Megaptera novaeangliae) populations typically undertake seasonal migrations, spending winters in low latitude breeding grounds and summers foraging in high latitude feeding grounds. Until recently, a broad scale understanding of whale movement has been derived from whaling records, Discovery marks, photo identification and genetic analyses. However, with advances in satellite tagging technology and concurrent development of analytical methodologies we can now detail finer scale humpback whale movement, infer behavioural context and examine how these animals interact with their physical environment. Here we describe the temporal and spatial characteristics of migration along the east Australian seaboard and into the Southern Ocean by 30 humpback whales satellite tagged over three consecutive austral summers. We characterise the putative Antarctic feeding grounds and identify supplemental foraging within temperate, migratory corridors. We demonstrate that Antarctic foraging habitat is associated with the marginal ice zone, with key predictors of inferred foraging behaviour including distance from the ice edge, ice melt rate and variability in ice concentration two months prior to arrival. We discuss the highly variable ice season within the putative foraging habitat and the implications that this and other environmental factors may have on the continued strong recovery of this humpback whale population.Humpback whale ( Perhaps the most classic concept of migration is the long-distance movements of many birds and mammals characterised by breeding at one end of the migratory pathway and feeding at the other. Most humpback whale (Megaptera novaeangliae) populations undertake seasonal migration, spending the winter in low latitude breeding grounds and the summer foraging in high latitude and productive feeding grounds2. Historically, our understanding of whale migration has been informed via examination of whaling records and Discovery mark recoveries , which provide a coarse description of the spatial and temporal characteristics of movement. More recently, non-lethal methods such as photo identification (for example4) and genetic analyses (for example5) have provided similar point location data. These data, however, provide no detail on the movements in between mark and recapture.Migration is a large-scale class of animal movement driven by resource quality or availability 6. Whales are no exception with the majority of movement occurring in remote, often inhospitable, areas with no survey effort7. The development of satellite tagging technologies has provided an extremely valuable, non-lethal technique to collect high-resolution movement data over biologically relevant time scales1. For example, satellite tags recently revealed novel migratory pathways of New Caledonia humpback whales that utilise seamount habitats both during the breeding season and while migrating8. Combining historical whaling and sightings data, Branch et al.9 hypothesised that pygmy blue whales  migrate between Australia and Indonesia along Australia’s western coastline – a migratory path which has since been confirmed using satellite telemetry10. In these and other cases (for example11), satellite telemetry provided new detailed movement information and identified habitat important to the conservation and management of whale species.Achieving a more detailed understanding of movement has been difficult for marine mammals because they are highly mobile, diving animals that spend relatively little time at the surface13. Concurrently, statistical techniques have evolved and analytical methods such as state space modelling (SSM) are increasingly being applied to tag based movement data16. SSM methods combine a process model  with an observation model (for the tracking data), to provide an estimation of the unobserved behavioural state of the animal17. This approach enables inference about the nature of that behaviour – for example, two-state models commonly differentiate more localised search  behaviour from more directed transiting behaviour (migration18). These inferred behaviours can be examined relative to the animal’s biophysical environment, enabling telemetry data to provide an understanding of the ecological factors influencing animal movement and habitat selection (e.g.19). The influence of directly measured and/or remotely sensed factors that may generate or concentrate resources can therefore be explored; for example environmental variables such as sea ice concentration20, bathymetric depth and gradient21, chlorophyll-a concentration22 and sea surface temperature23 have all been demonstrated to influence the “search” behaviour of cetaceans.Improvements in tagging technology, particularly advances in sensor, storage and transmission capabilities, have brought improvements in the amount and quality of data received24. The east Australian humpback whale population, designated as breeding stock E1 by the International Whaling Commission (IWC), migrates along the east coast of Australia and was hunted to near extinction in the 1950s and early 1960s26. However this population is now considered 58–98% recovered at a population size of 24,545 whales  with no evidence that the observed exponential rate of growth is slowing down27. Over multiple years (2008–2010) we deployed satellite tags on east Australian humpback whales near both the feeding and breeding grounds to collect the first high resolution movement data for this population. We aimed to describe migratory movement and identify foraging habitat. Using a SSM to infer foraging behaviour we investigate one factor that we hypothesise supports this population’s sustained recovery: access to productive feeding grounds. Here we identify the environmental factors that characterise the key Antarctic foraging habitat, discuss usage of ice associated habitats in the face of change and the role of supplemental feeding in temperate grounds.Obtaining a detailed understanding of the movement of wide ranging species such as humpback whales is important for informing management policy in the face of environmental variability and long term change associated with anthropogenic forcing−1 and when south of 60 °S slowed to 2.19 ± 0.74 kmh−1.Thirty humpback whales were tracked during three austral summers  while the other individuals continued transit into the Southern Ocean.The 21 whales tagged off the eastern Australian coast migrated south along the coastline and across the Bass Strait  during the month of October Fig. . Throughth November 2008 (n = 8) and the 21st November 2010 (n = 3), respectively. The mean date of arrival at 60 °S was the 10th December in 2008 (median: 7th December 2008) and the 4th December in 2010 (median: 23rd November 2010). All humpback whales with transmitting tags were in the Antarctic feeding grounds by January  where location data continued to be transmitted until May .In total eleven humpback whales tagged in east Australian waters travelled south of 60 °S Fig. , with fi18) were very well discriminated for the two states. The turn angles clearly showed frequent reversals during search (mean turning angles concentrated around 176° to 192°) as opposed to few when transiting , which undertook the longest migrations, as compared with those animals tagged on the Antarctic foraging grounds (γ1 ~ 0.5). Overall, whales spent on average 29.7 ± 27.8% (0–76.9%) of their time in search behaviour; 14.1 ± 22.7% when north of 60 °S (n = 21) and 29.8 ± 23.6% (n = 20) when south of 60 °S. There was relatively low uncertainty in the behavioural state estimates for the two Australian deployment campaigns with 15.6% (Eden) and 13.7% (Sunshine Coast) of estimates falling between 1.25 and 1.75; this was somewhat higher for the Antarctic deployment at 34.6%.The state-space model clearly distinguished between two behavioural states for the migrating whales  tagged during migration in east Australian waters had ‘search’ behaviour identified at locations within temperate waters; these were all animals tagged off Eden. Three of these whales undertook short search periods (of three days) near the tagging location from October 24, 2008, whilst four of these whales undertook search extending into the Bass Strait and/or along the east coast of Tasmania ranging in patch duration from four to 35 days  and the 18th of December in 2010 . The majority of search behaviour was concentrated between 145–175 °E  varied in relation to a suite of environmental variables . A movement not previously represented within the Discovery mark datasets is that of a lone individual who migrated along a westward trajectory through the Bass Strait and onto Antarctica, venturing as far west as 166 °W (management area VI).Satellite tracked humpback whales tagged off eastern Australia travelled south along three different migratory trajectories, detailed for the first time in this study. The majority of animals initially travelled southwards, following the eastern mainland and Tasmanian coastlines. One third of these whales headed in an easterly direction into the Tasman Sea. Of these seven individuals, three tags transmitted long enough to record movement as far as 165 °E, and the New Zealand coastline, before changing direction to transit to Antarctica. The simple information gained from historic Discovery marks hints at these migratory routes, with humpback whales marked off eastern Australia then captured off New Zealand and Antarctica  of the individuals tagged off eastern Australia. Temperate search behaviour was demonstrated only amongst those individuals tagged off Eden, Australia where supplemental feeding on small schooling fish and krill has been regularly reported30. However by temporarily suspending migration to forage, these whales may be able to meet up to 3.4 times their daily energy requirements. This may supplement the energy needed for migration and, in the process, begin to refuel energy reserves prior to reaching their Antarctic feeding grounds31. Riekkola et al.29 inferred supplemental feeding in endangered Oceania humpback whales and hypothesised that supplemental feeding may occur when energetic requirements are not being met on Antarctic feeding grounds. Supplemental feeding has also recently been noted for individuals from other Southern hemisphere humpback whale populations including those off the west coast of Africa37 and both the east38 and west39 coasts of South America. Stable isotope analyses of baleen whale plates indicate that supplemental feeding may actually be quite a common strategy for east Australian humpback whales40.Traditionally, humpback whales are considered to adopt the “feast or famine” approach to migration that is typical of baleen whales: fasting when not occupying high latitude feeding grounds30. More recently, photo identification data has added further weight to the importance of IWC Antarctic management area V42. Our study largely supports this, with the majority of search behaviour of satellite tagged animals occurring between 145°–175 °E. However the tracking data demonstrates that east Australian humpback whales do undertake search behaviour across a wider range between 100 °E and 165 °W; i.e., encompassing IWC Antarctic management areas IV, V and VI. Search patches within Antarctic waters persisted for up to 37 days with individuals transiting amongst up to five patches. This type of movement path, with periods of more tortuous track segments connected by relatively directed track segments, is consistent with those exhibited by other satellite tagged humpback whales foraging on Antarctic feeding grounds off the West Antarctic Peninsula45, as well as within the Ross, Amundsen and Bellingshausen Seas29. Marine prey resources are patchily distributed at multiple spatial scales46. So, following the major migration transit south to the polar feeding grounds, animals will typically still need to search between dynamic favourable forage patches within and around the sea ice and Antarctic shelf regions (see e.g.47).The primary Antarctic foraging grounds of east Australian humpback whales was historically considered to be between 130 °E and 170 °W (IWC Antarctic management area V) as determined through Discovery mark recoveries48. Additionally, persistent cloud cover necessitates averaging of remotely sensed chlorophyll measurements (here across a three month period); leading to the loss of temporal information.Our study identified important environmental predictors characterising Antarctic foraging habitat of east Australian humpback whales - high variability (in relation to the mean) in ice concentration two months prior to arrival, at a distance of approximately 65 km from the ice edge, and high ice melt rates at the time of ice retreat. These factors are clearly associated with the marginal ice zone. Essentially these humpback whales foraged where the sea ice was present two months prior. This is the first time this area, located off the Victoria Land coast and in the western Ross Sea, has been clearly identified as core foraging habitat for the east Australian humpback whale population, and defined in terms of key biophysical characteristics. The chlorophyll climatology we constructed as a proxy for primary oceanic productivity was only found to be a significant environment predictor in 2% of the statistical models. This is likely due in part to the inability of satellites to measure productivity of closely ice-associated habitats49 and is outer pack ice that persists for ≤100 days in a climatological sense50. It is perhaps not surprising that the marginal ice zone and more specifically, low mean and high variance in ice concentration two months prior, plays an important role in conditioning foraging habitat. Various modelled estimates indicate that it takes approximately 15 to 20 days51, around 30 days52 and less than 90 days53 for the biological cascade that results from the release of new production (by way of ice melt) to trigger the accumulation of zooplankton grazers such as krill on which baleen whales foraging in Antarctica depend54. In fact, productivity peaks within the marginal ice zone one to two months following the point at which maximum open water in the area is achieved, which is a direct reflection of the time it takes for phytoplankton blooms to fully respond to newly created ice-free waters55. Rapid ice retreat has also been shown to enhance production, which concentrates secondary producers and their vertebrate predators56. However, changing ice dynamics are not favourable for all Antarctic marine predators (for e.g.57).The marginal ice zone is defined as the area of transition from dense pack ice (up to 100% cover) through to no ice cover50 and the area in which the East Australian humpback whales forage. The high variability in the sea ice season between 100 °E and 145 °E occurs primarily in the marginal ice zone region 50. East of 145 °E, high variability in the ice season occurs across the entire ice covered region with the exception of the coastal fast ice zone. Despite such large variability, the foraging habitat of the east Australian humpback whale population has undergone a trend of increasing ice season duration over a 30 year period, through earlier advance and later retreat of the ice edge 50. The region east of 145 °E undergoes earlier and rapid ice edge advance with strong ice production occurring in autumn. Due to fewer open water days there has been an overall trend of decreasing sea surface temperature and decreasing net primary productivity, but also a small area of net primary productivity increase located at approximately 64 °S to 66 °S and 160 °E to 165 °E58.Large spatio-temporal variability in sea ice seasonality is a fundamental feature of East Antarctica60. However, recent evidence suggests that at least in East Antarctica, this relationship may not be so direct and that the winter pack ice may actually be a food-poor habitat for krill61. The marginal ice zone provides a better feeding habitat for krill due to the presence of light, nutrients, grinding ice floes and proximity to open ocean/waves which promote high larval krill growth rates. The complex habitat structure of the marginal ice zone further provides protection important for larval krill survival. Phytoplankton availability in autumn may also govern krill recruitment success with early ice formation in autumn separating adult and larval krill and reducing food competition. As such, time lags in ice-related environmental predictors that date back to the previous autumn may need to be considered in future modelling attempts.The Antarctic foraging habitat of east Australian humpback whales could also be habitat favourable for krill recruitment and success. Traditionally, increased ice season duration and associated winter ice has been thought to favour krill maturation by providing a winter, ice algae food supply resulting in a high recruitment rate from the spawning season63 and whaling catch records64 demonstrate that historically, humpback whales were located (sighted and captured) in relatively high numbers within the same area identified as foraging habitat for the humpback whales tagged during 2008–2010. Our findings showed notably persistent space use, such that different whales moved through the same location or occupied habitat immediately adjacent over three consecutive austral summers. Site fidelity, or persistent space use, has been observed for other humpback whale populations66. In the patchy marine environment, foraging site fidelity may be attributed to the interplay of habitat quality and predictability; ultimately, familiarity with foraging habitat may present significant ecological benefits over the long term68. Additionally, for humpback whales, foraging site fidelity is maternally directed with individuals returning to foraging sites that, historically, they first visited with their mother prior to weaning71. This behavioural mechanism can act on population structure at an evolutionary time scale72 and may contribute to the lack of recovery for some whale populations that were nearly extirpated due to whaling73.The IWC’s International Decade for Cetacean Research (IDCR) and Southern Ocean Whale Ecosystem Research (SOWER) sightings surveysth century25, and may have numbered just 104 individuals when commercial whaling ceased in 196326. This population, assigned the nomenclature E1, is considered to be one of three meta-populations that comprise population E74. The other two meta-populations  together with individuals from the central South Pacific collectively comprise the Oceania population. The rapid recovery rate of the east Australian population is just shy of the theoretical upper bound of population increase75 at 11.1% (95% CI of 10.6–11.3%27). Whilst the E1 post-exploitation recovery is strong at between 58–98% recovered27, the Oceania group remains listed as endangered by the International Union for Conservation of Nature76 and is estimated to be well below 50% of pre-exploitation population size29. This group, which was decimated by illegal hunting of Soviet whalers77, mainly forage farther east throughout IWC Antarctic Management Areas V, VI and I (approximately 180 °E to 90 °W)78. Tracking data demonstrate a small degree of overlap in foraging range at the eastward limit of the satellite tracks presented in this study. Clearly these populations can mix on their foraging grounds78 which highlights the possibility that the rapid recovery of east Australian humpback whales may only be partly due to forage conditions . Conversely, the poor recovery of the Oceania population may simply just be the result of their near extirpation. However, Clapham and Zerbini79 hypothesise that the high population growth rate of east Australian humpback whales could be due to temporary immigration by Oceania humpbacks as a consequence of a mating system that results in whales migrating from low density to high density breeding grounds. In light of the annual pregnancy rates recently reported for two Southern Hemisphere humpback whale populations80, a revisit of appropriate calving intervals along with further genetic analyses are required to determine the validity of this hypothesis.East Australian humpback whales were hunted along both their migratory corridors and upon their Antarctic feeding grounds during the 2027. Easily counted and sampled whilst on their tropical breeding ground, this population could act as a “sentinel species” providing cost-effective monitoring of the Antarctic sea ice ecosystem81. Significant changes are already occurring in key Antarctic forage habitat, with evidence of long-term changes in the sea ice environment50 and associated declines in ocean temperature and net primary productivity58. The capacity for these long lived, large animals with late reproductive age to respond to change is not known, but presumably currently being challenged by rapid environmental change. The future brings potential impacts of even larger scale changes: for example, the possibility of a complete collapse in Antarctic krill populations due to ocean acidification by 230082. Highly mobile species which rely on different habitats during different life history stages are subject to multiple and varied threats across their range, which makes it difficult to understand and predict their ability to adapt84. Certainly as this population approaches recovery, ongoing monitoring will be required to identify and address the various impacts of human and economic expansion on migratory pathways and breeding habitat85. However, if we consider that migration has evolved as a successful strategy to manage environmental variability, and persisted as a behaviour throughout global change over millions of years84, then for east Australian humpback whales the act of migration may facilitate their ability to respond to change.The east Australian humpback whale population is perhaps the best monitored population of Antarctic krill consumers in the world, with post-whaling surveys initiated in 1978 and repeated every one to three years since86 and a purpose-designed projectile carrier at a pressure of 7.5–10 bar. A custom-designed, 80 mm anchor section is attached to a stainless steel cylindrical housing containing a location-only transmitter . This superseded anchor design resulted in the anchor section disarticulating upon deployment in order to achieve improved tag retention times while minimising impact87. The tags were sterilised with ethylene oxide prior to deployment and implanted up to 290 mm into the skin, blubber, interfacial layers and outer muscle mass of the whale.Satellite tags were deployed on adult humpback whales with a modified version of the Air Rocket Transmitter System ARTS, Restech88. Biopsy samples were stored in 70% ethanol and DNA subsequently extracted using a Tissue DNA purification kit for the Maxwell 16 DNA extraction robot (Promega Corporation). The sexes of the tagged whales were determined using a 5′ exonuclease assay of the polymorphisms in the sex-linked Zinc Finger genes as described by Morin et al.89.Upon deployment, a small amount of skin and blubber was simultaneously collected for genetic analyses. These were collected using a biopsy dart fired from a modified 0.22 Paxarms systemTags were deployed during three austral summers: 2008/09, 2009/10 and 2010/11. The two Australian deployment campaigns were on individuals undertaking their southern migration along the east coast near Eden  between October and November 2008 and off the Sunshine Coast  during October 2010. Satellite tags were also deployed on individuals whilst on their Antarctic feeding grounds during February and March 2010 .90).Tags were programmed to transmit to the Argos satellite system at various duty cycles and repetition rates for a maximum of 720 transmissions per day Table . These t93) from whales in each deployment campaign  was entered into a hierarchical switching state-space model which enables joint estimation over multiple individuals (hSSSM94). This model both accounts for spatial uncertainty in Argos location estimates (via an observation model) and estimates two discrete  behavioural states (via a movement process model). The hierarchical structure assumes that all individuals move according to a correlated random walk but that that the movement of each individual is characterised by a different diffusivity17. Using a Bayesian approach, the movement process parameters governing each state are estimated: the mean turning angles (θ) and a movement persistence parameter . Here, we nominally label the two states as transit (indicating more directed travel) and search  with the latter putatively indicating foraging behaviour although alternate behaviours, e.g., resting, breeding or social activities may be relevant for particular species in certain areas95.Argos location data  associated with each location state  estimate. From the posterior, the most probable (discrete) behavioural state can then be evaluated for each location, as well as a summary made across all samples e.g., as a posterior mean . For mapping purposes the behavioural state shown is the mean posterior estimate and following Jonsen et al.95 we assign estimates <1.25 as ‘transit’ (blue) and >1.75 as ‘search’ (red). All estimates between 1.25 and 1.75 are shown as ‘uncertain’ (grey). To calculate search patch duration we follow Bailey et al.33 where a patch is comprised of successive location estimates with an estimate greater than 1.75, ending when 3 or more consecutive location estimates have an estimate below 1.75.For each of the deployment campaigns separately, the hSSSM was fit across animals  using a 12 h time step. The hSSSM is fit via Markov chain Monte Carlo (MCMC) simulation implemented in JAGS using the R97). The most probable discrete behavioural state estimate  was modelled in response to environmental predictors selected based on their potential to influence the search behaviour of humpbacks whales by concentrating prey. The environmental variables examined, with full details of the data sources and resolution are given in Supplementary 98 was assessed using variance inflation factors (ensuring VIF <3) and correlation coefficients (ensuring r < 0.8 – Pearson correlation coefficient). The environmental variables included in the final full GAMM (see Supplementary bathyg), chlorophyll climatology (CHLa), distance to ice edge (dist ice), ice melt rate (melt rate), the coefficient of variation in ice concentration two months prior (ice cv lag 2) and mean ice concentration one month prior (ice mn lag 1). Deployment campaign was also included as a categorical predictor. We examined spatial and temporal autocorrelation in the Supplementary material . To account for the uncertainty in both behavioural states and locations we resampled from the state-space model posterior estimates, thereby adopting a type of sensitivity analysis or bootstrap approach to fitting the GAMMs; this approach to accounting for measurement error is also called multiple imputation. We fit the first GAMM using the hSSSM behavioural state and location estimates summarised as the posterior mean for each location (n = 1442). We then randomly sampled the retained MCMC chains to obtain 100 possible realisations (50 per chain) of these 1442 behavioural and location state estimates.There is uncertainty inherent in the behavioural state estimate (here the response variable in our GAMMs) for each whale location, and also for each location estimate itself. It is particularly relevant to propagate location uncertainty when sampling environmental variables from spatially gridded remote-sensing data, although this is relatively rarely considered (but seeb) estimate at each location of the new track realisation was modelled against the relevant set of environmental predictor variables . This enabled assessment of the strength of environmental influences accounting for uncertainty in the state-space model posterior estimates for behavioural state and location. We report the number of times each environmental variable was determined to be significant  under the resampling procedure (n = 100 iterations). P-values indicate approximate significance of GAMM smooth terms based on Chi-sq. statistics. The GAMM was fit using a binomial family with a logit link, with individual whale included as a random effect. The GAMM allows for the relationships between behaviour and environmental predictors to be flexible and non-linear. All statistical models were fit using the gamm497 library in R96 which makes use of the modular fitting functions provided by lme4100 and is appropriate for binary data. gamm497 follows the approach taken by package mgcv101 and represents the smooths using R penalized regression spline type smoothers, of moderate rank. We focussed on the Antarctic forage grounds, restricting our behavioural analysis to only those locations at or south of 60 °S .These new samples drawn from the posteriors were then used to refit the above model 100 times. The discrete behavioural state (This study was carried out in strict accordance with the approvals and conditions of the Antarctic Animal Ethics Committee for Australian Antarctic Science project 2941. Fieldwork was undertaken with the permission of the Australian Government under EPBC permits 2007–006 and 2007–007.Supplementary 1,2 and 3Supplementary 3


Canavalia ensiformis) urease. Here we describe the properties of Soyuretox, a polypeptide derived from soybean (Glycine max) ubiquitous urease. Soyuretox was fungitoxic to Candida albicans, leading to the production of reactive oxygen species. Soyuretox further induced aggregation of Rhodnius prolixus hemocytes, indicating an interference on the insect immune response. No relevant toxicity of Soyuretox to zebrafish larvae was observed. These data suggest the presence of antifungal and entomotoxic portions of the amino acid sequences encompassing both Soyuretox and Jaburetox, despite their small sequence identity. Nuclear Magnetic Resonance (NMR) and circular dichroism (CD) spectroscopic data revealed that Soyuretox, in analogy with Jaburetox, possesses an intrinsic and largely disordered nature. Some folding is observed upon interaction of Soyuretox with sodium dodecyl sulfate (SDS) micelles, taken here as models for membranes. This observation suggests the possibility for this protein to modify its secondary structure upon interaction with the cells of the affected organisms, leading to alterations of membrane integrity. Altogether, Soyuretox can be considered a promising biopesticide for use in plant protection.Ureases from different biological sources display non-ureolytic properties that contribute to plant defense, in addition to their classical enzymatic urea hydrolysis. Antifungal and entomotoxic effects were demonstrated for Jaburetox, an intrinsically disordered polypeptide derived from jack bean ( Ureases  are nickel-dependent enzymes that catalyze the hydrolysis of urea into ammonia and carbamate ,3. TheseCanavalia (C.) ensiformis, namely jack bean urease (JBU) and Canatoxin (CNTX), and then later observed for the embryo-specific and ubiquitous soybean ureases , and eluted at 1 mL/min flow rate.15N-enriched Soyuretox for NMR spectroscopy was achieved using a similar protocol. In particular, 20 mL of pre-inoculum of Soyuretox-transformed cells were maintained overnight at 37 °C and 150 rpm, in LB medium supplemented with 1% glucose and 50 μg/mL carbenicillin and 40 μg/mL chloramphenicol. The cells were inoculated into 1 L of M9 medium  containing 1.25 g/L of (15NH4)2SO4, 4 g/L glucose, 50 μg/mL carbenicillin and 40 μg/mL chloramphenicol and maintained at 37 °C at 150 rpm until an OD600 of 0.6–0.7 was reached. Protein expression was thus induced by adding 0.23 mM IPTG and kept overnight at 21 °C and 150 rpm. The cells were collected by centrifugation at 4,500× g for 30 min, resuspended in the lysis buffer, lysed using a French Press , and purified following the two chromatographic steps described above.Production of −1·cm−1). The typical final yield was ~5–10 mg per liter of culture. Jaburetox was produced and purified as previously described [The purity of the fractions was checked by SDS-PAGE  and gels were stained with ProBlue Stain . The fractions containing the polypeptide were pooled and concentrated using Amicon devices with 3 kDa cut-off membrane. Protein concentration was measured by the Bradford method  or by abescribed .v/v nonfat milk solution in TBS buffer (10 mM Tris-HCl at pH 7.5 containing 0.15 M NaCl) for 2 h. After washing, the membrane was incubated for 2 h with rabbit anti-Jaburetox polyclonal antibodies (1:7500 dilution), followed by incubation with anti-rabbit IgG antibodies coupled to alkaline phosphatase . The colorimetric reaction was developed with NBT (nitroblue tetrazolium)  and BCIP  (5-bromo-4-chloro-3-indolyl-phosphate). Bovine serum albumin  was used as a negative control.Western blotting  was carrSoyuretox samples (50 µM) were prepared in 50 mM sodium phosphate buffer at pH 8.0. To check if Soyuretox undergoes structural changes when in contact with sodium dodecyl sulfate (SDS), solutions of the polypeptide were prepared in 50 mM sodium phosphate containing SDS at final concentrations of 0.1, 0.5, 1.0, and 10 mM. Measurements were made on a Jasco 810 spectropolarimeter  in 0.1 cm optical path cuvettes in the λ range of 190 to 250 nm. The spectrum of the buffer was subtracted from all measurements.15N-labeled Soyuretox (0.5 mM) in 50 mM sodium phosphate buffer containing 1 mM EDTA and 1 mM TCEP at pH 8.0, 10% D2O (v:v), were used for NMR spectroscopy. Soyuretox solutions were prepared in the absence and presence of SDS at final concentrations of 0.1, 1.0, 1.5, and 10 mM. The critical micellar concentration of SDS was taken as 1.5 mM in buffer [Samples of n buffer . SoyuretC. ensiformis major urease isoform structure (PDB ID: 3LA4) [C. ensiformis major urease (PDB ID 3LA4) [Ten structural models for Soyuretox were built with MODELER9v14  using thD: 3LA4)  as templD: 3LA4)  and theoD: 3LA4) . The SoyD: 3LA4) . BrieflyD: 3LA4)  and GROMD: 3LA4)  for 500 D: 3LA4) , and couD: 3LA4)  was applD: 3LA4) . TemperaD: 3LA4) , respectD: 3LA4) . SoyuretD: 3LA4)  taking aID 3LA4) . Their hID 3LA4)  and depiID 3LA4) . SequencID 3LA4) .C. albicans, C. parapsilosis and S. cerevisiae were kindly provided by Dr. Valdirene Gomes, Universidade Estadual do Norte Fluminense, Campos dos Goytacazes, RJ, Brazil. The growth assays were performed as previously described [4 cells/mL) in U-bottom microplates, in Sabouraud broth at 28 °C, for 24 h. The dialysis buffer was used as negative control. For C. albicans, a dose-response curve was tested using Soyuretox at 0.1, 0.5, 5.0, 10, and 15 µM. Growth was assessed after 24 h of incubation at 28 °C, by colony forming units (CFU/mL): 20 µL of Soyuretox-treated yeast cultures were serially diluted 10-fold in saline, plated in Sabouraud agar by the drop plate method, and further incubated for 24 h at 28 °C. The experiments were performed in triplicate and the results are shown as means ± standard deviations (SD) of three independent bioassays.The yeasts escribed  with minC. albicans suspensions (104 cells/mL) were incubated with 1 or 5 µM Soyuretox and 10 µL of 10% nitro blue tetrazolium  for 24 h at 28 °C. After incubation, the supernatant was discarded, and the plate was dried at room temperature. The production of reactive oxygen species (superoxide anions) by the Soyuretox-treated and control cells was evaluated by the amount of formazan crystals formed. Formazan formed in each well was solubilized in 120 µL of 2 M KOH  and 140 µL absolute dimethyl sulfoxide , and the absorbance was read at 620 nm using a Reader 490 EZ-Biochrom microplate reader.Detection of reactive oxygen species was performed as follows: aliquots of C. albicans cells were used for immunofluorescence assays. Yeasts (105 cells) were incubated with 1 µM Soyuretox for 24 h in microplates. After transferring the content of each well to Eppendorf tubes, 8% v/v formaldehyde was added at a 1:1 (v:v) ratio, followed by 1 h incubation at room temperature. Cells were washed with PBS buffer by centrifugation (500× g), and then submitted to a permeabilization-blocking treatment with 0.1% Triton X100, 3% BSA in 20 mM sodium phosphate, 150 mM NaCl, pH 7.0, for 30 min. After incubation with the anti-Jaburetox primary antibody (1:750 dilution) for 1 h, the cells were washed and incubated with anti-rabbit IgG ALEXA 488 (green) conjugated antibodies  for 1 h. Controls were not incubated with either the primary or secondary antibodies. Finally, the cells were stained with 0.1 μg/mL DAPI . For fluorescence microscopy, the cells were visualized in a Zeiss Axiovert 200 microscope equipped with an AxiocamMRc camera, and images were captured using the AxionVisionRel 4.8 software. In all cases, the control was performed in the presence of buffer (10 mM Tris-HCl at pH 7.0).R. prolixus were kindly supplied by Dr. Denise Feder, Universidade Federal Fluminense, Niteroi, RJ, Brazil. The in vivo hemocyte aggregation assays were performed by administering Soyuretox to the insects through injection or feeding [R. prolixus saline solution . The saline solution alone was injected as negative control. After 6 h, the hemolymph of the insects was collected and diluted in an anticoagulant solution . Free cells and aggregates were counted in a light field microscope hemocytometer. A cluster of 5 or more cells was considered an aggregate [Fifth instar nymphs of  feeding ,69. FiftR. prolixus nymphs were artificially fed for 30 min on parafilm-coated acrylic plates containing a solution of the polypeptide diluted in saline plus 1 mM ATP, kept at 37 °C. The concentration of the polypeptide in the feed solution was calculated to give a dose of ~0.1 µg/mg of insect body weight, considering the volume usually ingested by nymphs at this stage. Animals of the control group fed only in saline solution containing 1 mM ATP. The hemolymph of the insects was collected 18 h after the meal and diluted in anticoagulant solution. The numbers of aggregates and of free cells were counted as described above.Fifth instar R. prolixus nymphs was collected with a micropipette after cutting one of the insect’s legs. The pooled hemolymph was mixed with R. prolixus saline at a 1:1 (v:v) ratio. Soyuretox was added to the diluted hemolymph pool at final concentrations of 50, 100, 200 and 500 nM, followed by incubation for 1 h at room temperature under gentle agitation. The saline solution alone was used as negative control. The number of aggregates was counted as described above.Hemolymph of fifth instar D. peruvianus after buffer and polypeptide injection into the insect’s hemocoel. D. peruvianus fifth-instar nymphs were anesthetized by cooling at 4 °C for 5 min, fixed in a plate and injected into the hemocoel with 5 μL of Soyuretox in 50 mM NaPB pH 7.5, at 50 ng per mg of insect body weight (insect average weight ca. 30 mg). The experiments were repeated three times employing groups of 10 insects per condition.Soyuretox lethality effect was evaluated for 0, 24, 48, 72, and 96 h in D. peruvianus mortality was examined after oral administration. Fifth-instar nymphs of D. peruvianus were immobilized on a flat surface and their mouth apparatus were introduced into a glass capillary containing 3 μL of Soyuretox in 50 mM NaPB pH 7.5, to give a final dose of 50 ng ingested peptide per mg of insect body weight, as described [escribed . The expZebrafish embryos were obtained by natural mating of wild-type adults. Animals were kept in recirculating water systems  with osmosis-filtered water at controlled pH (7.0–7.5) and temperature (28 ± 2 °C). Nitrate, nitrite, ammonia and chloride levels were checked daily. Animals were maintained under day/night photoperiod cycle (14:10 h) and fed three times a day with commercial flakes  supplemented with live brine shrimp. The day before mating, two males and one female were placed in breeding tanks, separated overnight by a transparent barrier which was removed on the following morning. The fertilized eggs were collected for the experiments. All zebrafish protocols were approved by the institutional Animal Care Committee under the number 7659 (CEUA-PUCRS).Eggs were placed in six-well plates (10 eggs/well) and exposed to Soyuretox at concentrations of 10, 100 and 300 nM, or 5 mM sodium phosphate buffer or water as controls groups, for 4 h. After the treatment, the eggs were washed with water and kept in Petri dishes in water until 5 days post-fertilization (dpf). The medium was changed daily for all groups. Survival rate, determined by presence or lack of a heartbeat, was monitored daily.Morphological evaluation was performed in larvae at 5 dpf, under a stereomicroscope. Each animal was photographed individually, and body measurements were assessed by the NIS-Elements D software . The body length was taken as the distance from the larval mouth to the pigmented tip of tail; the size of eyes was determined by measuring the surface area of the eyes; and the ocular distance was assumed as the distance between the inner edges of the two eyes.The exploratory behavior of the larvae was evaluated at 5 dpf according to the procedure described by Colwill and Creton . Each lat-test and in vitro aggregation results were evaluated by one-way ANOVA followed by Newman-Keuls Multiple Comparison Test, and non-parametric ANOVA followed by Dunn’s Multiple Comparison Test. Mortality tests were accessed by the Kaplan-Meier (KM) method.Larval survival was analyzed by Kaplan-Meier analysis. Differences in locomotor and morphological parameters of larvae, considering time and concentration of Soyuretox were evaluated by one-way analysis of variance (ANOVA), followed by Bonferroni’s multiple comparisons tests. Yeast inhibition assay and oxidative stress were evaluated by one-way ANOVA, followed by Tukey’s multiple comparisons test. In vivo hemocyte aggregation was analyzed by Student’s C. ensiformis urease. Although collinear sequences equivalent to Soyuretox in distinct plant ureases diverged considerably, the data reported demonstrated that Soyuretox and Jaburetox possess conserved antifungal and entomotoxic domain(s) representative of Leguminosae ureases. Soyuretox can be regarded as safe to zebrafish larvae and in an environmental context. Altogether, our data bring into light the biotechnological potential of Soyuretox as a promising molecule for the development of transgenic plants with increased resistance to insect herbivory and fungal diseases.In summary, in the present work, Soyuretox, a recombinant polypeptide derived from the soybean ubiquitous urease, was described. Soyuretox was characterized as an intrinsically disordered polypeptide possessing potent antifungal and entomotoxic activities, and was compared with Jaburetox, the orthologue polypeptide from


The mammalian PBAF subfamily of SWI/SNF chromatin remodeling complexes plays a wide role in the regulation of gene expression. PHF10 is a subunit of the signature module of PBAF, responsible for its interaction with chromatin. PHF10 is represented by four different isoforms, which are alternatively incorporated in the complex. Two of PHF10 isoforms lacking C-terminal PHD domains contain a cluster of phosphorylated serine residues, designated as X-cluster. In the present study, we explore the phosphorylation of the X-cluster in detail. We identified additional phosphorylated serine residues and designated them as either frequently or rarely phosphorylated. The X-cluster consists of two independently phosphorylated subclusters. Phosphorylation of the second subcluster depends on phosphorylation of a primary serine 327. These two subclusters surround a sequence, which is predicted to be a nuclear localization sequence (NLS3). The NLS3 does not affect localization of PHF10 isoforms. However, it is essential for X-cluster phosphorylation and increased stability of isoforms that lack PHD. Conversely, the presence of NLS3 signal in isoforms that contain C-terminal PHD domains reduces their stability. Thus, phosphorylation of PHF10 isoforms regulates their cell level, determining the rate of incorporation in PBAF. This may alter the pattern of PBAF regulated genes. Summary: The sequential phosphorylation of the linker domain of PHF10 subunit of PBAF chromatin remodeling complex is triggered by two primarily phosphorylated serines and determines the different properties of PHF10 isoforms. Phosphorylation is the most widespread and the most studied post-translational modification in eukaryotes. Phosphorylation is involved in regulation of various cellular processes like transcription and catalytic activity; it can alter protein structure, conformation, stability, localization and protein interactions . PhosphoSites of phosphorylation in many proteins tend to form clusters with many closely located amino acids phosphorylated by the same kinase. Importantly, the phosphorylation events in clusters tend to be coordinated with phosphorylation of each amino acid, determined by the phosphorylation of the previous one .Phosphorylation is likely to be important for the regulation of the mammalian PBAF subfamily of SWI/SNF chromatin-remodeling complex, which plays an important role in the regulation of transcription . The PBAAmong the signature subunits of PBAF, only PHF10 was shown to be extensively phosphorylated . PHF10 iIn previous work, we have shown that each PHF10 isoform has its own unique phosphorylation pattern that is dependent on its domain structure . The impIn the present study, we expanded our understanding of the structure and function of X-cluster and its role in maintaining the stability of the PHF10-S isoforms. We demonstrate that the phosphorylation events in X-cluster are highly coordinated. The X-cluster consists of two independently phosphorylated sub-clusters that surround a potential nuclear localization signal (NLS3). The first sub-cluster contains two primary phosphorylated serines 297 and 301. The second sub-cluster includes the primary phosphorylated serine 327, its phosphorylation triggers phosphorylation of serines 331, as well as serines 323 and 335, which were also found to be phosphorylated. The potential NLS signal does not target the PHF10-S isoforms to the nucleus but is required for phosphorylation of surrounding amino acids. These findings show a complex mechanism for regulation of PHF10 stability, through highly modified novel domains.The domain structure of PHF10 is shown in . The isoPreviously we investigated phosphorylation of the X-cluster and showed  that CK1The sequence of the PHF10 linker domain is conservative and in the screenings of mouse and rat sequences the corresponding amino acid residues are also phosphorylated, which indicates their importance A. We hypThe phosphorylation of serines was first examined in HEK293 cells. We expressed recombinant non-mutated and mutated forms of the FLAG-tagged linker domain in HEK293 cells. In mutated forms, the serines that were the potential phosphorylation targets were replaced by alanine A. The wiin vivo. However, the level of their phosphorylation was much lower than that of serines 297, 301 and 327.The wild-type linker domain migrates as two bands in PAAG gel B, lane 1in vitro system, we have confirmed that in addition to the frequently phosphorylated serines 297, 301 and 327, the X-cluster contains serines 335 and 323 that are phosphorylated at lower levels.Next, to examine the phosphorylation of studied serines we performed a kinase assay. The linker domain of PHF10 as well as its mutated variants were expressed in bacteria, purified using His-tag columns and incubated with HEK293 lysates in the presence of [P]-ATP A. We detTo increase signal transduction through phosphorylation, phosphorylated residues could be organized in clusters . PhosphoTo determine whether phosphorylation of serines in the X-cluster depend on each other we first examined the frequently phosphorylated serines 297, 301 and 327. We produced recombinant forms of the linker domain, which contained mutations of serines in the first sub-cluster (297/301), or in the second (327). The signal in kinase assay decreased only partially when serines of only one sub-cluster were mutated, while the simultaneous mutation of serines 297/301 and 327 completely abolished phosphorylation . The nuclear localization sequences usually contain several positively charged amino acids (most frequently arginine and lysine), which are recognized by importin proteins. Phosphorylation of the X-cluster creates a strong negative charge on both sides of the NLS3 and therefore can mask NLS3 and prevent its recognition by importins A.First, we tested whether NLS3 sequence was functional and influenced the localization of isoforms. To this aim, we mutated the NLS3 in one of the PHDs containing PHF10 isoforms in which Х-cluster is not phosphorylated (PHF10-Ps). In mutated FLAG-tagged PHF10-Ps lysines in the NLS3 sequence were replaced by neutral alanines A.The endogenous PHF10-Ps, as well as its FLAG-tagged form, was present in both nuclei and cytoplasm. After overexpression of mutated Fl-PHF10-Ps-NLS, we did not detect any changes in the nuclear protein levels, indicating that the NLS3 did not affect translocation into the nucleus. Surprisingly, the amount of the mutated protein in the cytoplasm increased significantly . SimilarWe further suggested that X-cluster phosphorylation in isoforms lacking PHDs can affect the function of NLS3. Similar to PHF10-Ps, the endogenous PHF10-Ss isoform lacking PHDs was detected in both nuclei and cytoplasm, but the content of PHF10-Ss in the cytoplasm was much higher than that of PHF10-Ps  and 5A,CWe expressed the PHF10-Ss isoform with mutated serines 297/301 and 327 of X-cluster eliminating its phosphorylation [Ss(Xmut)] and performed immunostaining experiments. As expected, the phosphorylation was completely lost in mutated PHF10-Ss and it migrated as a single band on SDS-PAGE. Both fluorescent microscopy and western blot experiments showed a decrease of PHF10-Ss levels in the cytoplasm and in the nucleus B, confirIn order to reveal the function of NLS3 in PHF10-Ss isoform, we mutated the lysine residues in NLS3 sequence, similarly to PHF10-Ps-NLS A. The diTo better demonstrate that NLS3 is required for the phosphorylation of the surrounding serines of X-cluster, the protein extract from cells transformed with FLAG-PHF10-Ss or its mutant form were treated with Lambda-phosphatase. The effect of Lambda-phosphatase treatment on isoform mobility was analyzed on SDS-PAGE. Incubation of FLAG-PHF10-Ss with Lambda-phosphatase led to a substantial change in its mobility  consists of two independently phosphorylated clusters of serine residues. We identified the primarily phosphorylated serines and demonstrated that they are essential to trigger the phosphorylation of other serines in the cluster. The two clusters are separated by the predicted NLS (NLS3) sequence. However, our data show that the NLS3 does not target the PHF10 isoforms into the nucleus but is involved in maintaining their phosphorylation and stability. The NLS3 increases the rate of degradation of PHDs containing PHF10 isoforms. In PHF10 isoforms lacking C-terminal PHDs, the NLS3 is essential for phosphorylation and thus is essential for their high stability in the cell. Overall, these data demonstrate the complicated mechanism of maintaining the turnover rate of different PHF isoforms in the cell.The PHF10 isoforms that lack PHD domains are phosphorylated on six serine residues in the linker domain. The serines in the linker domain of PHF10 (X-cluster) are phosphorylated in PHF10-S isoforms. In turn, the X-cluster consists of two sub-clusters that are independently phosphorylated. The first sub-cluster contains serines 297 and 301, and the second – serines 323, 327, 331 and 335. The two serines of the first sub-cluster and serine 327 of the second sub-cluster are actively phosphorylated, independently of each other. Phosphorylation of serines 323, 331 and 335 depends on phosphorylation of serine 327 and does not happen if this residue is mutated into alanine. Thus, phosphorylation of serine 327 serves as a primer, required for phosphorylation of other serines in the sub-cluster.In sequentially phosphorylated sites, the phosphorylation of residues happens in a strict order, when phosphorylation of one residue is required for phosphorylation of the next phosphorylated amino acid in the sequence . The numIn recent publications, triggered sequential phosphorylation is considered an important mechanism of regulation. Such phosphorylation is highly coordinated and could modify the speed and sensitivity of the cellular response to external stimuli . PhosphoPreviously we have shown that phosphorylation of 297 and 301 serines, as well as of serines 327 and 331 in the second sub-cluster is performed by the casein kinase 1 (CK1). In line with this data, it was demonstrated that serines and threonines, organized in clusters and phosphorylated by the same kinases, are usually located in unstructured domains , and evoPreviously we have shown that phosphorylation of the X-cluster leads to increased stability of PHF10-Sl and PHF10-Ss isoforms, compared to PHF10-Pl and PHF10-Ps, in which the X-cluster is not phosphorylated. One of the functions of cluster phosphorylation was shown to be a regulation of protein stability and degradation . In lineIn our experiments, the same mutation of the NLS3 motif of PHF10-Ss had the opposite effect, leading to a decreased stability of the protein. However, we have found that this mutation also completely abolished the X-cluster phosphorylation that, as was shown before, protects PHF10-Ss from degradation. It is difficult to clearly understand the way NLS3 motif influences phosphorylation as it is not indicated in databases whether it has any additional functions. One may suggest that altering the charge of mutant NLS3 may influence the secondary structure of PHF10-Ss protein.Short isoforms PHF10-Ps/-Ss are localized both in the nucleus and in the cytoplasm, but the PHF10-Ss is mostly localized in the cytoplasm. From our data, it became apparent that the difference in the distribution between compartments is determined by the different speed of degradation.The amount of PHF10-Ps and PHF10-Ss in the nucleus was similar. As we have shown previously  recombinProteins with closely located phosphorylation sites combined into clusters have a limited number of functions. For cytoplasmic proteins, such site organization is typical for cytoskeleton proteins and proteins that regulate translation . It is pIn the nucleus, the PHF10-Ss and PHF10-Sl isoforms are also phosphorylated on X-cluster serines. Thus, which isoforms, PHF10-P or PHF10-S, that are incorporated into the complex can determine the interactions of the whole complex with DNA and histones, and the complex functioning during chromatin remodeling.2. HEK293 cells were lysed in Lysis Buffer: 10 mM HEPES (pH 7.9) containing 5 mM MgCl, 0.5% Nonidet P-40, 0.45 M NaCl, 1 mM DTT, a protease inhibitor cocktail (PIC) (Roche) and a 1% Phosphatase inhibitor cocktail 3 (PhIC) (Sigma-Aldrich). The lysate was centrifuged at 10,000 rpm, 4°C, for 10 min, and the supernatant was diluted fourfold with the same buffer but without NaCl. The extract was treated with DNAse I  and RNase .HEK293 cells were grown in DMEM medium supplemented with 10% FBS (HyClone), 2 mM L-glutamine at 37°C, 5% CO2, 1 mM DTT; PIC; PhIC) on the ice, ground in Loose Dounce, centrifuged for 1 min and the supernatant was employed as the cytoplasmic fraction. The pellet was resuspended in Lysis Buffer, ground in Tight Dounce, incubated for 10 min on ice, centrifuged as mentioned above and diluted the same way. The probes were equalized using Qubit Protein Assay Kit (Thermo Fisher Scientific), mixed with 4X LB , and boiled for 10°C.Cells were lysed in FLB buffer , X-cluster serine mutants and NLS-3 mutant forms were cloned in pET22b vector and then recombinant 6His-linker domain proteins expressed in Escherichia coli and purified in Ni-NTA Sepharose . For the in vitro kinase experiments, 1 µg 6His-linker domain or the same amount of mutant forms were incubated with 100 µl of HEK293 extracts in the presence of gamma-31[P]-labeled ATP at 37°C. Then the samples were processed for autoradiography or western blot as specified.eviously . For in Mutations of serines into alanines were performed on the base of constructs described previously . SubstitFor immunoblot analysis, we used polyclonal antibodies against PHF10 described in , 2016 di


Ictidomys tridecemlineatus) experience dramatic changes in physiological and molecular parameters during winter hibernation. Notably, these animals experience reduced blood circulation during torpor, which can put numerous stresses on their hearts. The present study evaluates the role played by the epidermal growth factor receptor (EGFR) in signal transduction during hibernation at low body temperature to evaluate signaling mechanisms. By investigating the regulation of intracellular mitogen activated protein kinase (MAPK) pathway responses, anti-apoptosis signals, downstream transcription factors, and heat shock proteins in cardiac muscle we aim to determine the correlation between upstream tyrosine phosphorylation events and downstream outcomes.Thirteen-lined ground squirrels  also increased strongly during the early arousal stage with p-p38 levels also rising during prolonged torpor. However, of downstream MAPK effector kinases that were measured, only p-Elk-1 levels changed showing a decrease during interbout arousal (IA). Apoptosis markers revealed a strong reduction of the pro-apoptotic p-BAD protein during entrance into torpor that remained suppressed through torpor and IA. However, active caspase-9 protein rose strongly during IA. Levels of p-AKT were suppressed during the transition phases into and out of torpor. Of four heat shock proteins assessed, only HSP27 protein levels changed significantly (a 40% decrease) during torpor.I. tridecemlineatus has natural mechanisms of tissue protection during hibernation that is largely due to cellular regulation through phosphorylation-mediated signaling cascade. We identify a possible link between EGFR and MAPK signaling via p-ERK, p-p38, and p-JNK in the cardiac muscle of these hibernating mammals that correlates with an apparent reduction in caspase-9 apoptotic signaling. This reveals a piece of the mechanism behind how these mammals are resilient to cardiac stresses during hibernation that would otherwise be damaging.We show evidence of EGFR phosphorylation correlating to activation of MAPK signaling and downstream p-ELK1 suppression during hibernation. We also demonstrate a reduction in p-BAD mediated pro-apoptotic signaling during hibernation with active caspase-9 protein levels increasing only during IA.  Mammals that hibernate are an example of nature’s ability to adapt to environmental challenges. In the face of winter stresses, including low ambient temperatures and little or no access to food, hibernators have developed strategies to survive by greatly reducing their normal energy needs, entering a torpid state and letting body temperature (Tb) fall to near-ambient (often as low as 0–5 °C). Hibernation is characterized by cyclical periods of torpor  and brief periods of arousal  when animals rewarm to euthermia (~37 °C). By reducing their metabolic rate during torpor to levels as low as 2–4% of euthermic values and relying on large reserves of body fat accumulated during summer feeding, hibernators can survive for many months without eating . The strIctidomys tridecemlineatus) over their torpor/arousal cycle. Coordination of cytoprotective responses would surely be a necessity to achieve appropriate pro-survival actions  and cell preservation processes  during cold torpor  is a cell-surface site for the integration and transduction of signals from multiple types of stimuli  and is known to be an important signaling center in many pathological states such as diabetes, cancer, and cardiovascular dysfunction . EGFR isd torpor .Signaling through receptor tyrosine kinases at the plasma membrane typically promotes increased metabolism and cell growth. During hibernation, it is important that these receptors be poised to mediate a reduction in metabolic activity while retaining readiness to respond rapidly during arousal from torpor. Signal transduction cascades and the control that they exert over transcription factors have been shown to contribute to the hibernating phenotype by providing a mechanism to sense the external environment and alter gene expression programs accordingly . EGFR isI. tridecemlineatus. The hearts of hibernators must naturally adjust to low temperatures and to the reduced perfusion that would be considered ischemic during euthermia or in non-hibernating species , weighing 150–300 g, were wild-captured by a United States Department of Agriculture-licensed trapper  and transported to the NIH. Animal housing and experimental procedures followed the guidelines set by the NINDS ACUC. Animals were individually housed in shoebox cages at 21 °C and fitted with a sensor chip  injected subcutaneously while anesthetized with 5% isofluorane. Animals were fed standard rodent diet and water ad libitum until they gained sufficient lipid stores to enter hibernation. To enable a natural transition into torpor, animals were transferred to an environmental chamber at ∼5 °C in constant darkness. Tb, time and respiration rate were monitored and used to determine sampling points. All animals had been through a series of torpor–arousal bouts prior to sampling. Tissue samples were shipped to Carleton University on dry ice. Tissues were stored at −80 °C until use. EC designates “euthermic control” animals, these euthermic squirrels had a stable Tb (∼37 °C) in the 5 °C cold room, could enter torpor but had not done so in the past 72 h; EN designates “entrance” into hibernation with a falling Tb of 18–31 °C; LT indicates “long term” torpor with a stable Tb at 5–8 °C for at least 5 days; EA designates “early arousal” from torpor with a rising Tb of 9–12 °C, and IA designates the “interbout arousal” where animals were fully aroused for >18 h with Tb restored to 37 °C  in heart muscle of thirteen-lined ground squirrels at specific time points. The multiplex EGFR profiler kit was used to determine the relative phosphorylation levels of EGFR in response to hibernation at the following residues: pT654, pT669, pY845, PY1045, pS1047, pY1068, and pY1086. The 10-Plex MAPK/SAPK Signaling Panel (Cat#48-660MAG) was used to determine phosphorylated protein levels of specific MAPK/SAPK signaling factors. Targets evaluated include MEK1 (Ser221), ERK/MAP Kinase 1/2 (Thr185/Tyr187), p38 (Thr180/Tyr182), JNK (Thr183/Tyr185). The 7-plex early apoptosis magnetic bead panel was used to determine active apoptosis components that include p-JNK (Thr183/Tyr185), p-BAD (Ser112), p-Akt (pS473), and active caspase 9 (Asp315). The 5-plex heat shock protein magnetic bead panel (Cat#48-615MAG) was used to measure the total protein levels of HSP27, p-HSP27(S78), p-HSP27(S78/s82), HSP60, HSP70, and HSP90alpha.Luminexg for 4 min and supernatants were collected as total soluble protein lysates. Protein concentration was determined using the Bradford assay with the Bio-Rad prepared reagent and then further diluted to an appropriate concentration using assay buffer. The protocol for multiplex analysis followed manufacturer’s directions as described in ® instrument with xPonent software .Positive and negative controls provided by the manufacturer were used with each assay panel and were prepared according to the manufacturer’s instructions. Extracts of frozen tissue samples were prepared as per manufacturer’s instructions . BrieflyTotal protein extraction from tissue samples, SDS-polyacrylamide gel electrophoresis, protein transfer to PVDF membranes, and immunoblotting was performed as described previously . MembranN = 3–4 independent samples from different animals) and were analyzed using a one-way ANOVA and a post hoc Tukey test (SigmaPlot 12 software). Differences between means were considered significant at p < 0.05.Bead-based assays used the net median fluorescence intensity of a population of measurements (with a minimum bead count of 50 with subtraction of background wells) to determine relative protein levels. Immunoblot protein bands were visualized using a ChemiGenius Bio Imaging System  and quantified by densitometric analysis with the associated GeneTools Software . Immunoblot band density was standardized against the summed intensity of a group of Coomassie-stained protein bands from the same sample lane, the latter chosen based on their lack of variation between experimental conditions and because they were not located close to the protein band of interest. This method of standardization was employed due to the lack of change in the expression of most proteins found in hibernator tissues across the torpor–arousal cycle. Data are expressed as mean ± SEM , entry into hibernation with a falling Tb of 18–31 °C (EN), LT torpor with a stable Tb at 5–8 °C for at least 5 days (LT), EA from torpor with a rising Tb of 9–12 °C (EA), and fully aroused for >18 h with Tb restored to 37 °C (IA) .Of the seven EGFR phosphorylation sites that were assessed, four showed significant changes as compared with EC controls over the course of the torpor arousal cycle: pT654, pY1045, pY1068, and pY1086. Phosphorylation on T654 showed an increasing trend throughout hibernation and peaked at 1.3-fold higher in EA, compared with EC before dropping by ∼50% after animals were fully aroused back to euthermia . At Y104Phosphorylated protein levels of several members of the MAPK-signaling pathway, representing the three main MAPK groups , also changed significantly in ground squirrel heart over the course of a hibernation bout . PhosphoTranscriptional activity is regulated by posttranslational modifications, such as protein phosphorylation, at several distinct sites on transcription factors. MAPKs are a class of protein kinases that can phosphorylate transcription factors. Known transcription factors targets of MAPKs (and the sites that they phosphorylate) include p-CREB (Ser133), p-ELK1 (Ser 383), p-c-Jun (Ser73), p-ATF2 (Thr 71), and p-p53 (Ser15). These are either directly phosphorylated by MAPKs or by MAPK phosphorylation of an intermediate kinases . Hence, we assessed phosphorylated protein levels of five well-known stress-activated transcription factors in cardiac muscle over the course of hibernation using luminex and immunoblotting . OverallExtracellular signaling through EGFR can also lead to the regulation of anti-apoptotic signals mediated through the action of the AKT or JNK protein kinases or by the proteins BAD (Bcl-2-associated death promoter) and caspase-9. Relative levels of these four proteins were assessed for four stages of the torpor–arousal cycle  . A cruciMitogen activated protein kinase action can also affect the expression of heat shock proteins that are pivotal to the stress response. The present study was designed to further our understanding of the molecular regulation of heart survival during hibernation at low Tb in thirteen-lined ground squirrels. EGFR signaling is known as a molecular integration site in pathological states such as cardiovascular dysfunction and cancer and the inhibition of this receptor has become a common treatment in the management of the latter . This trThe activation of EGFR generates tyrosine-phosphorylated recognition motifs for the binding of signaling proteins containing SH2 domains. The data for EGFR phosphorylation  demonstrMitogen activated protein kinase signaling pathways are known to be crucial to the regulation of the cellular stress response and have been previously implicated in hibernation and torpor . InteresTo further assess the potential effects of EGFR induced signaling, this study investigated the downstream phosphorylation of some transcription factors, anti-apoptotic signals, and HSPs that are known to be involved in ischemia protection and hypertrophy. The phosphorylation of MSK1 (a downstream kinase) and selected transcription factors that are downstream of MAPK signaling  were assessed in heart over the five timepoints across the hibernation cycle. Interestingly, among these targets there was a change in the phosphorylation state of only one, ELK1, that was strongly suppressed during IA . ELK1 isAkt is typically a downstream kinase responding to insulin and regulates various anabolic processes such as cellular growth. It has been shown to play an important role in other ground squirrel tissues during hibernation and is required for heart growth . In cardHeat shock proteins represent essential targets of MAPK pathways; for example, HSP27 is a terminal substrate of the p38 pathway and is known to be dephosphorylated during ischemia . In ischIctidomys tridecemlineatus has natural mechanisms of transcriptional regulation during hibernation through changes in the phosphorylation of receptor tyrosine kinases such as EGFR and their downstream signaling pathways. The current data suggest that the phosphorylation of MAPKs, such as ERK1/2, act as potential mediators of upstream EGFR signaling during hibernation. Further investigation also revealed a trend toward reduced apoptotic signaling since p-BAD protein levels were greatly suppressed during torpor and caspase nine levels only increased significantly during IA. Thus, the phosphorylation on EGFR sites, that can activate MAPK signals during early and IA, followed by the decrease in EGFR phosphorylation on sites that promote receptor internalization during late torpor, provides a possible upstream mechanism for this MAPK signaling. The subsequent inhibition of apoptosis through phosphorylation cascades and MAPK signaling in hibernator heart seems to agree, if ultimately opposite to data for other tissues  making EGFR phosphorylation a notable addition to the mechanism of cardiac protection during hibernation.10.7717/peerj.7587/supp-1Supplemental Information 1Data values used in study.Click here for additional data file.10.7717/peerj.7587/supp-2Supplemental Information 2Full Blot for pCreb EC EN LT time points. Box represents quantified bands.Click here for additional data file.10.7717/peerj.7587/supp-3Supplemental Information 3Full Blot for pCreb EC EA IA time points. Box represents quantified bands.Click here for additional data file.10.7717/peerj.7587/supp-4Supplemental Information 4Full Blot for pELK1 EA IA LT time points. Arrow indicates quantified bands.Click here for additional data file.10.7717/peerj.7587/supp-5Supplemental Information 5Full Blot for pELK1 EC EN LT time points. Arrow indicates quantified bands.Click here for additional data file.


Generally, proteases in medicinal plants had different therapeutic effects such as anti-inflammatory effect; modulate the immune response and inhibitory effect toward tumor growth. In this study, protease was purified and characterized from miswak roots, as medicinal plant and natural toothbrush.2+ had strong inhibitory effect on enzyme activity compared with other metal ions. Kinetic of inhibition for determination the type of protease was studied. Iodoactamide and p-Hydroximercuribenzaoic acid (p-HMB) caused strong inhibitory effect on enzyme activity indicating the enzyme is cysteine protease.Physical and chemical characterization of cysteine protease P1 were studied such as pH optimum (6.5), optimum temperature (50 °C), thermal stability (50 °C) and Km (3.3 mg azocasein/ml). The enzyme digested some proteins in the order of caseine > haemoglobin > egg albumin >gelatin > bovine serum albumin. HgThe biochemical characterization of this enzyme will be display the suitable conditions for using of this enzyme in toothpaste in the future and the enzyme may be used in other applications. Euphorbia microsciadia [B. subtilis substantially dehaired cow skin [Proteases has been characterized from plants such as pea roots , ginger osciadia  and sweeosciadia . Plant Posciadia –8. Bromeosciadia . In humaosciadia , 11. Othosciadia . Papaya osciadia . The parcow skin .Salvadora persica), natural toothbrush, included salvadoside and salvadoraside [Chemical compositions of miswak  was used and the elution buffer was Tris-HCl pH 7.2 in presence of gradient of NaCl ranged from 0.0 to 0.3 M. The same buffer was used for gel filtration  in absence of NaCl.The molecular mass of purified protease was determined by two techniques, gel filtration and sodium dodecyl sulphate (SDS-PAGE) .2+, Ni2+, Co2+, Pb2+, Hg2+, Cu2+ and Zn2+) were studied. Kinetics of inhibition (phenylmethylsulfonyl fluoride (PMSF), 1,10 phenanthroline, ethylenediaminetetraacetic acid (EDTA), p-HMB and iodoacetamide) for determination the types of protease were carried out.Physical and chemical characterization of protease with respect to pH optimum (pH 4–9), optimum temperature (30–80 °C), thermal stability (30–80 °C), substrate specificity  and effect of metal ions  were eluted by gradient steps of NaCl at 0.0, 0.1 and 0.2 M, respectively  on the activity of the protease P1 was detected  were detected for cysteine proteases from horse gram , Curcumama longa , Euphorb–45 kDa w nivulia .The substrate specificity of protease P1 showed that the enzyme acted on caseine, haemoglobin, egg albumin, gelatin and bovine serum albumin. Similar digestion was detected for horse gram cysteine protease toward the substrates . The Km Euphorbia microsciadia was detected at 40 °C and 45 °C, respectively [The maximum activity of protease P1 was detected at pH 6.5. However, horse gram cysteine protease showed higher activity at pH 5.5 . The aciectively , 28.2+ was strong indicated that the protease P1 is cystein protease. This agreed with cysteine proteases from Euphorbia microsciadia and horse gram which inhibited by Hg2+ [p-HMB and iodoacetamide caused strong inhibitory effect on the activity of the protease P1 indicating the enzyme is cysteine protease. Similar inhibitory effects were detected for cysteine proteases from Euphorbia microsciadia [All metal examined caused partial inhibitory effect on the protease P1. However, the inhibitory effect of Hg by Hg2+ , 28. Theosciadia , horse gosciadia .From the above findings the miswak protease P1, as cysteine protease, could be used in toothpaste for oral hygiene. However, Pleszczyńska et al.  studied 2+. Depending on cysteine proteases such as papain are used in oral gel, miswak protease P1may be digested the protein residues in the oral when the miswak is used as natural toothbrush. The biochemical characterization of this enzyme will be display the suitable conditions for using this enzyme in toothpaste in the future and the enzyme may be used in other applications.The study indicated that the purified miswak protease P1 is cysteine protease depending on the study of the inhibition by cysteine protease inhibitors and Hg