Anti-ANX-A1 neutralising monoclonal antibodies (Mabs) enhanced the release of pro-inflammatory mediators in response to compound 48/80.4.2, tryptase and β-hexosaminidase from mast cells challenged with compound 48/80. Anti-ANX-A1 neutralising Mabs prevented the inhibitory effect of these drugs.Nedocromil and ketotifen potently inhibited the release of histamine, PGD5.−/− mice were insensitive to the inhibitory effects of nedocromil or ketotifen but cells retained their sensitivity to the inhibitory action of hu-r-ANX-A1.BMDMCs derived from Anx-A16.2, but not histamine, release. BMDMCs derived from fpr2/3−/− mice were insensitive to the inhibitory effects of nedocromil on PGD2, but not histamine release.The fpr2/3 antagonist WRW4 blocked the action of nedocromil on PGD7.Compound 48/80 stimulated both p38 and JNK phosphorylation in CBDMCs and this was inhibited by nedocromil. Inhibition of p38 phosphorylation was ANX-A1 dependent.8.We conclude that ANX-A1 is an important regulator of mast cell reactivity to compound 48/80 exerting a negative feedback effect through a mechanism that depends at least partly on the FPR receptor. •Mast cell ‘stabilisers’ release anti-inflammatory ANX-A1 from mast cells.•Anti-ANX-A1 neutralising Mabs blocked their inhibitory effect.•−/− mice are insensitive to nedocromil or ketotifen.Mast cells from Anx-A1•2 release depends upon the fpr2/3 receptor.The effect of ANX-A1 on PGD•ANX-A1, acting partly through FPR, is a key regulator of mast cell reactivity. The method has been described in detail in a previous publication To generate primary bone marrow-derived murine mast cells (BMDMCs), femur bones from Anx-A1All animal work was performed according to the UK Home Office regulations  and was approved by the Queen Mary University of London Ethics Committee . Human cells were prepared according to a protocol approved by the East London & The City Local Research Ethics Committee .2.32 atmosphere. Drugs, antibodies or the FPR2/ALX antagonist  tested in this system were added 5 min prior to the activation of compound 48/80. Samples were centrifuged at 1000 g for 5 min before the cell-free supernatants were collected to measure histamine and PGD2 release. Cell lysates and supernatant were prepared for Western blots. Sample aliquots were stored at − 80 °C for further analysis. Drugs such as the anti-allergic nedocromil or the glucocorticoid dexamethasone were added as required. In some cases the drug-treated cell aliquots were incubated with a well-characterized specific neutralising anti-ANX-A1 , or an equivalent concentration of an isotype matched irrelevant , mAb.CBDMCs/BMDMCs were stimulated with compound 48/80  for 10 min at 37 °C with 5% CO2.4A commercially available enzyme immunoassay was used to detect and quantify histamine-released in the supernatant . The assay was conducted following the manufacturer's protocols. A standard curve ranging from 0.39–50 nM histamine was prepared using the reagent provided and the optical density was then read within 60 min in a microplate reader  at 405 nm. In some cases, the total cell content of histamine was established by freeze-thawing of cells prior to challenge.‘Spontaneous histamine release’ was expressed as a percentage of the amount (nmol) of histamine released in the absence of challenge divided by the total histamine content of cells.The ‘net histamine release’ was expressed as a percentage of the amount (nmol) histamine released during challenge (corrected for the spontaneous release) divided by the total histamine content of cells (corrected for the spontaneous release).2.52 released in the supernatant. The assay was conducted following the manufacturer's protocols. A standard curve ranging from 78 to 10,000 pg/ml PGD2 was prepared using the reagent provided and the optical density was then read within 60 min in a microplate reader  at 405 nm.A commercially available enzyme immunoassay  was used to detect and quantify PGD2.627 ANX-A1-P and PKC were assessed as previously described Ser2.75 cells in a 1.5 ml Eppendorf tube. Cells were treated with the stipulated drugs for 5 min prior to stimulation with compound 48/80. 2% PFA in 0.1 M PBS was added into the Eppendorf tube for 10 min on ice to fix the cells. Since the CBDMCs are non-adherent cells, each immunostaining steps were performed in 1.5 ml Eppendorf tube and centrifuged at 1000 g for 5 min between each methodological step and the supernatant was aspirated carefully with a vacuum without disrupting the pellet. After fixation, the cells were washed with 0.1 M PBS. Cells were then permeabilised with 0.1% Triton X-100 in PBS for 5 min. Non-specific secondary antibody binding site were blocked by incubation of 10% FCS in PBS for 30 min. Primary antibodies  was diluted with 1% FCS in PBS and left to incubate at 4 °C overnight. The cells were washed twice with 1% FCS in PBS and incubated with fluorescent-labelled secondary antibody  for 1 h at room temperature. The cells were then washed with PBS and stained with 100 ng/ml DAPI (Invitrogen) in ddH2O for 5 min. The cells were washed with 30 μl ddH2O and dropped onto the slide using a pipette. Cover slips were carefully mounted onto the slides using the Prolong Gold Mountant (Invitrogen). Slides were then analysed and micrographs were taken using the confocal microscope (Leica) at 63 × magnification.CBDMCs were plated at a density of 2 × 102.8Where the data satisfied the conditions (e.g. Gaussian distribution), we used ANOVA with the Bonferroni post-hoc correction. In some cases where only two columns of data were analysed, we used Student's T test (unpaired).2.9Nedocromil sodium was generously supplied by Sanofi-Aventis , dexamethasone was obtained from Sigma-Aldrich . The drugs were made freshly on the day of every experiment.33.1We have previously observed that PKC activation is responsible for the phosphorylation of ANX-A1 in the U937 cells GCs such as dexamethasone activate PKC by a non-genomic receptor based mechanism probably involving PI3 kinase We confirmed our previous observation that nedocromil action is dependent upon PKC by testing the effect of an inhibitor of PKC on the effect of nedocromil on the appearance of ANX-A1 Ser-27 in CBDMCs. Using a densitometry analysis n = 3;  we foundWe chose 5 min time point to treat CBDMCs with nedocromil as we have shown previously that the optimal effect of nedocromil was seen by 5 min at which time the amounts of ANX-A1 Ser-27 had increased by 2.1 ± 0.20 fold and the amounts of phospho-PKC by 3.6 ± 0.23 fold We did not measure inhibition of PP2A by nedocromil directly in this study  and where it is subsequently inactivated by PP2A prior to recycling and/or ubiquitination by the cell The CBDMCs were stained for total PKC  and total ANX-A1 . CBDMCs were treated with dexamethasone (2 nM) alone or in combination with nedocromil (10 nM) across various time points. In this experiment, nedocromil produced barely detectable activation of PKC alone compared with untreated cells (not shown) and similar cytoplasmic distribution of total PKC and ANX-A1 was seen at 5, 10 and 20 min (left hand composite). PKC was observed mainly in the cytoplasmic or peri-nuclear region apparently associated with vesicles. Some ANX-A1 was clearly localised at the plasma membrane but there was no co-localisation with PKC. However, when CBDMCs were treated with dexamethasone for 5 min , some co-localisation at membrane level between total PKC and ANX-A1 at 5 min but this effect was not maintained. Following exposure to both nedocromil and dexamethasone (right hand composite) substantial amounts of PKC translocated to the plasma membrane and co-localised with ANX-A1 and this effect was still observed at 20 min.3.2We next tested the ability of compound 48/80 alone to release histamine from CBDMCs. Since preliminary experiments suggested that 20 μg/ml compound 48/80 already promoted maximal mast cell degranulation (60–80%) and we wanted to avoid possible pitfalls associated with maximal stimulation of cells, we investigated the effect of lower concentrations of compound 48/80 ranging from 0.01–10 μg/ml.The time course of the response to compound 48/80 (0.05 μg/ml) was tested in conjunction with nedocromil in From these experiments we conclude that 48/80, in addition to activating CBDMC degranulation, also stimulates ANX-A1 phosphorylation and that this is itself prolonged by the action of nedocromil.3.3To determine the role of ANX-A1 in promoting the stability of mast cells, the effect of a specific neutralising monoclonal anti-ANX-A1 mAb was tested. We have previously reported CBDMCs were pre-incubated for 20 min with 20 μg/ml neutralising anti-ANX-A1 mAb 1B, or an equivalent concentration of an irrelevant isotype-matched mAb prior to treatment with nedocromil and compound 48/80. We also tested ketotifen as we have previously reported that this drug, which is chemically distinct from the cromones, exerts similar effects to nedocromil.2. We also measured the net β-hexosaminidase (We observed  that 10 minidase  and trypminidase  followin2 by 59% (p < 0.01). Nedocromil also inhibited β-hexosaminidase release by 44%  and reduced the release of tryptase from 65.5% to 10.1% (90.8 ± 0.90 units). However, in the presence of the neutralising anti-ANX-A1 monoclonal antibody, these inhibitory actions of nedocromil were completely abolished.Pre-treatment with nedocromil prior to compound 48/80 stimulation significantly (p < 0.001) inhibited the release of histamine by 73.5% and PGD2 from 1432.77 ± 175.77 to 1515.91 ± 127.84 pg/ml (p < 0.01).Interestingly, examination of the data suggested that the neutralising (but not the isotype-matched control) monoclonal anti-ANX-A1 mAb alone exhibited a tendency to enhance the release of mediators in the presence of compound 48/80. For example, in The amount of intracellular tryptase remaining after compound 48/80 was decreased from 65.5 ± 6.83% to 53.6 ± 6.73  in the presence of the neutralising anti-ANX-A1 monoclonal (but not the isotype-matched control).The release of β-hexosaminidase was also increased in the presence of the neutralising anti-ANX-A1 monoclonal from 57.37 ± 1.60 to 67.96 ± 4.44 pNA units (p < 0.01). In this connection, we also observed ; panel B3.42 (61.04% and 58.1% respectively) release in wild type BMDMCs but not in cells derived from Anx-A1 null mice. However, inhibition of mediator release could still be achieved by reconstituting the system with hu-r-Anx-A1 itself, which inhibited histamine and PGD2 release by 66.5% and 69.34% respectively.To further confirm the role of Anx-A1 in the inhibition by nedocromil and ketotifen we tested the activity of these drugs on BMDMCs derived from Anx-A1 null mice. 3.52 release.In many systems, the response of the cell to ANX-A1 is dependent upon the presence of the FPR receptor system e.g.  and thes2 release stimulated by compound 48/80, but while the effect on PGD2 release was greatly reduced in the cells treated with WRW4, the inhibition of histamine release by nedocromil was not altered.2 release.Panels C and D show experiments in which BMDMCs were prepared from fpr2/3  null mice or WT controls and stimulated with compound 48/80. Here, a similar pattern was observed. Whilst the effect of nedocromil on histamine release was still present in the fpr2/3 null cells, it no longer produced a concentration dependent inhibition of PGD3.6Both JNK and p38 have been identified as important components of signalling cascades in leading to mediator release in human blood basophils We have previously reported that activation of FPR2 homo-dimerisation in transfected HEK293 cells activated p38 MAPK pathway whereas activation of FPR1/FPR2 heterodimers activated a JNK mediated signalling pathway To test whether these signalling factors were important in the inhibitory effect of nedocromil on mast cells and whether this was Anx-A1 dependent we measured the phosphorylation of p38 and JNK in CBDMCs following compound 48/80 treatment using Western blotting techniques.4Our previous publications in this area highlighted a novel mechanism of action for the cromone anti-allergic drugs. Using a U937 cell model we demonstrated In a subsequent study, we demonstrated that the ability of cromones to inhibit CBDMC degranulation stimulated by IgE–anti-IgE cross-linking was prevented by immuno-neutralisation of Anx-A1 and was wholly absent in the BMDMCs from Anx-A1 null mice Whilst the cromones are known mainly as anti-allergic drugs, they have many other pharmacological actions including inhibition of leukocyte migration. We further showed It has been known for many years that both PKC and PP2A are translocated to the membrane during mast cell activation whether this is caused by antigen cross-linking at the high affinity IgE receptor or agents that bypass this receptor In this study PP2A translocation and activity was assessed indirectly in mast cells by monitoring the length of time that PKC is resident at the membrane following cell activation. Our idea that nedocromil prolongs PKC activation by decreasing the activity of PP2A enzyme was further supported by our observation here that this drug prolongs the translocation of PKC to the cell membrane. This is also consistent with previous reports in the literature that treatment of mast cells with cromones increases the phosphorylation of proteins thought to be involved in the exocytotic mechanism Here we have observed that in addition to glucocorticoid/cromone treatment, compound 48/80 itself activates PKC, promotes the phosphorylation and release of ANX-A1 and that this too, is potentiated by nedocromil.This finding points to a way by which the intensity of the degranulating stimulus is matched by an increased release of inhibitory ANX-A1 so as to regulate the overall magnitude of the secretory response. It also raises an interesting question: could nedocromil (or other cromone) actually potentiate mast cell degranulation provoked by agents such as compound 48/80 in the absence of ANX-A1?Some support for this idea arises from our findings using immuno-neutralisation of ANX-A1. This biologic not only abrogates the inhibitory effect of both nedocromil and ketotifen but also, interestingly, we observed that there was a strong trend for the neutralising mAb alone to promote mediator secretion when the mast cells were stimulated. This certainly would fit with the general conclusion that this is an endogenous regulatory molecule.Whilst we have previously published on the importance of ANX-A1 in the inhibitory action of cromones in mast cells stimulated by IgE–anti IgE or DNP-BSA/anti-DNP-IgE cross-linking Of interest therefore is the notion that under conditions where ANX-A1 becomes ‘depleted’ – for example, after repetitive challenge with degranulating agents, glucocorticoids or cromones – then the inhibitory response of the latter agents will be temporarily lost. We have indeed observed this in the case of prolonged hydrocortisone stimulation of macrophages 2 release in BMDMCs is entirely congruent with the neutralising monoclonal experiments and the fact that the ANX-A1 null cells still respond to Anx-A1 establishes that the receptor system is still present on these cells. In fact, the role of the FPR receptor family in the response of mast cells to nedocromil is of particular interest. In many systems, ALX/FPR2 is the chief subtype through which ANX-A1, and other anti-inflammatory regulators such as Resolvin D1 and Lipoxin A4, exert their inhibitory actions The results of the experiments in which we tested the ability of nedocromil and ketotifen to inhibit histamine and PGDSince either deletion of Anx-A1 altogether (as in the KO mouse) or its removal using neutralising monoclonals clearly establish that this protein is a vital component of nedocromil action, one would anticipate that removal of the principal receptor for Anx-A1, FPR2 (or its murine homologue) fpr2/3 would prevent the action of this drug as well.2 generation and release, it failed to completely block inhibitory action on histamine release.Our observations here suggest that this is only partially true. Whilst the blockade of the FPR2 receptor in CBDMCs or its removal in the fpr2/3-null BMDMCs clearly prevented the action of nedocromil on PGDN-Ac-2-26, an N-terminal peptide of ANX-A1, produced hetero-dimerisation of FPR1/2, and activated a different, JNK-dependent, signalling pathway.This apparent paradox might be understood in terms of the possible involvement of other members of the FPR family. In a previous study, we observed that the inhibitory effect of nedocromil on the migration of PMN in a cell chamber model of migration was dependent upon the presence of both FPR1 and FPR2 receptors N-Ac-2-26, which acts as a ligand at both FPR1 and FPR2/ALX It is therefore possible that the effect of ANX-A1 on mast cells is the result of a combination of signalling produced by the full-length protein as well as a clipped fragment such as 2 in RBL-2H3 cells The signalling data that we present also poses further questions. Many authors have commented on the roles of MAPK pathways in mast cell activation  reduced the basal phosphorylation of Anx-A1 as well as that induced by nedocromil (10 nM). Above: Representative blot showing the stimulation of phosphorylation by nedocromil and inhibition by the PKC inhibitor. Below: Densitometry data collected from 3 independent experiments expressed as mean ± SEM; *p < 0.05 and **p < 0.01 relative to control or samples treated with nedocromil alone.5 cells per well and the stipulated groups were treated with 10 μg/ml Anx-A1 neutralising antibody or an irrelevant isotype control. Subsequently, the cells were pre-treated with nedocromil (10 nM) for 5 min followed by compound 48/80 (10 μg/ml) stimulation for 10 min. Nedocromil produced consistent inhibition of β-hexosaminidase release, but this was abrogated in the presence of the immuno-neutralising mAb, but not the control reagent. In panel B, the cells were incubated with the Anx-A1 neutralising antibody (or an irrelevant isotype control) only. The supernatants were collected from the samples and assessed for β-hexosaminidase by colorimetric assay. Interestingly, the addition of the Anx-A1 neutralising antibody alone (but not the irrelevant isotype control) was sufficient to release β-hexosaminidase from the CMDMCs. Data are expressed as mean ± SEM from n = 3 experiment and were analysed using one-way analysis of variance (ANOVA), followed by a Bonferroni post-hoc test, *p < 0.05, **p < 0.01, ***p < 0.001 vs unstimulated. In panel A, §§p < 0.01 relative to compound 48/80 treatment alone as determined by Student's T test.The inhibition by nedocromil on β-hexosaminidase release from CBDMCs stimulated with compound 48/80 is dependent upon Anx-A1. In panels A, CBMCs were plated at a density of 2 × 10The inhibitory effect of nedocromil on compound 48/80 stimulated tryptase release from CBDMCs is Anx-A1 dependent. Panel A. Confocal examination of tryptase release in CBDMCs. The cells were stained with antibodies to tryptase (green). DAPI was used to stain the nucleus (blue) and the cells were exposed to 10 μg/ml compound 48/80, 10nM nedocromil and 20 μg/ml of anti-Anx-A1 neutralising mAb. Stimulation with compound 48/80 led to distinct degranulation of the mast cells. Tryptase was confined in the cytoplasm when CBMCs were treated with nedocromil but in the presence of Anx-A1 neutralising antibody, more tryptase were released from the cell. These images are representative of 3 independent experiments. Confocal images were taken at × 63 oil magnification. Scale bars: 10 μm. Panel B. The densitometry analysis of the western blot shows that in the presence of compound 48/80, the tryptase level in the cell lysate is significantly reduced (p < 0.05). When the CBMCs were pre-treated with nedocromil (10 nM), the tryptase is retained in the cells. In the presence of Anx-A1 neutralising antibody alone or combined with nedocromil, the tryptase release is significantly (p < 0.05) enhanced. Data are expressed as mean ± SEM .The following are the supplementary data related to this article.The authors declare none.


Steinernema feltiae freezing under a cryo-stage microscope. The IJs froze when the water surrounding them froze at −2°C and below. However, they avoid inoculative freezing at −1°C, suggesting cryoprotective dehydration. Freezing was evident as a sudden darkening and cessation of IJs' movement. Freeze substitution and transmission electron microscopy confirmed that the IJs of S. feltiae freeze intracellularly. Ice crystals were found in every compartment of the body. IJs frozen at high sub-zero temperatures (−1 and −3°C) survived and had small ice crystals. Those frozen at −10°C had large ice crystals and did not survive. However, the pattern of ice formation was not well-controlled and individual nematodes frozen at −3°C had both small and large ice crystals. IJs frozen by plunging directly into liquid nitrogen had small ice crystals, but did not survive. This study thus presents the evidence that S. feltiae is only the second freeze tolerant animal, after the Antarctic nematode Panagrolaimus davidi, shown to withstand extensive intracellular freezing.Taking advantage of their optical transparency, we clearly observed the third stage infective juveniles (IJs) of  Steinernema feltiae is a temperate species and is globally distributed S. feltiae is a cold tolerant species The entomopathogenic nematode S. feltiae IJs could survive sub-zero temperatures S. feltiae IJs based on morphological changes at the cellular level after freezing them under various conditions. Two techniques have been used: cold stage microscopy and freeze substitution/transmission electron microscopy. Nematodes are transparent and freezing within their bodies can be observed under a cold microscope stage In our previous experiments, we showed that Freeze substitution is a process in which the ice within the frozen specimen is replaced by an organic solvent, such as methanol or acetone, at a very low temperature; below −70°C We used freeze substitution of nematodes after different freezing regimes involving different temperatures and exposure times. The aim was not to prevent ice formation but to locate the position and pattern of ice crystals within the frozen nematodes, which may account for differences in their freezing survival.Steinernema feltiae was grown in Galleria mellonella larvae at 23°C using the method described by Kaya and Stock −1 and freezing was initiated by seeding with a small ice crystal at the edge of the coverslip. Nitrogen gas was supplied to the sample chamber of the cold stage to prevent condensation. The freezing process was visualized using a 10× objective lens and images were captured at regular intervals by a Canon Powershot A640 Digital Camera, controlled by Axio Vision v. 4.6 software (Zeiss) run on an Insite PC. Freezing of nematodes was observed as a sudden darkening. Unfrozen nematodes did not change their appearance and remained clear. After seeding, the sample was allowed to stay frozen for either 3 h at −1°C or 30 min at all other test temperatures, and then rewarmed to 2°C at 0.5°C min−1. Nematodes were removed from the cold stage once it had reached room temperature and transferred to watch glasses in ATW. Survival was determined after 24 hours. The experiment was replicated three times.The freezing process was observed on a microscope cold stage similar to that described by Wharton & Rowland −1, seeded with an ice crystal and held at −3°C or −10°C for 75 min. Slow-frozen samples were cooled from 2°C to −1°C at 0.5°C min−1, seeded with an ice crystal and held at −1°C for either 75 min or overnight. At the end of the freezing time, it was checked that the samples were frozen and they were then transferred to liquid nitrogen for transportation before freeze substitution.To see the differences in morphology of IJs after freezing, different freezing regimes were used. Each sample consisted of 10 μl of nematode suspension in an Eppendorf tube  transferred to a cooling block, the temperature of which was controlled by fluid circulating from a Haake Phoenix II-C35P programmable refrigerated circulator (Thermo Fisher Scientific). No thermocouple was inserted into samples processed for freeze substitution. Fast-frozen samples were cooled from 2°C to −3°C or −10°C at 0.5°C min−1 to 2°C and transferred to ATW in watchglasses at room temperature. Survival was determined after 24 hours by counting the proportion moving after a mechanical stimulus.Freezing survival was determined from samples subjected to the same freezing regimes as above, but with a NiCr/NiAl thermocouple inserted in each tube. Thermocouples were interfaced to a Macintosh computer via a Powerlab A/D interface . The temperature records were analysed using a computer programme  to ensure that the exotherm, and hence the freezing of the sample was completed. The duration of the exotherm was used as an indication of its freezing rate. Samples were rewarmed at 0.5°C minNematode suspensions in Eppendorf tubes were also plunged directly into liquid nitrogen without prior freezing and processed via freeze substitution to serve as a frozen control. Nematodes were also prepared for electron microscopy using a conventional preparation technique to compare the normal nematode morphology with those that were frozen. This consisted of fixing them in 4% formaldehyde, 2.5% glutaraldehyde, 1% osmium tetroxide followed by dehydration in ethanol, and embedding in Spurr resin.Samples in liquid nitrogen were transferred into a Reichert automatic freeze substitution (AFS) apparatus and substitution fluid (methanol containing fixatives) added. Freeze substitution and embedding were then carried out as reported previously The following parameters were measured after transmission electron microscopy.a) were calculated using a stereological method based on point counting a = ∑Pc/∑Ps .Areal Fractions , extracellular ice , and those that were unfrozen. All images taken (13–46 per treatment) were used for quantifying ice crystal pattern and shrinkage of nematodes . However, measurements of the diameter of the five largest ice crystals and areal fractions  were confined to those sections which were well infiltrated with resin and were clear enough to measure these parameters accurately (13–24 per treatment).The Statistical Package for Social Sciences (SPSS) ver. 15.0 was used for statistical analyses At −2°C and below, nematodes were clearly observed to freeze by exogenous ice nucleation by the surrounding ice, as indicated by a sudden darkening of the nematodes. Exogenous ice nucleation did not occur at −1°C and nematodes remained clear and unfrozen, although the medium surrounding them was frozen . NematodS. feltiae IJ sectioned through the intestinal region after preparation for transmission electron microscopy by chemical fixation is shown in The morphology of an unfrozen The morphology of specimens frozen in liquid nitrogen and prepared by freeze substitution for electron microscopy is similar to that of non-frozen controls except for its reticulated appearance due to the presence of small ice crystals throughout the tissues, including cell organelles . The cirNematode survival was high after freezing at −1°C or −3°C for 75 min and −1°C overnight but there was no survival after freezing at −10°C . Three d3, 58 = 16.45; P<0.05). Post hoc comparison tests showed that ice crystal diameters in nematodes frozen at −10°C were significantly different from those frozen at −1°C and −3°C. However, ice crystal diameters did not differ significantly between nematodes frozen at −1°C overnight, −1°C and −3°C for 75 min  among the freezing regimes  and was more prominent in the slow freezing regimes (−1°C: 50–77.8%) than the fast freezing regimes  as shown in Overall there was a significant difference between the diameters of the five largest ice crystals measured between freezing regimes , similar to the cold tolerance strategy of Heterorhabditis zealandicaInoculative freezing did not occur at −1°C in t freeze . This inS. feltiae have been reported by Brown and Gaugler S. feltiae, were freeze tolerant and froze as the temperature decreased after initiating freezing at −0.5°C. However, it is not clear from their study at which temperature the nematode froze, and they did not provide any pictures of the freezing event, so their nematodes may not have frozen at −1°C. The results of the present study are similar to those reported by Wharton et al. Panagrolaimus davidi, which does not freeze if the medium is seeded at −1°C and undergoes cryoprotective dehydration, but freezes below −2°C. A small amount of shrinkage, that may indicate cryoprotective dehydration, was observed in S. feltiae frozen at −1°C. Some nematode species freeze even at −1°C and cannot avoid inoculative freezing At −2°C, and lower temperatures, all the nematodes turned dark and stopped moving, suggesting that their survival in the cooling block experiments at these temperatures −1 for vitrification to occur Freeze substitution enabled the location of ice crystals to be determined, appearing as white spaces after the frozen water was replaced by substitution fluid and then by resin inside the frozen nematodes. Overall, fast-frozen samples had large and slow-frozen samples had small ice crystals. Ice crystals were observed both in intracellular and extracellular compartments. Nematodes frozen directly in liquid nitrogen had small ice crystals but did not survive . They haS. feltiae is not as strong as that described in an Antarctic nematode P. davidiPanagrellus redivivusSlight shrinkage is evident in some treatments. This indicates partial dehydration and was noticeably greater in the samples frozen overnight at −1°C than the rest of the treatments. This is an indication of cryoprotective dehydration as water is lost from liquid water inside the nematode to the surrounding ice, as a result of the difference in vapour pressure Rana sylvatica, produce cryoprotectants during the freezing process itself as soon as ice nucleation is initiated which helps the organism survive sub-zero temperatures S. feltiae for an extended time (overnight) at −1°C may improve survival by allowing them to synthesize cryoprotectants.Some organisms, such as the freeze tolerant wood frog Nematodes frozen at −3°C and −10°C were observed to have ice crystals in both intracellular and extracellular compartments and a different appearance from samples frozen in liquid nitrogen. Nematodes survived freezing at −3°C but not at −10°C. Ice crystals were observed in most of the compartments of the infective juveniles, including the pharynx, muscle cells, intestinal cells, secretary-excretory cells and epidermis. The pseudocoel was not clearly visible.S. feltiae is one of the rare organisms that have been shown to survive intracellular freezing. Most freezing tolerant animals can survive only extracellular ice formation and for them intracellular freezing is lethal P. davidi is the only organism previously reported to survive extensive intracellular freezing After thawing the infective juveniles were motile, pathogenic to their insect host as before and produced the next generation mammalian cells frozen predominantly by heterogeneous ice nucleation indicating this nematode may have aspecialized cell membrane. Survival of intracellular freezing may depend upon the pattern of ice formation. The size of ice crystals in samples frozen at −3°C was significantly smaller than those frozen at −10°C which may account for their survival. More than 97% of nematodes survived at −3°C, whereas all the nematodes died at −10°C where all or the majority of ice crystals formed were large.S. feltiae. Recrystallization inhibition has been reported in another species of entomopathogenic nematode S. carpocapsae, which was second in RI activity only to P. davidi when compared to four other species The formation of small ice crystals at −3°C could be due to the presence of an ice active protein, such as a recrystallization-inhibiting protein that inhibits the growth of the crystals S. feltiae infective juveniles to survive freezing.Although ice crystal size varied across the treatments, areal fractions were not significantly different, indicating no effect on the proportion of the body frozen. Fewer but larger ice crystals appear to be lethal to the nematodes irrespective of the proportional area they occupy. The formation of small ice crystals allows S. feltiae resist inoculative freezing at high sub-zero temperature (−1°C). At lower sub-zero temperature intracellular freezing occurs, which the nematodes can survive, but only down to about − 4°C. The species however, cannot completely control their ice crystal formation, since some large crystals, as well as small crystals, are formed even at −3°C. Survival of intracellular freezing is associated with the smaller size of ice crystals formed during slow freezing, which may be controlled by a substance that inhibits recrystallization.In conclusion, infective juveniles of


In the Materials and Methods section, there is an error in the second and third sentences of the section titled “Flow cytometric analysis for receptor expression.” The correct sentence is: Cells were then stained with anti-human GPR120 mAb  or isotype-matched control mAb (Santa Cruz Biotechnology) for 30 min on ice. Next, the cells were incubated with Alexa Fluor 488 anti-goat IgG  for 30 min on ice.


Micropterus dolomieu, white sucker Catostomus commersonii, and redhorse sucker Moxostoma species were collected in the summer, a period of low flow and low reproductive activity. Smallmouth bass were the only species in which testicular oocytes were observed; however, measurable concentrations of plasma vitellogenin were found in male bass and white sucker. The percentage of male bass with testicular oocytes ranged from 10 to 100 %, with the highest prevalence and severity in bass collected in the Susquehanna drainage. The percentage of males with plasma vitellogenin ranged from 0 to 100 % in both bass and sucker. Biological findings were compared with chemical analyses of discrete water samples collected at the time of fish collections. Estrone concentrations correlated with testicular oocytes prevalence and severity and with the percentage of male bass with vitellogenin. No correlations were noted with the percentage of male sucker with vitellogenin and water chemical concentrations. The prevalence and severity of testicular oocytes in bass also correlated with the percent of agricultural land use in the watershed above a site. Two sites within the Susquehanna drainage and one in the Delaware were immediately downstream of wastewater treatment plants to compare results with upstream fish. The percentage of male bass with testicular oocytes was not consistently higher downstream; however, severity did tend to increase downstream.Fishes were collected at 16 sites within the three major river drainages  of Pennsylvania. Three species were evaluated for biomarkers of estrogenic/antiandrogenic exposure, including plasma vitellogenin and testicular oocytes in male fishes. Smallmouth bass The online version of this article (doi:10.1007/s10661-014-3868-5) contains supplementary material, which is available to authorized users. Micropterus dolomieu in the Potomac drainage raised concern about the potential effects of exposure to endocrine-disrupting or modulating chemicals (EDC) in the Chesapeake Bay watershed  was first noted in smallmouth bass (SMB) during the microscopic examination of tissues in response to fish mortalities observed in 2002–2003 in the South Branch Potomac River. Since then, mortalities have also occurred in the Shenandoah and the Monocacy Rivers, both tributaries of the Potomac River, and moderate to high prevalence of TO has been documented at these sites as well  to document biological effects, specifically, prevalence and severity of TO and other indicators of reproductive endocrine disruption at sites in the three major river basins in Pennsylvania; (2) to compare these findings to chemical concentrations and potential sources and land use; and (3) to compare species responses.Fish were collected from a total of 16 sites within the three river drainages in Pennsylvania from 2007 to 2010  of two species. The rationale was to compare two species at each site that represented different food and habitat preferences. Species included SMB, a member of the Centrarchidae family, representing carnivorous, pelagic species. SMB have been shown to be a sensitive indicator species for EDC studies, expressing signs of estrogenic exposure such as TO and plasma vitellogenin (Vtg) in males . In the Ohio River drainage, SMB and RHS were collected in the summer of 2008 (July 23–August 5). All these species are spring spawners, and hence in summer, post-spawn is the least reproductively active period. However, this sampling period was chosen to represent low flow conditions. Pre-spawn SMB were compared at one site in the Susquehanna drainage (Juniata River) and one site in the Ohio drainage (upstream Allegheny) in spring 2010 (April 21–28). Exposure to agriculturally associated chemicals is likely to be highest during spring high flow periods after field application and when runoff is most likely.g and 4 °C for plasma separation. Plasma was removed, aliquoted into cryovials, and stored at −80 °C until assayed for Vtg.Fish were collected by either barge or boat electrofishing and held in large, aerated buckets of river water until processed (less than 1 h). Fish were euthanized with tricaine methanesulfonate  and bled from the caudal vein using heparinized 3-cc syringes with 23 gauge needles. Blood was placed in Vacutainer tubes  containing sodium heparin and held on wet ice until centrifuged (same day) for 10 min at 1,000 × 3 in millimeter) × 105. A complete necropsy-based fish health assessment as described by Goede and Barton (Each fish was weighed (to the nearest gram), measured (to the nearest millimeter), and observed for gross lesions and abnormalities, liver and gonad were removed and weighed to the nearest 0.1 g. Condition factor (Ktl) was calculated by the formula: ((body weight − gonad weight in grams) / lengthd Barton  was compPieces of gonad were fixed in Z-Fix™  for histological analysis. Gonadosomatic index (GSI) was calculated as follows: (gonad weight / body weight) × 100. Plasma Vtg concentrations in SMB and WS were measured using a direct enzyme-linked immunosorbent assay (ELISA) with species-specific monoclonal antibodies and were carried out at the Leetown Science Center or the University of Florida, Center for Human and Environmental Toxicology, as described by Denslow et al.  and BlazFive to seven cross sections were taken along the gonads of both male and female fish and processed for histological evaluation; provided enough tissue was present. The tissue pieces were embedded in paraffin, sectioned with a microtome at 6 μm, and stained with hematoxylin and eosin Luna . SectionDiscrete water and bed sediment samples were collected at the time of fish sampling. Methodology and occurrence data are presented in Reif et al. . In Sprip) level of 0.05 was used for all statistical analyses.If data were normally distributed, means were compared using Tukey’s multiple comparison test. Data not normally distributed were compared using nonparametric ANOVA followed by Dunn’s multiple comparison test to compare site medians. For upstream/downstream comparisons, a two-tailed Mann–Whitney test was performed. A Spearman’s rank correlation (rho) was used to test for the relation between TO or plasma Vtg and land cover characteristics or chemical concentrations . A significance (p < 0.0001) longer, heavier, and older than all other sites (Table p = 0.0199), with bass from upstream having a lower mean Ktl than those from the middle, while those from the downstream site were not different than either of the other two sites. The two Susquehanna sites were not different in a pairwise comparison  in length and weight than those from all sites except the downstream Monongahela site. In pairwise comparisons, there was no difference in SMB collected at upstream and downstream sites on the Monongahela River. However, bass from the upstream Allegheny site were longer (p = 0.0274), heavier (p = 0.0417), and older (p = 0.0180) than those from the downstream site . Weights of SMB collected at Allegheny were less than those collected at the Juniata site (p < 0.0001) as were condition factors  among sites. The Susquehanna upstream and downstream, Juniata, and downstream Swatara sites had a higher severity than all the Ohio sites which were all similar to one another. Testes from male bass collected at the Schuylkill, Swatara upstream, and Swatara mid-sites had mean severity ratings that were intermediate and not different from either the Ohio sites or the other Susquehanna sites (Table p < 0.0001) than those collected in Juniata  to 100 % in the Juniata River (summer and spring). On the Susquehanna mainstem, 63 % of the SMB had measurable Vtg at the downstream site, while only 21 % had measurable amounts at the upstream site Fig. . Mean cop = 1.000) or severity (p = 0.3705), percentage of males with Vtg (p = 0.0815), or mean Vtg of those individuals with measurable concentrations (p = 0.1429). Sites on Swatara Creek had significantly different (p = 0.048) TO severities with an increasing gradient from upstream to downstream. However, only the middle site had males with measurable Vtg concentrations.A comparison of SMB collected at upstream and downstream sites on the mainstem Susquehanna showed no significant difference in TO prevalence (p = 0.3899) in GSI among female SMB collected in the Ohio or Susquehanna drainages. Although not statistically different, it is interesting that female GSI were higher at the upstream than at the downstream sites in all cases  or female (p = 0.1651) SMB collected in spring 2010 at the Allegheny and Juniata sites . Condition factors of females were not significantly different among the three sites. A similar trend was seen in male WS with those collected at the upstream Brodhead Creek site being shorter and lighter than the other two sites (p < 0.0001), and condition factors were not different (Table p < 0.0001) than those collected downstream.WS were collected at three sites in the Delaware drainage and five sites in the Susquehanna drainage. There were size differences between males and females, and so they were analyzed separately. At the sites for which age data was available, the females tended to be older. Females also tended to be larger at most sites with the exception of the Schuylkill River site at which the males were larger. Within the Delaware drainage, female WS collected at the Schuylkill River site and the downstream Brodhead Creek site were similar in length and weight, while those collected at the upstream Brodhead Creek site were significantly shorter and lighter (nt Table . Suckersp < 0.05) and lighter (p < 0.01) than those collected at the mid-Swatara site or downstream Swatara site. All others were similar in length and weight. Female WS collected at the downstream Swatara site were heavier and longer (p < 0.05) than those collected at the mid-site and upstream site. There were no differences among the sites in condition factor.Within the Susquehanna drainage, male WS collected at the upstream Swatara Creek site were shorter  were similar to those from the upstream Swatara site, while WS collected at Yellow Breeches were similar to those collected at the downstream Swatara site Table .Table 7RTO were not observed in WS from any site; however, no males were collected at Conodoguinet and only four at the Swatara downstream site. Vtg was detected in the plasma of male WS from the Schuylkill River, Brodhead Creek, and upstream and mid-Swatara Creek sites. None of the six males collected at Yellow Breeches or the four collected at the downstream Swatara Creek had detectable Vtg. At Brodhead Creek, there was a higher prevalence of male WS with detectable Vtg at the downstream site when compared to the upstream site. The mid-Swatara site also had a higher prevalence of males with detectable Vtg than the upstream site Fig. . Both ofRHS were collected at all five sites in the Ohio River drainage and ranged in length from 180 to 605 mm. Histologically, most sucker testes showed no signs of development with the predominant cells being spermatogonia and somatic supporting cells. All but two of the RHS were 264 mm or greater which by previous studies were 3 years of age or greater and should have been mature adults Meyer . No TO w2) and Brodhead Creek (671 km2) to large drainage areas such as the Ohio  and mainstem Susquehanna  Rivers (Table p = 0.0170) and severity  but not with the percentage of males with Vtg  when comparing the 12 sites at which SMB were collected. These watersheds also varied greatly in the number of WWTP and sewage discharges (Table p = 0.0794) or severity  or the percentage of SMB males with Vtg . No significant correlations with land use characteristics and Vtg in male WS were noted.At all sites, general water quality parameters  were within ranges not expected to adversely affect the species of interest at the time of collection. Water temperature ranged from 20.4 to 27.3 °C at sites in the Delaware drainage, from 21.4 to 28.1 °C at sites in the Susquehanna drainage, and from 25.0 to 27.2 °C at sites in the Ohio drainage. Dissolved oxygen concentrations ranged from 6.1 to 10.6 mg/L, and pH values ranged from 7.1 to 8.5 in all drainages. Watersheds above which fish were collected ranged from small drainage areas such as Yellow Breeches (534 kmp = 0.0238), severity , and the percentage of male SMB with measurable plasma Vtg . No correlations were noted between chemical contaminants and Vtg concentrations in male WS.Concentrations of selected hormones, as well as other chemicals known to be associated with reproductive endocrine disruption, were measured in discrete water and bed sediment samples collected at the time of the fish collections  was the Ohio River site. Estimated concentrations of the plant sterol 3β-sitosterol were found at most sites with the highest present at the Conodoguinet, two Susquehanna, Swatara up and mid, and the two Monongahela sites  versus the Allegheny site (Ohio drainage) in spring 2010. Interestingly, the percentage of males with Vtg and the plasma concentrations from these two sites were similar in the bass collected in the spring 2010.SMB were the only species in which TO were observed in our study. Bass, in general, appear to be sensitive to estrogenic chemical exposure, particularly with regard to induction of TO. This was also evident in a national monitoring program conducted in nine US river basins from 1995 to 2004. TO were not observed in any of the 774 male carp collected, while 18 % of the 390 largemouth bass and 33 % of the 70 SMB collected at the same sites were observed with TO , and many of the nonylphenol compounds were found  in Colorado. Intersex prevalence was 18–22 % at these sites , suggesting species sensitivity differences  and agricultural runoff. While the prevalence of male SMB with TO was not higher at sites immediately downstream of WWTPs, the severity of TO and percentage of males with plasma Vtg generally increased at downstream sites. This is similar to observations on SMB collected at sites upstream and downstream of WWTPs in two tributaries of the Potomac River  was observed between TO and total hormone/sterol bed sediment concentrations  indicated a positive correlation of TO prevalence with agricultural use and number of animals  in the catchments above the sites, while severity positively correlated with those land-use factors as well as with WWTP flow, total animal feeding operations, and poultry houses ESM 2(DOCX 27 kb)Below is the link to the electronic supplementary material.


Here, we find that endogenous AMPK activation in mouse primary neurons induced an increase of tau phosphorylation at multiple sites, whereas AMPK inhibition led to a rapid decrease of tau phosphorylation. We further show that AMPK mice deficient for one of the catalytic alpha subunits displayed reduced endogenous tau phosphorylation. Finally, we found that AMPK deficiency reduced tau pathology in the PS19 mouse model of tauopathy. These results show that AMPK regulates tau phosphorylation in mouse primary neurons as well as in vivo, and thus suggest that AMPK could be a key player in the development of AD pathology.Neurofibrillary tangles (NFTs) are the pathological hallmark of neurodegenerative diseases commonly known as tauopathies. NFTs result from the intracellular aggregation of abnormally and hyperphosphorylated tau proteins. Tau functions, which include the regulation of microtubules dynamics, are dependent on its phosphorylation status. As a consequence, any changes in tau phosphorylation can have major impacts on synaptic plasticity and memory. Recently, it has been demonstrated that AMP-activated protein kinase (AMPK) was deregulated in the brain of Alzheimer’s disease (AD) patients where it co-localized with phosphorylated tau in pre-tangle and tangle-bearing neurons. Besides, it was found that AMPK was a tau kinase  Two main upstream kinases have been reported: LKB1 (liver kinase B1) and CaMKKβ . Although in the brain AMPK is mainly expressed in neurons1AMP-activated protein kinase (AMPK) is a master energy sensor whose activation following energy stress conditions helps maintain cellular energy levels. In this context, AMPK acts by phosphorylating key enzymes in metabolic pathways as well as transcriptional factors and cofactors. AMPK is a hetero-trimeric complex composed of a catalytic α subunit and two regulatory β and γ subunits. Several genes encode these subunits  giving rise to a multitude of complexes. The regulatory γ subunit possesses four CBS (cystathionine-beta-synthase) domains from which three of them are able to bind adenine nucleotides thereby allowing the kinase to sense and respond to AMP, ADP and ATP intracellular levels. In particular, AMP binding induces a conformational change in the α-subunit activation loop of the kinase that allows phosphorylation of the activating residue Thr262 and Ser356 phosphorylation was shown to alter the microtubule binding properties of tau11121415161721in vitro52+ concentration thus leading to CaMKKβ activation and subsequent AMPKα1 phosphorylation23272930Tauopathies - the best known of them being Alzheimer’s disease (AD) - are a subset of neurodegenerative diseases characterized by a common pathological process, aggregation of the microtubule-associated tau protein. Among these disorders, such aggregation leads to neurofibrillary tangles (NFTs), Pick bodies or argyrophilic grains. In all of these histopathological hallmarks, tau proteins are found abnormally and hyper-phosphorylated. Tau main functions include microtubules stabilization, neurite outgrowth, and facilitation of organelles axonal transport including mitochondria78in vivo. To this end, we used a pharmacological approach to mimic energetic stress conditions to activate AMPK. We then evaluated the involvement of AMPK in vivo in the mouse brain by assessing endogenous tau phosphorylation levels in the brain of mice conditionally knock-out (KO) for the α2 subunit of AMPK and in AMPKα2 KO mice crossed with the PS19 tau transgenic mouse model of tauopathy.The goal of our study was to determine whether AMPK is involved in tau phosphorylation in differentiated primary neurons and 172) co-localized both in neurites and cell body  and anti-p-Thr172AMPK primary antibodies. PLA amplification occurs only if the antigens are located within 40 nm of proximity. PLA staining which is visualized as fluorescent dots was observed in cell body and neurites thereby reflecting AMPK-tau interactions  at Ser79, a direct cytosolic target of AMPK  or Ser491 (for AMPKα2 subunit) thereby inactivating the kinase and AMPK-mediated tau phosphorylation. While this phosphorylation was reported to be necessary for AMPK inhibition, it is not sufficientWe previously demonstrated that AMPK was highly deregulated in the brain of AD and other major tauopathies patients where activated AMPK co-localized with phosphorylated tau in pre-tangle and tangle bearing neurons. An abnormal staining of AMPK was also observed in neurons not yet presenting tau hyper-phosphorylationin vivo studies have only assessed the impact of AMPKα2 subunit deletion on tau phosphorylation, we cannot exclude the possibility that the AMPKα1 subunit might also be involved in tau phosphorylation, and further studies using AMPKα1 KO mice are warranted. It will also be interesting to determine to which extend tau pathology is modulated in double AMPKα1/α2 KO mice. Unfortunately the double AMPKα1/α2 KO is embryonic lethalWhile our in vivo in the mouse brain. AMPK shares at least the same properties as the main tau kinases that act in AD. Indeed, evidence suggests that AMPK participates in APP processing32462425485In conclusion, our present study suggests that AMPK mediates tau phosphorylation in neurons and 79-ACC were obtained from Cell Signaling Technology. Anti-AMPK α1 and anti-AMPK α2 antibodies were from R&D Systems. Anti p-Thr172AMPK antibody and p-Thr172AMPK blocking peptide were obtained from Santa Cruz Biotechnology396/Ser404)202)231)262/356)Antibodies directed against AMPKα1/α2, ACC, pSerAICAR, metformin, PD98059, H-89, LY294002 were purchased from Tocris; roscovitin and MARK/Par1 inhibitor from Merck; Compound C was from Santa-Cruz Biotechnology and rapamycin was from Cell Signaling Technology.−/−)−/−:TauP301S and AMPKα2+/+:TauP301S controls. Males and females were used at 6 or 8 months of age as indicated. Following sacrifice, brains were quickly isolated, and hemi-brains were either snap-frozen or immersion-fixed in 4% paraformaldehyde (PFA) for histochemical analyses.All animal experiments were performed according to procedures approved by the Feinstein Institute for Medical Research Institutional Animal Care and Use Committee. PS19 tau transgenic mice expressing P301S human mutant tau protein under the direction of the mouse prion protein promoter were obtained from The Jackson Laboratory 3, 2 mM EGTA, proteases and phosphatases inhibitors (complete Mini Roche). Protein concentrations were determined using BCA assay.Brains were homogenized in an appropriate volume of homogenizing buffer, a solution of TBS , pH 7.4 containing 10 mM NaF, 1 mM NaVOFor heat stable tau, NaCl was added to a 500 μl aliquot of homogenate to a final concentration of 250 mM, and β-Mercaptoethanol was added to 5% final concentration. Homogenates were mixed, heated at 100 °C for 15 minutes, mixed again by vortexing, cooled on ice for 30 minutes, vortexed again and spun down at 20,000 × g for 10 minutes at 4 °C. The supernatant, containing heat stable tau fraction was retained and used for subsequent experiments.To obtain insoluble tau preparation6 cells/well. Culture medium was completely replaced after 16 h, and new medium (1:3 of starting volume) was added every 3 days until the end of the culture period. Drug treatments were realized directly in the conditioned media.Primary neurons were obtained from C57BL6 wild-type mice as described previously® i3 .Cell toxicity was assessed by the measurement of lactate dehydrogenase release as per manufacturer’s instructions . Absorbance measurements were obtained using a SpectraMax2, 2 mM EGTA, 30% glycerol, 0.1% Triton X-100, 80 mM Pipes, pH 6.8 supplemented with proteases and phosphatases inhibitors. Homogenates were ultracentrifuged at 100,000 × g at 27 °C for 20 min, supernatants were collected as cytosolic fractions. The remaining pellets (microtubule fractions) were washed once and recovered in an equal volume to that of cytosolic fraction by sonication in RIPA buffer and homogenization at 4 °C for 1 h. Samples were mixed with loading buffer, and equal volumes were loaded and analyzed by Western blotting.Cell fractionation was performed as previously describedCell extracts were separated by SDS-PAGE and transferred to nitrocellulose membranes as described previously396/404), CP13 (pSer202) and 2E12 (pThr231) at a concentration of 6 μg/ml in coating buffer  for at least 48 h at 4 °C. Plates were washed 3 times in wash buffer, and blocked for 1 h at room temperature using SuperBlock (ThermoScientific). Plates were washed 5 times and 50 μl of appropriate sample, recombinant tau or PHF-tau diluted in 20% SuperBlock in 1× TBS were added to the plate. 50 μl of the total detection antibody DA9-HRP diluted 1:50 in 20% Superblock in 1× TBS was added to the samples. Plates were then incubated overnight at 4 °C. Plates were washed 9 times and 100 μl of 1-step ULTRA TMB-ELISA (ThermoScientific) was added for 30 minutes at room temperature. Finally, 100 μl of 2 M H2SO4 was added to stop the reaction, and plates were read using an Infinite m200 plate reader (Tecan) at 450 nm.Low tau sandwich ELISA were performed as described previously172AMPK, 1:100 dilution) O/N at 4 °C, followed with anti-IgG secondary antibodies coupled to Alexa Fluor 488 and 568 . Nuclei were visualized with DAPI. Images were acquired on a Zeiss Axio Imager Z2 microscope  equipped with a Hamamatsu ORCA-Flash4.0 digital camera  and ApoTome.2 system . P-Thr172AMPK blocking peptide was used as a specificity control for the p-Thr172AMPK antibody. In these conditions, p-Thr172AMPK antibodies (200 μg/ml) were pre-incubated O/N at 4 °C with a five-fold excess of blocking peptide (1 mg/ml) in PBS  before proceeding with the immunofluorescence protocol described above.Neurons grown on poly-D-lysin and laminin-coated glass coverslips were fixed in 4% (PFA) and permeabilized using 0.25% Triton X-100 before blocking in 1% BSA for 1 h at RT. Neurons were then incubated with primary antibodies directed against total tau , MAP2 (1:200 dilution), total AMPKα1/α2 (1:100 dilution), and activated AMPK (p-Thr172AMPK (1:100 dilution) were used for in situ proximity ligation assay. PLA was performed according to the manufacturer’s protocol (Duolink® Technology). Briefly, primary antibodies were incubated as described for immunofluorescence protocol, followed by incubation with secondary antibodies conjugated to oligonucleotides strands denoted PLUS and MINUS . The ligation and polymerization steps were conducted at 37 °C in a humid chamber followed by detection using complementary fluorescent-labeled oligonucleotides (Duolink® In situ Detection reagents Green). Negative controls for PLA were realized using the same conditions with either one or both of the primary antibodies lacking.To detect AMPK-tau interactions in PFA-fixed primary neurons, mouse monoclonal antibodies directed against tau  in combination with polyclonal rabbit antibodies directed against p-ThrImmunohistochemistry was performed as previously describedpost-hoc test using GraphPad Prism . All data are reported as mean ± SD or SEM, and a value of p < 0.05 was considered statistically significant.Group comparisons were made using one-way ANOVA with Bonferroni’s How to cite this article: Domise, M. et al. AMP-activated protein kinase modulates tau phosphorylation and tau pathology in vivo. Sci. Rep.6, 26758; doi: 10.1038/srep26758 (2016).


Gln3, responds to four distinct nitrogen environments: nitrogen limitation or short-term starvation, i.e., nitrogen catabolite repression (NCR), long-term starvation, glutamine starvation, and rapamycin inhibition of mTorC1. We have previously identified unique sites in Gln3 required for rapamycin-responsiveness, and Gln3-mTor1 interaction. Alteration of the latter results in loss of about 50% of cytoplasmic Gln3 sequestration. However, except for the Ure2-binding domain, no evidence exists for a Gln3 site responsible for the remaining cytoplasmic Gln3-Myc13 sequestration in nitrogen excess. Here, we identify a serine/threonine-rich (Gln3477–493) region required for effective cytoplasmic Gln3-Myc13 sequestration in excess nitrogen. Substitutions of alanine but not aspartate for serines in this peptide partially abolish cytoplasmic Gln3 sequestration. Importantly, these alterations have no effect on the responses of Gln3-Myc13 to rapamycin, methionine sulfoximine, or limiting nitrogen. However, cytoplasmic Gln3-Myc13 sequestration is additively, and almost completely, abolished when mutations in the Gln3-Tor1 interaction site are combined with those in Gln3477–493 cytoplasmic sequestration site. These findings clearly demonstrate that multiple individual regulatory pathways cumulatively control cytoplasmic Gln3 sequestration.A remarkable characteristic of nutritional homeostatic mechanisms is the breadth of metabolite concentrations to which they respond, and the resolution of those responses; adequate but rarely excessive. Two general ways of achieving such exquisite control are known: stoichiometric mechanisms where increasing metabolite concentrations elicit proportionally increasing responses, and the actions of multiple independent metabolic signals that cumulatively generate appropriately measured responses. Intracellular localization of the nitrogen-responsive transcription activator,  Saccharomyces cerevisiae yeast cell into a barren landscape, or anything in between, depending on its severity. Coping with such changes requires a rapid response that is adequate but not excessive. Given these demands, it is not surprising that Gln3 and Gat1, the nitrogen-responsive GATA-family transcription activators, are finely regulated and rapidly responsive  (A simple rain shower can convert a lush environment inhabited by a epressed . Cytoplaed (Ure2 . As nitron (NCR) .Sit4 phosphatase-dependent Gln3 dephosphorylation, nuclear localization, and Gln3-mediated transcription in nitrogen-rich medium —a serine/threonine protein kinase complex (mTorC1)—aberrantly elicits Gln3 and Tor associated protein, Tap42, which, in turn, interacts with TorC1, PP2A, and Sit4 phosphatases,  sphatase .Gln3 regulation, it was not until the six independent methods routinely and interchangeably used to downregulate mTorC1 activity were simultaneously analyzed with a single reporter, Gln3-Myc13, that the full complexity of overall nitrogen-responsive regulation was appreciated  region flanked by basic residues. Alteration of these serine residues partially abolished cytoplasmic sequestration of Gln3-Myc13, but did not affect its responses to rapamycin, Msx treatment, or limiting nitrogen. The fact that the rapamycin- and NCR-responsiveness of Gln3-Myc13 remained intact when aspartate or alanine was substituted for these serines argues that they are not the targets of rapamycin-activated Sit4 phosphatase, or limiting nitrogen, and hence represent a novel nitrogen-responsive target in Gln3 that is not part of the mTorC1-Sit4 or Ure2-mediated pathways. Importantly, alteration of these serines, combined with substitutions that destroy the Gln3-mTor1 interaction site, additively and almost completely abolished cytoplasmic Gln3-Myc13 sequestration. Thus, we have developed another tool needed to isolate and deconvolve the individual mTorC1-dependent and -independent regulatory pathways that act cumulatively to control cytoplasmic Gln3 sequestration.In all of the above investigations, an important Saccharomyces cerevisiae wild type, JK9-3da  or gln3Δ, KHC2  strains were transformed with wild type and mutant plasmids carrying gln3 amino acid substitutions. JK9-3da is the parent of TB50, which is the parent of TB123. TCY1 is the parent of KHC2. Unless indicated otherwise, JK9-3da was used as the transformation recipient. Fresh transformants were then analyzed for intracellular Gln3-Myc13 distribution. Growth conditions were those described in A600 nm = 0.5) in YNB (without amino acids or ammonium sulfate) minimal medium that contained the indicated nitrogen source at a final concentration of 0.1%. Appropriate supplements  were added to the medium as necessary to cover auxotrophic requirements. Where indicated, cells were treated with 200 ng/ml rapamycin (+ Rap) for 20 min, or 2 mM methionine sulfoximine (+ Msx) for 30 min. For rapamycin resistance assays, the medium was solid synthetic complete (SC), with or without 50 ng/ml rapamycin.gln3 amino acid substitution mutants were constructed using standard PCR-based methods, and the primer sets in 13 and ADH1 transcriptional terminator were derived from pKA62 .i.e., cytoplasmic only or nuclear only. Gln3 is often simultaneously located in both cellular compartments. Therefore, we quantified intracellular Gln3 distribution by manually scoring Gln3-Myc13 localization in 200 or more cells in multiple, randomly chosen fields. Scoring was performed exclusively using unaltered, primary .zvi image files generated by the camera, and viewed with Zeiss AxioVision 3.0 and 4.8.1 software. Cells were classified into one of three categories: cytoplasmic , nuclear-cytoplasmic , and nuclear . “Standard” images, that depict the characteristics of these categories, appear in Cell collection and immunofluorescent staining were performed as previously described  were processed in the following way using Adobe Photoshop and Illustrator programs: (i) Any alteration of an image was uniformly applied to the entire image presented. (ii) The only alteration of the images was to decrease the background fluorescence due to leak-through light not removed by the barrier filter of the microscope. This avoided any change or loss in cellular detail relative to what was observed in the microscope. The alteration  was applied to each image individually. The alterations were nearly the same from one image to another, but were not rigorously identical because the amount of leak-through fluorescence was not identical from one image to another.Images accompanying the histograms in subsequent figures were chosen on the basis that they exhibited intracellular Gln3-MycWestern blots were performed as described earlier  upon request. This will be done in accordance with NIH guidelines.Gln3 site required for its response to the cell’s overall nitrogen supply, i.e., NCR. To search for this site, we compared the gross phosphorylation profiles of large Gln3-Myc13 truncations in rapamycin-treated cells, and those provided with a repressive (glutamine) vs. a derepressive (proline) nitrogen source : the amount of the most rapidly migrating Gln3 species increased at the expense of the middle species; a normal response to rapamycin  , in which aspartate or alanine was substituted for various residues  . Furtherrevisiae . A portirevisiae . Given tresidues . The abicumented .Gln3 residues 421 and 439. It contained 28% hydrophobic, and 32% asparagine/glutamine residues. We replaced two or three hydrophobic residues in each of three constructs with charged aspartate residues , reasoning that, if this region interacted with other proteins, or its secondary structure was needed for a particular function, these alterations would damage it. In all three substitution mutants, Gln3-Myc13 localization was indistinguishable from wild type, irrespective of whether we assessed glutamine- vs. proline-grown cells, or cells treated with rapamycin or Msx ; Gln3L459D,L464D,M465D-Myc13 (pRR1323)]  and Gln3S469A,T471A,T473A-Myc13 (pRR964)  . These cGln3 positions 477 and 497  or alanine (pRR680) for 13 serine/threonine residues situated between  and 497 . These s and 497 . Asparta assayed , pRR682.ed cells , pRR680 ed cells , pRR680.he cells .Gln3, they failed to totally abolish the ability of Gln3-Myc13 to be retained in the cytoplasm when cells were grown in repressive medium. (iii) Neither aspartate nor alanine substitutions in this region had any effect on nuclear Gln3-Myc13 localization in Msx-treated cells, just as has occurred in all of the Gln3 C-terminal alterations previously reported  it retained its ability to respond to rapamycin, arguing that these residues were not required for a response to rapamycin inhibition of TorC1 kinase. (ii) As extensive as the alanine substitutions were in this region of reported    . Here, a pRR676) , pRR678. pRR676) , pRR676.ild type . AlthougGln3 residues 485 and 493 (pRR1040 and pRR1043). These residues were not flanked by basic amino acids  largely abolished cytoplasmic Gln3-Myc13 localization prompted us to query whether this result might be associated with an altered ability to phosphorylate Gln3. We particularly wanted to know whether these alterations affected rapamycin-elicited Gln3 dephosphorylation. To this end, we grew four of the above mutants  in proline or glutamine medium, and further treated glutamine-grown cells with rapamycin  differed from those obtained with the alanine substitutions (pRR680 and pRR676) . Gln3-Myc13 localization in these substitution mutants was indistinguishable from wild type  and Gln3S496A,S497A-Myc13 (pRR1173). The only convincing effect the alanine substitutions elicited was in ammonia-grown cells   . Both sulization . This phon  that is required to sequester Gln3-Myc13 in the cytoplasm when cells are growing in nitrogen-rich medium , a phenotype similar to that of substituting all 13 serine/threonine residues of Gln3477-493 (pRR680) was obtained. We speculate that the serine and threonine residues in Gln3477–493 are phosphorylated in cells provided with excess nitrogen, because substitution of a phosphorylation mimic, aspartate (pRR682 and pRR678), yielded the same level of cytoplasmic Gln3-Myc13 sequestration in nitrogen-rich medium as wild type, whereas alanine substitutions that cannot be phosphorylated yielded the mutant phenotype, i.e., loss of cytoplasmic Gln3-Myc13 sequestration and rapamycin hypersensitivity (pRR680 and pRR676). Further, the slowest migrating Gln3-Myc13 species in western blots of the aspartate substituted mutants cited above was lost when alanine was substituted for these serines. The most slowly migrating Gln3-Myc13 species has been repeatedly shown to be the one that is most phosphorylated. The Gln3 site(s) and process(es) that control the phosphorylation of these residues remains a mystery.Experiments presented in this work define a unique h medium . The sitGln3477–493 must not only be capable of being phosphorylated, but also must act in concert with Gln3 sites phosphorylated by mTorC1 for Gln3-Myc13 to be effectively sequestered in the cytoplasm, as supported by the additive phenotypes of pRR850, pRR680, and pRR1340. However, the combined phosphorylation of these two targets is still insufficient to fully determine whether Gln3 enters the nucleus or is retained in the cytoplasm. Otherwise, the Gln3 localization in the single and double mutant transformants just cited should not have responded as they did to limiting nitrogen (proline grown), or Msx treatment. Further, also note that cytoplasmic Gln3-Myc13 sequestration is still not totally abolished even in the double mutant transformant growing in glutamine medium  the electrophoretic mobility of Gln3-Myc13 increased when the mutant transformants were treated with rapamycin (Gln3477–493 is phosphorylated, it is also unlikely to be the target of the rapamycin-elicited, Sit4-dependent Gln3 dephosphorylation and nuclear localization because a wild type rapamycin response occurred when aspartate was substituted for all of the serine residues in this site (13 only partially relocated to the nuclei of rapamycin-treated wild type cells. Yet when cytoplasmic Gln3-Myc13 retention was diminished by alanine substitutions in Gln3477–493, nuclear Gln3 localization correspondingly increased. This is the behavior expected when multiple regulatory pathways operate cumulatively to bring about an overall result.One might conclude that apamycin . By simihis site . FurtherGln3 has been reported to potentially be a disordered molecule. It is fascinating that the three specific regulatory sites identified in Gln3 thus far occur in regions of the molecule that are predicted to be disordered. In the most extreme case, the Gln3477–493 residues required for cytoplasmic Gln3 sequestration are also those that exhibit the greatest probability of being disordered (see residues between the arrows in Gln3421–473 in this work, and in a priori be expected to exhibit something other than wild-type phenotypes. We speculate that the likelihood of the C-terminal region of Gln3 being disordered may be responsible for our ability to alter each of the individual regulatory sites in this region without telegraphing the effects of those alterations to the others. Only in the case of the short proline-rich region between the sites required for cytoplasmic Gln3 sequestration and rapamycin-responsiveness did we find substitutions that affected the functioning of two adjacent regulatory sites (Finally, this is the first time that ry sites . We specry sites  may init


Expandable screws have greater pullout strength than conventional screws. The purpose of this study was to compare the biomechanical stability provided by a new built-in expandable anterior spinal fixation system with that of 2 commonly used anterior fixation systems, the Z-Plate and the Kaneda, in a porcine partial vertebral corpectomy model.Eighteen porcine thoracolumbar spine specimens were randomly divided into 3 groups of 6 each. A vertebral wedge osteotomy was performed by removing the anterior 2/3 of the L1 vertebral body and the T15/L1 disc. Vertebrae were fixed with the Z-Plate, Kaneda, or expandable fixation system. The 3-dimensional spinal range of motion (ROM) of specimens in the intact state (prior to osteotomy), injured state (after osteotomy), and after internal fixation were recorded. The pullout strength and maximum torque of common anterior screws, the expandable anterior fixation screw unexpanded, and the expandable anterior fixation screw expanded was tested.After internal fixation, the expandable device and Z-plate system exhibited higher left bending motion than the Kaneda system , and the Z-plate and Kaneda groups had significantly higher left axial and right axial rotation ROM as compared to the expandable device group . The maximum insertion torque of the expandable device was significantly greater than of a common screw (5.10 vs. 3.75 Ns). The maximum pullout force of the expandable device expanded was significantly higher than that of the common screw and the expandable device unexpanded .The built-in anterior fixation system provides better axial rotational stability as compared to the other 2 systems, and greater maximum torque and pullout strength than a common fixation screw.The online version of this article (doi:10.1186/1471-2474-15-424) contains supplementary material, which is available to authorized users. Surgical procedures for the treatment of thoracolumbar spine diseases include anterior, posterior, and combined anterior and posterior fusion, and adequate immobilization of the spine is critical for successful fusion. The use of anterior thoracolumbar implants and anterior instrumentation for stabilization has become more common in the past decade for reasons including direct visualization and ready access to the anterior column, improved distraction, and placement of a large interbody fusion device , 2. At pWhile the screw-rod system is relatively stable biomechanically, the size is relatively large and the implant profile is relatively high, which can damage peripheral neurovascular structures during or after surgery , 4. In aExpandable screws have been used for improved graft fixation in anterior cruciate ligament reconstruction . RichterWe designed a built-in expandable anterior thoracolumbar fixation system to overcome the shortcomings of current anterior thoracolumbar systems. The fixation system is made of a titanium alloy, and composed of an expandable cylinder screw with an internal cone-shaped space. When an insert is placed into the internal space of the screw, the distal end of the screw expands. The purpose of this study was to compare the biomechanical stability provided by the new anterior fixation system with that of 2 commonly used anterior fixation systems, the Z-Plate and the Kaneda, in a porcine partial vertebral corpectomy model.A total of 42 fresh pig thoracolumbar spine T14-L3) specimens were obtained from a local slaughterhouse, all animal experiments were carried out with the approval of the Southern Medical University Animal Care and Use Committee in accordance with guidelines for the ethical treatment of animals and 18 out of the 42 specimens similar in size were selected for testing ROM. Specimens with an intact posterior ligament complex (PLC) were choosen. Congenital diseases, osteoporosis, tumors, and fractures were excluded by visual observation and anteroposterior and lateral radiographies  Built-in expandable anterior spinal fixation system   was loaded and then unloaded in each specimen, and the process was repeated 3 times. Data were collected after 3 cycles of loading/unloading to minimize the influence of viscoelasticity of the spine, and obtain relatively stable results of thoracolumbar motion. The loading was maintained for 30 s at 6.0 N.m, and creep motion of the specimen was allowed. The loading test was carried out at room temperature (20-30°C). An image of the device used to test spinal motion is shown in Figure For testing, the upper end of the embedded specimen was connected to the loading tray of a 3D motion testing device , and the lower end was fixed to the base of the testing device. Testing was carried out according to the method described by Wilke et al. . Flexion.m , and 30 The 3D ROM of specimens in the intact state (prior to osteotomy), injured state (after osteotomy), and after internal fixation were recorded. Briefly, 6 infrared cameras  were placed around the specimen. An Eagle-4 digital infrared camera was used to dynamically trace and capture the displacement of the spherical makers fixed on the specimen . Images of each moving vertebra with spherical makers on the same plane, which can accurately reflect the range of vertebral motion, were selected. The displacement of each segment was calculated using EVaRT software and based on the principle of optical 3D motion analysis.Image analysis and data conversion were carried out using the Geomagic software system. Angular displacement of each vertebra was calculated according to the coordinates of the 3 non-collinear markers on each vertebra. The range of motion was calculated by adding the angular displacement of each vertebra. The parameters of the ROM of adjacent thoracolumbar segments were: Neutral zone (NZ), the ROM between the spine location at zero load and the neutral location; Elastic zone (EZ), the ROM between the neutral location and the location at maximum load; Spine ROM, the ROM between the spine location at zero load and that at maximum load (the sum of NZ and EZ) . BecauseTwenty-four of the 42 spine specimens were divided into 4 groups  for testing pullout strength and maximum torque. In group A, the pullout strength and maximum torque of common anterior screws  was tested. In group B, the pullout force and maximum torque of the expandable anterior fixation screw  unexpanded was tested. In group C, the maximum torque of the expandable anterior fixation screw expanded was tested. In group D, the pullout force of the expandable anterior screw expanded was tested. Testing was performed in the Biomechanics Laboratory of Southern Medical University using a Bose ElectroForce® BioDynamic® test instrument, which automatically records the load signals.Screws were fixed according to their standard installation methods. In group A and B, the screw was fastened to the last half of the thread. In the group C and D, the screw was fastened to the whole thread, bone fragments were placed inside the screw, and the inner core was placed and the screw was expanded. The specimen was then embedded again. In cases in which the screw penetrated the contralateral cortex, the protruding portion of the screw was embedded with plasticine first and then embedded and fixed with denture powder to prevent the denture powder from fixing the screw.For torque testing, specimens were fixed to special fixtures after internal fixation, and the screw adjuster was fixed to the testing device Figure . The angAll testing was performed, and results confirmed by agreement of the 2 primary authors, each with more than 15 years of experience. ROM, maximum insertion torque, and maximum pullout strength were presented by mean and standard deviation. One-way ANOVA was performed to compare continuous data among independent groups. The least-significant difference (LSD) method was used for further multiple comparisons if one-way ANOVA provided statistically significant results. Paired t-test was performed to compare the ROM between normal vs. injured, normal vs. internal fixation, and injured vs. internal fixation status within groups. Data were examined with the Kolmogorov-Smirnov test for normality. Statistical analyses were performed using SPSS 15.0 statistical software . A 2-tailed p < .05 indicated statistical significance.The Kolmogorov-Smirnov test for normality indicated that all data were normally distributed , which is also comparable to Kaneda system in left bending (5.04). After internal fixation, the expandable device and Z-plate system exhibited higher left bending motion than the Kaneda . Furthermore, after internal fixation, the Z-plate and Kaneda groups had significantly higher left axial and right axial rotation ROM as compared to expandable device .The Kolmogorov-Smirnov test for normality indicated that all data were normally distributed  and F-max; namely, the greater BMD, the greater the fixation strength . Li et aa et al.  reporteda et al.  reportedk et al.  applied in vivo. Because the diameter of the screws used in the MAC system is large and can damage the vertebral body, we did not include MAC system in the study. In an adult thoracolumbar spine screws 6 mm or 6.5 mm are commonly used. In China, the diameter of most screws used with the thoracolumbar anterior single screw-rod system are 5.5 mm, which is why 5.5 mm screws were used in this study. The difference in screw diameter will have affected the pullout strength results. In this study the pull- out strength of a single screw was compared among the 3 systems. The results may be different clinically as in the Kaneda and Z-plate 2 screws are used in each vertebra which may increase the overall pullout force. We did not perform fatigue testing or 3-point bending testing of the new system. Lastly, a finite element analysis should be carried out to study the load-carrying capacity of the screw, and provide a theoretical basis for further improvement of the design.There are limitations of this study that should be considered. The number of specimens tested was small, and while fresh adult pig specimens closely resemble the human spine, the number of thoracic vertebra is not the same as in the human spine (the pigspine has 15 thoracic vertebra). Because this study used pig spine specimens, and the absolute values for torque and pullout force will be different than if testing were done The built-in anterior fixation system tested provides better axial rotational stability as compared to other 2 systems tested, and greater maximum torque and pullout strength than a common fixation screw. The system can be installed with less manipulation of surrounding tissue than rod or plate systems.Additional file 1: Table S1: Normality test for spinal range of motion. Table S2 Normality test for torque and pullout strength. (DOC 60 KB)Authors’ original file for figure 1Authors’ original file for figure 2Authors’ original file for figure 3Authors’ original file for figure 4Authors’ original file for figure 5Authors’ original file for figure 6Below are the links to the authors’ original submitted files for images.


Antifreeze (glyco)proteins are found in polar fish species and act to slow the rate of growth of ice crystals; a property known as ice recrystallization inhibition. The ability to slow ice growth is of huge technological importance especially in the cryopreservation of donor cells and tissue, but native antifreeze proteins are often not suitable, nor easily available. Therefore, the search for new materials that mimic this function is important, but currently limited by the low-throughout assays associated with the antifreeze properties. Here 30 nm gold nanoparticles are demonstrated to be useful colorimetric probes for ice recrystallization inhibition, giving a visible optical response and is compatible with 96 well plates for high-throughout studies. This method is faster, requires less infrastructure, and has easier interpretation than the currently used ‘splat’ methods. Using this method, a series of serum proteins were identified to have weak, but specific ice recrystallization inhibition activity, which was removed upon denaturation. It is hoped that high-throughput tools such as this will accelerate the discovery of new antifreeze mimics. To survive in the harsh cold environments at high altitude or in the Earth’s polar regions, Nature has evolved a series of mechanisms to enable extremophiles to survive, and thrive. One particular method of survival employed by freeze-avoiding67et al. have reported that simplified glycopeptides and glycolipids have potent IRI and can enhance cryopreservation11121415et al. introduced domain recognition software1920Therefore, the development of new IRI-specific molecules/macromolecules has been pursued. Ben et al.et al.28Interestingly, Kim In this manuscript, we report a study into the use of gold nanoparticles (AuNPs) as a simple, and rapid, tool for screening for IRI activity. Using well-defined synthetic polymers, with known IRI activity, but no (or weak) thermal hysteresis we show that the inhibition of gold particle aggregation correlates with IRI. The utility of this in a 96 well plate format is reported to enable higher-throughput screening than compared to the splat assay.N-vinyl pyrolidone), PVP, were selected as they have no significant IRI in the relevant concentration range30−1 are sufficient to halt ice crystal growth.To test the applicability, if any, of the reversible freezing of colloidal gold as a probe for IRI activity −1 is used from this point onwards (Supp. Info).30 nm mercaptosuccinic acid (MSA) functionalized gold particles prepared using literature methods22−1 were synthesised using RAFT/MADIX polymerization−1 all the PVAs prevented aggregation of the gold particles after a freeze/thaw cycle, in agreement with the ‘splat’ assay. When the concentration was decreased below 1 mg.mL−1 there were clear differences between the polymers of different molecular weight, as shown in the zoomed-in region in 244) was capable of inhibiting aggregation as low as 0.1 mg.mL−1, but the shortest (PVA10) lead to AuNP aggregation in this concentration range, demonstrating that high molecular weight polymers have increased IRI activity.In order to ‘score’ the relative degree of aggregation post-thawing, the reduction in the absorbance peaks at 520 nm between pre- and post-thaw were measured to give a qualitative comparison between different additives see  with hig520 versus MLGS, 520, with lower absorbance values correlating with smaller MLGS values. Importantly, there were some outliers, giving rise to false positives in some cases. This highlights the potential as a screening tool, but still needs the complimentary splat test to confirm any findings. We believe these outliers are related to the relatively high concentration of the low molecular weight PVA’s which correlate to these data points.In an attempt to correlate these observable spectral features with our ‘gold standard’ splat assay, the whole data set was plotted as AbsVariable volume experiments were also conducted to ensure that this was a recrystallization-specific effect. Briefly, smaller volumes will thaw faster, reducing the opportunities for recrystallization, compared to larger volumes. A short screen of this (Supp. Info.) confirmed that 100 μL total volume per well (in a 96 well plate) lead to aggregation of the gold after freeze-thaw, but lower volumes lead to less aggregation. This also supports our hypothesis that recrystallization inhibition, not thermal hysteresis is the effect being probed and that the conducting these experiments in 96 well plates, as opposed to 384 well plates may be preferable.co-SA (35 kDa), α-cyclodextrin dextran (40 kDa), trehalose, poly(vinyl pyrolidone)  and bovine serum albumin (BSA). Of these only PAEMA-co-SA is known to have IRI activity and this is significantly less active than PVA, typically requiring concentrations of ~20 mg.mL−1 to have IRI activity, compared to 1 mg.mL−1 for PVA−1) to show significant ice inhibition and is only considered to be weakly active, with close to no activity in the concentration range used hereThe data presented above suggests that the AuNP method may be a useful screening assay for IRI activity. To probe this, a range of compounds with and without known IRI activity were screened; Poly(amino-ethyl methacrylate) 50% carboxylated with succinic anhydride, PAEMA-co-SA  kDa, α-cco-SA showed a reduction in aggregation across a range of concentrations, in line with its known IRI activity, which correlates well with its MLGS (Supp. Info). Using dye inclusion assays PAEMA-co-SA shows no activity, suggesting that it is not acting as a surfactant, which could provide stabilisation to the AuNPs and is a potential source of false-positives3036As expected, PAEMA-co-SA and PVA being amongst the few. The denatured BSA showed no IRI activity, indicating a remarkable tertiary-structure dependence, which has not been previously explored. This is of particular interest as many cryoprotectant solutions contain large quantities of serum proteins and this data implies that modulation of ice crystal growth may be an unexpected benefit in these systems. To determine whether this is a general property of serum proteins, human serum albumin (HSA) and ovine serum albumin (OSA) were also tested using both the AuNP and splat test methods. The AuNP screening method indicated some freeze-thaw resistance and the splat test confirmed that the OSA and HSA both had activity in line with the BSA, The splat test revealed that folded BSA does have real, if not very potent, IRI activity. This is surprising, as in our hands very few synthetic materials or proteins show any appreciable IRI with PAEMA-in vivo is as a carrier for various substances such as fatty acids and certain steroids, while also maintaining the oncotic pressure in the blood38et al. has shown that addition of increasingly hydrophobic alkyl chains to sugars can result in enhanced IRI which would seem to agree with this hypothesis for BSA activityBSA is often used as a negative control in antifreeze protein assays  this should be taken into account in the future as it clearly does have activity, although the features that cause this are not clear. One role of serum proteins This study shows that AuNP reversible freeze/thaw cycles and the subsequent aggregation appears to be linked to the rate of ice crystal growth  rather than the previously hypothesized TH activityHere the application of gold nanoparticles as convenient probes for ice recrystallization inhibition activity has been investigated. During thawing, extensive ice crystal growth occurs, reducing the available surface area for particles, meaning the gold particles are more likely to aggregate if crystal growth occurs. UV-visible spectroscopy can be employed to probe activity by measuring the characteristic red to blue shift of AuNP solutions as they aggregate. The degree of aggregation was found to correlate well with ice recrystallization inhibition activity determined by the (gold standard) splat assay. Previous studies had suggested that this reversible assay was due to freezing point depression  but here synthetic polymers without freezing point depression activity gave strong aggregation inhibition, confirming IRI not TH was the mechanism being probed.This assay has significant advantages in terms of speed and throughput compared to the splat test, which is the current ‘gold standard’ for ice recrystallization. This includes compatibility with 96 well plates for automated analysis and the use of a standard −20 °C lab freezer and easy colourimetric readout. Using this method, the IRI activity of serum proteins was identified for the first time and quantified by the ‘splat’ test and linked to the tertiary structure/folding of the proteins. At this time, the potential for false positives cannot be ruled out across all chemical space, but this represents a step towards a fully automated IRI activity screening platform.1H and 13C NMR spectra were recorded on Bruker DPX-300 and DPX-400 spectrometers using deuterated solvents purchased from Sigma-Aldrich. Chemical shifts are reported relative to residual non-deuterated solvent. Infrared data was recorded on a Bruker Vector 22 GI003097. The THF GPC system comprised of a Varian 390-LC-Multi detector suite fitted with differential refractive index (DRI), light scattering (LS) and ultra-violet (UV) detectors equipped with a guard column  and two mixed D columns of the same type. The mobile phase was THF with 5% triethylamine (TEA) eluent at a flow of 1.0 mL/min, and samples were calibrated against Varian Polymer Laboratories EasiVials linear poly(styrene) and poly(methylmethacrylate) standards (162–2.4 × 105 g/mol) using Cirrus v3.3. Characterization of AuNPs was carried out using differential light scattering (DLS) spectroscopy . Circular Dichroism (CD) spectra were recorded on a spectropolarimeter  using a data interval of 0.2 nm. Samples were dissolved in PBS buffer diluted two-fold with deionised water, and the spectrum was measured 16 times and averaged. The spectrum of a blank sample containing only buffer was then subtracted giving a final spectrum for each protein. UV-Visible spectroscopy was conducted on a microplate reader; Synergy HT multi-mode microplate reader, BioTek UK.4.3H2O), sodium citrate dehydrate (C6H5Na3O7.2H2O), mercaptosuccinic acid (MSA), dextran, α-cyclodextrin (MW 972.84 g.mol−1), trehalose, bovine serum albumin (BSA), human serum albumin (HSA), ovine serum albumin (OSA) and poly(ethylene glycol) (PEG) were purchased from Sigma Aldrich. Poly(amino ethyl methacrylate – co- succinic anhydride) was synthesized as previously reportedHydrogen tetrachloroaurate trihydrate (HAuCl−1. Dynamic light scattering confirmed that the gold particles were ~30 nm in size.Gold Nanoparticle (AuNPs) were synthesized according to the literature−1 (or higher concentrations as needed) in a 96 well plate. To this, 50 μL of as-prepared AuNPs were added. To determine the affect of the solution volume on the assay the quantities of test and AuNP solution were varied although the ratio remained 1:1. All experiments were undertaken in triplicate. Freeze thaw was achieved by placing the plate in a standard domestic freezer for 2 hours at −20 °C and leaving to thaw on the lab bench at room temperature. The UV-vis spectra were corrected for scattering by drawing a straight line between 450 and 680 nm and measuring the absorbance at 520 nm, between this line and the peak. The optimal concentration of gold is 80 ug.mL−1 (see Supp. Info).The standard assay involved the addition of 50 μL of test product prepared as a serial dilution from 10–0 mg.mL2 for 30 minutes. Photographs were obtained using an Olympus CX 41 microscope with a UIS-2 20x/0.45/∞/0-2/FN22 lens and crossed polarizers , equipped with a Canon DSLR 500D digital camera. Images were taken of the initial wafer  and after 30 minutes. Image processing was conducted using Image J, which is freely availableIce recrystallization inhibition was measured using a modified splay assayHow to cite this article: Mitchell, D. E. et al. Gold Nanoparticle Aggregation as a Probe of Antifreeze (Glyco) Protein-Inspired Ice Recrystallization Inhibition and Identification of New IRI Active Macromolecules. Sci. Rep.5, 15716; doi: 10.1038/srep15716 (2015).