Therefore, in the current study, we investigated the effect of ice slurry ingestion on Tbrain using proton magnetic resonance spectroscopy, which is a robust, non-invasive method. Eight male participants ingested 7.5 g/kg of either a thermoneutral drink  or ice slurry  for about 5 min following a 15-min baseline period. Then, participants remained at rest for 30 min. As physiological indices, Tbrain, rectal temperature (Tre), mean skin temperature, nude body mass, and urine specific gravity were measured. Subjective thermal sensation (TS) and thermal comfort (TC) were measured before and after the experiment. Tbrain and Tre significantly reduced after ingestion of ICE compared with after ingestion of CON, and there was a significant correlation between Tbrain and Tre. The other physiological indices were not significantly different between beverage conditions. TS and TC were significantly lower with ICE than with CON (p < 0.05). These results indicate that ice slurry ingestion can cool the brain, as well as the body’s core.We previously reported that ice slurry ingestion reduced forehead skin temperature, thereby potentially reducing brain temperature (T Siegel et al.1 were the first to investigate the effect of ice slurry ingestion on endurance exercise capacity in the heat, and reported that when participants ingested ice slurry (−1 °C) or cold water (4 °C) before exercise, their rectal temperature (Tre) was significantly reduced, and time to exhaustion improved by 19%. Thereafter, various studies have investigated the effect of ice slurry ingestion in different types of exercise3, with varied timing of ingestion , as well as on the central nervous system . These studies suggest that ice slurry ingestion has an ergogenic effect. Two mechanisms are proposed as contributing to this effect: (1) increased heat storage capacity through a reduction in core temperature and (2) a sensory effect, such as a reduction in ratings of perceived exertion or an improvement in thermal comfort .Ice slurry ingestion, which comprises an icy mixture that is consumed like a beveragebrain). Vanden Hoek et al.9 compared the core temperatures in swine after central catheter infusions of 50 mL/kg of saline ice slurry and 50 mL/kg of chilled saline, and reported that ice slurry significantly reduced the animals’ Tbrain. In humans, Siegel et al.10 suggested that oral ingestion of ice slurries possibly resulted in conductive cooling of the facial skin and brain. To verify this hypothesis, we previously investigated the effects of ice slurry ingestion on forehead skin temperature in the heat, and found a significant  reduction in temperature, suggesting a potential reduction in Tbrain through conductive cooling of the facial skin and brain with ice slurry ingestion11. However, our study used an indirect index . In addition, to the best of our knowledge, no studies have directly observed an alteration in Tbrain after ice slurry ingestion.Another possible factor for the ergogenic effect of ice slurry ingestion in preventing central fatigue is a reduction in brain temperature  solutions as models and revealed a reduction in the difference of NMR frequencies between NAA and water16. Moreover, experimental models20 showed a close correlation between temperature that was measured using MRS and those measured using implanted probes. However, this method has only been applied in clinical settings and not in the field of exercise physiology.Presently, magnetic resonance spectroscopy (MRS) is utilized as a non-invasive method to measure Tbrain using MRS. Given the nature of MRS, we conducted the experiment with participants at rest in a temperate condition.Therefore, the aim of this study was to investigate the effect of ice slurry ingestion on T2) participated in this study. Our study was approved by the Ethics in Human Research Committee of the Japan Institute of Sports Sciences (no. 024), and was performed in accordance with the Declaration of Helsinki. All participants signed an informed consent form prior to participation.Of the 10 healthy men recruited for this study, two were excluded because spectra measurement was difficult . Therefore, eight healthy men  and ingesting ice slurry . Throughout the study period, participants were asked to maintain their normal lifestyle activities, including their physical activity and nutritional habits. Participants were asked to ingest 500 mL of water 2 h before the experimental trials. Two trials were conducted on 2 consecutive days in a counterbalanced order and at the same time of day to eliminate any potential effects from circadian variations.Upon arrival in our laboratory, participants’ urine samples were collected, and nude body mass was obtained. Then, a rectal temperature probe was self-inserted, and skin thermistors were attached. Subsequently, subjects put on t-shirts and shorts. Participants entered a room that was maintained at 23 °C with 45% relative humidity , and were asked to lie supine on the magnetic resonance imaging (MRI) table. The head/neck coil was then attached. After a 15-min rest (baseline period), the participants ingested 7.5 g/kg of either CON or ICE ad libitum over about 5 min . After ingestion, participants remained at rest for an additional 30 min.Both beverages were conventional sports drinks  containing 6.2 g of carbohydrates and 49 mg of sodium and 20 mg of potassium per 100 mL as electrolytes. CON was heated in a thermostatic chamber  and ICE was made using a slurry machine .Given the difficulty of consuming beverages while lying supine, participants used a bottle  during the ingestion period. The beverage volumes were calculated and prepared by the research staff, who then filled two bottles, each with half of the beverage volume, with the respective beverage (CON or ICE). Before the baseline period, the participants were instructed to pick up one of the two bottles and ingest its contents. Both bottles were then positioned near the participant’s hand immediately before the ingestion period. The participants were instructed to ingest the beverage from either bottle while occasionally mixing the contents of the bottle. The bottles were collected after the contents were consumed. Despite these instructions, some ice remained in the bottle because of its structure; thus, the actual total volume of ingested ICE was lower than 7.5 g/kg. Individual variations were noted for the remaining ice volume, and the mean value for these four participants was 37.50 ± 48.32 g.1. Hydrogen MRS was performed with point-resolved spectroscopy localisation. Acquisition parameters were as follows: TR, 10,000 ms; TE, 135 ms; FA, 90°; data size, 2048; spectral width, 1,200 Hz; and excitation, 1. The voxel of interest (VOI) was set in the frontal cortex, which has been shown to correlate with cognitive function21, using the 3D-T1 image  and 64-channel head/neck coil. Three-dimensional (3D) T1-weighted images (magnetisation prepared rapid acquisition with gradient echo) were acquired using the following parameters: repetition time (TR), 1,900 ms; echo time (TE), 2.29 ms; inversion time, 850 ms; flip angle (FA), 8°; matrix, 356 × 256; field of view, 240 mm; and slice thickness, 1 mmage Fig. . The VOIift Fig. . We usedbrain, Tre, mean skin temperature (Tsk), nude body mass, and urine specific gravity were measured. Tbrain was measured every 30 s as described above and was averaged every 1 min. The change in Tbrain (ΔTbrain) was calculated using the following equation  and after beverage ingestion comprised the 30-min rest period, including the Post measurement. The Post measurement was defined as the end of beverage ingestion (10 min after Pre).where Pre was defined as the mean Tre was measured with a thermistor  that was inserted 12 cm from the anal sphincter with a disposable rubber sheath . The change in Tre (ΔTre) was calculated using the following equation , and Pre was defined as the recorded value immediately prior to beverage ingestion (the end of the 15-min baseline period).where the time when Tsk was calculated using the equation  using thermistors  that were secured with micropore tape. All thermistors were connected to a data collection device , and temperatures were recorded every 30 s. Tequation  by Rober23 (0 = very cold to 8 = very hot) and the modified Bedford24 scale (1 = very uncomfortable to 7 = very comfortable), respectively. TS and TC of the entire body and head were measured.Nude body mass was measured using a weighing machine  to the nearest 10 g, and urine specific gravity was determined using a digital urine specific gravity scale  before and after the experiment. Subjective thermal sensation (TS) and thermal comfort (TC) were measured before and after the experiment using the scale derived by Gaggebrain, Tre, Tsk, TS, TC, body mass, and urine specific gravity were performed using a two-factor (condition × time) analysis of variance with repeated measures. When a significant main effect was identified, the differences were compared using t-tests . Pearson’s correlation coefficients were calculated to assess possible correlations between ΔTbrain and ΔTre. All statistical analyses were performed using SPSS Statistics 17.0 software package . Statistical significance was accepted at p < 0.05. All physiological measurements after beverage ingestion are presented in 5-min increments from Post to 30 min (+30)  Fig. .Figure 2The datasets generated during and/or analysed in the present study are available from the corresponding author on reasonable request.brain during the experiment and ΔTbrain are shown in Fig. brain reduction than CON at Post, +10, and +15 . Moreover, ΔTbrain was significantly greater in the ICE condition than in the CON condition (p < 0.05).Changes in temperature over time are presented in Fig. re during the experiment and ΔTre are shown in Fig. re exhibited a significantly greater reduction with ICE than with CON from +5 to +25 . Additionally, compared to Pre, Tre was significantly lower from Post to +25 . Between conditions, ΔTre was significantly greater with ICE than with CON (p < 0.001). Further, ΔTbrain was significantly correlated with ΔTre .The changes in Tsk are shown in Fig. sk significantly increased at +20 and +25 in the CON condition and at Post and +5 in the ICE condition, but no differences between the conditions were observed.Changes in Tp < 0.001); however, no significant differences were detected between CON and ICE. Urine specific gravity significantly reduced after the experiment in both conditions (p < 0.05), but no significant differences were found between CON and ICE.The hydration state before and after the experiment is summarised in Table p < 0.001). Moreover, ICE yielded a significantly greater reduction in TC of the entire body after the experiment compared to before the experiment (p < 0.01). Further, TC of the entire body was significantly reduced in the ICE condition compared with in the CON condition (p < 0.05).Changes in TS and TC are summarised in Table brain has attracted attention. Some studies have suggested that ice slurry ingestion may reduce Tbrain27. Because directly measuring Tbrain in humans is difficult, only an indirect index can be used, particularly during exercise. Therefore, we measured brain temperature at rest using the MRS method to improve the validity of data obtained from the indirect index. Our results showed that ice slurry ingestion significantly reduced Tbrain, as well as Tre, TS, and TC. These results are important as they provide foundational data for future applied studies.The speculation that ice slurry ingestion can impair central fatigue during exercise in the heat by reducing Tbrain in humans through ice slurry ingestion using MRS. Two mechanisms could explain how ice slurry ingestion reduces Tbrain: inflow of cooled carotid blood and conductive cooling of the facial skin and brain10. Our previous report of reduced forehead skin temperature with ice slurry ingestion in a warm environment supports the latter mechanism11. Given the limitation of MRI, we could not measure forehead skin temperature using a thermistor probe in the present study; however, ice slurry ingestion may possibly reduce facial skin temperature, thereby reducing Tbrain as previously reported. Interestingly, the reduction in Tbrain observed in this study (−0.4 °C) was the same as that of head cooling achieved through fanning28. Therefore, ice slurry ingestion can cool the brain more easily than fanning, contributing to the development of effective body cooling strategies. Further investigation of this phenomenon is needed.To the best of our knowledge, the present study is the first to successfully reveal a reduction in Tet al.29 reported that despite different pre-exercise temperatures, environmental conditions, and running time, rats reached a point of fatigue at the same Tbrain, suggesting a critical level in Tbrain, as well as core temperature. If the same concept is applied in humans, reaching this critical level can be delayed by pre-cooling the brain with ice slurry ingestion, thereby extending the exercise time. Previous studies reported that a −0.4 °C reduction of core temperature can improve exercise performance27; hence, a −0.4 °C reduction of Tbrain may also improve exercise performance. Moreover, the reduction in Tbrain may explain the ergogenic effect of ice slurry ingestion during exercise. Previous studies reported that ice slurry ingestion during exercise improved performance without changes in core temperature5. The authors cited an improvement in subjective sensation, such as TC, as the contributing factor. Sensory improvement was possibly influenced by a reduction in Tbrain as core and skin temperatures were not significantly altered. Although TC became worse with ice slurry ingestion in the present study, we believe that this effect resulted from ingestion in a temperate environment, and heat production was likely not caused by shivering because the mean Tsk was unchanged and according to the participants’ perspective.Fuller 31, the liquid was expelled from a syringe into a capillary tube using a separate syringe pump. However, given that the ice slurry cannot be administered through a tube, the participants in this study picked up the ice slurry bottle by themselves and consumed the beverage ad libitum. Two limitations should be noted for this aspect. First, we cannot exclude the possibility that the movement of the participant’s head disturbed the acquisition of spectra. Indeed, Tbrain during beverage ingestion was excluded because of spectral issues. However, as the spectra after beverage ingestion was stable, similar to that before beverage ingestion, we believed that it was possible to investigate the effect of ice slurry ingestion on Tbrain. Second, some ice remained in the bottle despite pouring the ice slurry. Hence, we could not control the volume of beverage that the participants ingested. Therefore, ΔTre with ICE was smaller in the present study than that (0.66 °C) in a previous study in a temperate environment1. If the participants were able to ingest the required volume, ΔTre with ICE may be greater in the present study. Moreover, the present study found a significant correlation between ΔTre and ΔTbrain, indicating that the greater the ΔTre, the greater the ΔTbrain; thus, ΔTbrain with ICE might also be greater if participants ingest the required volume. Our results are robust because we show that even a lower volume of ice slurry can reduce Tbrain.In previous studies investigating the effects of beverage ingestion on MRI scansbrain during exercise. However, many studies have demonstrated that this method is challenging, especially during physical exertion in the heat, and can lead to measurement errors caused by dirt, inaccurate placement, and insufficient skill of the measurer36. Thus, it does not provide an accurate reflection of core temperature37. Conversely, measuring Tbrain before and after exercise using MRS is possible; therefore, investigating the effect of ice slurry ingestion on Tbrain when the body temperature increases from exercise is also possible.An indirect method using tympanic temperature was reported as an alternative to measuring Tbrain at the frontal cortex, which is related to cognitive function as the index of central fatigue. However, Tbrain in other brain regions may be different. For example, some studies have reported temperature gradients of up to 1 °C from cooler superficial to warmer deep brain tissues in humans13,39. Therefore, it is worthwhile to measure Tbrain in other deep regions that are related to thermoregulation, such as the hypothalamus.In the present study, we measured Tbrain, as well as Tre and TS in humans. Our findings can contribute to the development of effective body cooling strategies. However, further research is needed to clarify the underlying mechanism of the ergogenic effect of ice slurry ingestion.Ice slurry ingestion significantly reduced T


The twofold screw xylan is spatially close to cellulose, and has similar rigidity to the cellulose microfibrils, but reverts to the threefold screw conformation in the cellulose-deficient irx3 mutant. The discovery that induced polysaccharide conformation underlies cell wall assembly provides new principles to understand biomass properties.Exploitation of plant lignocellulosic biomass is hampered by our ignorance of the molecular basis for its properties such as strength and digestibility. Xylan, the most prevalent non-cellulosic polysaccharide, binds to cellulose microfibrils. The nature of this interaction remains unclear, despite its importance. Here we show that the majority of xylan, which forms a threefold helical screw in solution, flattens into a twofold helical screw ribbon to bind intimately to cellulose microfibrils in the cell wall.  et al. show that while xylan forms a threefold helical screw in solution it forms a twofold screw to bind cellulose microfibrils in the plant cell wall.The polysaccharide xylan binds to cellulose microfibrils in the plant cell wall, but the nature of this interaction remains unclear. Here Simmons  Plant secondary cell walls are strong composites of polysaccharides, lignin and proteins that are crucial for plant structure. They provide the most abundant renewable materials on Earth. This lignocellulosic material is therefore the focus of biofuel development2D-glucan chains fold in a twofold helical screw conformation (one 360° twist per 2 glycosidic bonds). The pattern of hydrogen bonding interactions is well understood from X-ray diffraction and magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) studies528910111213Arabidopsis plants with reduced xylan quantity show weakened walls and the plants are unable to develop a vascular system151920211911242526Arabidopsis secondary cell wallThe semi-crystalline cellulose microfibrils provide the best understood example of molecular architecture in lignocellulose. Its component β-1→4)--D-glucanArabidopsis molecular genetics and MAS NMR approaches, here we show that xylan interacts intimately with cellulose by adopting a twofold helical screw conformation. The change of xylan fold is induced by, and dependent upon, cellulose fibrils. This provides the first direct experimental evidence for the atomic-scale arrangement of two distinct polymers within plant cell wall biomass, and reveals new principles underlying the remarkable specificity of polysaccharide interactions. Our work provides an understanding of the molecular architectural basis for the biological and mechanical function of cell wall components in plant biomass.By combining 13C MAS NMR, a chamber to grow uniformly labelled Arabidopsis plants in 13CO2 was designed and constructed (2529Arabidopsis stems using cross polarization (CP) excitation, which emphasizes the more rigid components. In this experiment directly bonded nuclei appear at the same double-quantum (DQ) chemical shift that corresponds to the sum of the two single-quantum (SQ) chemical shifts. The spectrum shows broad line widths of ∼2 p.p.m. indicating the relative rigidity of the polysaccharides and a range of different environments. As expected, the major peaks in the spectrum are from glucose in the two main cellulose environments 89101112Arabidopsis stemsTo analyse the structure of xylan in never-dried cell walls by structed . Fresh a3f and Xn53f, respectively. It has been proposed that some xylan may fold as twofold screw in the cell wall192021in silico13C chemical shift prediction using gauge including projector augmented waves density functional theory (DFT)312f) in the CP-refocused INADEQUATE spectrum (2f) 13C chemical shift, covalently linked to an Xn5 signal (Xn52f), indicates that xylan forms a twofold screw in the cell wall.Xylan in solution forms a threefold helical screw19irx3 Arabidopsis mutant is deficient in secondary cell wall cellulose synthesis and is consequently dwarfed, but the plant continues to synthesize other cell wall components including xylan37irx3 stems confirmed depletion in this mutant of cellulose and the relative increase in other cell wall polysaccharides, including xyloglucan (Xg) and pectin  (irx3 compared with wild type. While twofold screw xylan (Xn42f peak) was by far the more prevalent of the two conformations in wild type -INADEQUATE spectra with a short recycle delay were acquired. The abundance of the Xn43f peak showed that relatively mobile threefold screw xylan is far more prevalent in irx3 than in the wild type  . Interesild type  and 2, itectable . In contild type . Indeed,irx3 cell walls gave us the opportunity to investigate further the Xn12f and Xn13f 13C chemical shifts, given the DFT prediction of substantial change in Xn1 (irx3 (a DP-PDSD spectrum where only threefold xylan is visible). Acetate methyl groups (21.6 p.p.m.) are spatially close to acetate carbonyl groups (173.6 p.p.m.) and also to Xn1–Xn5. Interestingly, the Xn13f shift in irx3 is 102.6 p.p.m. as seen in DP INADEQUATE and in solution xylan, but in wild-type Xn12f is significantly shifted to the higher 13C chemical shift of 105.2 p.p.m.. Because a clear increase in Xn12f shift from threefold to twofold was predicted by the calculations , cellulose domain 2  and twofold screw xylan  are not yet as strong as those showing spatial proximities within cellulose domains . Although spin diffusion in a fully 13C labelled system is complex, the presence of the relatively large Xn42f→C61 cross peak means that we can place an upper limit on the distance between the twofold screw xylan and cellulose: twofold screw xylan can be no farther than the width of a microfibril from the cellulose surface. Hence, the PDSD experiments indicate that the twofold screw xylan is bound to cellulose.To determine whether the twofold screw xylan is cellulose-bound, spatial proximities of molecules in the cell wall were investigated using a relaxation compensated 13C T1 relaxation time (SCH (0≤SCH≤1) which is sensitive to motion on the microsecond timescale. Both DP and CP DIPSHIFTirx3 samples. Cellulose-bound xylan would be expected to exhibit less motion (demonstrated by a larger order parameter) than unbound xylan. The order parameter of twofold xylan  is comparable to that of both cellulose domains , indicating relatively little motion. In contrast, for threefold xylan  the order parameter is significantly reduced, indicating more motion. Consistent with this, the T1 of Xn42f (82.5 p.p.m.) in wild type is very similar to cellulose values whereas Xn13f in irx3 has shorter T1 . Seeds were surface sterilized and sown on 0.8% (w/v) agar, 0.5 9 Murashige and Skoog salts including vitamins  and sucrose (1% w/v). Following stratification for 48 h at 4 °C in the dark, plates were transferred to a growth room . Optimaxx fibre rockwool  was cut into slabs and laid ∼8 cm deep in a growth tray. Hydroponics solution  was poured into the tray until half-way up. The rockwool was then covered with foil. Holes were pierced into the foil and rockwool and seedling were placed in, ensuring that roots made direct contact with rockwool. A growth chamber was constructed following Chen et al.2 using calcium oxide before 13CO2 was mixed back in at a concentration of 500 p.p.m. before entering the growth chamber. Plants were grown in a 50% humidity and 24 °C environment for 6–8 weeks. 13C enrichment was measured by analysing xyloglucan oligosaccharides yielded from xyloglucan endoglucanase digestion of stems using matrix-assisted laser desorption/ionization–time of flight mass spectrometry. Typical enrichments were 90–95%. Roughly 35 mg of the bottom third parts of five to ten stems were chopped and packed into a 3.2 mm Magic-Angle Spinning NMR rotor. For wild-type, five biological replicates (of at least five plants) were grown, each of which was analysed at least once; for irx3, two biological replicates (of at least five plants) were grown, each of which was analysed at least once. On the basis of the relative content of cellulose, pectin, xyloglucan and xylan observed in the wild-type and irx3 samples by NMR, we estimate the stem material contained a substantial but minority proportion of primary cell walls.k point grid of 2 × 2 × 1 and a fixed unit cell of 10 × 10 × 60 Å. The NMR shielding was calculated for the resulting structures using the gauge including projector augmented waves31The NMR shift calculations were carried out using the CASTEP code1H and 13C Larmor frequencies of 850.2 and 213.8 MHz and 500.1 and 125.8 MHz, respectively, using 3.2 mm double-resonance MAS probes. Experiments were conducted at room temperature at MAS frequencies of 12–14 kHz at the higher field and 10 kHz on the 500 MHz spectrometer unless otherwise stated. The 13C chemical shift was determined using the carbonyl peak at 177.8 p.p.m. of L-alanine as an external reference with respect to tetramethylsilane (TMS); 90° pulse lengths were typically 3.5 μs (1H) and 4.2 μs (13C). Both 1H−13C cross-polarization (CP) with ramped (70–100%) 1H rf amplitude1H nutation frequency of 70–80 kHz. Two-dimensional double-quantum (DQ) correlation spectra were recorded using the refocused INADEQUATE pulse sequence which relies upon the use of isotropic, scalar J coupling to obtain through-bond information regarding directly coupled nuclei2529τ spin-echo evolution times for a (τ–π–τ) spin-echo of 1.2 to 4.4 ms and the SPINAL-64 1H decouplingt1) was 4.5 -5.0 ms with a recycle delay of 1.5 and 1.9 s for CP and DP experiments, respectively. Intermolecular contacts were probed using 2D 13C−13C 1H driven spin diffusion (PDSD) experiments39F1) points with exponential line broadening of 80 Hz (CP) and 40 Hz (DP) in F2 and squared sine bell processing in F1. The 13C spin lattice relaxation time, T1, was measured at 125.8 MHz using saturation recovery following a comb of 30 pulses and echo acquisition. The dipolar order parameter, SCH, was determined using the 2D 13C−1H dipolar chemical shift (DIPSHIFT) experiment1H homonuclear coupling was suppressed using the frequency switched Lee-Goldberg sequence1H nutation frequency of 80 kHz. Measurements were taken with CP and with DP excitation with recycle delays of 2 and 20 s, respectively. All spectra obtained were processed and analysed using Bruker Topspin version 3.2.Solid-state MAS NMR experiments were performed on Bruker  850 and 500 MHz Advance III solid-state NMR spectrometers, operating at 13C magnetization under C−H dipolar coupling and MAS. The effective C−H coupling for CH2 units was obtained using the expressions reported by Terao et al.DIPSHIFT experiments were simulated using a home written program in Origin.c. The DIPSHIFT curves were calculated as a simple evolution of the http://dx.doi.org/10.17863/CAM.5896. The authors declare that all other relevant data supporting the findings of this study are available within the article and its The solid-state NMR data and scripts for DIPSHIFT simulations are available at How to cite this article: Simmons, T. J. et al. Folding of xylan onto cellulose fibrils in plant cell walls revealed by solid-state NMR. Nat. Commun.7, 13902 doi: 10.1038/ncomms13902 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables and Supplementary References.Peer Review File


IIbβ3 is the main integrin responsible for platelet activation and Rap1b plays a key role in integrin signalling. We investigated whether VASP is involved in the regulation of Rap1b in platelets since VASP-null platelets exhibit augmented adhesion to endothelial cells in vivo.Vasodilator-Stimulated Phosphoprotein (VASP) is involved in the inhibition of agonist-induced platelet aggregation by cyclic nucleotides and the adhesion of platelets to the vascular wall. αWashed platelets from wild type and VASP-deficient mice were stimulated with thrombin, the purinergic receptors agonist ADP, or the thromboxane A2 receptor agonist U46619 and Rap1b activation was measured using the GST-RalGDS-RBD binding assay. Interaction of VASP and Crkl was investigated by co-immunoprecipitation, confocal microscopy, and pull-down assays using Crkl domains expressed as GST-fusion proteins.Surprisingly, we found that activation of Rap1b in response to thrombin, ADP, or U46619 was significantly reduced in platelets from VASP-null mice compared to platelets from wild type mice. However, inhibition of thrombin-induced activation of Rap1b by nitric oxide (NO) was similar in platelets from wild type and VASP-null mice indicating that the NO/cGMP/PKG pathway controls inhibition of Rap1b independently from VASP. To understand how VASP regulated Rap1b, we investigated association between VASP and the Crk-like protein (Crkl), an adapter protein which activates the Rap1b guanine nucleotide exchange factor C3G. We demonstrated the formation of a Crkl/VASP complex by showing that: 1) Crkl co-immunoprecipitated VASP from platelet lysates; 2) Crkl and VASP dynamically co-localized at actin-rich protrusions reminiscent of focal adhesions, filopodia, and lamellipodia upon platelet spreading on fibronectin; 3) recombinant VASP bound directly to the N-terminal SH3 domain of Crkl; 4) Protein Kinase A (PKA) -mediated VASP phosphorylation on Ser157 abrogated the binding of Crkl.We identified Crkl as a novel protein interacting with VASP in platelets. We propose that the C3G/Crkl/VASP complex plays a role in the regulation of Rap1b and this explains, at least in part, the reduced agonist-induced activation of Rap1b in VASP-null platelets. In addition, the fact that PKA-dependent VASP phosphorylation abrogated its interaction with Crkl may provide, at least in part, a rationale for the PKA-dependent inhibition of Rap1b and platelet aggregation. IIbβ3, which is regulated by the small GTPase Rap1b. In the present study, we investigated whether Vasodilator-Stimulated Phosphoprotein (VASP) is involved in the regulation of Rap1b in platelets. To address this question, we activated platelets from wild-type and VASP-deficient mice and measured Rap1b activity. We found that activation of Rap1b was reduced in platelets from VASP-deficient mice. We identified a novel VASP binding partner in platelets, the adapter protein Crkl, which is important for Rap1b activation. Furthermore, we found that the interaction of Crkl and VASP is abrogated by PKA, which plays an important role in the inhibition of platelet function. Together, we propose that the control of the interaction of VASP and Crkl by PKA is important for Rap1b activation in platelets.Platelets, or thrombocytes, are the second most common kind of blood cells in the human body. Along with the coagulation factors, the main function of platelets is to stop bleeding by clumping and clotting blood vessel injuries (hemostasis). Hemostasis can be divided into three parts: platelet binding to the injured vessel (adhesion), platelet shape change and secretion of messengers (activation), and platelet bridging (aggregation). An important step in platelet function is the activation of the platelet receptor integrin αIIbβ3 integrin [2+ to its site on CalDAG-GEFI thus conferring GEF activity towards Rap1b [The central event in platelet adhesion at sites of vascular injury is the agonist-induced inside-out activation of αintegrin , a proceintegrin . Indeed,integrin . Rap1b cds Rap1b . Our preds Rap1b . CalDAG-ds Rap1b . C3G is ds Rap1b , 9. In pds Rap1b , 9. The ds Rap1b .2) derived from the endothelium whose effects are mediated through cGMP and cAMP, respectively [2 and NO inhibit platelet aggregation through their ability to block Rap1b activation [Circulating platelets are also continually exposed to inhibitory factors such as NO and prostacyclin , anti-Crkl (sc-319), and anti-Rap1b (sc-65) Abs were obtained from Santa Cruz Biotechnology; anti-CalDAG-GEFI and anti-Rap1GAP2 Abs were from ImmunoGlobe GmbH ; anti-VASP-pSer235 (clone 16C2) was from nanoTools . Alexa Fluor 633 phalloidin (Invitrogen) was used to visualize actin filaments. The protease inhibitor cocktail was purchased from Roche.6-tagged human VASP was purified from E.coli cultures and VASP was phosphorylated by PKA in vitro as previously described [Plasmids encoding the GST-Crkl fusion proteins were a generous gift of Dr. Stephan Feller  and proteins were purified on Glutathione-conjugated sepharose as detailed previously . Recombiescribed . Protein8) were lysed with an ice cold buffer composed of 50 mM Tris-HCl (pH 7.4), 1 % Triton X-100, 100 mM NaCl, 10 mM MgCl2, 20 % glycerol, 1 mM Na3VO4, and 1 mM pefabloc. The GST-RalGDS fusion protein, coupled to glutathione-sepharose beads, was used to pull down GTP-bound Rap1b as described [Platelets (2 × 10escribed . The bea8) were lysed in the following buffer: 40 mM Hepes-NaOH, pH 7.5, 90 mM NaCl, 1 % Igepal CA-630, and a protease inhibitor cocktail. Lysates were clarified by centrifugation for 10 min at 16,000 x g at 4 °C, and Crkl was subjected to immunoprecipitation using either anti-Crkl specific Abs or isotype control Abs, followed by incubation with protein G-conjugated sepharose beads. After extensive washing with the lysis buffer, the precipitated material was analyzed by Western blotting using anti-Crkl or anti-VASP Abs.For immunoprecipitation, human platelets (2 × 108) were either treated with a combination of forskolin (FSK) (5 μM) and okadaic acid (OA) (1 μM) for 20 min at 37 °C to stimulate PKA activity (+PKA) or left untreated (−PKA). Thereafter, cells were lysed as described above. Clarified lysates were incubated with 20 μg GST-Full-length-Crkl (GST-FL-Crkl) or equimolar amounts of GST-Crkl-SH2, GST-Crkl-SH3N, GST-Crkl-SH3C, GST alone (negative control), or GST-Spec-SH3 (positive control) coupled to glutathione sepharose beads. GST pull-down experiments with recombinant VASP were performed as above, using 500 ng of in vitro PKA-phosphorylated or non-phosphorylated His6-VASP and 5 μg GST-FL-Crkl or equimolar amounts of the other fusion proteins in the presence of 25 ng/μl BSA to block unspecific interactions. After extensive washing of the pelleted glutathione sepharose beads, precipitated material was analyzed by Western blotting using anti-VASP Abs.For pull-down assays using GST-fusion proteins, human platelets (2 × 106) were seeded on fibronectin-coated (25 μg/ml) 8-well glass bottom μ-slides (Ibidi) and fixed with 4 % formalin after 2, 5, 12, and 30 min incubation at room temperature. Cells were extensively washed with PBS, blocked/permeabilized with 5 % normal donkey serum in PBS containing 0.2 % Triton X-100, and incubated with 1:80 diluted primary Abs in 0.1 % BSA in PBS containing 0.2 % Triton X-100. After extensive washing, platelets were incubated with Alexa Fluor 488/546 conjugated donkey anti-mouse or donkey anti-rabbit Abs (Invitrogen), washed again, and mounted with 50 % glycerol in PBS containing 5 % DABCO (Sigma-Aldrich) as an anti-fading agent. Stained sections were investigated using a confocal laser scanning microscope  equipped with a 63-fold oil immersion objective. Images were acquired and prepared for presentation using the ZEN software .Washed human platelets , 2.8-fold with ADP  and 2.7-fold with U46619   or VASP-deficient mice (VASP KO) were stimulated with thrombin, the purinergic receptor agonist ADP, or the thromboxane A2 receptor agonist U46619 and Rap1b activation was measured by GST-RalGDS-RBD binding assay. We have previously shown augmented basal Rap1a activity in neutrophils from VASP-null mice . To our 5) Figs. . We exclActivation of monomeric GTPases is associated with their movement, together with their regulators (GEFs and GAPs), from the cytosol to the membrane fraction. We next investigated whether VASP controlled the movement of Rap1b, CalDAG-GEFI or Rap1GAP2, the dominant GAP for Rap1b in platelets . StimulaIn addition to activating factors, platelets circulating in vivo are continuously exposed to inhibitory factors such as endothelium-derived NO and prostacyclin . Most, iC3G is a GEF for Rap1b, which is activated through its recruitment by the docking protein Crkl. In adherent cells, VASP is localized to actin rich structures including the integrin-based focal adhesions and membrane protrusions such as filopodia, lamellipodia, and ruffles , 16, 20.Crkl-VASP interaction was further analysed by confocal immunofluorescence microscopy in platelets seeded on fibronectin-coated glass slides. To address the localization of Crkl and VASP during the dynamic adhesion and spreading of the platelets, cells were fixed 2, 5, 12, and 30 min after seeding and then processed for staining using anti-Crkl and anti-VASP specific Abs as well as fluorescent- conjugated phalloidin to stain actin fibres Fig. . Two min6-VASP, purified from E.coli. We found that GST-FL-Crkl and GST-SH3N-Crkl efficiently precipitated recombinant His6-VASP. We also observed a weak but distinct binding of VASP to the GST-SH2-Crkl fusion protein, but interaction of VASP with GST-SH3C-Crkl was negligible. Furthermore, we found that binding of His6-VASP to GST-FL-Crkl, GST-SH3N-Crkl, and GST-Spec-SH3 was comparable  in the linker, which conforms to the consensus binding motif of the VASP EVH1 domain (F/L/W/Y)PxϕP (where ϕ is a hydrophobic residue) [We made novel findings regarding the domains of VASP and Crkl involved in complex formation. We found that recombinant His6-VASP, but not VASP present in platelet lysates, bound to both the GST-SH3N-Crkl domain and the GST-SH2-Crkl fusion protein (however weakly). The SH2 domain of Crkl binds to phospho-tyrosine motifs , but we residue) . Thereforesidue) , 30, VASSH3 domains are the most abundant protein recognition motifs, comprising an estimated 409 copies in the human proteosome alone , mostly vβ3 and αIIbβ3 [vβ3 and αIIbβ3 bind vitronectin and the RGD motif of Von Willebrand factor, respectively [vβ3 leads to PKA-dependent phosphorylation of VASP which drives suppression of β1 integrin activation. This is achieved through disruption of the interaction between phosphorylated VASP and the Rap1-GTP-interacting adaptor molecule (RIAM) [vβ3 on β1 integrins, thus explaining the augmented adhesive capacities of VASP-null platelets to injured blood vessels [IIbβ3 outside-in signalling as well as secretion of platelet granules [Platelets express five types of integrins: three β1 integrins and the two β3 integrins αd αIIbβ3 . The β1 ectively . In fibre (RIAM) . Based o vessels  since wegranules .In summary, we propose that the formation of a ternary C3G/Crkl/VASP complex regulates, in parallel with CalDAG-GEFI, Rap1b-dependent platelet activation and this may explain why agonist-induced activation of Rap1b in VASP-null platelets is impaired. PKA-dependent phosphorylation of VASP on serine residue 157 abrogated Crkl binding, which may provide, at least in part, a rationale for the PKA-dependent inhibition of Rap1b activation and platelet aggregation.Crkl and VASP are dynamically interacting in actin-rich domains during platelet spreading and the interaction involves the N-terminal SH3 domain of Crkl. We propose that Crkl-VASP complex formation is important for C3G-dependent Rap1b activation in platelets. Conversely, Rap1b activation in VASP-null platelets is reduced. The interaction between Crkl and VASP is regulated by PKA-mediated VASP phosphorylation. This may, at least in part, explain the PKA-dependent inhibition of Rap1b and platelet aggregation.


For each crustacean species, the cooked extract had greater IgE reactivity than the raw . In contrast, there was a trend for lower PBMC proliferative responses to cooked compared with raw extracts. In crustacean-stimulated PBMC cultures, dividing CD4+ and CD56+ lymphocytes showed higher IL-4+/IFN-γ+ ratios for crustacean-allergic subjects than for non-atopics (p<0.01), but there was no significant difference between raw and cooked extracts. The percentage IL-4+ of dividing CD4+ cells correlated with total and allergen-specific IgE levels . Regulatory T cell proportions were lower in cultures stimulated with cooked compared with raw extracts . In conclusion, cooking did not substantially alter overall T cell proliferative or cytokine reactivity of crustacean extracts, but decreased induction of Tregs. In contrast, IgE reactivity of cooked extracts was increased markedly. These novel findings have important implications for improved diagnostics, managing crustacean allergy and development of future therapeutics. Assessment of individual allergen T cell reactivity is required.Crustacean allergy is a major cause of food-induced anaphylaxis. We showed previously that heating increases IgE reactivity of crustacean allergens. Here we investigate the effects of thermal processing of crustacean extracts on cellular immune reactivity. Raw and cooked black tiger prawn, banana prawn, mud crab and blue swimmer crab extracts were prepared and IgE reactivity assessed by ELISA. Mass spectrometry revealed a mix of several allergens in the raw mud crab extract but predominant heat-stable tropomyosin in the cooked extract. PBMC from crustacean-allergic and non-atopic control subjects were cultured with the crab and prawn extracts and proliferation of lymphocyte subsets was analysed by CFSE labelling and flow cytometry. Effector responses were assessed by intracellular IL-4 and IFN-γ, and regulatory T (CD4 In allergic individuals, allergen-stimulated T cells secrete IL-4, IL-5 and IL-13, Th2-type cytokines, which initiate and propagate the allergic IgE-mediated immune response [+ cells and impaired Treg function in food-allergic individuals [Shellfish, comprising crustacean and mollusc species, are a major cause of IgE-mediated adverse food reactions including anaphylaxis , 2. Unlientified –8. Ratioresponse , 10. On response . Regulatresponse , 13. A rividuals , 15.+, CD8+ and CD56+ cells, and Foxp3+ Treg proportions were compared. This analysis of the cellular response to differently processed crustacean allergens will inform development of safe and effective specific immunotherapy as well as monitoring bioassays.Food processing can influence recognition of food allergens by immune cells and the ensuing immune response . CookingInformed written consent was obtained from all subjects, with ethics approvals from the Alfred Hospital Research Ethics Committee (Project number 192/07) and Monash University Human Research Ethics Committee (MUHREC CF08/0225).A/L). Non-atopic control subjects had a negative skin prick test to a panel of common aeroallergens (including house dust mite), negative shrimp- and crab-specific IgE (ImmunoCAP), no IgE reactivity to shellfish extracts by IgE immunoblotting, and no clinical history of crustacean allergy.Peripheral blood samples were obtained from eight crustacean-allergic subjects  and four non-atopic controls  . AllergiPortunus pelagicus), black tiger prawn (Penaeus monodon), banana prawn (Fenneropenaeus merguiensis/indicus) and mud crab (Scylla serrata) were purchased from local markets. For raw blue swimmer crab (RC1), raw mud crab (RC2), raw black tiger prawn (RP1) and raw banana prawn (RP2) extracts, the outer shell was removed and muscle collected. For cooked blue swimmer crab (CC1), cooked mud crab (CC2), cooked black tiger prawn (CP1) and cooked banana prawn (CP2) extracts, the outer shell was retained during the heating process (20 minutes immersed in boiling PBS) before its removal and muscle tissue extracted. Finely cut muscle was blended in PBS pH 7.2 and incubated overnight at 4°C with constant mixing. After centrifugation at 13,000 rpm at 4°C for 20 minutes the supernatant was collected, dialyzed against PBS and filter sterilized before storage at -80°C in aliquots. Extract protein concentrations were determined using the Bradford assay kit  with bovine gamma globulin as a standard. All extracts were confirmed to be neither mitogenic nor toxic as described previously [Extracts were prepared as described previously . Brieflyeviously . EndotoxProteins of crustacean extracts, 15 μg/lane, were separated by electrophoresis under reducing conditions using 4–12% Bis-Tris gels . Pre-stained standards  were used as molecular weight markers. Proteins were resolved at 200 V for 35 minutes using an Xcell II mini-cell apparatus (Invitrogen) and stained with Coomassie brilliant blue.Proteins (3 μg/lane) from raw and cooked mud crab extracts  were separated by SDS-PAGE as above and assessed for IgE reactivity by immunoblotting using pooled shellfish-allergic serum as described previously , 23, 24.IgE ELISA was based on a method described previously . Briefly7 cells/mL PBS) were labelled with 0.5 μM carboxyfluorescein succinimidyl ester (CFSE) (Life Technologies) and cultured at 2.5 x 105 cells/well in 96-well microplates with culture medium alone (‘no antigen’ negative control), tetanus toxoid as positive control  or each of the eight crustacean extracts (50 and 100 μg/mL). Culture medium comprised RPMI-1640  supplemented with 2 mM L-glutamine (Life Technologies), 100 U/mL penicillin-streptomycin (Life Technologies) and 5% heat-inactivated AB+ human serum . All conditions were plated in duplicate. Cultures were incubated for 7 days at 37°C in 5% CO2.PBMC were isolated from heparinized whole blood by Ficoll  gradient. PBMC (102 prior to antibody staining. Cells from duplicate cultures were pooled and incubated with an antibody cocktail containing aqua live/dead dye (Life Technologies) and fluorochrome-labelled anti-human CD4, CD8 and CD56  for 30 minutes at 4°C in the dark. Cells were then fixed with 1% paraformaldehyde and stained with fluorochrome-labelled anti-human IL-4 and anti-human IFN-γ (BD Biosciences) diluted in 0.3% saponin  for 1 hour at 4°C in the dark. For Treg analysis, cells were stained with aqua live/dead dye and an antibody cocktail containing fluorochrome-labelled anti-human CD4, CD25 and CD127 (BD Biosciences) for 20 minutes at room temperature in the dark. Cells were then fixed and permeabilized  for 30 minutes at 4°C in the dark, blocked with 2% rat serum (Sigma-Aldrich) in 1X permeabilization buffer (eBioscience) for 15 minutes at 4°C in the dark and incubated with fluorochrome-labelled anti-human Foxp3 (eBioscience) for 30 minutes at 4°C in the dark. Data were acquired using an LSR-Fortessa (BD Biosciences) and analysed using FlowJo 7.6.4 (TreeStar) software.For intracellular cytokine staining, cultured cells were re-stimulated with 10 ng/mL phorbol 12-myristate 13-acetate  and 250 ng/mL ionomycin  for 5 hours in the presence of Brefeldin A  for the last 4 hours at 37°C in 5% COGraphpad Prism 6 software  was used for statistical analyses and a p-value of <0.05 was considered significant. Statistical methods are indicated in Figure legends.SDS-PAGE analysis of raw and cooked crustacean extracts showed a range of proteins from approximately 5–188 kDa . In raw To assess whether allergen content contributed to differences in IgE reactivity, IgE reactive proteins within the raw and cooked mud crab extracts were compared using mass spectrometry . In the IgE ELISA showed that, for all species, cooked extracts had significantly higher IgE binding than the corresponding raw extracts . While bTo assess cellular immune responses to crustacean extracts, PBMC proliferative responses were first examined. Extract concentrations of 50 and 100 μg/mL, and 7 days of culture were found to be optimal for reliable detection of lymphocyte proliferation for raw and cooked extracts in preliminary experiments (data not shown). Crustacean extracts induced marked total lymphocyte proliferation above that observed in ‘no antigen’ control cultures . Interes+, CD8+ and CD56+ lymphocyte subsets was selectively analyzed  and RC2 (p <0.05) extracts (data not shown), but differences were less marked and not significant for the CFSElo CD8+ subset. Cooking did not significantly affect cytokine reactivity of the extracts for either subject group.Analysis of the intracellular cytokine profile of lymphocytes proliferating in response to crustacean extracts showed an inverse relationship between the percentage IL-4oduction Fig 6). . + and I+ cells within the proliferating CD4+ subset in response to prawn and crab extracts  and all crab extracts  to calculate the median percentage of cytokine positive cells; + cells (p<0.05 or p<0.01). Similar relationships were observed for dividing CD56+ cells with regard to cytokine responses and total IgE, and IFN-γ responses and prawn-specific IgE (data not shown). There were no correlations between cytokines produced by dividing CD8+ cells and total or specific IgE. Proliferation of total and subsets of lymphocytes showed no correlation with total or specific IgE levels (data not shown).There was a significant positive correlation between total IgE and specific IgE levels and the percentage of IL-4+CD127loCD25+Foxp3+ phenotype (gating strategy shown in +CD25+CD127loFoxP3+) has been shown previously to correspond with functional Tregs [hi subset, cells with a Treg phenotype showed no differences in proportions in response to any of the extracts or between subject groups, and proportions were similar to those seen for the ‘no antigen’ control (data not shown). However, a wider range of Treg proportions was seen within the CFSElo cell population and some interesting differences observed. Non-atopic subjects displayed a trend for higher proportions of Tregs in response to each of the crustacean extracts, and Treg proportions were generally lower in cultures stimulated with cooked extracts compared with raw for crustacean-allergic and non-atopic subjects  following heating in the presence of endogenous or exogenous sugars by the Maillard reaction (non-enzymatic attachment of sugars to proteins) has been shown to increase IgE binding and T cell reactivity to other food allergens , 32. Gly+ T cell reactivity to shrimp tropomyosin has been studied [Despite the clear changes in protein profiles and IgE reactivity of crustacean extracts induced by heating, there were no marked differences in PBMC proliferative responses to raw versus cooked extracts. In fact there was a trend for greater cellular reactivity of the raw extracts. This could in part be explained by the difference in allergen composition between raw and cooked extracts, with raw extracts being shown in this study to contain a large number of individual allergens whilst cooked extracts predominantly contain tropomyosin. Therefore the raw extracts may be able to stimulate a wider repertoire of allergen-specific T cells than cooked extracts. To date only CD4 studied . Further studied .+ or CD8+ T cells. We have previously demonstrated that patients generate IgE antibodies against both heat stable and heat labile allergens of crustaceans, with the heat stable tropomyosin being a major allergen [Our findings suggest that several prawn and crab allergens are able to induce good T cell responses, since PBMC proliferative responses to the mixed allergen content of raw extracts was similar to that for the ‘tropomyosin-enriched’ cooked extracts. The effect of cooking on allergen uptake by innate immune cells and the very different nature of T and B cell antigen epitopes also need to be considered, T cell epitopes comprising short linear peptides produced by APC processing while B cell epitopes are often conformational. The heat-induced changes that increased IgE reactivity of crustacean allergens apparently had no substantial effect on the net allergen peptide presentation to CD4allergen . The relin vitro in response to allergen-derived peptides presented by APC [As noted previously for other food allergens , 34, thed by APC –37. Argu+, CD8+ and CD56+ lymphocyte subsets from crustacean-allergic and non-atopic subjects. CFSElo proliferating cells were analysed as these were the cells, either directly or indirectly, responding to in vitro stimulation with crustacean proteins. As expected, the production of IL-4 by dividing CD4+ lymphocytes was higher for crustacean-allergic subjects than non-atopic subjects, and vice versa for production of IFN-γ. These differences were shown more clearly by comparing IL-4+/IFN-γ+ ratios, better reflecting the Th2 polarisation of the immune response to allergen by allergic individuals and Th1 bias by non-atopics, and supported by the strong correlation with clinically relevant serum prawn- or crab-specific IgE levels. Proliferating CD8+ and CD56+ lymphocytes were also shown to contribute to the cytokine milieu in raw and cooked crustacean-stimulated PBMC cultures. Further studies are required to determine the precise phenotype of the CD8+ and CD56+ lymphocyte subsets responding (including use of a wider range of NK and NKT cell markers) and whether their responses were directly in response to extract stimulation or bystander effects from cytokines produced by the allergen-responsive CD4+ T cells in culture. Of particular interest, in terms of influencing clinical outcome, would be determining whether the whole crustacean extracts contain NK or NKT cell ligands that could engage directly with these cell subsets and stimulate their production of Th2 polarising cytokines. Cytokine production by both NK and NKT cells have been shown to play a role in promoting development of the allergic immune response [To assess effector cell phenotype, the intracellular cytokines IL-4 and IFN-γ were analysed in CD4response –41.lo) population of CD4+CD25+CD127loFoxp3+ cells in allergen-stimulated PBMC cultures. This phenotype has been shown to correspond with functional Tregs [Better defined is the role of Tregs in mediating immune tolerance to allergens , 36. Typal Tregs . There w+ T cell epitopes of allergens [Together, our findings inform the development of more reliable diagnostic assays for crustacean allergy as well as the design of an appropriate allergen-specific therapeutic. Conventional allergen immunotherapy using whole allergen is likely not suitable for treating crustacean allergy due to the high risk of IgE-mediated adverse reactions . The mosllergens , 44. Our


Many antimicrobial and anti-tumour drugs elicit hormetic responses characterised by low-dose stimulation and high-dose inhibition. While this can have profound consequences for human health, with low drug concentrations actually stimulating pathogen or tumour growth, the mechanistic understanding behind such responses is still lacking. We propose a novel, simple but general mechanism that could give rise to hormesis in systems where an inhibitor acts on an enzyme. At its core is one of the basic building blocks in intracellular signalling, the dual phosphorylation-dephosphorylation motif, found in diverse regulatory processes including control of cell proliferation and programmed cell death. Our analytically-derived conditions for observing hormesis provide clues as to why this mechanism has not been previously identified. Current mathematical models regularly make simplifying assumptions that lack empirical support but inadvertently preclude the observation of hormesis. In addition, due to the inherent population heterogeneities, the presence of hormesis is likely to be masked in empirical population-level studies. Therefore, examining hormetic responses at single-cell level coupled with improved mathematical models could substantially enhance detection and mechanistic understanding of hormesis. Hormesis is a highly controversial and poorly understood phenomenon. It describes the idea that an inhibitor molecule, like an anti-cancer or anti-microbial drug, can inadvertently stimulate cell growth instead of suppressing it. This can have a profound effect on human health leading to failures in clinical treatments. Therefore, getting at the mechanistic basis of hormesis is critical for drug development and clinical practice, however molecular mechanisms underpinning hormesis remain poorly understood. In this paper we use a mathematical model to propose a simple and yet general mechanism that could explain why we find hormesis so widely in living systems. In particular, we discover that hormesis is present within a fundamental structure that forms a basic building block of many intracellular signalling pathways found in diverse processes including control of cell reproduction and programmed cell death. The benefits of our study are two-fold. Having simple molecular understanding of the causes of hormetic responses can greatly improve the design of new drug compounds that avoid such responses. Moreover, due to the fundamental nature of the newly proposed mechanism, our findings have a potential broad applicability to both anti-cancer and anti-microbial drugs. Hormesis is a phenomenon describing biphasic dose response relationships that exhibit low-dose stimulation and high-dose inhibition . Many meDespite the overwhelming body of research, some dating back a century, that documents hormetic responses to a broad range of compounds, their clinical significance has only relatively recently come to the fore . The conThe vast majority of targets for antibiotics, antifungals and anti-tumour drugs fall into the following categories: enzymes, receptors, transporters and DNA/RNA and the ribosome . HoweverAn area of research where understanding of the mechanisms giving rise to hormesis is particularly lacking involves enzyme-targeting drugs. Known as enzyme inhibitors, they are designed to block enzyme activity leading to disruption of bacterial cell wall , fungal mitogen-activated protein kinase (MAPK) pathway [In recent years kinase inhibitors, a subset of enzyme inhibitors, have been shown to be very effective therapeutic agents in a broad range of diseases, including cancers. Amongst other enzyme inhibitors, significant attention has been focused on those inhibiting the  pathway –24, whic pathway .The observations of hormesis in MAPKs as a result of inhibition of BRAF oncogene are widespread: low doses of RAF inhibitors designed to cease tumour proliferation  can causIn this paper we put forward a novel, simple but general mechanism driving hormetic responses in systems where an inhibitor acts on an enzyme. We develop a mathematical model based on a basic building block in intracellular signalling, namely a dual phosphorylation-dephosphorylation motif, to which a kinase inhibitor is applied. In a broader context, dual-phosphorylation can be found in diverse processes such as circadian rhythms , virulenThe model demonstrates that under certain conditions the steady state amount of the double-phosphorylated protein substrate in the cycle can substantially increase at low inhibitor doses compared to the base level without inhibition. Therefore the dose-response curve of the double-phosphorylated substrate possesses a hallmark of hormesis: it is upward sloping at low inhibitor doses and downward sloping at high inhibitor doses. The existence of hormesis in our model depends on the mechanism of inhibition and the dissociation rates of the kinase-substrate-inhibitor complexes. We also found that the magnitude of hormetic responses depends on the substrate-kinase ratio in a non-monotone way.The benefits of our study are two-fold. Our mechanism is based on a principal component of intracellular signalling pathways, and as such has a potential broad applicability. Moreover having simple molecular understanding of the causes of hormetic responses can greatly improve the design of new drug compounds that avoid such responses.P) and subsequently into a double-phosphorylated form (CPP), through a chain of reactions facilitated by a kinase (kin). Conversely CPP is converted back to CP which is subsequently converted to C, through a chain of reactions facilitated by a phosphatase (pho). In the distributive mechanism, the kinase(phosphatase) facilitates at most one phosphorylation (dephosphorylation) in each molecular encounter [We consider a simple dual phosphorylation-dephosphorylation motif, whereby a distinct kinase protein is phosphorylating a separate protein substrate. Multiple phosphorylations can occur in close proximity or in diverse sites on a protein and here we focus on the former, instances of which can be found in activation of conventional MAPK enzymes , cell-cyncounter .P · kin according to the following inhibition scheme:ki and ei in Eqs df and dr in e2, e4 and backward e−2, e−4 rates [Therefore our dual phosphorylation-dephosphorylation motif can be described by the following reaction kinetic equations, which are a simplification of the reaction scheme described in :C+kin⇌k−4 rates .The model describing the time evolution of the substrate, kinase, phosphatase and inhibitor concentrations is based on the law of mass action and assumes the total conservation of mass holds for all four compounds. The details of the system of 9 differential equations and the corresponding analysis are presented in PP, [In the absence of an inhibitor, the double phosphorylation motif PP, , 58, 59.PP*) in the absence of an inhibitor, we find that CPP can exhibit biphasic (or hormetic) response to an inhibitor as illustrated in PP increase monotonically, while for sufficiently large inhibitor doses, the computed steady-state values of CPP monotonically decrease.In the case of a single stable steady state  the strong dissociation effect of intermediate substrate-kinase-inhibitor complexes C · kin · inh and C(C2) large dissociation rate of kinase-inhibitor complexes.e−2, e−4 > 0  will exhibit a monotone decreasing dose-response  will exhibit a hormetic response (In addition the above conditions (C1-C2) can also be used to forecast the presence of a hormetic dose response in the second case under our consideration, namely when in the absence of an inhibitor the motif PP,1*  and CPP,* . InGiven the fundamental nature of the dual phosphorylation-dephosphorylation motif and the relative simplicity of the derived conditions necessary to observe hormesis, why was this mechanism previously overlooked in theoretical literature? A further examination of the (C1) condition could provide a potential answer. In general, when considering partial competitive enzyme inhibition  as we doin vitro tests of our model predictions. First, biochemical assays involved with in vitro studies are not standardised and vary between research groups, making comparisons between already published observations difficult. Second, testing our model predictions requires measurements of single and double phosphorylation outputs, this could be problematic as antibody specificity required to distinguish these outputs might not readily be available. This would particularly be relevant for systems where phosphorylation sites are situated close together. Third, ensuring that the condition for observing hormesis e2, e4 > 0 is satisfied experimentally is challenging as kinase biochemical assays would not usually include phosphatase activity. Furthermore varying rates of reactions individually or measuring fluxes in such systems is equally difficult.Once a new mechanism is proposed to explain a particular biological phenomenon, ideally it should be put to test. However, there are a number of difficulties associated with in vitro systems, we next consider whether these difficulties could be overcome with a cell-based experimental systems. In particular, our model predicts that hormetic dose-response could be a wide-spread feature of MAPK pathways when exposed to enzyme inhibitors. However we argue here that the non-trivial biphasic dose-response associated with hormesis might often be overlooked when performing experiments at cell population level, as we now discuss.Having discussed difficulties associated with testing our model in reductionist PP. This means that tumour cells within a population can be grouped into two types: type-1 cells with ‘high’ CPP and type-2 cells with ‘low’ CPP. In reality these heterogeneous cell phenotypes can emerge not only due to multistability of the system [Consider the case where in the absence of an inhibitor, the double phosphorylation motif e system , 54 but e system , 67. In e system .PP  will exhibit a monotone decreasing dose-response  will exhibit a hormetic response  to the steady-state values of CPP in the presence of the inhibitor  at the IC50 concentration estimated for the entire population Click here for additional data file.


Ice nucleation and growth is an important and widespread environmental process. Accordingly, nature has developed means to either promote or inhibit ice crystal formation, for example ice-nucleating proteins in bacteria or ice-binding antifreeze proteins in polar fish. Recently, it was found that birch pollen release ice-nucleating macromolecules when suspended in water. Here we show that birch pollen washing water exhibits also ice-binding properties such as ice shaping and ice recrystallization inhibition, similar to antifreeze proteins. We present spectroscopic evidence that both the ice-nucleating as well as the ice-binding molecules are polysaccharides bearing carboxylate groups. The spectra suggest that both polysaccharides consist of very similar chemical moieties, but centrifugal filtration indicates differences in molecular size: ice nucleation occurs only in the supernatant of a 100 kDa filter, while ice shaping is strongly enhanced in the filtrate. This finding may suggest that the larger ice-nucleating polysaccharides consist of clusters of the smaller ice-binding polysaccharides, or that the latter are fragments of the ice-nucleating polysaccharides. Finally, similar polysaccharides released from pine and alder pollen also display both ice-nucleating as well as ice-binding ability, suggesting a common mechanism of interaction with ice among several boreal pollen with implications for atmospheric processes and antifreeze protection. The formation of macroscopic ice crystals via nucleation and growth is of fundamental importance in various environmental systems such as in cloud, formation processes of Earth’s atmosphere234In the atmosphere, on the contrary, ice nucleation is normally rate-limiting for ice crystal formation. In deep-convective clouds and in cirrus clouds, ice nucleation does sometimes occur homogeneously, but often heterogeneous ice nucleation is the dominant pathway in mixed-phase clouds and in cirrus clouds161681011121314et al.The physical basis for the distinction between high and low efficacy ice-nucleating particles is still a matter of intense research as are the properties and nature of so-called active sites, which are the locations on the particles where critical ice embryo formation occurs171822021222324Pseudomonas putida was shown to also act as an ice-nucleating proteinPseudomonas syringae also exhibits ice-binding ‘antifreeze’ ability by showing both ice shaping and thermal hysteresis26Organisms have developed a large diversity of molecular compounds to sustain life at extreme conditions2345252833323334Betula pendula we performed ice nucleation experiments with microliter-sized water droplets using an optical freezing array (BINARY) and with picoliter-sized droplets using differential scanning calorimetry (DSC), see Methods. The results of the microliter-sized droplet experiments agree closely with previous measurements124 INM are released per individual pollen particle, see Thom ≈ −38 °C . This technique has been developed for purification of antifreeze proteins, i.e., molecules that bind to ice, from other non-binding proteins and solutes3−1, see also the corresponding Hippophae rhamnoides chitinases45Ice crystals growing slowly in aqueous solutions of non-ice-binding solutes  just below the ice melting temperature show a round shape as this reduces the crystal’s total surface area and thus its overall interface energy 444. Ice 43 as a function of time, resulting in a linear increase of r3 with time, just as expected for an Ostwald-ripening process according to Lifshitz-Slyozov-Wagner (LSW) theory473 can be analysed from linear fitting (solid lines in kl0 is obtained as a function of the dilution degree of the stock solution dd = c(solution)/c(stock solution), see kl0 is large in the control solution , while it becomes smaller with increasing birch pollen molecule concentration, i.e. for larger dd values. This behaviour is equivalent to that of antifreeze (glyco)proteins previously investigated with IRRINAci representing the efficacy of a particular ice recrystallization inhibition agentdd = 0.78, but the corresponding concentration ci cannot be calculated easily, as we do not know the concentration of ice-binding molecules in the stock solution. We did, however, determine that this stock solution contains ~1.1 nmol L−1 of ice-nucleating molecules, from which we can infer an upper limit of ci, see related discussion in We have performed ice recrystallization experiments using the IRRINA assay47The experimental data presented above reveal that molecules released from birch pollen show ice-binding properties similar to antifreeze (glyco)proteins. In fact, the investigated samples exhibit at least three of the four hallmarks of ice-binding activity known from antifreeze (glyco)proteins: (i) they can be purified from multicomponent solutions by binding to slowly growing macroscopic ice; (ii) they induce a characteristic shaping of individual ice crystals; and (iii) they reduce the rate of ice recrystallization in polycrystalline ice samples. In preliminary thermal hysteresis measurements using DSC and cryo-microscopy similar to previously reported protocols49−1, see discussion below, and they are similar to that of birch pollen washing water reported in the literature, which was, however, not assignedWe have investigated the birch pollen molecules using Fourier Transform Infrared (FTIR) spectroscopy in attenuated total reflection mode (see Methods). The spectra in −1 to 900 cm−1 are characteristic of saccharide moieties52−1 and about 1520–1590 cm−1, respectively, see magenta spectrum of antifreeze protein AFP III for comparison38−1 and 1409 cm−1, which are characteristic for the asymmetric and symmetric stretches of carboxylates, respectively. The bands are slightly upshifted from ~1550 cm−1 and ~1400 cm−1 by attachment to saccharide moietiesIn order to gain more information on the chemical nature of the birch pollen molecules, we analysed the spectrum of an ice-affinity purified birch sample (solid red line in −1 (brown)−1, which is present in the hyaluronate spectrum (blue).A common group of natural acidic polysaccharides are glycosaminoglycans, which consist of disaccharide chains of an amino sugar and an acidic sugar. In −1 and 1409 cm−1 are clearly reduced in intensity in favour of a band at 1741 cm−1, characteristic for protonated carboxylic acids (red arrow). The remaining band at ~1660 cm−1 may be attributed to residual water in the sample−1, further pointing to the absence of significant amounts of amide moieties.While these comparisons suggest that the birch molecules do not belong to the class of glycosaminoglycans, we can use the latter spectra to support the fact that they contain acidic sugars. When birch washing water is acidified with dilute aqueous HCl to pH2 before drying , the carboxylate bands at 1614 cm−1 guanidinium chloride, or treatment with the enzymes pronase E, papain, and trypsin2626In summary, the analysis of the IR spectra shown in 2 + (aq) ions are added: the monovalent carboxylate groups form complexes with the bivalent Ca2 + (aq) ions, thereby linking different polysaccharide molecules to form a macroscopic gel572 + (aq) to ice affinity-purified birch #C washing water resulted in flocculation, similar to alginate  and Scots pine (Pinus sylvestris). These pollen are known to be good ice nucleators266071226383960It has been reported previously that washing water of several kinds of pollen induce heterogeneous ice nucleationLobelia telekii are supposedly carbohydratesBacillus thuringiensis was found to inhibit ice nucleationUpis ceramboides exhibited a pronounced thermal hysteresis due to ice bindingPolysaccharides constitute a common class of molecules that interact with ice in nature either by triggering ice nucleation or by inhibition of ice nucleation and growth. For example, ice nucleators found in the Afro-alpine plant It seems, therefore, reasonable to propose that the active site of the ice-nucleating molecules and the ice-binding site of the ice growth-inhibiting molecules may not be distinct, but in fact may originate from the same molecular moieties. Our observations do not contradict but indeed support the consideration that the same type of molecule can show both ice-nucleating as well as ice-binding ability, in agreement with the notion recently discussed by Davies that apart from the size of their active site there is no principal difference in the mechanistic interaction of antifreeze or ice-nucleating proteins with iceIt has not escaped our notice that the two seemingly opposing properties of pollen polysaccharides opens the question of their primary biological function: is it the ability of ice-nucleating polysaccharides to trigger precipitation in the atmosphere, or is it the antifreeze protection by ice-binding polysaccharides during periods of night frost in early springtime, or are both functions important by triggering extracellular ice nucleation and inhibiting intracellular ice growth? Antifreeze protection by extracellular ice nucleation is supported by a correlation between the ice nucleation ability of pollen species and the time of year they are released60Furthermore, it is certainly notable that, to the best of our knowledge, the pollen polysaccharides are the first known ice-binding molecules that can safely be assumed to be present in the atmosphere. Although we consider it to be relatively unlikely that ice-binding polysaccharides from pollen will significantly affect the shape of atmospheric ice crystals, ice-nucleating polysaccharides from pollen may affect atmospheric ice nucleation. While the atmospheric concentration of entire pollen particles is comparably low due to their large size and corresponding settling velocitiesBetula pendula; item#: RM527, LOT#: P1329870-1/-2, P3317516-1, and P1329872-1 termed here batch #A, batch #B, and batch #C, respectively. #A was collected on March 30, 2011 in France, contained 2.16% plant parts, and 0.39% contamination by spores or other pollen. #B was collected on April 18, 2013 in France, contained 3.04% plant parts, and no contamination. #C was collected in March, 2011 in France, contained 0.98% plant parts, and 0.29% contamination by spores or other pollen.), European alder  and Scots pine ). Bi-distilled water was prepared freshly each day, all other chemicals were obtained commercially: sucrose ; glucose ; NaCl ; Span 65 ; heparin sodium salt (Sigma Aldrich); hyaluronic acid sodium salt (Sigma Aldrich); dextran ; alginic acid sodium salt (Alfa Aesar); microcrystalline cellulose (Sigma Aldrich); natural type 3 antifreeze protein, AFP III, from polar fish of suborder Zoarcoidei (A/F-Protein); methylcyclohexane  and methylcyclopentane ; Na2B4O7·10H2O, HCl  and NaOH .Pollen were obtained from GREER as non-defatted samples: European white birch  by manual shaking in a test tube for a few minutes. Pollen washing water for DSC or BINARY freezing experiments was prepared by filtration of the pollen suspension through a 0.2 μm polyethersulfone syringe filter (VWR). Pollen washing water for ice affinity purification required large amounts of pollen material. Several aliquots of a total of about 100 g of birch pollen (batches #B or #C) were suspended consecutively in a total of about 500 mL freshly bi-distilled water . After each suspension procedure (15–60 minutes of stirring with a magnetic stirrer) the pollen grains were filtered out of the suspension through a 5 μm cellulose nitrate membrane filter (Sartorius) and washed with water several times. The resulting raw filtrate was used for ice affinity purification. Normally, pollen suspensions and washing water were prepared freshly on the day of measurement, but when necessary filtrated washing water and ice affinity purified washing water samples were stored in a freezer (at −18 °C) to prevent degradation or aging and were used within a few weeks. We did not observe any significant inactivation during that time.−1 and ice melting temperatures usually at a heating rate of +1 °C min−1 . Ice nucleation temperatures (Thom and Thet) were determined as the onset of the corresponding exothermic signal, and ice melting temperature (Tm) as the inflection point of the corresponding endothermic signal. The inverse emulsions were prepared by mixing 2 mL of an organic phase, consisting of 7 wt% of the surfactant Span 65 dissolved in a 1:1 by volume mixture of methylcyclohexane and methylcyclopentane, and 2 mL of the aqueous phase of interest. This mixture was stirred with a high-speed disperser (IKA Ultra-Turrax T25 basic) at room temperature for 10 min at 20,000 rpm to generate an emulsion containing aqueous droplets about 1–10 μm in size. About 7 mg of an emulsion was transferred into a hermetically-sealed aluminium DSC pan for immediate measurement.Ice nucleation behaviour of pollen washing water in inverse (water-in-oil) emulsions was investigated with a differential scanning calorimeter (TA Instruments Q100). Exothermic processes such as homogeneous or heterogeneous ice nucleation were measured as the heat flow difference between the sample pan and an empty reference pan. The calorimeter has been calibrated exhaustively as a function of temperature and cooling/heating rate40−1 until all droplets were frozen. Freezing of the droplets was detected by the associated change in droplet transparency using a digital camera (QImaging MicroPublisher 5.0 RTV).To investigate the freezing behaviour of both pollen suspensions and washing water over a broad concentration range an optical freezing array (BINARY: Bielefeld Ice Nucleation ARraY) was usedThe ice-binding molecules contained in the raw birch pollen filtrate washing water were purified using the method of ice affinity purification (IAP)−1 most of these crystals were melted until finally only one or two ice crystals were obtained in the field of view. The very slow growth of these crystals was observed at a very small cooling rate of −0.01 °C min−1 with digital microphotographs taken every 6 s, see previous papers for more details4748Different types of samples were used in the ice-shaping experiments. For birch pollen, raw washing water after filtration and concentrating at 90 °C for 3 hours at ambient pressure was used as well as solutions obtained after ice affinity purification, see above.  The birch pollen molecules were analysed in a 30 wt% aqueous sucrose solution, and a pure 30 wt% aqueous sucrose solution was also analysed as a control. For alder and pine pollen the raw washing water after filtration and concentrating at room temperature and reduced pressure was used without further treatment. 5 μL of a sample solution was embedded as a thin film between two glass coverslips (14 mm diameter) and sealed with silicone grease. This sample cell was then placed into a temperature-controlled cryostage  attached to an optical microscope (Olympus BX51) equipped with a digital camera (QImaging MicroPublisher 5.0 RTV). First, the sample was rapidly cooled to −30 °C (for purified samples) or −40 °C (for raw filtrate samples) to form polycrystalline ice within the film, and subsequently the sample was slowly heated to a temperature just below the melting point until only a few ice crystals remained. (The absolute temperature differed between samples due to differences in solute concentrations.) In further cooling and heating cycles at very small cooling and warming rates of ±0.1 or ±0.01 °C min−1 to −50 °C in order to produce a polycrystalline ice film. After heating to −8 °C the sample temperature was held constant for 2 hours while microphotographs were taken every 15 s. The size of all ice crystals in the field-of-view was obtained using image analysis and the corresponding cubic mean radius was analysed as a function of time using LSW theory; for detail see previous publications47The kinetics of ice recrystallization in aqueous 45 wt% sucrose solutions containing birch pollen molecules was studied using the Ice Recrystallization Rate INhibition Analysis (IRRINA) assay47−1 using either 1024 scans (IFS 66 s) or 512 scans (IFS 66 v). Absorbance values were divided by the corresponding wavelength to correct for the wavelength dependence of the penetration depth of the evanescent wave.Fourier transform infrared (FTIR) spectroscopy was performed in attenuated total reflection (ATR) mode on one of two FTIR spectrometers (Bruker IFS 66 v and IFS 66 s) equipped with an ATR unit containing a diamond reflection element with nine active reflections  and a mercury cadmium telluride detector as described previouslyCentrifugal filters with a 100 kDa molecular weight cut-off  were used to separate molecules of different size present in the birch pollen washing water. Initially, the stock solution was centrifuged for about 30–60 min  at 5,000 rpm, the supernatant was shaken occasionally, about every 5–10 min. Thereafter, the filtrate was removed from the tube and stored separately. Then several aliquots of pure water (~300–500 μl each) were added and centrifuged subsequently  to wash off any residual smaller molecules from the supernatant. Afterwards the remaining supernatant was diluted with water up to the initial volume. The entire procedure was performed independently with three stock solutions: with washing water of birch #B and #C, and with a concentrated, twice ice affinity-purified sample of birch #B.Figure source data of the x-y-plots shown in this paper have been deposited in the public institutional repository (PUB) of Bielefeld University (doi: 10.4119/unibi/2907691).How to cite this article: Dreischmeier, K. et al. Boreal pollen contain ice-nucleating as well as ice-binding ‘antifreeze’ polysaccharides. Sci. Rep.7, 41890; doi: 10.1038/srep41890 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Phalcoboenus australis) in the Falkland Islands in response to seasonal changes in the availability of seabird carcasses. We banded more than 450 individuals on Saunders Island, deployed archival and satellite GPS data loggers on 17 individuals, and monitored movements within and between two feeding areas on Saunders Island, a “marine-subsidized” site near seabird colonies and an anthropogenic “human-subsidized” farm site 16 km to the southeast.The extent to which seasonal changes in food availability affect small-scale movements in free-ranging populations of birds of prey is relatively little studied. Here we describe a seasonal “micro-migration” of a farm-island population of striated caracaras (2 [mean ± SD]), whereas those at the human-subsidized site in winter maintained larger 95% kernel core areas (3.8 ± 4.6 km2). Two of 6 satellite-tagged individuals that summered at known caracara breeding colonies 80 km WNW of Saunders Island were subsequently resighted in winter at the human-subsidized site.During 67 observation days between 2010 and 2015, resightings of 312 banded caracaras were greater at the marine-subsidized site during austral summer than winter, and the total daily resightings varied significantly between spring versus summer, summer versus winter, autumn versus spring, and autumn versus winter. Resightings were higher at the human-subsidized site in austral winter than summer and the total daily resightings varied significantly across all bi-seasonal comparisons. Resightings indicated that at least 12 of 197 birds (6.1%) moved between the human- and marine-subsidized sites at least once during the same winter, 15 of 335 birds (4.5%) did so in spring, none of 164 birds did so in summer, and 16 of 297 birds (5.4%) did so in autumn. Individuals fitted with archival GPS data loggers at the marine-subsidized site in summer maintained highly localized 95% kernel core areas (0.55 ± 0.12 kmOur results suggest that seasonal shifts in food resource availability drive seasonal micro-migrations in a farm-island population of striated caracaras, and that farm sites can be critical in providing nutritional resources for caracaras when naturally occurring marine-subsidized resources become less available. Our results have important implications for striated caracara spatial ecology and conservation, as increased winter survival could improve the status of this globally Near-Threatened population. Capreolus capreolus) ) at the site during 5–20 days. Five birds fitted with retrievable GPS data loggers at the farm site maintained larger 100% MCP areas of use (28.03 ± 19.29 km2) over 3–9 days at the site  [15, 17Patinigera sp.), and on at least one occasion, caracaras were observed eating an octopus  .Striated caracaras are aggressive and opportunistic scavenging birds of prey that breed singly and in colonies near and on the perimeters of large colonies of nesting seabirds where they feed upon seabird eggs, nestlings, and vulnerable adult seabirds, as well as upon the nearby carcasses and faeces of marine mammals and livestock carcasses . Our sumBreeding success is high in striated caracaras in the Falkland Islands , and fooHistorically, overwintering caracaras are believed to have depended heavily upon the carcasses and feces of pinniped populations that bred on the islands prior to the latter’s decimation by sealers during an extended “seal rush” during late 1700s and mid 1800s , 29. SinOur principal study site, Saunders Island, is not a breeding site for caracaras, but rather is a non-breeding nursery island for a population of more than 100 caracaras, most of which are juveniles and sub-adults. A lesser number of adult caracaras on the island include year-round, non-breeding residents  and adult breeders that return from breeding sites on other islands to overwinter at the farm site on Saunders. Our observations indicate that the seasonal to-and-fro movements of caracaras between seabird colonies on the island and the island’s farm site are best characterized as instances of “partial migration” (sensu ) in whicCapreolus capreolus) exhibit partial to-and-fro migration in which some but not all individuals migrate to-and fro short-distances between principal home ranges and episodic winter refuges [To our knowledge this type of non-altitudinal, short-distance, “micro-migration,” has not been reported in other birds, however, a somewhat similar pattern has been described in at least one mammal. In Europe, GPS-tracked roe deer ( refuges . That caUnderstanding the extent to which a species may benefit nutritionally from human subsidies during times of nutritional stress is critical to understanding the impact of human actions on that species’ conservation status. Understanding risks posed to a species when using such human-dominated landscapes is equally crucial. Strange 1996 , Woods , and theMost avian migrations, including those of birds of prey, entail multi-day, if not multi- week or multi-month journeys, many of which involve long-distance linearized movements, requiring pre-migratory fat loading, refueling en route, atmospheric-assisted soaring flight, or all three methods of assistances , 32. The


That year marks the onset of a rapid deterioration in the capacity of the GICs firn to refreeze meltwater. Consequently, GICs runoff increases 65% faster than meltwater production, tripling the post-1997 mass loss to 36±16 Gt−1, or ∼14% of the Greenland total. In sharp contrast, the extensive inland firn of the GrIS retains most of its refreezing capacity for now, buffering 22% of the increased meltwater production. This underlines the very different response of the GICs and GrIS to atmospheric warming.Melting of the Greenland ice sheet (GrIS) and its peripheral glaciers and ice caps (GICs) contributes about 43% to contemporary sea level rise. While patterns of GrIS mass loss are well studied, the spatial and temporal evolution of GICs mass loss and the acting processes have remained unclear. Here we use a novel, 1 km surface mass balance product, evaluated against  Mass loss of Greenland's glaciers and ice caps (GICs) is a major contributor to contemporary sea-level rise. Here, the authors identify the year 1997 as a tipping point in GICs mass loss induced by a rapid deterioration of the refreezing capacity of inland snow, resulting in irreversible long-term mass loss. We selected five GICs regions (black boxes) to highlight the agreement and differences of the downscaled SMB product with observations . Note that the Greenland's GICs area of ∼81,400 km2 used in this study is smaller by ∼8% than previous estimates212) in the original GIMP DEM.A direct comparison with mass loss estimates from independent ICESat/CryoSat-2 measurements over the period 2004–2015 demonstrates that the downscaled SMB product with albedo correction successfully reproduces GICs mass changes , includi−1 and spatially integrated over (a) Greenland's GICs and (b) the GrIS. −3) and the underlying ice (∼900 kg m−3). Decades of increased melt have reduced pore space to such a degree that enhanced refreezing can no longer compensate for increased meltwater production. Because it would take decades to regrow a healthy firn layer, we interpret 1997 as a tipping point in the mass balance of Greenland's GICs.−1; see −1), with an insignificant trend of −0.01±0.22 Gt yr−2. Note how, even in this earlier period, melt (95 Gt yr−1) persistently and significantly exceeded precipitation (58 Gt yr−1), stressing the importance of the refreezing process for maintaining the mass balance of these Arctic GICs close to zero (−1) exceeded melt (556 Gt yr−1) by a wide margin , while precipitation remains constant. A similar pattern emerges for the GrIS; here, a negative trend in precipitation (−3.5±2.6 Gt yr−2) somewhat reinforces the decrease in SMB (−10.4±4.0 Gt yr−2), but again the latter is dominated by the increase in runoff (6.9±3.7 Gt yr−2). But Between 1997 and 2015, the integrated GICs SMB decreased at a rate of 1.1±0.6 Gt yrhmax), prior to and after 1997. hmax, and is now situated well above the peak in the hypsometry .ry 0.62, . In combry 0.62, . Figure 62, hmax , but rem62, hmax . Therefo62, hmax  and runo−1) before 1997 and a tripling in mass loss (7.6±1.4 Gt yr−1) afterwards (−1), but with a significant contribution from the interior (∼100 mm w.e. yr−1). This implies that all but the very highest parts of the GICs accumulation zones now regularly experience runoff, that is, the firn layer is no longer capable of refreezing all meltwater that is produced in summer. To analyse spatial differences, we examined the changes in runoff, refreezing fraction (RF/ME) and 2-m air temperature in 12 marginal regions of Greenland (1997–2015 minus 1958–1996), covering 85% of all Greenland's GICs . All reg25terwards . Figure −3) and no additional water can be stored. At this point, any additional water is assumed to run off. The model also includes a snow albedo scheme using prognostic snow grain sizeOutput of the Regional Atmospheric Climate Model (RACMO2.3) is used as input for the downscaling procedure142, ∼8% less than previous estimatesWe used a downsampled version of the GIMP DEMTo correct for the systematic bare ice albedo overestimation of RACMO2.3 in low-lying glaciated regions at 11 km, we used a 1 km version of the 500 m MODerate-resolution Imaging Spectroradiometer (MODIS) 16-day Albedo product (MCD43A3). Bare ice albedo is defined as the average of the 5% lowest surface albedos recorded for 2000–2015. At 1 km, bare ice albedo values range from 0.15 for dark ice surfaces at the ablation zone edges and local glacier tongues, to 0.55 under persistent snow cover in the accumulation zoneA total of 965 SMB measurements collected at 101 stake sites−3 above the ELA. Elevation changes for 2003–2009 and 2010–2014 were derived from ICESat and CryoSat-2 measurements, following the methods described in refs The daily, 1 km SMB product is statistically downscaled from the output of RACMO2.3 at 11 km resolution (1958–2015) to the 1 km GIMP topography and ice mask , using eb11 km  are calculated using a linear function of the high-resolution topography (h1 km) as:Daily regression parameters are calculated for the dependence of modelled SMB components on the 11 km RACMO2.3 topography. A local regression slope, mm w.e., . To fullThe downscaled product based on elevation dependence only is hereafter referred to as version v0.2.add) observed in low-lying regions compared to the downscaled product v0.2.To correct for bare ice albedo overestimation in RACMO2.3 at 11 km, melt and runoff (v0.2) are adjusted to account for additional ice melt (MEadd (mm w.e. per day) is the additional amount of ice melt calculated at 1 km (α (dimensionless) is the difference between the averaged bare ice albedo retrieved from the set of regression cells used to downscale runoff at 11 km and the MODIS albedo product at 1 km; SWdirect 1 km is the modelled daily cumulated downward shortwave radiation bi-linearly interpolated to 1 km; Lf is the latent heat of fusion (3.337 × 105 J kg−1). To account for the slope of the glacier surface, the dimensionless correction factor for a tilted plane ξ is applied to the direct component of modelled downward shortwave radiation. This correction is required as RACMO2.3 models radiation assuming a horizontal surface. This factor ranges from 0 for north sloping glaciers to values larger than 1 for south-oriented slopes.where ME at 1 km ; Δα .A daily ratio add is then scaled by a factor fscale (0.682), obtained by computing a least-squares fit minimizing ΔSMB using both GICs and GrIS ablation measurements. The justification for including GrIS observations is that GICs SMB is not independent of the GrIS as the regression parameters were extrapolated from the ice sheet margins onto the GICs. The fact that fscale<1 indicates a slight overestimation of the melt adjustment calculated in equation Assuming that the residual misfit between reconstructed and observed SMB (ΔSMB) for the different GICs ablation sites is ascribable to underestimated runoff in narrow ablation zones, RUv1.0) is obtained by adding RUadd to the downscaled runoff (RUv0.2):The adjusted amount of runoff (RUv1.0) is obtained in a similar manner:The corrected melt (MEv1.0) is estimated as a residual between adjusted melt, runoff and rainfall:Refreezing , daily precipitation totals from v0.2 were adjusted in regions presenting a positive annual cumulative SMB in v1.0:To eliminate the systematic negative SMB bias of RACMO2.3 in Greenland's accumulation zonesv1.0 is the daily adjusted total precipitation v1.0, PRv0.2 is the daily bi-linearly interpolated total precipitation v0.2, PRv0.2a is the annual cumulative bi-linearly interpolated total precipitation v0.2 and σSMB is the accumulation zone SMB bias in the downscaled product v1.0.where PR−1 (∼40%) is obtained by summing the mean ablation bias integrated over the GICs ablation zones  and the mean accumulation bias over the whole GICs area , to account for a potential precipitation bias in the ablation zones.Assuming that the remaining discrepancies in −1 (∼20%). These calculations were repeated for 250 mm w.e. SMB bins, which yielded similar results.Average biases for the GrIS accumulation (22 mm w.e.) and ablation zones (170 mm w.e.) have been calculated in a similar fashion and provided a product SMB uncertainty of 52.5 Gt yrWe applied the segmented regression method described in ref. https://landsat.usgs.gov/.The daily, 1 km SMB data set v1.0 presented in this study is freely available from the authors without conditions. The background in How to cite this article: Noël, B. et al. A tipping point in refreezing accelerates mass loss of Greenland's glaciers and ice caps. Nat. Commun.8, 14730 doi: 10.1038/ncomms14730 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, Supplementary Discussion and Supplementary References