Ursus maritimus) to hunt for seals and hampered bears’ ability to meet their energetic demands. In this study, we examined polar bears’ use of an ancillary prey resource, eggs of colonial nesting birds, in relation to diminishing sea ice coverage in a low latitude region of the Canadian Arctic. Long-term monitoring reveals that bear incursions onto common eider (Somateria mollissima) and thick-billed murre (Uria lomvia) nesting colonies have increased greater than sevenfold since the 1980s and that there is an inverse correlation between ice season length and bear presence. In surveys encompassing more than 1000 km of coastline during years of record low ice coverage (2010–2012), we encountered bears or bear sign on 34% of eider colonies and estimated greater egg loss as a consequence of depredation by bears than by more customary nest predators, such as foxes and gulls. Our findings demonstrate how changes in abiotic conditions caused by climate change have altered predator–prey dynamics and are leading to cascading ecological impacts in Arctic ecosystems.Northern polar regions have warmed more than other parts of the globe potentially amplifying the effects of climate change on biological communities. Ice-free seasons are becoming longer in many areas, which has reduced the time available to polar bears ( AlteredUrsus maritimus) is an apex predator in the circumpolar Arctic that relies on sea ice as a platform to hunt seals and other marine mammals. The progressively earlier break-up of annual sea ice in low latitude regions of the Arctic has reduced the amount of time available to bears to hunt for seals and amass the fat reserves they require to sustain themselves on shore during the ice-free season  and to calculate daily probabilities of bear presence [BearDay] .Information about temporal changes in the frequency of polar bear incursions onto bird colonies was obtained from two locations. East Bay Island , situated off the eastern coast of Southampton Island, Nunavut, supports approximately 5000 common eider breeding pairs and is the largest known eider colony in the Canadian Arctic . Cape Pembroke , located on the northeastern tip of Coats Island, Nunavut, provides nesting habitat for approximately 30 000 thick-billed murres . Biologists were present at East Bay Island during eider incubation (18 June–1 August) from 1997 to 2012 and at Cape Pembroke during murre incubation (10 July–10 August) from 1988 to 2011 . At both locations, bear observations were recorded in daily log books. We used these data to estimate the cumulative number of days on which one or more bears was present each year [Thttp://dynaweb.cis.ec.gc.ca/IceGraph20) [ceCover] to characterize winter ice season length and the timing of spring break-up at good-to-poor and poor-to-non-habitat thresholds, as well as IceCT on specified dates corresponding to eider and murre nesting phenology .Sea ice coverage information was obtained using the Canadian Ice Service's IceGraph Tool 2.0 (Graph20) . We querGraph20) ,35. We totalBear and the various IceCover indices against Year, and against each other, we derived IceCover–Year residuals and used the resulting detrended variable [IceCover′] to assess statistical relationships.When assessing the influence of a climatic factor (e.g. ice coverage) on a biological process (e.g. bear incursions) the co-occurrence of temporal trends can lead to spurious correlations. This is because of the possibility that the trend in biological process results from a relationship with an overlooked casual factor rather than a relationship with the climate factor itself . We predearDay as a binary response variable  and classified year as a categorical random effect [YearRE] to account for the non-independence of daily observations within years. Fixed effect covariates included Year (classified as a continuous variable), Day of the breeding season, Day2 and the best fitting representation of detrended IceCover′ from the regressions described above. Data for East Bay Island and Cape Pembroke were pooled and we included Site as a categorical fixed effect. Our global model included YearRE in additive combination with these fixed effects, as well as a Day × Site interaction. We then compared the global model to a series of reduced models, the most basic of which was a YearRE-only model that served as the null model in our candidate set. Model selection was based on maximum-likelihood methods, evidence ratios and Akaike's information criterion corrected for sample size (AICc)  to control for sampling at some locations in multiple years. Fixed effect covariates included the number of eider nests [sqrtNests], island size [Area], distance to the mainland [Mainland], maximum inter-step distance over open water that a mammalian predator coming from the mainland would have to cross to reach the island [Crossing], distance to the nearest Inuit community if travelling by boat [Village] and ice concentration within a 25 km radius of the island on 25 June [Ice25] . Our candidate set of models included all additive combinations of the predictor variables, as well as an IslandRE-only null model. To avoid over-parametrization, we did not evaluate interactions between variables.For our analysis, we summarized the number and proportion of islands with either direct or indirect evidence of predator presence by species group [BestSuccess] in relation to the types of predator sign encountered on islands , as well as survey zone [Zone], nest abundance [sqrtNests] and date of survey [Date]. We again used GLMMs and treated island as categorical random effect [IslandRE]. We used our analyses to determine colony and landscape attributes associated with documented predator presence and to estimate rates of nest loss when different combinations of predator sign were encountered.We also estimated the proportion of nests remaining active at the time of survey  indicated that Year, Day, D2ay and detrended IceCover′ were significant predictors variables (log(L) = −507.67, K = 6, AICc = 1027.40. iw = 0.72). The global model, which included the same predictors, as well as Site and Site × Day was also supported by the data (log(L) = −506.70, K = 8, ΔAICc = 2.12. iw = 0.25) .The best fitting model in our candidate set of GLMMs investigating variation in daily probability of bear incursions [BβearY = 0.152, 95% CI: 0.111–0.192) , which was reflected in raw data plots illustrating summary proportions parsed by site and fit with a linear model . We also estimated an increasing probability of bear incursions with advancing date during the incubation period , but a declining probability late in the breeding season when eggs begin to hatch or are lost to predators . Summary proportions fit with a quadratic equation indicated that bear incursions peaked in mid-July during late-incubation . We also found that the daily probability of bear incursion was higher on East Bay Island than Cape Pembroke ; however, we estimated a negative interaction between site and day, suggesting later arrival and a greater rate of increase with advancing date at Cape Pembroke . In accordance with the main prediction of our study, we estimated a negative association between bear incursions and detrended ice cover .Model averaged regression coefficients indicated an increasing probability of bear incursions with advancing year ((b)Our broad-scale geographical sampling conducted in 2010 through 2012 included 230 islands supporting more than 32 500 common eider nesting pairs . These years coincided with the three lowest early summer ice coverage extents on record in Northern Hudson Bay Narrows and three of the four lowest ice coverage extents on record for Hudson Strait. However, variation in ice coverage was apparent among locations and across years, with 2012 being a relatively heavy ice year (IceCT > 70% on 25 June) in the Foxe Peninsula, South Baffin and Frobisher Bay survey zones .During the course of our surveys, we observed 22 polar bears (including four cubs) on 16 islands with eider colonies. Indirect evidence for the presence of bears was observed on a further 63 islands and polar bear presence (direct + indirect evidence) was estimated on 34% of islands in total . We obseearSign in relation to colony and landscape attributes indicated that nest abundance, distance to the mainland, distance to the nearest village and ice conditions were important predictor variables (log(L) = −117.74, K = 6, AICc = 247.86, wi = 0.33); however, several other models also were supported by the data . In accordance with a priori predictions, model averaged regression coefficients indicated a negative association between BearSign and ice concentration  and positive associations with nest abundance , distance to mainland  and distance to the nearest village .Our GLMM examining variation in the prevalence of BoxSign, several models had a similar fit to the data . Multi-model inference and model averaging indicated that the probability of encountering fox sign increased with island size  and decreased with crossing distance . Regression coefficients could not be distinguished from zero for the other variables included in our analysis . Logistic regression of pair-wise relationships between the predictors and response variable illustrates patterns for both bears and foxes within the range of conditions that we sampled  on 11 July 2011. We counted 536 eider nests, among which 334 were active. Fresh bear faeces containing egg shell fragments were found on the island and we counted greater than 30 gulls, many of which were actively hunting eider eggs. We returned to the same island 2 days later and observed a bear on the island eating eggs and more than 50 gulls present. We resurveyed the colony after the bear left of its own accord and counted 24 active nests. This example was not unique, but was among the most definitive cases of near total nest destruction on a colony.estSuccess, varied substantially in relation to the types of predator sign encountered, as well as nest abundance and survey date (log(L) = −38.49, K = 7, AICc = 91.50, wi = 0.68) . Model averaged regression coefficients indicated lower nest success on islands with both gull and bear sign  or both gull and fox sign  compared to islands with gull sign alone . The proportion of nests remaining active decreased with advancing date (βateD = −0.091 95% CI: −0.148 to −0.035) as our surveys were conducted during incubation and simultaneous with the arrival of nest predators. We also estimated positive association between nest success and nest abundance within a colony  despite the fact the bear sign was positively correlated with nest abundance. Models including a survey zone effect were not well supported by the data. Raw data summaries were consistent with modelled results. We estimated that nest success (±s.e.) was three times lower on islands with both bear and gull sign (proportion of nests surviving to the date of survey = 0.22 ± 0.01) compared to islands with gull sign alone (0.66 ± 0.01) and was roughly 1.5 times lower on islands with both fox and gull sign (0.39 ± 0.02) compared with islands with gull sign alone (The proportion of nests remaining active at the time of survey, which was our index of Ngn alone .Table 24.Predictions about biological responses to climate change focus largely on the environmental tolerances of individual species . The cirChanging climatic conditions can also lead to shifts in interspecific interactions that influence population and community dynamics . DepredaThe negative association between bear incursions and sea ice conditions that we documented held after controlling for co-occurring temporal trends, thus constituting strong support for a causal effect of ice coverage on bears’ decision to consume eggs. However, year-effects remained evident, suggesting that variables not included in our models also influenced observed patterns. For example, increasing polar bear numbers or declining seal numbers could contribute to the increased prevalence of bears on bird colonies. Available data are insufficient to estimate polar bear  populatiBroad-scale geographical sampling of common eider colonies during years of record low ice coverage indicate that nest depredation by polar bears is extensive and that when bears raid eider colonies nesting success is severely reduced. Population modelling suggests that the species’ population growth rate is more sensitive to variations in adult survival than variations in annual productivity; however, retrospective analyses based on field data indicate that survival rates have proved relatively invariant over time, whereas fluctuations in reproductive success have been a primary driver of changes in population size ,46. ClimWe measured considerable spatial- and habitat-related variation in bear prevalence on eider colonies. Eiders nesting on islands nearer to shore and in closer proximity to Inuit villages were less vulnerable to depredation by bears than eiders nesting on islands further from the mainland and nearer seal hunting grounds. In addition, eiders nesting solitarily or in small aggregations were largely ignored by bears. These observations suggest that distributional and density-dependent factors set a lower threshold for bears’ interest in eider eggs that we predict will moderate population impact.While ground nesting species such as common eiders may be particularly vulnerable to nest depredation by polar bears; bears have also proved an emerging conservation concern for cliff nesting thick-billed murres. Gaston & Elliot  attributRelationships between changing ice conditions and access to eggs by different predator species are also potentially important. Foxes are traditional nest predators for eiders and eiders nest on islands in large part to minimize fox depredation . In thisOur results demonstrate how the direct effects of sea ice loss on polar bears are having unanticipated indirect effects on breeding birds. While the nutritional benefits to bears are not known, our results clearly demonstrate that nest depredation is not limited to a few bears or a handful of nests. Our results are consistent with assertions that polar bears are experiencing difficulty meeting their energetic demands in locations where ice-free seasons have grown significantly longer. Perhaps most importantly, they highlight the importance of incorporating interspecific interactions into predictions about the ecological impacts of changing environmental conditions in a rapidly warming Arctic.


Reperfusion injury results from pathologies of cardiac myocyte physiology that develop when previously ischemic myocardium experiences a restoration of normal perfusion. Events in the development of reperfusion injury begin with the restoration of a proton gradient upon reperfusion, which then allows the sodium-proton exchanger (NHE) to increase flux, removing protons from the intracellular space while importing sodium. The resulting sodium overload drives increased reverse-mode sodium-calcium exchanger (NCX) activity, creating a secondary calcium overload that has pathologic consequences. One of the attempts to reduce reperfusion-related damage, NHE inhibition, has shown little clinical benefit, and only when NHE inhibitors are given prior to reperfusion. In an effort to further understand why NHE inhibitors have been largely unsuccessful, we employed a new mathematical cardiomyocyte model that we developed for the study of ischemia and reperfusion. Using this model, we simulated 20 minutes of ischemia and 10 minutes of reperfusion, while also simulating NHE inhibition by reducing NHE flux in our model by varying amounts and at different time points. In our simulations, when NHE inhibition is applied at the onset of reperfusion, increasing the degree of inhibition increases the peak sodium and calcium concentrations, as well as reducing intracellular pH recovery. When inhibition was instituted at earlier time points, some modest improvements were seen, largely due to reduced sodium concentrations prior to reperfusion. Analysis of all sodium flux pathways suggests that the sodium-potassium pump (NaK) plays the largest role in exacerbated sodium overload during reperfusion, and that reduced NaK flux is largely the result of impaired pH recovery. While NHE inhibition does indeed reduce sodium influx through that exchanger, the resulting prolongation of intracellular acidosis paradoxically increases sodium overload, largely mediated by impaired NaK function. Myocardial ischemia, commonly observed when arteries supplying the heart become occluded, results when cardiac tissue receives inadequate blood perfusion. In order to minimize the amount of cardiac damage, ischemic tissue must be reperfused. However, reperfusion can result in deleterious effects that leave the heart muscle sicker than if the ischemia had been allowed to continue. Examples of these reperfusion injuries include lethal arrhythmias and an increased region of cell death. Some of the early events that result in reperfusion injury include changes in pH and an overload of sodium inside the cell. During reperfusion, the sodium-proton exchanger (NHE) removes protons from the cell in an effort to restore normal pH, in turn importing sodium ions. Many strategies have been attempted to prevent reperfusion injury, including inhibition of the NHE, with little clinical effect. Using a mathematical model that we developed to study ischemia and reperfusion in cardiac cells, we found that NHE inhibition produces more severe sodium overload, largely due to adverse consequences of the delayed pH recovery produced by NHE inhibition. These results suggest that NHE inhibition alone may not be a viable strategy, and that therapies which prolong intracellular acidosis may be problematic. Ischemia-reperfusion (IR) injury represents a constellation of pathological events that occur when previously ischemic myocardium experiences a restoration of normal tissue perfusion. IR injury, which can manifest as dangerous arrhythmias such as ventricular tachycardias and fibrillation, reduced myocardial force development, or an increased region of cell death, is likely to become even more clinically relevant in coming years owing to an aging population and the impact of aging on susceptibility to ischemia/reperfusion injury Because of the danger that ischemia-reperfusion related events pose, there has been great interest in this problem for several decades. A large number of studies, directed at furthering the understanding of ischemia-reperfusion injury and examining many potential therapeutic targets, have been undertaken During ischemia, as the available oxygen is depleted, cells switch to anaerobic metabolism, with reduced ability to synthesize ATP. As anaerobic metabolism progresses, metabolic acidosis develops. This acidosis is exacerbated by the rise in the partial pressure of carbon dioxide in the ischemic region Increased NHE flux and decreased NaK activity conspire to produce an overload of intracellular sodium  and sodium-bicarbonate symporter (NBC), two of the so-called acid exchangers, the sodium-calcium exchanger (NCX), sodium-potassium exchanger (NaK), fast-sodium current  overloads are similar in all simulations, these overloads are clearly the most severe in the R0 simulation, which also manifested the lowest mean and 10-minute reperfusion  values . When coExamining the two simulations in which NHE inhibition began at reperfusion is helpful, in that prior to the onset of inhibition all simulations experienced the same history. As NHE inhibition severity is increased from 50 percent (R50) to 100 percent (R0), the peak sodium concentrations during reperfusion increased from 19.71 to 20.21 mM  and mean to 6.99 , suggestGiven the counterintuitive relationship between pH recovery and sodium overload, the next question we asked was which subcellular components appear to be responsible? There are 8 components though which sodium moves in and out of the cell in our model, and their relative contributions in each simulation are displayed in On the other hand, NaK current, as presented in overload .et al, found that NHE inhibition impaired intracellular pH recovery. However, sodium overload following the acid load was reduced, not increased, as was the case with our NHE inhibition simulations. This fundamentally different behavior is largely the result of adding pH dependence to the NaK, which appears to be absent in the older model.It is interesting to compare these findings with those from another modeling study in which acid-loading was employed as an analog to ischemia-reperfusion The only other sodium flux source that appeared to play a role in sodium overload during reperfusion was the NBC, which moves sodium, as well as bicarbonate, into the cell. We observed that as the strength of NHE inhibition was increased, which suppresses pH recovery, intracellular bicarbonate concentrations were decreased  and NBC in vitro and in vivo. Whether such improvement was the result of increased NaK activity remains to be shown, but the prospect appears promising nonetheless.Given the apparent failure of NHE inhibition to attenuate sodium overload during reperfusion, and the prominent role that NaK plays in intracellular sodium regulation, we investigated whether the NaK could be manipulated in order to improve sodium overload. To this end, we performed two additional simulations, the results of which are provided in Figure S2 in NHE inhibition and the resulting suppression of pH recovery during reperfusion appears to worsen sodium load, and in simulations where we removed pH-related inhibition of the NaK, there was a reduction in sodium load relative to control . HoweverEarlier it was noted that there are several possible reasons for the failure thus far in developing an effective therapy for the treatment of ischemia-reperfusion injury. Within the context of NHE inhibition, it is important to remember that many of the subjects involved in clinical trials are afflicted with conditions such as cardiac hypertrophy and heart failure. There is evidence linking increased NHE activity to heart failure, both in the clinical setting and in experimental models The preceding discussion concerning sodium and calcium overload has been of a qualitative nature, but a logical followup question is exactly how much sodium and/or calcium overload is pathological? This is a difficult question to answer, as it likely depends on the state of many other components in the cell Again, these numbers provide only a rough guide, and the results of the ouabain study did not occur in the setting of true ischemia. However, what we found particularly interesting in the present study was that at best NHE inhibition resulted in a very small improvement in peak [The model presented here was used for the simulation of abrupt, complete stop-flow ischemia and abrupt-onset reperfusion. However, with some relatively simple modifications to the simulation protocols, it is possible to simulate ischemia of varying severity and following different time courses .in vitro. Reactive oxygen species generation is also believed to play a major role, as is apoptosis. These will be interesting potential additions in the future once suitable mathematical representations are devised, especially if longer reperfusion times are to be modeled, where apoptosis likely plays a more significant role.This model is by no means an attempt to capture all of the mechanisms of ischemic pathophysiology. For example, Comparison of our simulation results with experimental data in Many calcium-handling components are sensitive to changes in cellular pH, and some of these relationships are represented in our model. In addition, our model includes ATP-dependencies of components that directly and indirectly affect sodium and calcium balance, such as the NaK, SERCA, and ATP-inactivated potassium efflux channels. The importance of these influences have been alluded to above and are being explored in current work.The pathophysiology of ischemia-reperfusion injury is very complex, taking place within the context of a system that is highly coupled with nonlinear relationships between many of the components. Because such systems frequently exhibit nonintuitive behavior, we believe that the model developed in this study will prove useful in illuminating underlying mechanisms. For example, here we have used the model to propose a possible explanation for why NHE inhibition, a seemingly straightforward approach to limiting sodium-mediated reperfusion injury, has shown no meaningful clinical efficacy to date, and what efficacy has been observed requires the administration of NHE blockers prior to the onset of ischemia. Our investigations indicate that NHE inhibition can paradoxically exacerbate sodium and calcium overload. NHE inhibition suppresses intracellular pH recovery during reperfusion, which decreases the ability of the sodium-potassium exchanger to remove sodium from the cell and favors influx of sodium through the sodium-bicarbonate symporter. Most of any observed benefit of NHE inhibition appears to come from decreased sodium load resulting from reduced NHE activity prior to the onset of ischemia. Suppressing pH recovery during reperfusion is also likely to negatively impact other cellular systems, such as those responsible for handling calcium, that we have not examined here.Text S1Contains names and constant values (where applicable) for all model variables, all model equations, additional information regarding experiments used to inform model parameters, a summary of the ischemia-reperfusion simulation protocol, and additional figures pertaining to simulations relevant to model validation.(PDF)Click here for additional data file.


The AMP-activated protein kinase (AMPK) is a cellular energy sensor that is conserved throughout the eukaryotic domain. AMPK exists as complexes comprising catalytic α subunits and regulatory β and γ subunits, which are activated >100-fold by phosphorylation of the α subunit at Thr-172 by upstream kinases. Binding of AMP to one site on the Y subunit causes 10-fold allosteric activation by AMP, while binding of either AMP or ADP to a second site promotes phosphorylation and inhibits dephosphorylation of Thr-172. Since both effects are antagonized by ATP, the kinase is activated by falling cellular energy status, when it phosphorylates downstream targets that switch on catabolic pathways generating ATP, while switching off processes consuming ATP. Catabolic processes switched on including uptake and metabolism of glucose and fatty acids, and mitochondrial biogenesis. ATP-consuming processes switched off include biosynthetic pathways for lipids , carbohydrates , proteins and ribosomal RNA, and progress through the cell cycle.By switching on glucose and fatty acid uptake and metabolism and switching off gluconeogenesis, AMPK should reverse the key metabolic defects in insulin resistance and type 2 diabetes. Consistent with this, AMPK is a target, although probably not the only target, for the anti-diabetic drug metformin. Because AMPK activation inhibits most biosynthetic pathways and causes cell cycle arrest, it should also have a cytostatic, tumor suppressor effect. The discovery in 2003 that the major upstream kinase that phosphorylates Thr-172 is the tumor suppressor kinase, LKB1, strengthened this view. The finding also led to retrospective analyses suggesting that metformin protects against the development of cancer.If AMPK is a tumor suppressor, one would expect that it would be down-regulated in many tumors. This indeed occurs through loss-of-function mutations in the upstream kinase LKB1, particularly in non-small cell lung cancer and cervical cancer. The majority of cases of breast cancer also appear to have defects in AMPK activation, although the mechanism is not known. We have found that AMPK is down-regulated in cells infected with hepatitis C virus (HCV). HCV may switch off AMPK to prevent its inhibitory effects on lipid and protein synthesis, required for viral replication. HCV infection activates the PKB/Akt pathway, leading to phosphorylation of the α1 subunit of AMPK on Ser-485, thus inhibiting AMPK activation via phosphorylation at Thr-172. It is possible that this contributes to the elevated risk of hepatocellular carcinoma observed in humans with chronic HCV infection.in vivo. It is possible that some of the beneficial effects of salicylate derivatives, including amelioration of insulin resistance and protection against colon cancer, might be mediated by AMPK.Finally, we have found that AMPK is directly activated by salicylate, and have evidence that at least some of the metabolic changes induced by the drug in vivo are mediated by AMPK. Salicylate is a natural product of plants whose medicinal use was described in ancient manuscripts. Its use has largely been replaced by derivatives such as salsalate and aspirin, but both are rapidly broken down to salicylate


In an effort to circumvent resistance to rapamycin – an mTOR inhibitor - we searched for novel rapamycin-downstream-targets that may be key players in the response of cancer cells to therapy. We found that rapamycin, at nM concentrations, increased phosphorylation of eukaryotic initiation factor (eIF) 2α in rapamycin-sensitive and estrogen-dependent MCF-7 cells, but had only a minimal effect on eIF2α phosphorylation in the rapamycin-insensitive triple-negative MDA-MB-231 cells. Addition of salubrinal – an inhibitor of eIF2α dephosphorylation – decreased expression of a surface marker associated with capacity for self renewal, increased senescence and induced clonogenic cell death, suggesting that excessive phosphorylation of eIF2α is detrimental to the cells' survival. Treating cells with salubrinal enhanced radiation-induced increase in eIF2α phosphorylation and clonogenic death and showed that irradiated cells are more sensitive to increased eIF2α phosphorylation than non-irradiated ones. Similar to salubrinal - the phosphomimetic eIF2α variant - S51D - increased sensitivity to radiation, and both abrogated radiation-induced increase in breast cancer type 1 susceptibility gene, thus implicating enhanced phosphorylation of eIF2α in modulation of DNA repair. Indeed, salubrinal inhibited non-homologous end joining as well as homologous recombination repair of double strand breaks that were induced by I-SceI in green fluorescent protein reporter plasmids. In addition to its effect on radiation, salubrinal enhanced eIF2α phosphorylation and clonogenic death in response to the histone deacetylase inhibitor – vorinostat. Finally, the catalytic competitive inhibitor of mTOR - Ku-0063794 - increased phosphorylation of eIF2α demonstrating further the involvement of mTOR activity in modulating eIF2α phosphorylation. These experiments suggest that excessive phosphorylation of eIF2α decreases survival of cancer cells; making eIF2α a worthy target for drug development, with the potential to enhance the cytotoxic effects of established anti-neoplastic therapies and circumvent resistance to rapalogues and possibly to other drugs that inhibit upstream components of the mTOR pathway. The phosphatidylinositol 3-kinase - protein kinase B - mammalian target of rapamycin (PI3K-Akt-mTOR) pathway regulates cell growth and proliferation. The deregulation of the pathway underlies oncogenic transformations and its modulation by anti-neoplastic treatments affects their outcome. The mTOR's inhibitors - rapamycin, and its derivatives - decrease cancer cell proliferation and have been tested as anti-cancer agents in clinical trials –3. RapamRapalogues bind their intracellular receptor FK506 binding protein 12 (FKBP12), forming a complex that inhibits mTOR complex 1 (mTORC1) by binding mTOR's FKBP12 rapamycin-binding domain . MoreoveTherefore, in rapamycin-sensitive cancer cells, delineating rapamycin downstream effectors that modulate tumor growth and response to anti-neoplastic treatment is likely to lead to discovery of new compounds that will inhibit tumor growth and/or enhance its sensitivity to established therapies. Such molecules are expected to circumvent the resistance of cancer cells to drugs that target upstream components of the PI3K-Akt-mTOR pathway while having only a partial effect on its global activities. MET to the ribosome which then scans the capped mRNA. Following recognition of the initiation codon and GTP hydrolysis, the inactive eIF2·GDP is released and recycled into an active eIF2·GTP complex via interaction with the guanine nucleotide exchange factor eIF2B [51 turns eIF2 from a substrate of eIF2B into its competitive inhibitor, leading to a reduction in the level of eIF2·GTP·Met·tRNAMET complex and to attenuation of global protein translation. Importantly, because the cellular level of eIF2 is in excess of eIF2B, a slight increase in eIF2α phosphorylation can sequester a major fraction of eIF2B [In the present study we report that inhibition of mTOR leads to increased phosphorylation of eIF2α - a subunit of eIF2. To date, contrasting reports have been published regarding the involvement of mTOR in eIF2α phosphorylation –16. Howeor eIF2B . Under nor eIF2B . Howeverof eIF2B . In mammof eIF2B , and itsof eIF2B . Salubriof eIF2B ,21. The molecular outcome and physiological relevance of increased eIF2α phosphorylation has been extensively studied during endoplasmic reticulum (ER) overload where it is generally thought to exert a protective role. In response to increased ER load PKR-like ER-localized eIF2α kinase (PERK) is activated and a transient increase in eIF2α phosphorylation ensues . This leExposure of mouse embryonic fibroblasts (MEF) to anti-cancer agents - such as doxorubicin or histone deacetylase inhibitors - also led to increased phosphorylation of eIF2α, albeit a sustained rather than a transient one ,26. In tOur studies implicate sustained and excessive eIF2α phosphorylation in inhibition of DNA repair, development of senescence and decreased expression of a surface marker associated with capacity for self renewal, thereby providing a rationale for association with increased sensitivity to anti-neoplastic treatments such as ionizing radiation and histone deacetylase inhibitors (HDACi). Our results suggest that the development of drugs that increase eIF2α phosphorylation may provide additional means for enhancing the sensitivity to established anti-neoplastic therapies and/or circumventing resistance to drugs that target upstream components of the PI3K-Akt-mTOR pathway.3 per cm2 and maintained as described before [137 irradiator (Gammacell 1000 Elite/3000 Elan) at a dose rate of 475 cGy/minute or in X-ray irradiator (Polaris sc-500 series II) at a dose rate of 100 cGy/minute. Rapamycin, Vorinostat , Ku-0063794  and salubrinal  were added to the cultures from stock solutions in dimethyl sulfoxide (DMSO). Control cultures received equal amounts of the vehicle. DMSO concentration in the medium did not exceed 0.08%. Drugs were added to the culture immediately following radiation. MCF-7 and MDA-MB-231 breast cancer cell lines, from American Type Culture Collection , were plated at a density of 4 ·10d before . Cells wPlating for colony survival assay, colony counting, and calculation of surviving fractions was performed in triplicates as described before . Unless Preparation of cell lysates and analysis of treatment-induced changes in protein level and phosphorylation was done as described before  with minControl and salubrinal treated cells were detached with non-enzymatic cell dissociation solution , incubated with human serum and FcR blocking reagent and then with fluorescent isothiocyanate (FITC)-anti-ESA or with isotype control . 7-aminoactinomycin D  served as viability dye. Detection of cell staining was performed by FACSCalibur using Quest software  as described by Keshet et al. for staining of surface MDR1 . Cell staining kit from Cell Signaling Technologies was used for cell staining and photomicrographs of stained cells were obtained with Nikon TS100 Eclipse inverted microscope and Nikon DS camera. Non-homologous end joining (NHEJ) repair was assayed in HeLa cells and homologous recombination repair (HRR) was assayed in U2OS cells stably transfected with pEJSSA and pDR-GFP respectively ,32. The heIF2α S51A and heIF2α S51D in pcDNA3.CD2 ,37 were t test or one sample t test after logarithmic transformation was employed for statistical analysis. Unpaired Student p < 0.05 was considered statistically significant.50 - ~10 nM). In contrast, the triple negative MDA-MB-231 are insensitive to the drug (IC50 -5900 nM) [50 of ~50 nM, in the MDA-MB-231 cells concentrations up to 2 µM did not significantly affect cell survival (Table S2 in It has been previously demonstrated that the estrogen dependent MCF-7 breast cancer cells are highly sensitive to rapamycin IC5900 nM)  due to r00 nM  duAt nM concentrations rapamycin led to increased phosphorylation of eIF2α which was much more pronounced in MCF-7 than in MDA-MB-231 . IonizinTo determine the relevance of increased eIF2α phosphorylation to cell survival we treated MDA-MB-231 cells with salubrinal – an inhibitor of eIF2α dephosphorylation. Salubrinal led to a dose-dependent increase of eIF2α phosphorylation that was associated with increased clonogenic death ; Table 1Similar to the effect of salubrinal, transient transfection with the phosphomimetic eIF2α S51D variant decreased - in a plasmid-dose-dependent manner - clonogenic survival relative to that observed in cells transfected with the non-phosphorylatable S51A variant. At high plasmid concentrations  survival44+/CD24-/low/ESA+ on their surface [44+/CD24-/low, sorting for cell surface ESA expression in these cells can serve as an indicator for changes in size of cellular fraction with self renewal capacity [It has been reported that tumorigenic breast cancer stem cells are enriched with a sub-population of cells expressing CD surface , and tha surface . Becausecapacity . As noteColonies of irradiated and salubrinal treated cells contain enlarged and senescent looking cells. We counted these cells in colonies formed following exposure to 1 Gy, to 4.5 µM salubrinal to the combination of radiation and salubrinal and in untreated controls. Interestingly, even though combined treatment of 4.5 μM salubrinal and 1 Gy did not lead to enhanced clonogenic death  it did lRadiation led to increased level of BRCA1, a protein that participates in DNA repair following radiation damage . The incSimilar to radiation, the HDACi – Vorinostat also leads to increased eIF2α phosphorylation. This increase has been thought of as a mean of cellular protection against damage . HoweverKU-0063794 is a specific mTOR inhibitor which competes with ATP binding for the catalytic site of the enzyme . Its addSaccharomyces cerevisiae rapamycin increases eIF2α phosphorylation via activation of GCN2 [Our previous finding that inhibition of mTOR increased sensitivity of MCF-7 cells to ionizing radiation , prompte of GCN2 . While t of GCN2 , and nM  of GCN2 . Nonetheless, very recently Mounir et al. demonstrated that inhibition of PI3K in MEF leads to inhibition of Akt with the consequent activation of PERK and phosphorylation of eIF2α . RelevanIt remains to be determined if the phenomenon we demonstrated in our study e.g. increased eIF2α phosphorylation following inhibition of mTOR by rapalogues or by Ku-0063794, results from inactivation of mTORC1, mTORC2 – Akt pathway, or whether additional mTOR mediated pathways are involved. We have however clearly shown that prolonged incubation of cancer cells with mTOR inhibitors can lead to sustained elevation of eIF2α phosphorylation. It is important to note though that deregulation of mTOR leads to ER overload and to increased phosphorylation of eIF2α , showingA chemical screen for compounds that protect cells against ER stress has led to the discovery of Salubrinal – an inhibitor of eIF2α dephosphorylation . SalubriEnhanced phosphorylation of eIF2α following exposure to salubrinal can alleviate ER stress caused mainly by accumulation of unfolded proteins in the ER. However, in the case of fatty acids, excessive and sustained phosphorylation of eIF2α may aggravate cell damage by interfering with activation of stress response and consequently with fatty acid oxidation thus leading to accumulation of lipid droplets and to enhancement of the initial damage ,44 . MEF homozygous for eIF2α A/A have also been employed in many studies to demonstrate the role that eIF2α phosphorylation plays in the regulation of other pathways and in cell survival . DecreasOur experiments with salubrinal and with the eIF2α variants show that sustained increase in eIF2α phosphorylation is detrimental to the cells' survival. We also showed that level of eIF2α phosphorylation that is tolerated by non-irradiated cells is harmful in irradiated ones. The fact that treatment with salubrinal decreases expression of surface ESA, suggests that increasing eIF2α phosphorylation is likely to affect the self renewal capacity of the cells. Of interest is the finding that enhancing eIF2α phosphorylation in irradiated cells abrogates the radiation-induced increase in BRCA1 level. It is quite possible that the translational machinery is damaged in irradiated cells in more than one way and that increasing eIF2α phosphorylation beyond a certain level further hinders its activity; it is also possible that excessive eIF2α phosphorylation in the presence of radiation-induced eIF2α kinase(s) leads to proteasomal degradation of BRCA1 and possibly of other DNA repair proteins. This hypothesis is in line with the recent findings of Raven et al. who demonstrated that increased PKR or PERK activity in the presence of an increased eIF2α phosphorylation leads to proteasomal degradation of cyclin D1 . While this work was in progress Kim et al. showed that in cells that do not express caspase-3, radiation increased ER stress leading to activation of PERK and increased phosphorylation of eIF2α, autophagy and sensitivity to radiation . We haveFinally, to the best of our knowledge, our experiments connect, for the first time, excessive phosphorylation of eIF2α during genotoxic stress with inhibition of DNA repair. Pertinent to our experiments is a recent study by Chen et al. who demonstrated that rapamycin inhibits both homologous and non-homologous end joining DNA repair in MCF-7 cells . ImportaIn conclusion we have demonstrated that eIF2α is a downstream effector of the mTOR pathway, and that excessive phosphorylation of eIF2α interferes with DNA repair processes and negatively affects survival of cancer cells. Our results suggest that targeting eIF2α will both potentiate the effects of established anti-neoplastic therapies and help circumvent resistance to rapalogues.File S1Tables S1, S2, S3, S4, S5 and S6.(DOC)Click here for additional data file.Figure S1eIF2α variants do not alter radiation-induced phosphorylation of endogenous eIF2α.Cells were transfected with plasmids expressing non-phosphorylatable eIF2α S51A (SA) or the phosphomimetic eIF2α S51D (SD) and processed for analysis of eIF2α phosphorylation 48 hours post-irradiation. (TIF)Click here for additional data file.


Antifreeze proteins (AFPs) evolved in many organisms, allowing them to survive in cold climates by controlling ice crystal growth. The specific interactions of AFPs with ice determine their potential applications in agriculture, food preservation and medicine. AFPs control the shapes of ice crystals in a manner characteristic of the particular AFP type. Moderately active AFPs cause the formation of elongated bipyramidal crystals, often with seemingly defined facets, while hyperactive AFPs produce more varied crystal shapes. These different morphologies are generally considered to be growth shapes. In a series of bright light and fluorescent microscopy observations of ice crystals in solutions containing different AFPs, we show that crystal shaping also occurs during melting. In particular, the characteristic ice shapes observed in solutions of most hyperactive AFPs are formed during melting. We relate these findings to the affinities of the hyperactive AFPs for the basal plane of ice. Our results demonstrate the relation between basal plane affinity and hyperactivity and show a clear difference in the ice-shaping mechanisms of most moderate and hyperactive AFPs. This study provides key aspects associated with the identification of hyperactive AFPs. These proteins have the ability to bind to ice crystals, and are therefore named ice-binding proteins (IBPs)  or ice-sTf) below the equilibrium melting temperature (Tm), a non-colligative phenomenon termed thermal hysteresis (TH) [Tf, the bound proteins are insufficient to contain the crystal, and sudden, fast ice-growth occurs.By adsorption to a particular set of ice planes or sites, AFPs inhibit crystal growth normal to the bound surfaces. The crystals continue to grow on unprotected planes, if such exist, and those planes are minimized until they eventually vanish and growth ceases in all directions . The abisis (TH)  that is sis (TH) . Ice cryTmAFP from Tenebrio molitor [MpAFP [Since the first observation of AFGPs in the serum of Antarctic fishes , many AF molitor , along wr [MpAFP , and a mr [MpAFP  reveal tr [MpAFP . Recentlr [MpAFP . This sur [MpAFP .c-axes that are similar but distinguishable [TmAFP produces ice crystals reminiscent of the shape of lemons, or ellipsoids of revolution [Tf [c-axis, suggesting low or no binding to the basal planes. On the other hand, ice in solutions of hypAFPs ‘burst’ normal to the c-axis, and any growth by addition of water molecules to the basal planes is considerably slower than to the prism planes, suggesting an affinity of the AFPs to the basal planes [TmAFP.Directly connected with ice growth arrest is the ability of AFPs to modify ice growth habits. Adsorption to particular ice planes results in distinct ice shapes characteristic of the type of AFP. For instance, ice in the presence of type I, II or III AFPs forms bipyramidal shapes with elongated uishable . The hypvolution . Specifivolution  or AFP avolution –32. A sytion [Tf . The bipl planes . Differel planes . In thesl planes , and in Lolium perenne (LpIBP) [LpIBP has different shaping properties than other moderate or hyperactive AFPs and propose an explanation for its behavior. On the basis of these findings, the definition of hyperactivity can be broadened.Here we report the differential effects of AFPs on the growth and melting habits of ice. We include in this study the IBP from ryegrass,  (LpIBP) , which i2.2.1.TmAFP constructs and the AFGPs, all proteins were prepared and/or purified in the Davies laboratory at Queen's University, Ontario, Canada. All proteins were dissolved in 20–50 mM ammonium bicarbonate, pH ∼ 8.The proteins used in this study and listed below were either prepared by recombinant methods or were directly purified from the organism. Apart from the Moderate AFPs:Pseudopleuronectes americanus (winter flounder), isoform HPLC6. The peptide was prepared by solid-phase peptide synthesis that included the C-terminal amidation of Arg37, and was purified by reversed-phase HPLC [— Type I AFP from ase HPLC .Hemitripterus americanus (sea raven). The protein was expressed with a His-tag and a flanking tobacco etch virus (TEV) cleavage site [Drosophila melongaster S2 cells. After Ni-NTA affinity chromatography, the His-tag was removed by TEV protease digestion and the AFP was recovered by ice-affinity purification [— Recombinant type II AFP from age site  in Drosofication .Macrozoarces americanus (ocean pout), isoform HPLC12. The protein was expressed in Escherichia coli and purified and refolded from inclusion bodies as previously described [— Type III AFP from escribed .Gadus ogac) [— Antifreeze AFGPs. The protein sample was a gift from Dr Garth Fletcher. The sample contained primarily the 2650 Da isoform consisting of four glycotripeptide repeats. It was extracted from the blood plasma of rock cod (us ogac) .Lp IBP. The protein was expressed with a His-tag in E. coli and purified by heat treatment followed by ice-affinity purification [— fication  and Ni-Nfication .Hyperactive AFPs:T. molitor, isoform 4–9 (TmAFP). We used both a GFP fusion of the protein (GFP-TmAFP) and an untagged version [E. coli and purified on a Ni-NTA affinity column. The GFP-TmAFP was further purified by ice affinity [TmAFP fusion followed by digestion with TEV protease [— AFP from the beetle  version . Both foaffinity . The untprotease , a seconprotease .Choristoneura fumiferana (spruce budworm), linked to GFP (GFP-sbwAFP). The fusion protein was expressed in E. coli and recovered from the insoluble fraction [— AFP from fraction  by refolfraction , as prevfraction .Marinomonas primoryensis, fused to GFP (GFP-MpAFP). The calcium-dependent AFP domain fused to GFP was expressed with a His-tag in E. coli and purified by Ni-NTA affinity chromatography, as described [— AFP from the Antarctic bacterium escribed .Hypogastrura harveyi (snow flea), fused to GFP (GFP-sfAFP). The fusion protein was expressed with a His-tag in E. coli and purified by two cycles of adsorption to ice [— AFP from n to ice , as prevn to ice .2.2.Tm to 0.05°C above it. Each experiment was repeated at least 10 times.The morphologies of ice crystals during growth and melting were monitored using a homemade nanolitre osmometer, as previously described . Briefly2.3.TmAFP solutions. The confocal microscope is equipped with 488 and 633 nm illumination lines and filters for the detection of GFP and Cyanine 5 (Cy5). The experimental cell used in this apparatus is the same as the one used in the nanolitre osmometer [TmAFP and Cy5-dUTP (PerkinElmer) as a free dye. The Cy5 dye was previously shown to have no interaction with ice [TmAFP image, as explained in detail by Pertaya et al. [We used confocal microscopes  to image ice crystals in GFP-conjugated smometer  but withwith ice  and its a et al. .3.3.1.Tm but within the TH gap, crystals in all solutions containing moderate AFPs tested here developed facets and edges that readily turned into truncated bipyramids with hexagonal symmetry  without apparent growth of the truncated tips, but if the temperature was lowered to close to the Tf, the tips were rapidly sharpened to eliminate the basal plane. For example, in solutions containing approximately 20 µM AFP type III, the tips of 10 by 15 µm crystals (length versus width) grew along the c-axis at a rate of approximately 1 µm min−1 at 0.04°C below Tm, but when the temperature was lowered to 0.05°C or 0.06°C below Tm the growth rate increased to approximately 6 µm min−1 or approximately 27 µm min−1, respectively. These rates were substantially reduced as the crystal lengthened and the basal planes shrank.At temperatures below  figures  and 2. Tc-axis , as previously noted [c-axis is due to effective protection of the prism and/or pyramidal planes relative to the basal planes [LpIBP, which will be discussed in §3.4.Once the tips of the bipyramidal crystals have grown, they appeared to remain intact as long as the temperature was kept within the TH gap. Occasionally, some local growth was observed at temperatures within the hysteresis regime close to the freezing point. This effect was more pronounced in AFGP samples. The well-known sudden rapid growth, or ‘burst’, usually initiated from the tips of the bipyramids when the temperature exceeded the TH point. Spicular ice growth was then observed along the direction of the ly noted . In the l planes . An exce3.2.c-axis direction  until they become elongated before melting in the direction of the basal plane can be observed . Ice crystals in the presence of TmAFP melt in the prismatic direction and the basal planes shrink until they are eliminated and become only tips . This process gives rise to the previously noted lemon shape [figure 4c, right panel). In GFP-MpAFP and GFP-sfAFP solutions, the basal planes do not necessarily shrink down to a tip, and the shapes obtained are not as sharp and as symmetric as those observed with TmAFP (and GFP-TmAFP) . The asymmetry along the direction of the c-axis commonly observed in GFP-MpAFP and GFP-sfAFP solutions may result from minor temperature gradients in the samples. As it is difficult to maintain the location of the sample droplet in the middle of the oil and to control the location of the crystal inside the sample, small temperature changes of a few hundredths of a degree within the sample are present. We note that despite the temperature gradients along the sample area, the temperature of the metal plate in which the sample is held is controlled with an accuracy of 0.002°C. This accuracy is the same for every position in the droplet.on shape . Once thh). The tendency of the crystals to lie with the c-axis horizontally (figures 4a–d) or vertically (figures 4e–h) depends on the c : a axial length ratio. Elongated crystals lie horizontally while flat crystals lie vertically due to gravity. Therefore, the typical observed melting shapes of different hypAFP consistently dictate the orientation of the crystals. In many experiments, the initial crystal has a wide basal plane before melting and therefore is oriented vertically, but as the melting progresses, the area of the basal plane diminishes, the axial ratio is inverted and the crystal turns .With GFP-sbwAFP, the crystals usually float with their basal planes oriented normal to our line of observation and it is therefore difficult to follow melting to the direction of the basal planes. This orientation implies that the crystals are relatively flat with large, exposed basal planes. Nevertheless, formation of hexagonal shapes is clearly observed as the crystals melt h. The te figures  and 4a–d figures  and 4e–hTm the shapes do not change and the crystals do not grow, at least at the level of resolution we can detect (around a micrometre). The bottom limit of the growth arrest is the freezing point, at which sudden fast growth occurs normal to the c-axis. The twofold symmetry of the crystal burst is observed when it is aligned along the c-axis, as usually seen with GFP-MpAFP, GFP-sfAFP and TmAFP . The growth morphologies are dependent on the supercooling level and therefore on the TH activity of the sample. Ice protected by highly active protein samples is supercooled to several degrees at the lower limit of the freezing hysteresis, and the growth resembles a dendritic explosion . Diluted protein solutions have low TH activity, and therefore, the degree of supercooling is small. The growth pattern in these cases is slower and smoother as expected from the growth habit of ice in water [The ice shapes obtained during melting are stable within the TH range. When cooled below nd TmAFP . The hexin water .Figure 3.4.LpIBP, it grows into a hexagonal bipyramid in a manner similar to moderately active AFPs (figures e and 2b). Accordingly, melting of these crystals commences from the tips (e). Although the formation of the bipyramids and their melting pattern is as expected for a moderate AFP, the growth and melting rates seem to be slower than those of the moderate fish AFPs. A major difference with this plant IBP is the burst pattern at temperatures below Tf. It commences with the flattening of the two bipyramidal tips into an expansion of the basal planes. Growth continues in a stepwise manner, mostly in the direction of the a-axes, while maintaining the faceted morphology of the crystal in the process . This pattern suggests that the protein has some affinity to both prismatic and basal planes, which is in accordance with ice hemisphere and growth experiments and the recently solved crystal structure of the protein [When an ice crystal is cooled in the presence of  figures e and 2b.the tips e. Althou protein .3.5.TmAFP, the melting shape of the ice crystal becomes lemon-like as the crystals shrink in size and the basal plane becomes progressively smaller. Fluorescence confocal microscopy images confirm that hyperactive TmAFP has affinity to both prismatic planes and basal plane of ice (figures 7b). The stronger fluorescence at the junctions between the crystal faces and edges relative to the rest of the crystal contour suggest that these junctions contain exposed areas of primary prism planes onto which the protein accumulates, as described in detail previously by Pertaya et al. for sbwAFP [a,b).By using GFP-tagging, it is possible to directly visualize the binding of AFPs to ice. This enables us to observe where the protein is located on the ice crystal. In the presence of GFP- figures  and 7b. r sbwAFP . The flu4.et al. [c-axis in the presence of moderate AFPs and normal to the c-axis with hypAFPs. That study established an underlying observable difference between the moderate and the hyperactive AFPs and suggested that in addition to binding to prism or pyramidal planes, hypAFPs bind to the basal planes of ice, a hypothesis that was derived primarily from the crystal structures of sbwAFP and TmAFP [TmAFP [TmAFP presented here clearly demonstrates that the basal planes, as well as some other planes, are covered by the protein (figures 7b). Basal plane affinity was directly demonstrated previously for GFP-labelled sbwAFP by a series of experimental observations [TmAFP [The modifications of ice growth patterns induced by AFPs have been studied for decades in order to understand the activity of AFPs and the differences between them ,27,28,31et al.  have shond TmAFP ,20. Etchnd TmAFP . Similarnd TmAFP . Ice etcnd TmAFP ,48, typend TmAFP  and AFGPnd TmAFP  gave no P [TmAFP  are more figures  and 7b. rvations ,45. In arvations . We notervations  as well rvations  and GFP-rvations  or hypers [TmAFP ) actuallTf further supports the concept that hypAFPs protect the basal planes of ice while moderate AFPs do not. The ability of hypAFPs to cover ice from the basal plane and at least one more orientation with a significant component orthogonal to the basal plane prevents ice crystals from growing and precludes the possibility of controlling crystal shape during growth within the TH gap. This concept is shared among all hypAFPs studied here regardless of the hypothesis that different hypAFPs types bind ice from different crystallographic orientations. Moderate AFPs do not prevent ice growth in the c direction, and therefore growth along the c-axis occurs within the TH gap. Elongated bipyramidal shapes are then formed, as previously noted in TH measurements [c-axis direction) on the c : a ratio may be related to the layer-by-layer growth mechanism of the basal planes, which requires step-nucleation events. As the surface area of the basal planes becomes smaller, the probability for a two-dimensional nucleation event is reduced, and a lower temperature or a longer time period is required to evoke such events.Our study of the characteristics of ice growth and melting at temperatures above urements ,54 and gurements ,31. The c-direction gives rise to the eye-shaped melting habit. These eye-shaped crystals have exposed unprotected planes and are therefore unstable, leading to either complete melting of the crystal or formation of the bipyramidal growth shape, if the temperature is lowered at the right moment. On the contrary, the melting shapes obtained by hypAFPs are stabilized by AFP protection from all directions, and therefore can supercool throughout the TH gap or superheat for a certain range of temperatures (approx. 0.4°C for MpAFP) [It is evident that for the formation of distinct shapes, the proteins interact with ice from some crystallographic planes more than others. This concept is true for both growth and melting, although it might be more difficult to envision the fact that defined shapes can form during melting, at a time when molecules are leaving the ice front. Nevertheless, both moderate and hyperactive AFP molecules interact with ice and form unique shapes during melting. In moderate AFP solutions, the ice protection on planes parallel to or inclined to the crystallographic r MpAFP)  without a). The eye-shaped region in the centre of the crystals is the area of the original crystal before it grew to form the bipyramid. The strong fluorescent signal in this region is due to protein present inside, originating from the fast freezing process at approximately −20°C of the sample before it was melted to obtain single crystals. At these low temperatures, fine crystals are grown and GFP-AFP type III is accumulated typically on prismatic planes and some other planes slightly tilted from them [TmAFP remain in their melting shapes and have strong fluorescent layers around them ([b). This protein layer protects these crystals from both growth (within the TH range) and melting [To emphasize the differences between the growth and melting shapes, we introduced fluorescent images of crystals that contain contours of both types of shape. Ice grown from the melt in GFP-type III AFP solutions has a strong fluorescent signal inside the eye-shaped melting forms and around the bipyramidal growth shapes a. The eyrom them . The higrom them ,45. For nd them . In figure 7b, the melting shape of the crystal after burst is clearly observed by the fluorescent layer surrounding it. The directionalities of the melting shapes of crystals in moderate AFPs solutions are compared with the melting shapes observed with hypAFPs in figure 7c. Owing to basal plane affinity of hypAFPs, the crystals grown in hypAFP solutions melt normal to the c-axis and the basal planes shrink without change of the distance between the two basal faces. This is in contrast to the melting habits in solutions of moderate AFPs. Therefore, the longitudinal axis of the melting shapes obtained with hypAFPs is the crystallographic c-axis, while the longitudinal direction of the melting shapes obtained in moderate AFP solutions is normal to the crystallographic c-axis.In comparison with the bipyramidal growth shapes obtained with moderate AFPs within the TH gap, the growth shape of ice in hypAFP solutions is observed only when the TH gap is exceeded , aides in the protection of the plants from ice damage and from excessive supercooling.The moderate ryegrass  studies . The rec studies  and ice- studies  of this  hypAFPs –22. Neve hypAFPs ,42. This protein ,35,63. Ito LpIBP . This muoperties  relativeThe differences in the ice-shaping patterns between hypAFPs and moderate AFPs demonstrated here have significant implications for the utility of AFPs in a variety of fields such as cryopreservation and cryosurgery. For example, it was shown that although type I AFP increased the viability of red blood cells after a freeze–thaw cycle, lethal effects were observed at high concentrations of this AFP owing to extensive growth of extracellular ice ,66. Cell5.LpIBP to the generalization that relates basal plane affinity with hyperactivity and sharply divides AFPs between ‘hyperactive’ and ‘moderate’ hints that perhaps this classification needs to be broadened. The findings of this study, particularly that ice reshaping occurs during melting, may enable the construction of better models to describe the interactions of AFPs and ice.Although the ice-shaping properties of hypAFPs have been studied, they have been uniformly referred to as growth shapes, analogous to those formed by moderate AFPs. The fact that these shapes are formed during melting has been generally ignored. Our work demonstrates that the crystal shapes obtained with hypAFPs are obtained during melting and not during growth. This phenomenon is directly related to the ability of hypAFPs to protect the basal plane of ice crystals in addition to other planes. Therefore, in hypAFPs solutions, changes in ice morphology do not occur in the TH regime, and the shaping occurs during melting owing to the influence of the proteins on interfacial melting rates. In agreement with these observations, melting simulation results show that a lemon-like shape can be obtained during melting if the basal planes of the crystals are covered and melting in this direction is suppressed . For mod


Free-living microbial eukaryotes are an enigmatic group of organisms. Many have been discovered through traditional cultivation and microscopic observation, with continued observations leading to the acknowledgment that many small eukaryotic species are potentially dispersed at a global level Finlay, . While cIn recent years, investigations into the diversity of microbial eukaryotes have revealed a massive diversity that had been previously overlooked. While knowledge has been gained in more accessible environments, extreme environments that may show drastic habitat preference for microbial eukaryotes have been less sampled. Within pelagic environments, mixing was thought to lead to a more even distribution of organisms, however, the empirical evidence of habitat selection has often led to the concept of moderate endemicity where only a few species are cosmopolitan, and others are endemic  in the deep ice in contact with the upper water was extremely similar, yet the taxon abundances were dissimilar, with shallow water holding more Fungi than the deepest ice. Diversity increased through the ice, with the most recent years of shallow ice containing lower diversity than deeper ice, suggesting that inputs are different between layers, or organisms are showing adaptation to the habitat over time. Detailed analysis of the sequences show that some species could be interpreted as originating in the water column and entering the deep overlying ice, likely during freezing, suggesting that the deep ice more closely resembles the community in the shallow waters. Snow had low diversity and the sequences detected in it are suggestive of potential contaminants, including humans and temperate plants. As such, eukaryotic sequences, such as those associated with pollen from Activity is inferred in this study from the lack of phototrophs detected. Typically, abundant diatom species are observed in ice and water samples, however, at the end of the polar night, the light starved community had decreased to lower levels. Many of the detected organisms were related to known mixotrophs and diatom predators were detected, suggesting that the primary production within the community was low and impacted by grazing (Bachy et al., The glimpse by Bachy et al.  into the


The Rho GTPases Rac1 and Cdc42 regulate a variety of cellular functions by signaling to different signal pathways. It is believed that the presence of a specific effector at the location of GTPase activation determines the route of downstream signaling. We previously reported about EGF-induced Ser-71 phosphorylation of Rac1/Cdc42. By using the phosphomimetic S71E-mutants of Rac1 and Cdc42 we investigated the impact of Ser-71 phosphorylation on binding to selected effector proteins. Binding of the constitutively active (Q61L) variants of Rac1 and Cdc42 to their specific interaction partners Sra-1 and N-WASP, respectively, as well as to their common effector protein PAK was abrogated when Ser-71 was exchanged to glutamate as phosphomimetic substitution. Interaction with their common effector proteins IQGAP1/2/3 or MRCK alpha was, however, hardly affected. This ambivalent behaviour was obvious in functional assays. In contrast to Rac1 Q61L, phosphomimetic Rac1 Q61L/S71E was not able to induce increased membrane ruffling. Instead, Rac1 Q61L/S71E allowed filopodia formation, which is in accordance with abrogation of the dominant Sra-1/Wave signalling pathway. In addition, in contrast to Rac1 transfected cells Rac1 S71E failed to activate PAK1/2. On the other hand, Rac1 Q61L/S71E was as effective in activation of NF-kappaB as Rac1 Q61L, illustrating positive signal transduction of phosphorylated Rac1. Together, these data suggest that phosphorylation of Rac1 and Cdc42 at serine-71 represents a reversible mechanism to shift specificity of GTPase/effector coupling, and to preferentially address selected downstream pathways. The small Rho GTPases are monomeric GTP-binding proteins that play a role in a variety of cellular processes that depend on the actin cytoskeleton, such as morphogenesis, endocytosis, phagocytosis, cytokinesis and migration. They act as nucleotide-dependent switches cycling between an active, GTP-bound state and an inactive, GDP-bound state. In their GTP-bound state, they interact with downstream effectors to initiate downstream signaling. The binding of Rho GTPases to their effector proteins occurs by distinct binding motifs. Most effector proteins harbor the common Cdc42/Rac1 interactive binding motif (CRIB), which is present in the p21-activated kinase (PAK) and the Wiskott-Aldrich syndrome protein (WASP) 64ydRIRplSYp73. Most Rho GTPases, e. g. Cdc42 and RhoA/B/C/G share this sequence. The Ser-71-phosphorylation of Rac1 results in reduced GTP-binding without affecting GTPase activity Phosphorylation of Rho GTPases is an additional mechanism to modulate the activity of these proteins, mainly leading to their functional inactivation. Phosphorylation was first described for RhoA, which can be phosphorylated by PKA/PKG at Ser-188 Phosphorylation of Rac1/Cdc42 induces a specific phenotype of cells. Treatment of cells with the epidermal growth factor (EGF) induces Rac1/Cdc42 phosphorylation which is accompanied by the formation of filopodia C. difficile TcdA, which catalyzes inactivation of Rho GTPases including Cdc42 by mono-glucosylation served as negative control. These results indicate that the Rac1 S71E-induced filopodial phenotype is not caused by concomitant activation of Cdc42. It is well established that filopodia formation can be enforced by suppression of Rac1 signaling, either directly through microinjection of mixtures of active Cdc42 and inactive Rac1 To investigate the mechanism leading to filopodia formation in more detail, we checked whether overexpression of Rac1 S71 somehow induces activation of Cdc42. Neither overexpression of wildtype Rac1 nor of Rac1 S71E nor the control mutant Rac1 S71A induced activation of Cdc42 . The conrac1−/−) to check for pSer-71 staining of Rac1 and Cdc42. As shown in rac1−/− fibroblasts do not express detectable levels of Rac1. Cdc42 expression, however, was comparable to the parental cell line fl/flrac1 fibroblasts. Beta-actin served as loading control. In contrast to control fibroblasts, Rac1-deficient cells were completely devoid of pSer-71-Rac1 and pSer-71-Cdc42 staining, even after treatment of cells with 100 ng/ml EGF. This strongly argues that EGF mainly drives phosphorylation of Rac1, at least in fibroblasts. With respect to this finding, we assumed that the observed effects in the present study are mainly due to Rac1 phosphorylation.Unfortunately, it is not possible to dissect between Ser-71 phosphorylation of Rac1 and Cdc42. To date there is no antibody that differentiates between pSer-71-Rac1 and pSer-71-Cdc42. We therefore took advantage of murine fibroblasts lacking the Rac1 gene (g fraction) of HEp2 cells (g fraction). The majority of activated Akt kinase was detected within the cytosol but also to some extend within the membrane fraction. Furthermore, Overexpression of Rho-GDI in HEp2 cells did not reduce level of active pRac1 as tested in pull down assay with PAK p21 binding domain (PAK-PBD)  . In contPAK-PBD) . UnspeciPAK-PBD) . Rac1 S7Based on these findings, we performed further pull down experiments from HEp2 cell lysates where GST-fusion proteins of wild type and mutant (S71E) Rac1 and Cdc42 (data not shown) were used as baits to identify possible interacting proteins. Therefore, the specificity of binding to effector proteins was checked by using GTPγS-loaded wild-type GTPases. Additionally, constitutive active (Q61L) mutants of Rac1 and Cdc42 were compared with respect to their specificity. Comparison of GTPγS loaded GTPases with constitutive active ones was done to evaluate the Q61L mutants for further experiments. In fact, GTPγS-loaded Rac1 and the constitutively active (Q61L) mutant interacted with the Rac1-specific effector protein Sra-1 but not with the Cdc42-specific effector N-WASP . In contPull down experiments with constitutively active GTPases were performed in triplicate. Results shown in Since Rac1 S71E showed active conformation as tested by PAK-PBD pull downs and bound to effector proteins IQGAP and MRCK, we investigated whether phosphomimetic Rac1 and Cdc42 are able to activate specific pathways. We choose the NF-κB pathway because the activation by Rac1 and Cdc42 is well described Rho GTPases are involved in various signaling pathways that regulate diverse cellular functions We previously reported that Ser-71 phosphorylation of Rac1/Cdc42 is not accompanied with general loss of function C. sordellii Lethal Toxin TcsL-82 from strain IP82, which glucosylates Rac1 but not Cdc42 This hypothesis is substantiated by the predominant morphotype of cells with increased pSer-71 Rac1 showing massive formation of filopodia see  - a morpBesides Rac1, we also analyzed the effector binding properties of the phosphomimetic mutant of Cdc42 to check, whether phosphorylation at Ser-71 of these highly homologous GTPases might uncover a common mechanism for regulation. The two most important features of Rac1/Cdc42 phosphorylation were as follows: 1) Phosphorylation at Ser-71 only leads to partial functional inactivation of these GTPases, and 2) residual functional activity of pRac1 and pCdc42 appears to be harmonized with respect to interaction with here investigated effectors.The question about activity status of Ser-71 phosphorylated GTPases resulted from different observations. Kwon and coworkers observed that GTP-binding was abrogated when serine-71 was exchanged to alanine and, thus, concluded that modification of this amino acid interferes with activation of Rac1 phox, phospolipase C-β2, and others has to be tested in separate studies to describe their binding and activation The set of effectors investigated in this study is sufficient to exemplify the bivalent feature of pRac1 (S71) as discussed above. The interaction with other effectors, such as p67fl/flRac1 fibroblasts were generously provided by Cord Brakebusch, Kopenhagen, Denmark.G418 was obtained from PAA, Germany. Monoclonal anti-pPAK1 (Ser144) was from abcam, UK. Polyclonal anti-pPAK1/2 (Thr423/Thr402), polyclonal anti-IQGAP1, polyclonal anti-N-WASP(30D10), polyclonal anti-PAK1, and monoclonal anti phospho-Rac1/Cdc42 (Ser71) were from Cell Signaling, Germany; monoclonal anti-IQGAP2 (Clone BB9), Upstate, MA, USA; polyclonal anti-MRCK alpha, Santa Cruz Biotechnology, CA, USA; anti-VASP, rhodamin-phalloidin; peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG, Rockland, PA, USA; polyclonal Sra-1 antiserum was generously provided by Theresia Stradal , anti-HA (clone HA-7), and guanosine-5′-[γ-thio]triphosphate tetrasodium salt solution (GTP[γS]), Sigma, MO, USA; guanosine-diphosphate (GDP), human epidermal growth factor (EGF) was from R&D Systems, Germany; oligonucleotides were synthesized by Eurofins, Germany; Quickchange XL site-directed mutagenesis kit, Stratagene, Germany; Fugene 6 and Fugene HD transfection reagent, Roche, Germany; reporter lysis buffer, Promega, Australia; SuperSignal West Femto Substrate, Pierce, Rockford, USA; the construct for PAK-PBD domain (aa 56–272) was generously provided by John Collard (Netherlands). Exchange of serine for glutamate is an established method to mimic phosphoserine. Mutagenesis of Ser-71 to Glu-71 in Rac1 and Cdc42 was performed according to a standard protocol supplied with the site-directed mutagenesis kit Quickchange XL using 5`-GATTACGCCCCCTAGAATATC CGCAAACAGATGTG-3` (sense) and 5`-CACATCTGTTTGCGGATATTCTAGGGGGCGTA ATC-3′ (antisense) primer for Rac1 and 5`-GATTACGACCGCTGGAATATCCACAAACAGA TGTA-3` (sense) and 5`-TACATCTGTTTGTGGATATTCCAGCGGTCGTAATC-3′ (antisense) primer for Cdc42 amplification. The appropriate expression vectors (pGEX Rac1/Cdc42 and pcDNA3.1 3x HA Rac1/Cdc42) were used as template for PCR.Likewise, the mutagenesis of Ser-71 to Ala-71 in Rac1 and Cdc42 was performed using 5`-GATTACGCCCCCTAGCCTATCCGCAAACAGATGTG-3` (sense) and 5`-CACATCTGTTTG CGGATAGGCTAGGGGGCGTAATC-3′ (antisense) primer for Rac1 and 5`-GATTACGACCG CTGGCTTATCCACAAACAGATGTA-3` (sense) and 5`-ACATCTGTTTGTGGATAAGCCA GCGGTCGTAATCT-3′ (antisense) primer for Cdc42 amplification. Mutagenesis of Gln-61 to Leu-61 in Rac1 and Cdc42 was performed using 5`-GGGATACAGCTGGACTAGAAGA TTATGACAGATT-3` (sense) and 5`-AATCTGTCTAATCTTCTAGTCCAGCTGTATCCC-3′ (antisense) primer for Rac1 and 5`-GATACTGCAGGGCTAGAGGATTATGACAGA-3` (sense) and 5`-TCTGTCATAATCCTCTAGCCCTGCAGTATC-3′ (antisense) primer for Cdc42 amplification. Rac1 S71E/Cdc42 S71E, Rac1 S71A/Cdc42 S71A and Rac1 Q61L/Cdc42 Q61L were expressed in E. coli after standard protocol for glutathione-S-transferase fusion proteins.Escherichia coli and purified following standard procedures for glutathione-S-transferase fusion proteins. GST-GTPase fusion proteins bound to glutathione-beads were used for pull down experiments.GTPases for fishing experiments were expressed in 32P]GTP in 700 µl binding buffer  at 15°C for indicated times. The samples were then applied to a nitrocellulose membrane that had been rinsed with 3 times with 1 ml wash buffer. The filters were immediately washed 3 times with 1 ml of ice-cold wash buffer  and soaked in scintillation solution for 30 min. GTPase bound radioactivity was counted by liquid scintillation spectrometry.Recombinant Rac1, Rac1 S71E, Cdc42 and Cdc42 S71E (2.5 µg of each) were incubated with 13,5 µCi [γ-22HEK293 cells were cultured in DMEM with sodium pyruvate , supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicilline, 100 µg/ml streptomycine at 37°C in humified air with 5% COfl/flRac1 fibroblasts were kindly provided by Cord Brakebusch and are homozygous for the loxP-flanked Rac1 allele described before −/−Rac fibroblasts will be described elsewhere. fl/flRac1 and −/−Rac1 fibroblasts were maintained in DMEM containing 10% (v/v) fetal bovine serum, 2 mM glutamine, 1 mM Na+ pyruvate and 1% non-essential amino acids .Cell lysates or precipitates from pull down assays were subjected to SDS-PAGE and subsequent transfer onto nitrocellulose. After blocking with Tris-buffered saline containing 3% (w/v) milk powder and 0.2% (v/v) Tween-20, the nitrocellulose membrane was incubated with the appropriate primary antibody diluted 1∶1.000 in TBS-T supplemented with 3% (w/v) bovine albumine over night at 4°C. The membranes were washed three times with TBS-T and incubated with the corresponding HRP-conjugated secondary antibody diluted 1∶5.000 in TBS-T for 30 min. Bound antibodies were visualized by incubation with SuperSignal West femto chemiluminescence substrate (Pierce).5 cells were fixed in ice cold ethanol (70%) for 30 min. After washing once with 1% bovine serum albumin in PBS, the total DNA content was stained with propidium iodide . RNA was removed by incubating cells with 0.5% RNase for 30 min. Subsequently, cells were subjected to FACS-analysis . A fluorescence area (FL2) of 400 was set to correlate with a 2n-set of chromosomes within the G1-phase. Cells found in the sub-G1-phase were considered as apoptotic and necrotic due to the decrease in DNA content.Flow cytometry was performed to estimate number of cells within different cell cycle phases. The DNA content was measured using the fluorescent nucleotide acid dye propidium iodide. Therefore, cells were suspended by trypsinization and app. 5×10Protein samples were separated by SDS-PAGE and transferred onto nitrocellulose membrane. After blocking with 5% (w/v) nonfat dry milk in TBS-T  Tween 20) the membrane was incubated overnight with the primary antibody at 4°C. After washing with TBS-T it was incubated for 45 min at room temperature with the appropriate horseradish peroxidase-conjugated secondary antibody. Detection was performed by means of enhanced chemiluminescence.HEK-293 cells were transiently cotransfected with 50 ng of NF-κB luciferase reporter plasmid p3ENhκBcona-Luc containing three tandem repeats of NF-κB sites from the immunoglobulin Gκ promoter and 1 µg of the HA-tagged Rho GTPases. At 40 h post transfection, cells were washed with PBS and lysed in reporter lysis buffer. Luciferase activities were measured in cleared lysates with a luciferase assay system in accordance with manufacturer’s instructions. NF-κB activity was calculated as per cent of induction compared to that of pcDNA3.1_Cdc42 Q61L (constitutively active Cdc42) transfected cells.2.Pull down experiments with the PAK-PBD domain to detect active Rac1, Cdc42 or P-Ser-71 Rac1/Cdc42 were performed as previously described by Schoentaube 2, 10% glycerine, 100 mM NaCl, 1% NP40, 0.5 mg/ml BSA). After 5 min incubation on ice the lysates were centrifugated at 16,000 g for 5 min. The supernatant was split into four samples of 0.5 ml each and used for precipitation of desired proteins in parallel to guarantee identical protein load in all samples. Therefore, 20 µl of bead slurry with bound GST-fusion protein of the respective bait (app. 15 µg each) were added to each sample and rotated at 4°C for 60 min. The beads were collected by centrifugation at 10.000 g and washed twice with Fish-buffer and subjected for SDS-PAGE.For pull down experiments HEp2 cells grown in 75 cm2 flasks were lysed in 2 ml ice-cold Fish-buffer  and the generated peptides were extracted and solved in 10% acetonitril containing 0.2% trifluoroacetic acid. Peptides were cocrystalized with alpha-Cyano-4-hydroxycinnamic acid (4 mg/ml) in 50% acetonitril and 0.2% trifluoroacetic acid on an anchor target . After crystallization the peptides were analyzed in an MALDI-TOF/TOF mass spectrometer . Masses were determined by external calibration using suitable standard peptides . MS and MS/MS spectra were generated and analyzed with the BioTools  and MASCOT  software package.