in retinal whole-mounts and sections were visualized using a monoclonal antibody against βIII-tubulin .514-phosphorylated CRMP2 and phosphorylated ribosomal protein S6 were visualized using polyclonal antibodies . Secondary antibodies  included donkey-anti-mouse, anti-goat and anti-rabbit IgG antibodies conjugated to Alexa Fluor 488, 594 or 350 . Sections and whole-mounts were cover-slipped with Mowiol and analyzed using either a fluorescent  or confocal laser scanning microscope .Whole-mounts and sections were immunohistochemically stained with antibodies against S9-phosphorylated GSK3β  S21-phosphorylated GSK3α  and total GSK3α/β . Transduction with AAV-Cre was verified by GFP co-expression or staining of the HA-tagged Cre-recombinase using a polyclonal HA antibody . GFP expression was amplified by staining with a polyclonal antibody . T44, regeneration of axons was quantified over the entire length of the optic nerve and chiasm for all animals as previously described32. In brief, eyes were isolated with optic nerves attached and prepared for histology 21 days after surgery as described above for immunohistochemistry. The number of CTB-labeled axons extending 0.5,1, 1.5, 2, 2.5, 3, and 3.5 mm from the injury site was quantified on at least six nerve sections per animal. For this purpose, pictures of optic nerve sections were taken under ×200 magnification using a fluorescent microscope . Subsequently, axons were counted and normalized to the cross-sectional width of the optic nerve at the respective measuring points. Each experimental group included 6–13 mice.As described before45, for quantification of surviving RGCs, retinal whole-mounts were stained with an antibody against βIII-tubulin . Retinae were divided into four quadrants. In each quadrant, 4–5 non-overlapping pictures were taken using a fluorescent microscope , proceeding from the center to the periphery. The average number of βIII-tubulin-positive RGCs per picture was determined and normalized to an area of 1 mm². Values were averaged per retina and then across all similarly treated animals to obtain a group mean ± SEM. Six to ten retinae were analyzed per experimental group.As described previously4, CRMP2Thr514/Ala, or GFP downstream of the CMV promoter was used. AAV-293 cells (Stratagene) were co-transfected with the respective pAAV-MCS plasmid, pAAV-RC (Stratagene) encoding the AAV rep and cap genes and the helper plasmid pHGTI-Adeno1 encoding E24, E4, and VA. Purification of virus particles was performed as described previously46. Mainly RGCs are transduced upon intravitreal injection of AAV248 as this virus serotype is highly neurotropic49 and RGCs are the first neurons to be encountered by the virus.For recombinant AAV2 (afterwards referred to as AAV) production, pAAV-MCS plasmid  carrying either cDNA for Cre-HA 27, mice were subjected to ONC in vivo. The specific mTOR inhibitor rapamycin  or the vehicle  was injected intraperitoneally starting 1 week before surgery. Five days after ONC, retinal cultures were prepared as described previously50. In brief, tissue culture plates  were coated with poly-D-lysine  for 1 h at room temperature, rinsed three times with distilled water and air-dried. Some of the culture plates were additionally coated with inhibitory CNS myelin extract (200 µl/well), obtained as described previously36. Myelin was applied at a preoptimized concentration of ~12.5 μg/ml and dried overnight. To prepare low-density retinal cell cultures, mice were killed by cervical dislocation. Retinae were rapidly dissected from the eyecups and incubated at 37 °C for 30 min in a digestion solution containing papain  and L-cysteine  in Dulbecco’s modified Eagle medium . Retinae were then rinsed with DMEM and triturated in 2.5 ml DMEM containing B27-supplement  and penicillin/streptomycin . Dissociated cells were passed through a cell strainer  and 300 µl of cell suspension was added into each well. Rapamycin dissolved in DMSO was applied into cultures at 10 nM. The ROCK inhibitor Y27632 (Sigma) was added at a concentration of 10 µM to verify the specificity of growth-inhibitory myelin as described previously32. The previously characterized CRMP2 inhibitor (R)-lacosamide  was used at final concentrations of 5 µM. Retinal cells were cultured for 48 h and then fixed with 4% PFA (Sigma).As described previouslyAfter fixation with 4% PFA for 30 min at RT, cell cultures were processed for immunocytochemical staining with a βIII-tubulin-antibody . All RGCs with regenerated neurites were photographed using a fluorescent microscope  and neurite length was determined using ImageJ software. Also, the total number of βIII-tubulin-positive RGCs with an intact nucleus (6-diamidino-2-phenylindole (DAPI)) per well was quantified to test for potential neurotoxic effects. The average neurite length per RGC was determined by dividing the sum of neurite length per well by the total number of RGCs per well (on average ~500 RGCs). The resulting neurite length per RGC also includes the majority of RGCs which did not grow out any neurite (~90%) thereby representing neurite growth as well as neuritogenesis. Thus, obtained values are much lower than the average axon length of RGCs with extended neurites (~20–200 µm). Cultures were arranged in a pseudo-randomized manner on the plates so that the investigator would not be aware of their identity. Data represent means ± SEM of at least three independent experiments each with four replicate wells per treatment.t-test or analysis of variance (ANOVA) followed by Holm-Sidak or Tukey post hoc test using GraphPad Prism software.Significances of intergroup differences were evaluated using Student's Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Antifreeze proteins and ice-binding proteins have been discovered in a diverse range of extremophiles and have the ability to modulate the growth and formation of ice crystals. Considering the importance of cryoscience across transport, biomedicine, and climate science, there is significant interest in developing synthetic macromolecular mimics of antifreeze proteins, in particular to reproduce their property of ice recrystallization inhibition (IRI). This activity is a continuum rather than an “on/off” property and there may be multiple molecular mechanisms which give rise to differences in this observable property; the limiting concentrations for ice growth vary by more than a thousand between an antifreeze glycoprotein and poly, for example. The aim of this article is to provide a concise comparison of a range of natural and synthetic materials that are known to have IRI, thus providing a guide to see if a new synthetic mimic is active or not, including emerging materials which are comparatively weak compared to antifreeze proteins, but may have technological importance. The link between activity and the mechanisms involving either ice binding or amphiphilicity is discussed and known materials assigned into classes based on this. Current strategies are based on adding large volumes of (low molecular weight) cryoprotective agents such as glycerol or DMSO which enable slow freezing or vitrification.–13 Antifreeze (glyco) proteins, (AF(G)Ps) have been found to improve cryopreservation outcomes, due to their property of ice recrystallization inhibition ,–16 but also potentially by stabilizing membranes.,18 Considering this, there has been significant recent interest in developing synthetic materials to mimic AF(G)Ps. AF(G)Ps have three main properties resulting from their ice interactions; non-colligative depression of the freezing point ), dynamic ice shaping (DIS); IRI activity. The latter (IRI) is of particular interest, as it was discovered in 2003 that synthetic mimics of AFGPs can retain IRI activity, but not thermal hysteresis or DIS, even after major structural modifications, suggesting this property may be easier to mimic (as it may not require ice-face binding) and it has application in cryopreservation.–23 What is clear, however, is that the synthetic mimics tend to be far less active than the natural antifreezes, yet the activity appears to be sufficient to enable beneficial cryopreservation outcomes. In short, it might not be that the most active IRI material is the most useful  and recent results suggest that IRI activity can be induced by more than one mechanism, including those which do not include ice binding.,24Antifreeze, ice-binding and ice-nucleating macromolecules have been discovered in a diverse range of extremophile organisms from fish to insects to plants. These are produced to enable the species to survive by promoting freeze-tolerance and freeze-avoidance and have been extensively reviewed. whereas we intend to include examples of, and activity data for, emerging materials at all activity levels.Considering the interest in introducing IRI activity into synthetic materials, the aim of this article is to provide a concise comparison of the IRI activities of a range of biological and synthetic compounds. Here we use the “splat” assay, which is a common and easily accessible technique to evaluate IRI activity, but by no means the only method. We also summarize the currently known materials with IRI, providing a snapshot of the emerging field. Based on this, we also propose a “rough guide to activity,” grouping materials from potent to very weak activity to enable researchers to decide if a new material is active or not, relative to those already published and especially for non-protein IRI active materials. It should be noted that Koop et al. have proposed a similar map based on growth rate constants, but only included highly active peptide (and PVA) ice growth inhibitors,2 This assay enables quick assessment of IRI activity by monitoring the growth of ice grains that have been nucleated rapidly at low temperatures (≈−80 °C) to ensure only growth (not nucleation) events are probed. This is a kinetic assay, hence shorter time points will always give smaller crystals. It is essential to compare materials under similar conditions to allow reliable assessment of more/less activity. In the present work, 30 min of annealing is used, as this is sufficient for screening, especially for identifying materials with lower or no activity, which quickly trend toward the control. More active IRIs (such as the antifreeze proteins) can inhibit ice growth for hours, but the majority of mimics are far less active than this, however all materials, no matter how active, will only slow ice growth, none can stop it completely. The temperature of the assay and the use of saline are also crucial. There must be sufficient NaCl  to ensure a eutectic phase such that there is liquid water between the boundaries. False positives can occur when using pure water or too low saline, and hence even poly(ethylene glycol) (PEG) which is used as a negative control appears to inhibit recrystallization when conducted in water (see  An adaptation of this method uses concentrated sucrose solutions (>20 wt%) to generate isolated ice crystals. Due to the separation of crystals image analysis is facilitated. Using this method, Koop and co-workers have analyzed the kinetics of ice growth in the context of LSW theory to generate inhibition constants,,28 but it is a slower method than the aforementioned “splat” assay, albeit one that provides detailed insight. Davies et al. have employed rapid freezing of capillaries to measure IRI, with the advantage that the samples can be stored for later use. However, this method is well suited for potent IRI active materials that inhibit all growth (as it is an end-point assay), so it is not suitable for those materials with weak activity as discussed here.The first, and most widely used, method for measuring IRI is the “splat” assay as shown by Knight.ater see . The temThis article contains both new, and previously published IRI activity, focusing on that obtained by the “splat” assay where possible to allow critical comparison.38, AFP type III (AFPIII), PVA, and PEG (as a negative/weakly active) control is plotted in −1, but for PVA20  up to 1 mg mL−1 is required—a 200-fold difference. The AFPIII (which is not glycosylated) showed activity between the two, inhibiting all growth at ≈0.1 mg mL−1. This difference in activity highlights the remarkable properties of AF(G)Ps and the immense challenge of reproducing their function. PEG, which is not reported to have any ice-binding/interacting effects, cannot fully inhibit growth even at 100 mg mL−1. This not only shows the vast range of potential IRI activities, but also highlights the key challenge in the discovery of useful new materials in that any macromolecular material will slow ice growth if a sufficiently high concentration is added.To demonstrate the large range of IRI activity between some commonly used macromolecular inhibitors, the concentration dependence on the MLGS for AFGP There is still uncertainty as to how exactly AFGPs bind ice though, either through their glycans, or the peptide backbone. Hyperactive AFPs, which display a larger thermal hysteresis gap can also bind the basal face.,35 Experimental and modeling evidence supports PVA’s unique activity being due to its hydroxyls precisely matching the spacing on the prism plane of ice, and a balance of enthalpy/entropy compensation leads to increased affinity and hence inhibition–38 due to a multivalent or “zipper” effect  confine the third.This small set alone shows the importance of screening with a negative control, such as PEG, as essentially any material can claim to have IRI activity but the magnitude of this is the crucial parameter. A detailed discussion of AFGP/AFP’s high activity is beyond the scope of this feature article, but in both cases it appears to be due to specific recognition of the primary prism face of ice, which is the fastest growing face under standard conditions.” effect . This al” effect  showing 4 Safranin-O has no obvious structural similarities to AFPs but in aqueous solution self-assembles into fibers which can present a hydrophobic face that is similar to the solution structure of some AFPs. Safranin-O can inhibit all ice growth at just 1 mg mL−1, similar to PVA. Addition of a phenyl group disrupts the self-assembly and turns off the activity, supporting a mechanism where an extended hydrophobic face is essential.The previous section summarizes the activity range of materials with characterized (or hypothesized) ice-binding interactions. As an interesting example of unusual IRI active materials, Drori et al. showed that safranin-O (a dye) is a potent IRI .[43] Saf,45 In solution, the structure of ZrAc produces a coordination polymer with regularly spaced hydroxyls and acetate groups, which could either hydrogen bond to ice  or interact via hydrophobic interactions (as with AFPs). −1. Other metal acetates, to the best of our knowledge, do not have this effect but it does support a hypothesis that the design of supramolecular IRIs is indeed possible, if not easy. There is also a report of graphene oxide inhibiting ice growth in this concentration range . Graphene oxide is also known to affect ice nucleation,48; such dual activity has also been observed for some AFPs.In solution, zirconium acetate (ZrAc) can polymerize to form extended structures and it has been reported to have both ice shaping and IRI activity.5α-galactose (in place of the native disaccharide) and replacing alanine with glycine resulted in peptides that had definite IRI but no thermal hysteresis. This finding suggested that it was possible to achieve potent IRI, but without the need to target a specific plane of ice, that is, there are multiple molecular level mechanisms which can give rise to the same macroscopic effect. The same group has reported a series of modified glycopeptides with potent IRI, with the distance between backbone and glycan being important as well as backbone hydrophobicity.,52 Whilst the molecular binding details are still under investigation, there is overwhelming evidence for AFPs binding to ice faces and strong evidence of molecular level interactions have been determined.,55–59 However, for the facially amphipathic molecules, with no evidence for ice binding, an alternative molecular level mechanism which can give rise to IRI is required. One proposal is that these can sit at the semi-ordered water layer /bulk water interface, rather than directly interact with ice faces.,62 If the compounds can disorder this region, they will not fit well with bulk water, nor semi-ordered water later and hence the barrier to adding more water to the ice crystal is higher and slows recrystallization.The previous examples are materials which have characterized ice-binding activity, either from direct measurements, or by observation of ice crystal shaping, and in most cases this affinity for the primary prism plane of ice is what drives their observable activity. In 2003, Ben et al. discovered that simplification of the AFGP structure to present only  Lysine-based surfactants with increasing hydrophobic modifications show increased IRI activity, and clearly have no ice-binding site. These carry a net charge confirming that a sugar unit is not an essential moiety for IRI.,65 It should be noted, however, that removing the glycan unit from an AFGP completely removes its ability to bind to ice effectively, as shown by Nishimura et al., which demonstrates that conclusions drawn for one class of IRI may not be valid for others.Ben and co-workers synthesized a range of alkylated galactose derivatives and assessed their IRI. Increasing the alkyl chain length from seven to nine carbon atoms lead to an increase in activity  until mi Using the “splat” assay, Nisin shows IRI at low pH , which was erased at higher pHs. This provided more evidence for the hypothesis that the presentation of a spatially segregated hydrophobic face is crucial for activity. The actual IRI seen, however, was rather weak requiring 5 mg mL−1 to inhibit 50% of ice growth. AFPs and AFGPs also show facial amphiphilicity and simulations suggest this facial amphiphilicity is crucial for AFGPs but not necessarily for AFPs.,69 These experiments provided crucial data that a second structural feature, after ice binding, can be used to introduce IRI into a synthetic material; spatially segregated hydrophobic/hydrophilic domains, but without micellization. Mitchell et al. further used self-assembling metallohelices to probe this concept. The helices show patchy hydrophobicity and no obvious ice-binding sites, yet can inhibit ice growth completely at around 5 mg mL−1, making them one of the more potent synthetic inhibitors reported  semi-rigid polymers can be obtained. ROMP-derived polymers have limited rotation due to the alkene (and often cycle) structure in the backbone, hence hydrophobic groups can be placed opposite hydrophilic, to install facial amphiphilicity.,72 Graham et al. synthesized ROMP glycopolymers bearing a galactose residue on one face, and either an oxo or a fulvo (more hydrophobic) residue on the opposing face. IRI assays showed that the fulvo polymers were far more active than oxo, confirming that the amphiphilicity rather than the presence of a glycan is the key structural feature. It also supported the hypothesis that segregation of these groups is crucial. No ice shaping was seen for these materials, suggesting no ice binding.To avoid the issue of aggregation upon sequential increases in hydrophobicity, Mitchell et al. exploited Nisin A as a pH switchable IRI. Nisin A is an antimicrobial peptide that upon lowering the pH assumes a facially amphipathic structure, with segregated hydrophobic/hydrophilic domains.reported . A key f6 However, in the case of synthetic polymers, most are flexible coils due to a saturated backbone with sp3 bonding, and cannot present specific “patches” of hydrophobicity. There has been interest in the introduction of IRI activity into polymers based on the statistical incorporation of hydrophobic groups, which may offer a route to low-cost IRI active materials. The most studied in this context are poly(ampholytes)—polymers bearing mixed positive and negative charges. Matsumura et al. reported that carboxylation of poly(ε-lysine), to give a 50:50 mix of charges, produced polymers that were potent cryopreservatives when used in conjugation with traditional cryoprotective agents.,76 Later studies showed that this ratio of charges was essential for both cryopreservation and for IRI activity. Matsumura has investigated poly(ampholytes) derived from dimethylaminoethyl methacrylate (DMAEMA) and methacrylic acid (MAA), which have some IRI activity as well as being effective cellular cryoprotectants. Copolymers of DMAEMA and MAA achieved around 50% inhibition at 100 mg mL−1 , however upon the incorporation of as little as 5% butyl methacrylate or octyl methacrylate into the chain, a MGS of 5% was observed at the same concentration, suggesting an increase in activity  To study this in more detail, Stubbs et al. synthesized a panel of regioregular poly(ampholyte)s using maleic anhydride copolymers, which ensure an alternating, rather than random, copolymer structure. Sequential increases in hydrophobicity were introduced by ring opening of the anhydride using alcohols. There was a clear trend that increased hydrophobicity increased the IRI activity. It is crucial to note here that the magnitude of activity is far less than the previous classes (AF(G)Ps/PVA) with some ice growth still occurring at 20 mg mL−1, hence care must be taken when discussing potency. These results, when taken together, suggest that while hydrophobicity is important, the spatial segregation of these domains needs to be carefully controlled to access IRI active materials. Similar observations have been made by copolymerization of vinyl pyrrolidone with increasingly hydrophobic units. Homo poly(vinyl pyrrolidone) has essentially no activity but incorporation of hydrophobic units did increase IRI, albeit at a level which was only just above that of PEG.It is clear that correct 3D placement of hydrophobic groups can modulate IRI activity and may be crucial in natural AF(G)Ps, in addition to their remarkable ice-binding properties.6.1−1 levels. There is also significant evidence for their selective and strong binding to specific planes of ice, which is crucial for their activity. Next, we include those that inhibit in the range up to ≈1 mg mL−1, which includes PVA, ZrAc, and safranin-O. These materials also shape ice and have potent IRI activity, with at least some of their activity arising from ice-face binding . After this, there are the materials that have no evidence for ice binding (at the moment) but are still relatively potent. It is crucial to note that ice binding could be occurring, but that it is so weak or transient that macroscopic ice shaping is not seen. However, these materials are facially amphiphilic with segregation of the domains crucial for their function, enabling them to slow ice growth fully, typically in a range <10 mg mL−1. It should be highlighted that these are approximate definitions, for example, the highly active lysine surfactants of Ben have similar activity to the ice binders; hence the need for appreciation that IRI is a continuum not a binary property. Next is the broad zone of “weak inhibitors,” which includes poly(ampholyte)s and hydrophobically modified polymers, with the upper boundary being the activity of PEG. Materials in this range do not appear to bind ice to any appreciable extent and in many cases cannot completely inhibit ice growth over any time period. Nonetheless, that does not rule out potential applications, where slowing ice is crucial or where high concentrations can be applied.Considering the examples shown above, we propose a rough guide for activity considerations when evaluating a new material for IRI, especially those which are far less active than AF(G) Ps −1 have been tested, which is clearly not a specific effect. There is no suggestion here as to which region is worthy of the most study, nor which is most promising for a specific application, but rather to help as a guide in defining materials’ relative function, and to ensure that false positives due to simple colligative effects are not misreported in the literature as the field grows.We feel this (simplistic) display provides a framework to discuss new inhibitors, especially those where ultra-high concentrations of up to 100 mg mL7In conclusion, we present a concise evaluation of a range of IRI active macromolecules covering several orders of magnitude in terms of activity. We show that even though AFGPs and AFPs are clearly far more active than synthetic mimics, their role in cryopreservation is not just linked to absolute activity but also availability, cost, and possible toxicity/immunogenicity. We propose a grouping of materials into different activity magnitudes especially at the lower end of the activity scale see , and hig8A 10 μL droplet of the macromolecule in PBS solution was dropped from 1.4 m onto a glass microscope coverslip, which is on top of an aluminum plate cooled to −78 °C using dry ice. The droplet freezes instantly upon impact with the plate, spreading out and forming a thin wafer of ice. This wafer is then placed on a liquid nitrogen cooled cryostage held at −8 °C. The wafer is then left to anneal for 30 min at −8 °C. Three micrographs were taken of the wafer and the longest grain crystals as well as the total number of crystals in the image are counted using ImageJ, and reported as a percentage of area compared to PBS control.Supporting Information is available from the Wiley Online Library or from the author.Supporting information


We identify late Paleozoic ice age (LPIA) subglacial megalineations from field and geospatial imagery of the Twyfelfontein area of northern Namibia, and present the results of a geomorphometric analysis of those data. Asymmetric 0.1–1.5 km-long megawhalebacks indicate a paleo-ice flow to the northwest. We infer that an ice stream draining the LPIA Kaokoveld ice sheet existed within the proto-Huab River valley and that was comparable to ice streams in modern Antarctica. Recognition of a paleo-ice stream in northern Namibia supports interpretations of glaciogenic sedimentary successions (Itararé Group) in southern Brazil that suggest the presence of major, terrestrial glacial outlet systems in southern Africa during the LPIA. We infer the presence of a late Paleozoic ice age (LPIA) paleo-ice stream in the Huab River valley based on the occurrence, shape, and size of elongate bedforms exposed on a Carboniferous-Permian glacial erosional surface. Many parts of Gondwana exhibit bedrock features including striations and glacial valleys formed during the LPIA, including Argentina , souther3 km in length and with catchments of ≤106 km2 ).The megalineations parallel the strike of bedding in the Swakop Group everywhere except around the closures of a kilometer-scale, doubly-plunging synform south of Khorixas . The asyWe measured to maximum lengths and widths of 93 megalineations around Twyfelfontein using freely available satellite imagery in Google Earth Pro . Axial lThe bedforms at Twyfelfontein inhabit a narrow range of lengths  northwest, probably during the LPIA. There were probably numerous advances and retreats of ice during the LPIA, and we do not attempt to ascribe these features to a specific glacial event.n > 104 bedform features) suggests an elongation value >8 is more reasonable where a continuum exists with drumlins [The overwhelming majority of Twyfelfontein bedforms are megawhalebacks  Fig 3B)Fig 3B) adrumlins , 24. In drumlins  break-oudrumlins , that (2drumlins ), an arb2–103 m a-1, and MSGLs (E >10) at >103 m a-1 [We infer that the megalineations at Twyfelfontein can be considered MSGLs or as transitional between elongate drumlins and MSGLs. Therefore, they record passage of an ice stream of some significant ice flow velocity. The relationship between ice stream velocity and underlying bedforms is limited to geophysical studies of active ice streams where the velocity is known . All th03 m a-1 ; rates cNorthern Namibia and then-adjacent parts of what is now SE Brazil were located close to the South Pole in Early Mississippian and moved to about 60°S through the Pennsylvanian and Permian . The regThere is abundant evidence of LPIA sediment supply from southern Africa in the Itararé Group, contemporaneous with the Dwyka Group, within the Paraná Basin. The Itararé Group records glacial advance and retreat at the ice margin followed by the onset of open marine conditions . NorthwePlate reconstructions juxtapose SE Brazil and northern Namibia during the LPIA supporting links between the Kaokoveld ice sheet and its distal deposits in the Paraná Basin e.g., , 23, 26), 2623, 2We have identified a field of large, subglacial sculpted bedforms in northern Namibia that lead us to infer the past existence of major ice stream draining a LPIA ice cap in southern Africa. The location and paleo-ice flow directions of the whalebacks and megawhalebacks indicate northwestward flow of ice along the paleo-Huab River valley that likely discharged into the shallow marine environment several hundred kilometers downstream in modern Brazil.S1 Dataset(XLSX)Click here for additional data file.


Variations in tropical sea surface temperatures (SST) have pronounced impacts on the stratospheric polar vortex, with the role of El Niño being the focus of much research interest. However, the Indo–Pacific warm pool (IPWP), which is the warmest body of seawater in the world, has received less attention. The IPWP has been warming in recent years. This paper presents for the first time the remarkable nonlinearity in Northern Hemisphere (NH) stratospheric circulation and temperature response to IPWP warming  in boreal winter. The magnitude of NH stratospheric vortex weakening is strong and significant in case of moderate IPWP Niño, but is weak and insignificant in strong IPWP Niño case. This phenomenon is robust in both the historical simulations and observations. An idealized model experiments forced with linear varying SST forcing in the IPWP region isolate the nonlinearities arising from IPWP Niño strength. Westward extension of precipitation into the Maritime Continent drives attenuation and westward shift of extratropical waves during strong IPWP Niño events. Linear wave interference analysis reveals this leads to weak interference between the climatological and anomalous stationary waves and thereby a weak response of the stratospheric vortex. These findings imply a distinct stratospheric vortex response to the IPWP Niño, and provide extended implications for the surface climate in the NH. The tropospheric Rossby wave anomaly associated with ENSO warming, or El Niño, which projects onto the climatological stationary wave over the North Pacific, intensifies the vertical propagation of planetary scale Rossby waves upwards into the stratosphere in the Northern Hemisphere (NH) during the boreal winter and therefore leads to a markedly warmer and weaker polar vortex12. However, for the cold phase of ENSO, La Niña, reanalysis data reveal a weak and insignificant polar stratospheric cooling14, though a recent study has reported a robust strengthened and cooled vortex associated with La Niña15. Possible explanations for this asymmetric response to the opposite phases of ENSO include inherent differences in extratropical teleconnections19 and the background SST in the cold tongue area20.Previous studies have demonstrated the importance of tropical sea surface temperatures (SST) in modulating the stratospheric polar vortex, and the El Niño–Southern Oscillation (ENSO) is one of the most discussed aspects of this relationship22. Recent projections indicate that very strong El Niño events, also called extreme or super El Niño events, are expected to increase in frequency in the future under global warming27. A major impact on stratospheric circulation has been observed during the three unusually strong El Niño events that have occurred during the satellite era; i.e., the 1982/83, 1997/98, and 2015/16 El Niño events28. Domeisen et al.2 reviewed on the question that whether strong El Niño events lead to a proportionately stronger stratospheric response compared with moderate El Niño events, where discrepancy was noted. Garfinkel et al.29 shown stronger response to extreme El Niño comparing with that to moderate El Niño in North Pacific Sea Level Pressure field with weak nonlinearity in the location of the extrema; Weinberger et al.30 suggested that stratospheric response to extreme El Niño is weaker than proportionate amplitude of El Niño strength. But some idealized experiments have different results. Zhou et al.31 found that the magnitude of the simulated stratospheric response to extreme El Niño events was four times stronger than that to moderate ones in late winter and early spring; Jimenez-Esteve and Domeisen32 showed that strong El Niño with double amplitude of its moderate counterpart yields more than twice the impacts of moderate El Niño in North Pacific atmosphere.Each El Niño event can differ in magnitude, which is commonly measured by the corresponding SST anomalies in the eastern tropical Pacific33. When isolated from ENSO variability, the Indian Ocean warming induces increased precipitation over the Indian Ocean, drives teleconnections to the NH extratropics, and leads to positive northern annular mode (NAM) response in an opposite sign of El Niño impacts34. Thus it partially attenuate El Niño impacts on the stratospheric polar vortex during the El Niño winter35.The SST anomalies in different tropical regions have different impacts on the stratosphere. The tropical Indian Ocean, for example, has become a research focus due to sustained warming in recent years36, being one of the major suppliers of atmospheric releasing heat and water vapor, leads to the most intense global air convection and climate influences globally37, yet has received less attention than ENSO in the regard of stratospheric impacts. Since the 1980s, the IPWP has experienced a continuous expansion, with significant increases in SST39. Similar to the definition of ENSO events40, anomalous warming/cooling of the IPWP  is identified when 5-month running mean of the SST variations in the IPWP (TI(IPWP)) exceed half the standardized deviation (0.5σ)41. It is well appreciated that SST variations in the IPWP is correlated with ENSO through the “atmospheric bridge”42. El Niño-induced surface changes in surface heat fluxes can lead to warming in the IPWP43; On the other hand, the extension, displacements and intensity variations of the IPWP are known to affect the onset, intensity, and period of ENSO44. The IPWP Niño/Niña events can occur with or without ENSO events, each case accounting for about half episodes of IPWP Niño/Niña events from 1980 to 2015 according to Zhou et al.41. When isolated from ENSO variability, the IPWP can significantly influence the stratospheric circulation and temperature41 and is even more efficient in pumping water vapor from the troposphere into the stratosphere than ENSO45.However, the IPWP, spanning the tropical western Pacific and eastern Indian oceans where the sea surface temperature (SST) exceeds 28 °C year-round46, influencing the tropopause temperature with smaller zonal asymmetries than that of ENSO and thereby leading more upward transport of water vapor into the stratosphere45. Temperature perturbations by adiabatic heating associated with IPWP Niño can radiate away into the mid-latitude troposphere, which bring about extratropical teleconnection47. During boreal winter, anomalous SST warming associated with IPWP Niño can excite NH extratropical teleconnections that project onto the positive phase of the Pacific–North America (PNA) pattern in mid–high latitudes, intensifying the upward propagation of planetary waves into the stratosphere and, in turn, warming and weakening the NH stratospheric vortex41. Note that the wave pattern excited by IPWP Niño is located further west than the PNA pattern, a fact that may be related to the different wave source locations from that of ENSO. Asymmetric response between the IPWP Niño and IPWP Niña–i.e. a same-signed response during opposite IPWP phase–has already been shown41, which is linked to asymmetries in extratropical IPWP teleconnections. However, it remains unclear whether a nonlinear relationship exist between the NH stratospheric response and IPWP Niño strength.Enhanced convection excited by the IPWP Niño launches equatorial planetary waves in the form of Kelvin and equatorial Rossby wave responsesThis study constitutes the first attempt to investigate the nonlinearities in the NH stratospheric polar vortex response to IPWP Niño in both observations and atmospheric model. We first show the relationship between the SST anomalies associated with IPWP Niño events and the polar stratospheric response in CMIP5 historical simulations, for identification of the nonlinearity and turning point of the stratospheric response. Using this turning point as a threshold, we confirm the nonlinearity by comparing the composite stratospheric circulation and temperature anomalies during strong and moderate IPWP Niño winters based on reanalysis datasets. Finally, we isolate the nonlinearities arising solely through changes in IPWP Niño strength by forcing a linear change in the amplitude of the SST forcing in time-slice model simulations.(IPWP), which indicates the strength of the IPWP Niño, versus the December–January–February (DJF) polar cap temperature anomalies between 70°N and 90°N and from 100 to 50 hPa 2, better describes the relationship between IPWP Niño strength and the stratospheric response than does a linear fit. This is measured by the adjusted R2 (See the Method section), which is larger in case of polynomial fit  than in case of linear fit . Importantly, an inflection point occurs near the 1σ threshold. When IPWP Niño strength is below the 1σ threshold, the intensity of the polar stratospheric temperature and zonal wind anomalies increases with IPWP Niño strength. The correlation coefficient between IPWP Niño strength and the DJF polar vortex temperatures is r = 0.57, which suggests that the stronger the IPWP Niño event, the warmer the stratospheric anomaly. Correspondingly, the correlation coefficient for the zonal wind anomalies is r = −0.59; i.e., the stronger the IPWP Niño event, the weaker the polar vortex. In addition, the linear fit crossed zero close to, but lower than, the 0.5-SD threshold, supporting the use of 0.5σ to define IPWP Niño winters. However, when IPWP Niño strength exceeds the 1σ threshold, the response reverses so that it weakens with increasing IPWP Niño strength. An inflection point occurs near the 1σ threshold for both stratospheric temperature and circulation response.To obtain a wide range of IPWP Niño events, we used CMIP5 outputs from a coupled climate system. Considering the good performance of the Community Earth System Model (CESM) Whole Atmosphere Community Climate Model, version 4 (WACCM4) (see Simulations for more information) in representing stratospheric variability, we analyzed simulations using CESM-WACCM4 from historical experiments covering the period 1850–2005 (see the Methods for details). Figure hPa Fig. , as wellhPa Fig. . A clearNow we have seen the nonlinear stratospheric response in CESM-WACCM4 historical experiments. Note that this relationship is based on a single CMIP5 model (CESM-WACCM4), and can be different in other models, though not extended in this paper. However, two questions must be addressed here. Does the nonlinearity really exist? If so, does it come from IPWP Niño strength? Thus, we next investigate the change in stratospheric response with increasing IPWP Niño strength in the observations to validate the nonlinearity in the CMIP5 simulations, and then isolate the signal and tracing the nonlinearities from tropics to extratropical stratosphere based on idealized modeling results.(IPWP) exceeds 1σ, with the moderate IPWP Niño group containing all of the remaining IPWP Niño events. The years included in each composite are listed in Table et al.41  response is used as a proxy for the intensity of tropical convection response. Low-level (850 hPa) convergence of zonal winds associated with Kelvin and Rossby waves towards the IPWP region coincides with upper level (250 hPa) divergence, leading to enhanced convection  over the IPWP region  using CESM-WACCM4 to mimic the IPWP Niño SST forcing with linear increasing strength, ranking as weak, moderate, strong, and very strong PWP Niño forcing Fig.   force opposite-signed extratropical response. Their findings suggest that the extratropical response in cases of strong and very strong IPWP Niño is very likely to have some cancellation between contribution from precipitation anomalies over the east part the IPWP region  and contribution from precipitation over the Maritime Continent . The essence of this argument involves the distinct extratropical response to convection over the two domains. In order to identify basic atmospheric processes associated with hearting in domain 1 and 2 separately, the linear baroclinic model (LBM) is used, with two heating fields restricted in domain 1 and 2 imposed separately in two runs (See Simulations for more information). Figure 53. The LBM solutions confirms the opposite-signed response between precipitation anomalies over the east and west part of the IPWP, which is likely to attenuate extratropical Rossby waves during strong IPWP Niño event.Many studies have found a large sensitivity of the extratropical response to the location and amplitude of the convective anomalies near the equatorZ* 250 hPa; Z* indicates that the zonal mean has been removed), with an anomalous deepened Aleutian low and an anomalous American high  and its climatological mean is measured by the pattern correlation rzp (See the Method for details). The entire depth of the troposphere and lower stratosphere is considered, in order to reveal a precursory planetary wave signal from the troposphere58,. The anomalous wave 1 projects weakly onto climatological wave 1 in weak IPWP Niño case (rzp = 0.45), but projects strongly onto climatological wave 1 in moderate IPWP Niño case (rzp = 0.78)    of extratropical planetary waves determines the stratospheric vortex response78) Fig. ; and theño) Fig. . This paño) Fig.  and the ño) Fig. . This waFigure The combination of analyses using the historical relationship, reanalysis composites, and idealized experiments allows us to draw conclusion on the nonlinearity of IPWP Niño’s impacts on the NH stratospheric vortex. Anomalous warming SST associated with IPWP Niño launches precipitation over this area, which drives extratropical waves that further weaken the polar vortex. However, a nonlinear relationship between the amplitude of the IPWP Niño strength and the NH stratospheric vortex response is identified.When the strength of the IPWP Niño is below the 1σ threshold, the intensity of the polar stratospheric temperature and zonal wind anomalies increases with increasing IPWP Niño strength. However, the response reverses and becomes weaker when IPWP Niño strength exceeds the 1σ threshold. This nonlinear relationship between IPWP SST anomalies and the NH stratospheric response is seen in CESM-WACCM4 CMIP5 historical simulations from 1850 to 2005. As 1σ is the point at which nonlinearity begins to develop in the stratospheric response, we used 1σ as the threshold to separate the IPWP Niño events into two groups (moderate and strong). By comparing the composition of the anomalous stratospheric temperature and circulations during moderate and strong IPWP Niño events, we found that the stratospheric circulation and temperature response is weaker during strong IPWP Niño events than during moderate events based on ERA-Interim for the period 1979–2017. That is, the stratospheric polar vortex is significantly warmer and weaker during moderate IPWP Niño events, whereas there is no significant signal during strong IPWP Niño events in the observations.Idealized model experiments with scaled IPWP Niño SST forcing have been performed to isolate the signal from IPWP Niño and to validate the nonlinearities arising from IPWP Niño strength. After the westward extension of precipitation into the Maritime Continent, there is some cancellation between the extratropical response to the west part convection over the Maritime Continent and the response to convection over the east warm pool. This leading to the attenuation of extratropical wave response, with a ~10° westward shift in the Aleutian low region during strong IPWP Niño events. Thus, the wave-1 component moves from strong in phase with the climatological wave in moderate IPWP Niño case into rather weak in phase in strong IPWP Niño case and is mostly confined in upper stratosphere. This linear wave interference produces a significant vortex response in moderate IPWP Niño case but a very weak response in strong IPWP Niño case.60, which is extended from 1979 to near-real time, available at http://apps.ecmwf.int/datasets/. For SST, we use the monthly-mean product from the Hadley Centre Sea Ice and Sea Surface Temperature (HadISST) dataset61, available at http://www.metoffice.gov.uk/hadobs/hadisst/data/download.html.SST, temperature and circulation data are involved in observational composite analysis. For upper-air atmospheric fields, we use the monthly mean European Center for Medium Range Weather Forecasting (ECMWF) reanalysis dataset (ERA-Interim)http://cmip-pcmdi.llnl.gov/cmip5/availity.html) allowed us to examine the stratospheric response to a wide range of IPWP Niño events. CESM-WACCM4 uses active ocean and sea ice components, and the model is forced using observed atmospheric composition changes from both natural  and anthropogenic  sources. The atmospheric component used was WACCM4. A representation of the QBO was achieved by relaxing the equatorial zonal wind between 86 and 4 hPa toward that observed58. To avoid the possible entanglement of QBO signals in our composite results from the fully coupled model, we regressed out the zonal wind at 50 hPa.The monthly output for the period 1850–2005 from historical simulations by CESM-WACCM4 in the CMIP5 archive (http://www.cpc.noaa.gov/data/indices/). All statistical tests were performed using the two-tailed Student’s t-test. To better represent the reversing manifestation of the nonlinearities, the composite results and model results are not scaled by IPWP Niño strength.We calculated the monthly anomalies by subtracting the long-term mean of each calendar month from each individual month. The linear trends were removed before analysis from the temperature, zonal wind, and geopotential height data using linear regression analysis. The quasi-biennial oscillation (QBO) index used was the standardized anomaly of equatorial 50-hPa zonal winds and it was used to regress out the QBO signal. The Niño 3.4 index was defined as the area mean SST anomaly over the region 5°S–5°N, 150°W–90°W  and its climatological mean, following the framework of Fletcher and Cassou34. The weights are based on the relative thickness of each of the vertical layers from 700 to 10 hPa. Since the The correlation is computed separately for the zonal wavenumber 1 and 2 components of Z * 60°N, which are filtered using a Fourier transform.To diagnose the linear interference of Rossby waves, we used the pressure-weighted correlation R2 (Eq. 3.30 of Chatterjee and Hadi62) is used to quantify the added value in using a polynomial best fit 2) instead of a linear best fit ). The adjusted R2 takes into account the likehood that a polynomial predictor will reduce the residuals by unphysically over-fitting the data. The polynomial fit could be preferred if the adjusted R2 for the polynomial fit is larger as compared to the linear R2.The adjusted −6 hPa , with vertical resolution of 1.1–1.4 km in both the TTL and the lower stratosphere (<30 km). It is unable to internally simulate the QBO signals but is forced using a 28-month fixed cycle (nudged QBO). The time-slice simulations presented in this paper were performed at a resolution of 1.9° × 2.5°, with interactive chemistry.The time-slice simulations were performed using CESM-WACCM4. The CESM-WACCM4, developed by the National Center for Atmospheric Research (NCAR), has 66 vertical levels extending from the ground to 4.5 × 1032, Cao et al.48, and Lin and Derome49, the performed experiments mimic a linear IPWP Niño forcing. Five 30-yr time-slice experiments are conducted, consisting of a climatological run (R1) and four experiments (R2–R5) with IPWP Niño SST forcing. The simulations use prescribed fixed SSTs following the 1958–2016 monthly SST climatology. The winter mean composite SST anomalous pattern for moderate IPWP Niño  is used as a diagnostic tool for studying the extratropical atmospheric response to idealized forcing


Mitrella kentii (Bl.) Miq. The compounds were evaluated for their ability to inhibit prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) production in human whole blood using a radioimmunoassay technique. Their inhibitory effect on platelet activating factor (PAF) receptor binding to rabbit platelet was determined using 3H-PAF as a ligand. Among the compounds tested, chrysin showed a strong dose-dependent inhibitory activity on PGE2 production , which might be due to direct inhibition of cyclooxygenase-2 (COX-2) enzymatic activity. Polycarpol, acetylmelodorinol and stigmasterol exhibited significant and concentration-dependent inhibitory effects on TXB2 production with IC50 values of 15.6, 19.1 and 19.4 µM, respectively, suggesting that they strongly inhibited COX-1 activity. Polycarpol and acetylmelodorinol showed strong dose-dependent inhibitory effects on PAF receptor binding with IC50 values of 24.3 and 24.5 µM, respectively.Acetylmelodorinol, chrysin and polycarpol, together with benzoic acid, benzoquinone and stigmasterol were isolated from the leaves of  Prostaglandin E2 (PGE2), a metabolite of AA through the cyclooxygenase-2 (COX-2) pathway, has received great attention because of its role and contribution to inflammation. It is also involved in a variety of other functions, such as vasodilation, febrile responses, and altered microvascular permeability -TXB2 were incubated at 4 °C for 18–24 h. After incubation, the mixtures were added with 200 µL of dextran charcoal and were incubated again for 10 min. The final concentrations of the samples in the mixture were 10.0, 5.0, 2.5 and 1.25 µg/mL. After centrifugation at 3,000 × g for 15 min at 4 °C, 3 mL of liquid scintillation cocktail was added to 300 µL of supernatant. The radioactivity was measured by liquid scintillation counter. Thromboxane BThe readings obtained for each set of triplicate were averaged. The net counts for all standards and samples were calculated by substracting the value of the antibody binding to the antigen in the sample (Bx) with non specific binding (Nc). The normalized percentage bound (% B/Bo) for each standard and sample (Bx) were calculated as follows:versus the corresponding picogram (pg) concentration of PGE2 and TXB2 were plotted using semi-logarithmic graph. The concentrations of PGE2 and TXB2 in each sample were determined by interpolation from the standard curve. Percentage inhibition of samples was obtained as follows:The % B/Bo for each standard et al. [3H-PAF (2.0nM) with or without unlabeled 25 µL of PAF (2.0 µM) and 25 µL of compound (200 µg/mL) or control solution. The final concentration of compounds in the reaction mixtures were 18.2, 9.1, 4.5, 2.3 µg/mL. Cedrol, a known PAF antagonist was used as a positive control 0.1% DMSO in saline was used as a control. The final concentration of DMSO in the reaction mixture was fixed at 0.1% to avoid interference with the receptor binding studies. The reaction mixture was incubated at room temperature for 1 h. The free and bound ligands were separated by a filtration technique using Whatman GF/C glass fiber filters. The radioactivity was measured by a scintillation counter. The difference between total amounts of bound 3H-PAF in the absence and the presence of excess unlabeled PAF is defined as specific binding of the radiolabeled ligand. The IC50 values of the compounds were obtained from at least three independent determinations. Percentage inhibition of the sample was obtained by the following equation: The assay was carried out according to the method as described previously by Jantan et al. . The proStatistical Package for Social Sciences (SPSS) software. Each sample was measured in triplicate and the data are presented as means ± standard deviation (SD). Probit programme was used to determine the IC50 values for the active pure compounds. The values were obtained from at least three determinations. Data were analysed using one way ANOVA. P < 0.05 was considered to be statistically significant.All the data were analysed using the Mitrella kentii, chrysin showed a potent dose-dependent inhibitory activity on PGE2 production in human blood induced by LPS, indicating that it might directly inhibit COX-2 enzymatic activity. The results of this study indicate that the strong inhibitory activity of M. kentii on TXB2 production was due to polycarpol, acetylmelodorinol and stigmasterol which exhibited significant and concentration-dependent inhibitory effect on COX-1 activity. The strong PAF antagonistic effect of the plant was contributed by polycarpol and acetylmelodorinol which showed strong and dose-dependent inhibitory effects on PAF receptor binding. Acetylmelodorinol, chrysin and polycarpol have the potential to be used as lead structures for the development of potent anti-inflammatory agents. However, further studies need to be carried out to study their mechanisms of anti-inflammatory actions such as the cell free assays for COX enzyme activity and to find derivatives with maximum inhibitory effects. Among the compounds isolated from the leaves of


The control of ice growth in ice slurry is important for many fields, including (a) the cooling of the brain during cardiac arrest, (b) the storage and transportation of fresh fish and fruits, and (c) the development of distributed air-conditioning systems. One of the promising methods for the control is to use a substance such as antifreeze protein. We have observed and report here growth states of ice particles in both quiescent and flowing aqueous solutions of winter flounder antifreeze proteins in mini-channels with a microscope. We also measured ice growth rates. Our aim was to improve the levels of ice growth inhibition by subjecting the antifreeze protein solution both to preheating and to concentrating by ultrafiltration. We have found that the ice growth inhibition by the antifreeze protein decreased in flowing solutions compared with that in quiescent solutions. In addition, unlike unidirectional freezing experiments, the preheating of the antifreeze protein solution reduced the ice growth inhibition properties. This is because the direction of flow, containing HPLC6 and its aggregates, to the ice particle surfaces can change as the ice particle grows, and thus the probability of interaction between HPLC6 and ice surfaces does not increase. In contrast to this, ultrafiltration after preheating the solution improved the ice growth inhibition. This may be due to the interaction between ice surfaces and many aggregates in the concentrates. The inhibition of ice growth has been the focus of significant recent research. This inhibition is important for many fields, including (a) cryotherapy , (b) theOne of the most promising methods for controlling the growth of ice is to use a substance which has the specific purpose of controlling ice growth. Antifreeze proteins (AFPs) or antifreeze glycoproteins (AFGPs) are appropriate substances for controlling ice growth, because they lower the temperature at which a seed crystal grows but do not alter the temperature at which the crystal is stable during melting at very low cooling rates. Furthermore, an appropriate concentration of AF(G)Ps is much lower than that of other solutes . This mi3 in oil or liquid paraffin in osmometers are typically frozen at −40 °C, and gradually heated until only a single crystal of approximately 7 μm in diameter is obtained [3 sample solution of winter flounder AFP was held in a glass tube, and they measured the nucleation rate in the tube. Coger et al. [Among various AF(G)Ps discovered to date, HPLC6, the major fraction of winter flounder AFP, has been widely investigated. Measurements of ice crystal growth and ice crystal morphology in solutions of this protein are classified into the following two types according to the cooling rates: (i) at a very low cooling rate (≤−1 °C/min) and (ii) at a low cooling rate >−1.5 °C/min). In type (i) measurements, an AFP solution of 0.01 mm °C/min. r et al. , Butler r et al.  Hagiwarar et al. , Hagiwarr et al.  and Miyar et al.  carried c-axis in the quiescent solution and the crystals being transported in the core region of flow. Onishi et al. [Although these findings are valid, it has not yet been confirmed whether they hold in the situation where the AFP solution flows along with ice crystals. Furthermore, experimental results for such flows are limited. Grandum et al.  showed ti et al.  found thWith this in mind, in the present study, we measure the growth of ice particles in the flow of HPLC6 solution in mini-channels. We discuss the effects of preheating or ultrafiltration of the solution on the ice-particle growth.The sequence of amino-acid residues for HPLC6 is as follows: DTASDAAAAAALTAANAKAAAELTAANAAAAAAATAR . It consists of 37 amino acid residues and has a molecular weight of 3242 Da. The secondary structure of HPLC6 is an α-helix. Four threonine residues are positioned at nearly identical distances on one line parallel to the helical axis. The distance between the oxygen atoms on the pyramidal faces of ice crystals, which were observed in the supercooled solution of HPLC6, is nearly identical to the distance between the threonine residues. It had thus been hypothesized that the hydrogen atoms of the threonine residues were bonded permanently to the oxygen atoms on the pyramidal faces in the ice crystal and that the water molecules were prevented from bonding to the ice surface by the Kelvin (or Gibbs–Thomson) effect . On the We purchased the synthetic polypeptide from GenScript Inc. .The HPLC6 solution was preheated at a constant temperature for a predetermined time and cooled in the temperature-controlled room before the measurements were carried out. A polypropylene bottle containing the sample liquid was installed in a drying chamber . The temperature was set to 80 °C and the duration was one hour. The HPLC6 concentration was 0.5 mg/mL. The preheating condition was determined based on our previous study on the inhibition of unidirectional freezing of HPLC6 solution .4 and 105. The centrifugal force was 14,000× g.The preheated HPLC6 solution was filtered to increase the solution concentration. We used a centrifuge  and ultrafiltration membranes. The molecular weight cut-offs of membranes were 10First, the temperature of the solution was maintained at −3 °C. Secondly, seed crystals of ice were added to the supercooled solution. The solution with ice particles was stirred with an agitator  for one hour. The temperature of the mixture was maintained at −1.2 °C.The apparatus consisted of an inverted biological microscope , a monochrome charge-coupled device (CCD) video camera , a bench-top cooling section and a syringe pump . The apparatus was placed in a temperature-controlled room maintained at 2 °C.X-axis was in the slurry-flow direction, the Y-axis was in the transverse direction, and the Z-axis was in the vertical (viewing) direction. The origin of the coordinates was located at the corner of the channel bottom surface.A silicone tube of inner diameter 2 mm and outer diameter 3 mm was attached to the channel exit. The tube was connected to the syringe pump to draw ice slurry from the channel. A syringe, which consisted of a polypropylene cylinder and a polyethylene piston, was used. The volume of the syringe was 1 mL. The flow rate of slurry was set at 0, 20 and 40 μL/min.The ice slurry with or without HPLC6 was introduced into the mini-channels from the cone-shaped entrance. The ice slurry was mixed manually with a small stirrer at the entrance.We adopted the following two types of channel cooling: The ambient air temperature was 0.5 °C lowered to subzero degrees (type A), and copper plates surrounding the channel were cooled by a thermoelectric cooler  (type B). In the case of type B cooling, the channel axis was in parallel with the axis of conductive heat transfer of the copper plates. The flow direction was opposite to the conduction. The copper plates and the thermoelectric cooler were adhered with heat conduction grease. The copper plates had slits of width 2 mm to illuminate and observe the channel. The temperature inside the cooler was set at −2.0 °C.The image-capturing condition is shown in In the case of ice slurry flow containing HPLC6, we adopted the identical mini channel, the cooling condition and the image-capturing condition. The concentration of HPLC6 was 0.25 mg/mL. The flow rate was 40 μL/min. The observation area was moved downstream so that the images of identical ice particles could be captured. The measured temperature around the observation area was −1.4 °C.In the case of flow of the mixture of ice slurry with the preheated solution of HPLC6, we used the mini-channel 2 under the type B cooling condition. We adopted the identical image-capturing condition. The concentration of HPLC6 was 0.25 or 0.50 mg/mL. The concentration of sodium chloride was 0.45 wt%. The measured temperature around the observation area was 1.0 °C.In the case of flow of the mixture of ice slurry with the ultra-filtrated solution of preheated HPLC6, the mini-channel and the cooling condition were identical with those used in the case of flow of the mixture of ice slurry with the preheated solution of HPLC6. We changed the volume of HPLC6 solution for its mixing with the ice slurry and the molecular-weight limit of ultrafiltration.a-axis because the growth rate along the a-axis is more than 100 times higher than the growth rate along the c-axis [e c-axis . The icea-axis direction.In the case of quiescent ice slurry with HPLC6 whose concentration was 0.125 mg/mL, the ice particles became plates with the shape of a hexagon when the image-capturing started see b. These z-axis  before the image-capturing started. A small ice particle whose c-axis is parallel to the images is also seen in this figure. After 200 s, the hexagonal shapes became larger, and the edge surfaces of large hexagonal plates became much wider than before. That is, ice particles grew in the a-axis and c-axis directions, and the edge surfaces inclined further. Consequently, the large plates seemed to become truncated hexagonal pyramids. The small ice particle became a truncated hexagonal bi-pyramid. Note that these truncated pyramids became pyramids eventually.In the case where the HPLC6 concentration was 0.25 mg/mL, as shown in z-axis. This is probably a result of slow flow of ice slurry in the mini-channel to introduce ice particles into the central part of the channel. Thus, it is surmised that the adsorption of HPLC6 on the pyramidal faces of ice particles had already initiated just after the ice slurry and HPLC6 solution were mixed.In the case where the HPLC6 concentration was highest, the ice particles had already had shapes of hexagonal bi-pyramid before the image-capturing started see d. The hea-axis direction, while the vertical axis shows the ice particle growth rate in the identical direction. (The growth rate in the case of elliptic plates was defined by the increase in the major axis.) It turns out that both the diameter and growth rate of ice particles in the case of the highest concentration of HPLC6 are lower compared with those in the cases of the lower concentrations. In the case without HPLC6, the diameter and the growth rate are scattered in wide ranges. Generally, the scattering of the diameter and growth rate decreased with an increase in the HPLC6 concentration.a-axis direction causes the enhancement of ice growth in the c-axis direction. Consequently, the hypothesis in which the pyramidal faces become the slowest surface through the ice crystal growth in osmometers stands even for the ice particles in the mini-channel.z-axis, or to the c-axes of the ice particles.In the case where the HPLC6 concentration was 0.25 mg/mL, as shown in In reference , the shoa-axis. a-axis. It is found that the average growth rate in the case where the preheated HPLC6 solution was used is higher than that in the case where the unheated HPLC6 solution was used. In addition, the standard deviation in the case where the preheated HPLC6 solution was used is higher than the unheated HPLC6 solution. These facts show that the interaction between HPLC6 and ice particles, such as the adsorption of HPLC6 on ice surfaces, was not strong in the case where the preheated HPLC6 solution was used. This is different from the experimental results for the unidirectional freezing of HPLC6 solution obtained by Miyamoto et al. [In the case where the preheated HPLC6 solution was used, on the other hand, ice crystals with the shapes of truncated hexagonal pyramids were observed, as shown in o et al. . The HPLTo improve the activity of ice growth inhibition in the case where the preheated HPLC6 was used, we carried out ultrafiltration for the solution. To examine the effects of the ultrafiltration, we measured the growth rates of ice particles in the flow of ice slurry in the cases where the filtrate and concentrate were used.a-axis. It is found that the average growth rates in the case where the filtrates of preheated HPLC6 solution were used are higher than those in the case where the unheated or preheated HPLC6 solution was used. The growth rate for the filtrate in the case of Mw = 105 is lower than that in the case of Mw = 104. This is because many aggregates whose molecular weight is higher than 104 passed through the membrane and were included in the filtrate. The average growth rates in the case where the small amount of concentrates of preheated HPLC6 solution were used are higher than those in the case where the unheated or preheated HPLC6 solution was used. In contrast with these, the average growth rates in the cases where the concentrates of preheated HPLC6 solution were used are the same, or lower, than those in the case where the unheated or preheated HPLC6 solution was used. In particular, the use of membrane whose molecular weight limit is 104 is found to be effective. This is because many small aggregates and large aggregates remaining in the concentrate interacted with ice particle surfaces effectively. It can be concluded that the combination of short-time preheating and ultrafiltration is effective for the inhibition of ice growth and enhancement of the supercooled states of the carrier fluid flow in ice slurry.(1)The ice growth inhibition by the antifreeze protein decreased in flowing solutions compared with that in quiescent solutions. This is because the slurry flow carried HPLC6 and the latent heat away from the surfaces of ice particles locally.(2)The preheating of the antifreeze protein solution before mixing with the ice slurry reduced the ice growth inhibition properties for the ice slurry flow. As the direction of flow, containing HPLC6 and its aggregates, to the ice particle surfaces can change as the ice particle grows, the probability of interaction between HPLC6 and ice surfaces does not increase with the ice particle growth.(3)The ultrafiltration after preheating the solution improved the ice growth inhibition. In a specific case, the inhibition was enhanced because many small aggregates and large aggregates remaining in the concentrate could interact with ice particle surfaces effectively.We have carried out observations of ice particle growth in both quiescent and flowing aqueous solutions of winter flounder antifreeze proteins in a mini-channel and measured ice growth rates. The main conclusions are as follows:


Sambucus sieboldiana agglutinin (SSA) lectin, a binder to sialylα2,6galactose residue, inhibited antibody binding to α2,6-sialylated transferrin (Tf) (SSA inhibition). SSA inhibition was not observed with other glycoforms, such as periodate-treated, sialidase-treated and sialidase/galactosidase-treated Tf, suggesting that the assay was glycoform-specific. SSA inhibition was also applicable for visualizing localization of α2,6-sialylated-Tf in a liver section. This is the first immunohistochemical demonstration of glycoform localization in a tissue section. SSA inhibition was utilized for establishing ELISA to quantify α2,6-sialylated carcinoembryonic antigen (CEA), a marker for various cancers. In addition, α2,6-sialylated-CEA was visualized in a colonic adenocarcinoma section by SSA inhibition. The method would further be applicable to a simple and rapid estimation of other α2,6-sialylated glycoproteins and have a potential aid to histopathological diagnosis.Antibodies are useful for detecting glycoprotein antigens, but a conventional antibody recognizes only a protein epitope rather than a glycan. Thus, glycan isoform detection generally requires time- and labor-consuming processes such as lectin affinity column chromatography followed by sandwich ELISA. We recently found antigen-antibody reactions that were inhibited by lectin binding to glycans on the glycoprotein antigen, leading to a convenient glycoform-specific assay. Indeed,  Glycosylation is the most extensive and complex post-translational modification and affect properties of the proteins and lipids to which glycan attach. Alteration of glycan’s structure and its site-specificity modulates biological phenotypes such as development, immunity, and disease susceptibility. Abnormal glycosylation is detected in various pathological processes; malignant transformation and immune abnormalities. Accordingly, glycoproteomics is drawing great attention as an emerging research field, along with genomics and proteomics ,2. It isLectins are carbohydrate-binding proteins that specifically recognize glycan structures and have been utilized for profiling glycoproteins in crude specimen ,6,7. ForSambucus sieboldiana agglutinin (SSA) lectin blotting reveals that the former binds to SSA as serum Tf, indicating both have α2,6-sialic acid residues. In contrast, Psathyrella velutina lectin (PVL) binds the latter, but not the former, indicating the presence of N-acetylglucosamine (GlcNAc) residue(s) on its glycans. The former was designated as serum-type Tf and the latter was brain-type Tf. Each of the glycoforms were purified and their glycan structures were analyzed by authentic analytical methods including mass spectrometry. We found that serum-type Tf has complex-type biantennary N-glycans carrying a sialylα2,6galactose sequence, which is a binding epitope for SSA lectin, and brain-type Tf has complex-type biantenary N-glycans carrying bisecting GlcNAc and core-fucose  were analyzed by SDS-PAGE followed by silver staining. CSF band patterns of major proteins are similar to those in serum a. Indeede-fucose b 14,15],15Sambuc4) to destroy SSA epitopes on the antibody. Periodate treatment did not affect Ab binding to antigens (data not shown). The detection antibody was HRP-conjugated anti-human Tf-Ab . We found that addition of SSA lectin  significantly attenuates the ELISA signal , a senile dementia, which is caused by abnormal CSF metabolism . The marA signal b,c 10]..4) to deN-glycan as serum-type Tf in CSF, was treated with 1 mM NaIO4 to oxidize the exocyclic glycerol side chain of sialic acid, abolishing binding to SSA. Serum Tf was also treated with sialidase to generate asialo-Tf. Asialo-Tf was further digested with β-galactosidase to generate agalacto-Tf. Tf glycoforms so prepared were subjected to lectin inhibition assay. SSA attenuates the ELISA signal of serum Tf but not those of other glycoforms, indicating that lectin inhibition is glycoform- or glycan-specific  decreases linearly in the presence of 0.2–2 μg/mL of SSA and then saturates in a range of 10–30 μg/mL. The maximum rate of inhibition under the aforementioned experimental conditions is approximately 60% [We also examined inhibitory potencies of other lectins—RCA120, PVL, PHA-E, and AAL (120–1250 μg/mL)—with the expectation that some inhibitors for brain-type Tf would be useful for iNPH diagnosis. RCA120 binds terminal Gal residues, which are carried by asialo-Tf. RCA120 inhibits asialo-Tf signal by 27% in ELISA . PVL is Automated latex-agglutination turbidimetric immunoassay (ALI) is a rapid and high-throughput method for detecting various antigens including Tf. Indeed, several hundred assays are performed daily in hospital care, indicating that application of lectin inhibition to ALI would facilitate clinical use of glycoforms analysis. For detecting Tf in ALI, Tfs are mixed with Tf-Ab-coated latex particles to form aggregates that increase turbidity a. The cor = 0.997). Dilution linearity of CSF was in the range of 0.125–0.50 μL. A recovery test reveals that 96–98% of added serum-type Tf in CSF was recovered. Day-to-day variation was less than 8% [Schemas of Tf isoforms used for SSA-ALI are indicated in  than 8% .r = 0.847, p < 0.001), indicating that SSA-ALI is useful for quantifying serum-type Tf as well as total Tf in CSF , does not, suggesting that cancer-specific α2,6-sialylation on CEA may play a role in malignant phenotypes such as cell invasion and metastasis. SSA appears to bind α2,6-sialylated-CEA and inhibit its ELISA signal . We therefore extracted CEA from formalin-fixed colon adenocarcinoma tissues by using PBS containing 1% NP-40 detergent. CEA concentration was determined by sandwich ELISA using two specific antibodies: CD66e Ab-2  and anti-human CEA . The extracts contained CEA in a range of 1.40–3.92 μg/g tissue (n = 3). Indeed, detergent extracts showed a fair amount of ELISA signal in the absence of SSA whereas the signal was markedly reduced in the presence of SSA (Carcinoembryonic antigen (CEA) is a glycoprotein marker that is widely used for diagnosing various cancers, especially colon adenocarcinoma. CEA carries α2,6-sialyl residues on its e of SSA b, suggesCEA is a member of the immunoglobulin (Ig) superfamily and, like other Ig-like molecules, it mediates aggregation of colonic adenocarcinoma cells, which appear to favor invasive nest formation. We examined localization of α2,6-sialylated-CEA in cancer invasion areas of colon adenocarcinoma tissue. Immunohistochemistry reveals infiltration of undifferentiated cancer cells b, suggesIn this review, we demonstrate that SSA inhibits antibody binding to α2,6-sialylated-Tf and α2,6-sialylated-CEA in a glycoform-specific manner. The SSA inhibition is also applicable for visualizing the localization of α2,6-sialylated-Tf in the liver and α2,6-sialylated-CEA in colonic adenocarcinoma tissue. This is the first immunohistochemical demonstration of glycoform localization in a tissue section. SSA inhibition requires binding of SSA to sialylα2,6galactose epitopes of glycoproteins, because removal of sialic acid by sialidase treatment markedly attenuated SSA inhibition signals. SSA forms tetramer (130 kDa), the size of which may partly account for hindrance of many antigen epitopes on Tf (75 kDa). This speculation may be supported by the observation that RCA120 (120 kDa), PVL (40kDa), and AAL (28kDa) lectin inhibited Tf-Ab binding to asialo-Tf and brain-type Tf by 27, 13 and −2%, respectively . In addiLens culinaris agglutinin (LCA) lectin, recognizing core fucose (13). The lectin inhibited monoclonal antibody binding to fucosylated alpha-fetoprotein, an early marker of hepatocellular carcinoma . However, LCA inhibited only 2 out of 30 monoclonal antibodies; one was inhibited by 36% and the other was 59%. Suzuki et al. reported that LCA binding to the core fucose sterically hinders neighboring antigen epitopes recognized by the two monoclonal antibodies. We demonstrated that SSA lectin inhibits polyclonal Tf-Ab binding, suggesting that binding complex of SSA and α2,6-sialylated glycans is flexible enough to hinder a substantial number of antigen epitopes on Tf. This speculation led us to examine SSA inhibition using site-directed peptide antibodies. One is Tf-596Ab against a peptide sequence, Cys596-Ala614, which was proximal to N-glycosylation sites. The other is Tf-120Ab against a peptide sequence, Val120-Cys137, which was distal to N-glycosylation sites. SSA inhibits binding of Tf-596Ab but affects Tf-120Ab only a little (N-glycans is important for SSA inhibition. SSA complexed with the N-glycans may inhibit most polyclonal Tf-Ab, but it may not inhibit some antibody clones that recognize ‘distal’ antigen epitopes. This may explain why SSA partially inhibits polyclonal Tf-Ab signals in immunohistochemical and biochemical assays. Further analysis will be required for addressing the exact molecular mechanisms of lectin inhibition.Partial inhibition of antibody binding to an antigen was also reported for a little , suggestIn conclusion, SSA lectin inhibition assay would be useful for detecting α2,6-sialylated glycoform of Tf and CEA even in crude specimens and tissue sections. The method would further be applicable to glycobiology and clinical medicine.