The Last Interglacial  was a period of warmer global mean temperatures and higher and more variable sea levels than the Holocene . Therefore, a better understanding of Antarctic ice-sheet dynamics during this interval would provide valuable insights for projecting sea-level change in future warming scenarios. Here we present a high-resolution record constraining ice-sheet changes in the Wilkes Subglacial Basin (WSB) of East Antarctica during the LIG, based on analysis of sediment provenance and an ice melt proxy in a marine sediment core retrieved from the Wilkes Land margin. Our sedimentary records, together with existing ice-core records, reveal dynamic fluctuations of the ice sheet in the WSB, with thinning, melting, and potentially retreat leading to ice loss during both early and late stages of the LIG. We suggest that such changes along the East Antarctic Ice Sheet margin may have contributed to fluctuating global sea levels during the LIG. Sedimentary records from offshore of the East Antarctic Ice Sheet support two episodes of ice loss during the last interglacial period (130-115 ka), leading to elevated global sea levels under global mean temperatures similar to the present day. These elevated sea levels are attributed to a combination of ocean thermal expansion, melting of mountain glaciers, and mass loss from the Greenland and Antarctic ice sheets, with at least some contribution from Antarctica probably being required12. Furthermore, in order to explain the multiple sea-level peaks during the LIG, multiple events of ice-sheet mass loss must have occurred. However, detailed records of ice-sheet behaviour during the LIG are lacking, limiting our ability to independently test the proposed global mean sea-level fluctuations or to resolve their geographic origin. The East Antarctic Ice Sheet stores freshwater with a sea-level equivalent of ~53 m, of which ~19 m resides in marine-based subglacial basins, including the Wilkes, Aurora, and Recovery basins13. Because those marine-based sectors are considered to be vulnerable to ocean warming14, even small oceanographic changes in those regions could have contributed substantially to past fluctuations in global sea level. Whether ice in the marine-based subglacial basins of East Antarctica partially melted during the LIG is critical for understanding future sea-level changes14, but is not currently resolved by global sea-level records or by local sea-level fingerprints from this interval15. For this reason, a focus on sedimentary records from the East Antarctic margin is needed.Global mean sea levels during the Last Interglacial (LIG) are thought to have reached ~6–9 m higher than present, with some records suggesting several peaks within that interval16. However, when and to what extent the ice-sheet margin in the WSB retreated during the LIG compared to its present position remains a matter of debate. A recent study using sediment provenance tracing in marine sediment core U1361A 18. However, due to limitations in the chronologies and temporal resolution of existing records18, the timing and magnitude of ice-sheet change in the WSB during the LIG remain uncertain.The ice sheet in the Wilkes Subglacial Basin (WSB) Fig.  has receNd values, the deviation of 143Nd/144Nd ratios from the Chondritic Uniform Reservoir value in parts per 10,000, and provide evidence on the locus of subglacial erosion17 (see Methods). Authigenic Be isotope ratios are reported as 10Be/9Be ratios and are considered to be an indicator of meltwater inputs25 (see Methods). In addition, to evaluate the extent of ice-sheet margin retreat in the WSB, we estimated changes in elevation at Talos Dome during the LIG based on the reported oxygen isotope (δ18O) records in Antarctic ice cores22. The chronologies for marine sediment cores GC1407 and U1361A are tuned to the Antarctic Ice Core Chronology 2012 (AICC2012)27, based on the onset and end of the LIG (see Methods).To better constrain the ice-sheet behaviour in the WSB during the LIG, we generated high-resolution records (300-year resolution) of detrital neodymium (Nd) isotopes and iceberg rafted debris (IBRD), supported by authigenic beryllium (Be) isotopes, in marine sediment core GC1407  in core GC1407 shows relatively low εNd values (around −16 to −15) during the Holocene and relatively higher values with more variability (−17 to −13) during the LIG  are recognised within the LIG (129–126 ka and 122–118 ka)  Fig. , suggestka) Fig. . Given trds Fig. , the sim17.This interpretation is based upon the ultimate source of detrital sediment input to marine sequences on the Antarctic margin being from the erosion of proximal and/or upstream coastal bedrock36, which can be expected to change with advance or retreat of the ice-sheet margin. Furthermore, the εNd values of the bedrock vary by region30 , while coastal bedrock proximal to core U1361A  has εNd values of −12 to −18 30. In contrast, εNd values in the WSB, inland of George V Coast, are inferred to be more radiogenic (−4 to −7), reflecting the Permian-Cretaceous rocks of the Ferrar Large Igneous Province (FLIP) and associated Beacon Supergroup37. At present, detrital sediments are dominantly transported from the coastal margin of the WSB to the location of core U1361A17. Since the LIG was also an interglacial climate state, it can be assumed that the sediment transport regime during the LIG was broadly similar to the modern day. Therefore, the high εNd values in core U1361A during the LIG  . If retreat of the ice-sheet margin in the WSB enhanced the supply of sediments from inland FLIP sources, relatively high detrital εNd values would also be expected in core GC1407, whereas the supply of detritus from the Adelie Coast would result in a lowering of εNd values. As such, we suggest that the relatively low εNd values in core GC1407 compared to core U1361A (~4 εNd units offset) probably indicate continuous detrital input from the Adelie Coast through the Holocene and LIG st) Fig. , especiaLIG Fig. . On the  19 Fig. , which i10Be/9Be ratio in marine sediments proximal to ice-sheet margins can be used to infer regional inputs from ice melt25. The production of 10Be in the atmosphere is followed by its deposition onto the ocean and ice sheets, such that the melting of ice sheets and icebergs releases the accumulated 10Be into the ocean, where it is scavenged by particles and accumulates in the authigenic fraction of marine sediments23. In contrast, 9Be is the stable naturally-occurring isotope found in bedrock and supplied via erosion and weathering. An increase in the 10Be/9Be ratio in marine sediments can therefore be interpreted as indicating a significant ice melting event23 (see Methods).To provide further evidence on the nature and magnitude of ice-sheet margin changes in the WSB that are implied by the two peaks in the detrital Nd isotope record, we measured authigenic Be isotopes in core GC1407. Changes in the 10Be/9Be record in core GC1407 provides strong support for ice mass loss from the WSB during the LIG. The 10Be/9Be ratios vary from 6.0 × 10−9 to 9.1 × 10−9 during the LIG, with a two-stepped increase leading to maximum values at ~120 ka , which provides a qualitative indicator of iceberg dischargeing Fig. , which s18O records from the Talos Dome and EPICA Dome C (EDC) ice cores39. Differences in the δ18O records between the Talos Dome and EDC ice cores have been proposed to provide an indicator for the advance and retreat of the ice-sheet margin in the WSB22. Because the Talos Dome site is located in the headlands of the WSB outflow region, it is sensitive to changes of the ice-sheet margin in the WSB22, unlike inland plateau sites such as EDC , the δlel Fig. . In contiod Fig. 39. The i6 ka, the22, we estimate the change in elevation at Talos Dome during the LIG by applying three different δ18O-elevation relationships derived from climate simulations and observations 44. Based on those three different relationships, the 1.5‰ increase in δ18O values at Talos Dome is equivalent to a drop in local ice-sheet surface elevation of 111–283 m. Although their study focused on the early LIG, Sutter et al. (2020)19 presented a model in which the elevation drop of 100–200 m at Talos Dome could be related to an inland retreat of the ice margin in the WSB by several hundred km 19 21. However, our records reveal evidence for extensive ice mass loss from the WSB during the late LIG (~120 ka), when air temperatures were comparable to the present  when air temperature rise in Antarctica reached its peakent Fig. . Hence, 45 and by a southward-shifted Polar Front in the Indian Ocean46. Both these conditions could have contributed to enhanced ocean forcing of ice-sheet mass loss, with the frontal shifts potentially helping to drive incursions of relatively warm Circumpolar Deep Water onto the continental shelves, which could lead to inland migration of grounding lines48. Notably, deep-ocean temperatures in the Southern Ocean also remained at elevated values from the early to the late LIG49  in the Southern Ocean that were approximately 2 °C warmer than the pre-industrial HoloceneG49 Fig. , pointin 51 Fig. . Althoug9. Sea-level variability during the LIG remains uncertain, but three broad scenarios have been proposed: (1) a high-stand during the early LIG9; (2) a high-stand for the initial several thousand years followed by a secondary rise around 120 ka6; and (3) bimodal sea-level peaks during the early and late LIG4 5, then significant ice mass loss from other sectors of East Antarctica would still be required, after accounting for contributions from mass loss of the marine-based West Antarctic Ice Sheet (~3.3 m) and a portion of the Greenland Ice Sheet (likely up to ~2 m)55. Within East Antarctica, the ice sheets in the Aurora and Recovery Subglacial Basins   in 25% acetic acid  in a water bath at 85 °C for four hours. Opal was then leached using 1 M NaOH in a water bath at 80 °C for 30 min. After those procedures, the leached samples were rinsed in ultrapure water three times and dried in an oven (50 °C) for ~24 h. Then, ~60 mg samples were completely digested with HNO3-HF-HClO4 at 130 °C for at least one day.To measure detrital Nd isotopes in core GC1407, ~200 mg of the bulk fine-grained (<63 µm) sediment fraction was first leached by sequential leaching methodsChemical separation of Nd was carried out as follows: (i) major elements were removed from the sample solution using a cation exchange resin column  with 1.8 M HCl; (ii) the REE fraction was extracted using 4.5 M HCl eluent; and (iii) the Nd fraction was isolated using a Ln-spec resin column  with 0.25 M HCl eluent. All reagents were trace metal grade reagents  and all procedures were conducted in a clean laboratory at the University of Toyama.3 and ultrasonicated for 1 h prior to analysis using either a first generation (Nu Plasma) or second generation (Nu Plasma II) Nu Instruments HR MC-ICP-MS in the MAGIC laboratories at Imperial College London. The sample introduction system consisted of a Glass Expansion MicroMist  nebuliser  connected to a DSN-100 desolvation nebuliser system. Measurements consisted of 50 cycles. All Nd isotope ratios were corrected for instrumental mass bias using the exponential law and a 146Nd/144Nd ratio of 0.7219. The reported 143Nd/144Nd ratios were corrected to a JNdi-1 143Nd/144Nd ratio of 0.512115 (ref. 59) using bracketing standards. A correction for 144Sm interference was applied, but all samples fell significantly below the threshold for accurate correction of 144Sm on the 144Nd signal (i.e. <1000 ppm). The external reproducibility of sample data was estimated from the within-session standard deviation (2 s.d.) of the JNdi-1 standards, while its accuracy is demonstrated by repeat analyses of rock standard BCR-2, which yielded 143Nd/144Nd = 0.512641 ± 0.000015 (n = 16), in excellent agreement with the literature value60.Samples were then dissolved in 0.16 M or 0.1 M HNO61, carbonate and biogenic silica were removed from sediment samples by reacting with 1 M acetic acid for 24 h, followed by heating to 85 °C in 2 M sodium carbonate for 5 h, with samples being agitated several times during each step. Samples were then suspended in 0.2% sodium diphosphate decahydrate solution and placed on a rotating wheel before analysis using a laser diffraction-scattering particle size analyser (Horiba LA-920). Repeated analysis was performed on a subset of samples in an arbitrary order over several days and the mean standard deviation of replicate analyses was ±0.1 µm.In preparation for grain-size analysisNd values from −17 to −13 during the last glacial-interglacial cycle . Therefore, the long-term glacial-interglacial trends in Nd isotopes probably reflect changes in the relative contribution of detrital sources among contourites and turbidites.The detrital Nd isotope record in core GC1407 (<63 µm fraction) shows large variations in ε61, shows large glacial-interglacial variations, with finer grain size during glacials and coarser grain size during interglacials  (17. The 250 µm–2 mm fraction was weighed to obtain a weight percent of IBRD (percent 250 µm–2 mm) relative to the bulk sediment. For this study, we did not calculate IBRD mass accumulation rates, but they would scale almost directly with the weight percent IBRD .Samples were processed for grain size analysis at Hokkaido University to provide evidence on the IBRD content. Dried samples were weighed and then wet-sieved to recover the coarse sand fraction (percent 250 µm–2 mm), which has previously been used to indicate IBRD in Antarctic studies2O2 to remove the organic matter, followed by treatment with HCl solution to remove the calcium carbonate. The samples were then wet sieved (45 µm mesh size), after which two types of permanent slides were made from the residue to quantify the abundance (Q-slide) and for faunal analysis (F-slide)62. Briefly, to prepare the Q-slides, all residues were transferred to a 200 mL beaker containing 100 mL of distilled water. The solution was then well mixed, after which a 500 μL sample was taken from the suspension using a micropipette and dropped onto a glass slide. The sample was then dried and mounted with Norland optical adhesive. The F-slides were made from the remaining residues in the beaker and then mounted with Norland. On average, 100 individuals were counted on each slide, and the abundance of C. davisiana is reported in percent as the number per 100 individuals.Freeze-dried subsamples were weighed (approximately 1–2 g) and then treated with 15% H2 content was analysed by X-ray fluorescence (XRF) analysis of slab samples conducted at 1-cm intervals using the TATSCAN-F263 at Kochi University. Samples were wrapped in thin prolene film and scanned with a scanning diameter of 1 cm.The SiO64 and revised by ref. 65. Authigenic Be was extracted from 2 g dry samples by soaking them in 20 ml of leaching solution (0.04 M hydroxylamine hydrochloride and 25% acetic acid) at 95 ± 5 °C for 6 h. A 2 ml aliquot of the resulting leachate was sampled for measurement of the natural 9Be concentration using inductively coupled plasma mass spectrometry (ICP-MS). The remaining solution was spiked with 300 µl of 9Be carrier (9.833 × 10−4 g/g 9Be) before Be purification by chromatography in order to determine accurate 10Be concentrations using accelerator mass spectrometer (AMS) measurements of 10Be/9Be ratios at Micro Analysis Laboratory, Tandem accelerator, University of Tokyo (MALT)67. Sample 10Be concentrations were calculated from the 10Be/9Be ratios normalized to the KN standard with a nominal 10Be/9Be ratio of 8.56 × 10−12 (ref. 68), and the reported authigenic 10Be concentrations were decay-corrected using the 10Be half-life of 1.387 ± 0.012 Ma70. Uncertainties on the authigenic 10Be/9Be ratios were calculated by propagating the uncertainties on the ICP-MS and AMS measurements.Samples for Be isotope analysis were prepared at the National Institute of Polar Research according to the chemical procedure established by ref. 65, sea-ice extent, ice-shelf coverage71, and freshwater discharge from melting ice65. Cosmogenic 10Be is produced in the upper atmosphere by the interaction of cosmic rays and is globally homogenised, with an atmospheric residence time of 1–2 years, before being deposited as meteoric 10Be on the Earth surface72. Although cosmic rays also interact with surface rocks and produce in-situ 10Be, the production rates for this process are orders of magnitude lower than for meteoric 10Be. In contrast, stable 9Be occurs naturally as a trace component of crustal rocks and is transported by glacial erosion and weathering to the Antarctic coastal margin25. Therefore, 10Be/9Be ratios can be applied in Antarctic margin sediments to constrain sedimentary processes and paleoenvironments, including freshwater supply during events of large-scale ice-sheet retreat. Since using the 10Be/9Be ratio corrects for variations related to particle size, retention behaviour, and dilution, this ratio has been proposed as a more effective meltwater proxy than using 10Be concentrations alone25.Beryllium isotopes have been used as tracers in various fields of earth sciences, providing proxies for geomagnetic field strength10Be and 9Be concentrations in core GC1407 show similar long-term changes, with higher values during interglacial periods (Holocene and LIG) and lower values during glacial periods (Marine Isotope Stages 2 and 6)  during the LIG73. Hence, the 10Be and 10Be/9Be variations in core GC1407 during the LIG likely reflect variations in the excess supply of 10Be to this region via freshwater input from melting ice. Rapid scavenging by particles in the upper water column would transport the 10Be to depth, enabling it to accumulate in the authigenic fraction of the marine sediments. Although other factors such as the scavenging of 10Be by biogenic opal could potentially influence the deep-ocean sedimentary record of 10Be, the SiO2 content in core GC1407 does not show significant fluctuations during the LIG , we use the authigenic 10Be/9Be record in core GC1407 as an indicator of regional freshwater inputs from melting ice.Authigenic 18O records have been extensively applied to determine chronologies for marine sediment sequences, but benthic foraminifera are not continuously preserved in cores GC1407 or U1361A. Therefore, we have identified the interval of the LIG in cores GC1407 and U1361A based on records of C. davisiana and productivity that have previously been used to determine chronologies in the Southern Ocean .Benthic foraminiferal δC. davisiana stratigraphy between cores GC1407 and RC11-120 , since the chronology of RC11-120 has been well constrained using its planktonic foraminiferal (Globigerina bulloides) δ18O record77 .First, we identified the interval of the LIG in core GC1407 based on the correlation of 17 and GC1407. In the Southern Ocean, the glacial-interglacial productivity pattern depends on latitude, with low and high productivity during glacial and interglacial periods, respectively, in the high latitudes of the Indian sector of the Southern Ocean75. Given the proximity of cores GC1407 and U1361A, their productivity patterns are expected to be similar through time. Indeed, the SiO2 content in core GC1407 and Ba/Al ratios in core U1361A (proposed as a productivity proxy in the study area17) show similar patterns on orbital to millennial timescales, including during the LIG interval  in the Southern Ocean and air temperature proxies (δD or δ18O) in the Antarctic ice cores can be used to tune the age scales of marine sediment cores, including during the LIG when there is a strong similarity between the marine and ice core records79. However, SST records are not available for cores GC1407 or U1361A. Therefore, the planktonic foraminiferal δ18O record in core RC11-120, which is taken as an indicator of SST, and the δD record from the Dome Fuji ice core80 were compared in order to transfer the age of core RC11-120 onto the AICC2012 age scale, using three tie points  Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary datasets 1-6


However, during the storage, it was revealed that there was a slight decline in constituents concentration reasoned to oxygen exposure and interaction with other food ingredients. Similarly, for total phenolic content, the highest amount was found in T3 as 545.38 ± 4.34 mg GAE/100 g. The quality attributes of prepared ice cream treatments were also found acceptable till the end of the study (60 days). Conclusively, the addition of microencapsulated anthocyanins powder in ice cream proved to stabilize black carrot anthocyanins and contributed positively to the sensory characteristics of ice cream.The present study was conducted to investigate the effect of storage on quality attributes of microencapsulated black carrot anthocyanins‐enriched ice cream. Purposely, black carrot anthocyanins were obtained using ethanolic extraction. Later on, extracts were acidified and microencapsulated with gum arabic and maltodextrin (1:1). Results showed that anthocyanin contents for T The present research demonstrated the potential of gum arabic and maltodextrin for the production of microencapsulated anthocyanins powder for further utilization in ice cream. The resultant encapsulates showed significant potential to stabilize the anthocyanins during processing and storage of ice cream. Conclusively, the addition of microencapsulated anthocyanins powder in ice cream proved to stabilize black carrot anthocyanins and contributed positively to the sensory characteristics of ice cream. It is largely cultivated and consumed in Pakistan. Its cultivars are divided into two groups; carotene or western carrots (Daucus carota ssp. sativus var. sativus) and anthocyanin or eastern carrots (Daucus carota ssp. sativus var. atrorubens Alef). Although orange‐colored carrots are more consumed, recently there is a growing trend toward consumption of black carrots (Daucus carota L.) owing to its higher anthocyanin contents and astonishing antioxidant potential  or micro‐ (50 nm to 2 mm) particulates. It supplies the phytochemicals effectively, masks the undesirable flavor of core material, and avoids the destruction of bioactive components  purchased from Merck  were provided by a local supplier.2.2Black carrots were washed and cleaned in order to remove adherent dust, dirt, and foreign materials in the Functional and Nutraceutical Food Research Section, National Institute of Food Science and Technology (NIFSAT), University of Agriculture, Faisalabad. Afterward, black carrots were stored at refrigeration temperature (4–6°C) until further use.2.32O , containing 0.01% HCl (37% v/v), and left with stirring at room temperature (25°C) for 2 h. Afterward, the liquid phase was separated and the extract was filtered through a 0.45 μm polytetrafluoroethylene (PTFE) membrane, followed by ethanol evaporation under vacuum. The extraction was carried out in triplicate.One hundred grams of black carrots were peeled and finely chopped. The sample was subjected to extraction with 100 mL EtOH:H2.4The resulting extract was purified by liquid–liquid extraction with a solvent mixture containing chloroform (50 mL), pentane (50 mL), and cyclohexane (50 mL), followed by concentration of aqueous phase via vacuum rotary evaporator at a temperature of 30°C and stored at 4°C in a dark place.2.5Microencapsulation of anthocyanins was carried out using maltodextrin and gum arabic by freeze drying following the prescribed method of Khazaei et al. . Wall ma2.62.6.1The pH of the ice cream was measured using a digital pH meter . All measurements were done in triplicate. The pH meter was first calibrated using buffer solutions of pH 4, 7, and 10. The ice cream sample was taken into beaker and adjusted to room temperature. Electrode of pH meter was immersed in ice cream sample and reading was recorded after stabilization of pH meter reading.2.6.2The acidity of samples was determined by titration method AOAC, . Acidity2.6.3The Overrun of ice cream was measured by a procedure outlined by El‐Samahy et al. . The met2.6.4Meltdown enables an assessment to be made of the firmness of ice cream at the time of consumption. The rate of melting of all samples of ice cream was calculated according to the volume of liquid which drained off from 25 to 30 g of ice cream samples and placed on a sieve with 2 mm openings for 10 min at 25 ± 2°C  were measured according to the following equation:Monomeric anthocyanin contents  in black carrot extract was measured using the Folin–Ciocalteu method   of the prepared ice cream was measured by Texture Analyzer  using the protocol of Aime et al. .2.6.9The products' surface color, L (lightness), a* , and b* , was measured according to the method of Ngadi et al. .2.7p < .05.All the data were subjected to analysis of variance (ANOVA) analysis using Statistix 8.1 software. The results were concluded significant at 3Frozen food items are widely consumed across the world for its taste and convenience. Addition of bioactive ingredients and natural colors in such foods can result in the formulation of a better‐accepted product. The physicochemical analyses of microencapsulated anthocyanins powder‐enriched ice creams were carried out consisting of pH, acidity, overrun, meltdown, specific weight, anthocyanins, total phenolic content, viscosity, firmness, and color. In this respect, the results regarding each trait are discussed as follows:3.1The effect of treatments and storage on pH of the microencapsulated anthocyanins‐enriched (MAE) ice cream was found significant. As evident from the average values (means) Table , there wpH has a direct influence on flavor perception in dairy products. According to Homayouni et al. , normal 3.23 (9% microencapsulated anthocyanins powder‐enriched ice cream) as 0.20 ± 0.01%, followed by T2 (6% microencapsulated anthocyanins powder‐enriched ice cream) and T1 (3% microencapsulated anthocyanins powder‐enriched ice cream) as 0.18 ± 0.01 and 0.16 ± 0.01%, respectively. However, the minimum value for the attribute was observed in T0 (control) as 0.15 ± 0.01%. An increase in acidity among treatments may be due to the addition of microencapsulated anthocyanins powder in ice creams. Over the storage, there was observed a non‐momentous increase in titratable acidity of ice creams which ranged between 0.17 ± 0.01 and 0.18 ± 0.01% from initiation to the termination of the research trial. The recorded values on the 15th and 45th days of storage were also 0.17 ± 0.01 and 0.17 ± 0.01%, respectively. The possible reason behind the increase in acidity during storage is the decline in pH. Among the treatments, the maximum decline in titratable acidity was observed in T0, T1, and T3 ranging from 0.15 ± 0.01, 0.16 ± 0.02, and 0.19 ± 0.01 at day 0, to 0.16 ± 0.01, 0.17 ± 0.01, and 0.2 ± 0.01% after 60 days of storage. However, the minimum drop off in titratable acidity was recorded in T2 as 0.18 ± 0.01 to 0.18 ± 0.0% from 0 to 60 days of storage.Table Acidity is the measure of the amount of acids in any food sample. Ideal acidity of ice cream is 0.198%  as 53.37 ± 1.60%, followed by T2 (6% microencapsulated anthocyanins powder‐enriched ice cream) and T1 (3% microencapsulated anthocyanins powder‐enriched ice cream), that is, 50.72 ± 3.04 and 47.17 ± 1.89%, respectively. However, the minimum value for the trait was recorded in T0 (control) as 44.34 ± 2.22%.The mean values for overrun (%) of enriched ice creams are exhibited in Table Increase in the volume of the ice cream with respect to the volume of the initial mix, incorporating a certain amount of air into mix, and expansion due to freezing is called overrun. Overrun is largely influenced by stabilizer, emulsifier, fat content, and processing conditions  as 29.19 ± 1.75%, followed by T1 (3% microencapsulated anthocyanins powder‐enriched ice cream) and T2 (6% microencapsulated anthocyanins powder‐enriched ice cream), that is, 27.50 ± 1.10 and 25.62 ± 1.28%, respectively. However, the minimum value for the trait was recorded in T3 (9% microencapsulated anthocyanins powder‐enriched ice cream) as 22.53 ± 0.68%.Tremendous variations took place in ice cream due to the addition of various concentrations of microencapsulated anthocyanins powder. The mean values for meltdown (%) of enriched ice creams are exhibited in Table Meltdown is one of the important properties of frozen dairy products. It is influenced by various factors including the amount of air incorporated, ice crystals, and networking of fat globules  as 2.62 ± 0.04 kg/gallon, followed by T1 (3% microencapsulated anthocyanins powder‐enriched ice cream) and T2 (6% microencapsulated anthocyanins powder‐enriched ice cream); that is, 2.57 ± 0.03 and 2.51 ± 0.05 kg/gallon, respectively. However, the minimum value for the trait was recorded in T3 (9% microencapsulated anthocyanins powder‐enriched ice cream) as 2.46 ± 0.03 kg/gallon.The mean values for the specific weight  of enriched ice creams are exhibited in Table The findings of the present study research are in general agreement with the earlier work of El‐Samahy et al. , who eva3.61 (3% microencapsulated anthocyanins powder‐enriched ice cream) as 43.16 ± 1.17 mg/100 g, followed by T2 (6% microencapsulated anthocyanins powder‐enriched ice cream) as 92.63 ± 1.17 mg/100 g. However, the maximum value for the TPC was observed in T3 (9% microencapsulated anthocyanins powder‐enriched ice cream) as 143.21 ± 1.14 mg/100 g. The inclining trend in the case of anthocyanins in T1, T2, and T3 is probably related to the inclusion of microencapsulated anthocyanins powder into the ice creams. Over the storage, it is evident from the table that the storage was affected differently with regards to anthocyanin contents of ice cream. Values regarding this trait were 69.81 ± 0.85 mg/100 g at 0 day while a gradual increase was noted on the 15th days of storage as 72.3 ± 1.09 mg/100 g. However, a clear decline was observed on 45th and 60th days of storage . A possible reason behind the decline in storage may be due to the exposure to oxygen and interaction with other food ingredients. Among the treatments, the same trend was recorded for the trait, that is, 143.64 ± 1.05, 92.28 ± 1.16, and 43.31 ± 1.21 mg/100 g at the initiation of the research trial and 146.98 ± 1.64, 95.43 ± 1.24, and 46.8 ± 1.47 mg/100 g at 15th day in T3, T2, and T1, respectively, while at the 60th day of storage, the values were 139.57 ± 1.05, 89.64 ± 0.96, and 39.53 ± 1.17 mg/100 g in T3, T2, and T1, respectively. However, no anthocyanins were recorded in T0 as evident from Table For microencapsulated anthocyanins powder‐enriched ice cream, the means regarding anthocyanin contents are given in Table The findings of the current study are in general accordance with the earlier work of Mazur et al.  who chec3.73 (9% microencapsulated anthocyanins powder‐enriched ice cream) as depicted by the mean value, that is, 545.38 ± 4.34 mg GAE/100 g, followed by T2 (6% microencapsulated anthocyanins powder‐enriched ice cream) as 352.76 ± 4.47 mg GAE/100 g. Likewise, for T1 (3% microencapsulated anthocyanins powder‐enriched ice cream), the mean value was 164.38 ± 4.45 mg GAE/100 g. However, there was no measured total phenolic content in T0 (control). The incline in TPC of T1, T2, and T3 seems to be due to the elevated levels of anthocyanins in enriched ice creams. Regarding the storage, it is evident from the table that the storage affected differently the total phenolic content of ice creams. A significant increase was recorded on the 15th day of storage; that is, the values noted on day 0 were 265.85 ± 3.24 mg GAE/100 g, while 275.35 ± 4.15 mg GAE/100 g on 15th day. However, a gradual decline in PPC was observed on 60th day of storage equal to 255.86 ± 3.04 mg GAE/100 g. Moreover, a reason for the decline in TPC over storage could possibly be due to the decrease in anthocyanin contents owed to environmental factors. Among the treatments, the same trend was observed in T1, T2, and T3 as 164.95 ± 4.6, 351.43 ± 4.4, and 547.02 ± 3.98 mg GAE/100 g at the initiation of the research trial, while 178.24 ± 5.61, 363.43 ± 4.72, and 559.74 ± 6.26 mg GAE/100 g were the TPC values on the 15th day of storage and 150.55 ± 4.47, 341.38 ± 3.67, and 531.52 ± 4.01 mg GAE/100 g at the termination of the study. Moreover, there were no measured anthocyanins content in T0 at any time during storage.From the mean values, it can be inferred that there were significant variations in total phenolic content of the treatments as shown in Table The results of the present study are in line with those of Rababah et al.  who repo3.83 (9% microencapsulated anthocyanins powder‐enriched ice cream) as 75.81 ± 2.253 cP, followed by T2 (6% microencapsulated anthocyanins powder‐enriched ice cream) as 68.88 ± 2.008 cP. Likewise, the average value for the trait was observed as 63.81 ± 1.715 cP for T1 (3% microencapsulated anthocyanins powder‐enriched ice cream). However, the minimum value for the attribute was recorded in T0 (control) as 58.76 ± 4.072 cP. The increase in viscosity for T1, T2, and T3 is attributed to the addition of microencapsulated anthocyanins powder in ice creams. Over the storage, there was observed a momentous decrease in viscosity of ice cream which ranged between 82.68 ± 2.784 and 52.03 ± 2.074 cP from initiation to the termination of the research trial. The recorded values on the 15th, 30th, and 45th days of storage were 74.16 ± 2.414, 66.26 ± 3.112, and 58.95 ± 2.176 cP, respectively. However, the storage decrease in viscosity may be due to the decrease in firmness. Among the treatments, the maximum decline in viscosity was observed in T0 and T1 ranging from 75.73 ± 6.985 and 80.23 ± 1.014 cP on day 0 to 43.16 ± 2.296 and 48.03 ± 1.814 cP after 60th day of storage, followed by T2 as 84.42 ± 0.586 to 54.31 ± 2.44 cP from initiation to termination day. However, the minimum drop in viscosity was recorded in T3 as 90.35 ± 2.55 to 62.60 ± 1.745 cP from day 0 to day 60 of storage.From the average values shown in Table Viscosity is a factor to measure resistance to flow usually in centipoise (cP). The factors affecting viscosity of ice cream are temperature, fat globule size, type, and amount of stabilizer. It also contributes to the mouthfeel and flavor of ice cream  as 4.11 ± 0.058 N, followed by T2 (6% microencapsulated anthocyanins powder‐enriched ice cream) and T1 (3% microencapsulated anthocyanins powder‐enriched ice cream) as 3.81 ± 0.061 and 3.49 ± 0.047 N. However, a minimum value for firmness was recorded in T0 (Control) as 3.08 ± 0.017 N. Increment in firmness of T1, T2, and T3 is attributed to the stabilizing effect of the enrichment of ice creams with microencapsulated anthocyanins powder that contains maltodextrin and gum arabic as wall materials. Over the storage, a steady decline in firmness was observed ranging from 3.72 ± 0.047 N on day 0 to 3.53 ± 0.045 N after 60 days of storage. The possible reason for this trend in firmness is due to the retrogradation of starch molecules and dextrin degradation. Similarly, a clear demotion in firmness values in T3 was also noticed ranging from 4.22 ± .06 to 4 ± 0.056 N on day 0 and day 60 of storage, respectively. Minimum values for firmness were recorded in T0 from 3.15 ± 0.018 to 2.99 ± 0.017 N.The mean values of firmness of enriched ice creams are exhibited in Table The firmness of ice cream is measured as the resistance of ice cream to deformation when an external force is applied. Both the ice crystal size and ice phase volume contribute to the firmness of ice cream. This factor may be used as a measure of ice crystal growth. Ice cream resistance to mechanical force provided by the tongue, teeth, and upper palate will give the overall perception of firmness  as evident from the mean (95.59 ± 1.31). Among the treatments, the decrease in L* values in T1 (3% microencapsulated anthocyanins powder‐enriched ice cream) and T2 (6% microencapsulated anthocyanins powder‐enriched ice cream) was monitored as 52.60 ± 1.05 and 44.05 ± 1.28, respectively. However, a minor increase was observed in T3 (9% microencapsulated anthocyanins powder‐enriched ice cream) at 45.19 ± 1.34. Over the storage, there were noted significant differences in the mean values of the L color parameter, there was a decrease in L* values as 60.03 ± 1.20 on day 0 and 59.76 ± 1.23 on the 15th day of storage, while a trivial increase was recorded on the 30th day of storage. However, a gradual decrease was observed on the 45th and 60th days of storage at 58.96 ± 1.18 and 58.06 ± 1.44, respectively. Among the treatments, the minimum decrease was observed in T0 ranging from 95.90 ± 1.68 to 94.61 ± 0.67 from initiation to the termination of the research trial during storage. A maximum decrease was recorded in T2 , while a gradual decrease in L* value was observed in T1 and T3 as 53.06 ± 1.08 to 51.76 ± 1.54 and 46.07 ± 0.97 to 43.63 ± 1.58 at initiation and termination of the research trial during storage, respectively. The findings of the present study are in general agreement with those of Patras et al.  as 32.19 ± 1.2, followed by T2 (6% microencapsulated anthocyanins powder‐enriched ice cream) and T1 (3% microencapsulated anthocyanins powder‐enriched ice cream) as 29.25 ± 1.2 and 28.19 ± 1.08, respectively. However, the lower a* values were observed in T0 (control) . Over the storage, there were recorded significant differences in the mean values for this color parameter. Table 1, T2, and T3, respectively. The mean values for T0 at the beginning to the end of the research trial were −1.04 ± 0.44 to −2.78 ± 0.7, respectively. The earlier studies of Kaur et al.  as evident from the mean . Among the treatments, the decrease in T2 (6% microencapsulated anthocyanins powder‐enriched ice cream) and T0 (control) was measured as 8.37 ± 1.04 and 6.51 ± 1.04, respectively. The minimum b * values were recorded in T1 (3% microencapsulated anthocyanins powder‐enriched ice cream) . During the storage, there was observed a steady decline in b* values as 8.11 ± 0.99 on day 0, followed by 7.80 ± 1.0 on 15th day, 7.04 ± 1.07 on 45th day, and 6.71 ± 1.20 on 60th day of storage. Among the treatments, a steady decline was recorded for T0 ranging from 7.16 ± 1.09 on day 0 to 5.90 ± 1.27 on day 60 of storage. Similarly, the b* values for T1, T2, and T3 were 5.14 ± 0.97 to 3.81 ± 1.50, 9.08 ± 0.95 to 7.67 ± 1.12, and 11.05 ± 0.96 to 9.46 ± 0.89 at initiation to termination of storage, respectively. The results of the current study are in concordance with those of Patras et al.  as 33.81 ± 1.24, followed by T2 (6% microencapsulated anthocyanins powder‐enriched ice cream) and T1 (3% microencapsulated anthocyanins powder‐enriched ice cream) as 30.44 ± 1.11 and 28.56 ± 1.11, respectively. However, the minimum value for chroma was observed in T0 (control) as 6.85 ± 1.06. Over the storage, there was observed a momentous decrease in chroma of ice cream which ranged between 25.82 ± 1.14 and 23.98 ± 1.10 from initiation to the termination of the research trial. The recorded values on the 15th and 45th days of storage were 25.42 ± 0.90 and 24.51 ± 1.20, respectively. Among the treatments, the maximum decline in chroma was observed in T3 ranging from 34.97 ± 1.29 on day 0 to 32.60 ± 0.81 on day 60 of storage. A steady decrease in chroma values was recorded in T1 and T2 as 29.67 ± 1.06 and 31.39 ± 1.07 at initiation to 27.44 ± 1.15 and 29.35 ± 1.04 on termination day. However, the minimum decrease in chroma was recorded in T0 as 7.24 ± 1.13 to 6.53 ± 1.40 from day 0 to day 60 of storage. The current findings are in general agreement with the preceding work of Patras et al.  and T1 (3% microencapsulated anthocyanins powder‐enriched ice cream) as 107.10 ± 3.46 and 8.95 ± 2.52, respectively. However, a clear demotion for hue angle was recorded in T3 (9% microencapsulated anthocyanins powder‐enriched ice cream) and T2 (6% microencapsulated anthocyanins powder‐enriched ice cream) as 17.74 ± 1.29 and 15.98 ± 2.14, respectively. Over the storage, there was observed a significant difference in the mean values for this attribute. Table 0 ranging between 98.09 ± 2.82z and 115.31 ± 3.57, respectively. However, the minimum values were recorded for T3 as 18.40 ± 0.97 to 16.89 ± 1.76 from initiation to the termination of the research trial, respectively. The findings of the current study are in accordance with the preceding work of Mazur et al. ; formal analysis ; methodology ; software ; writing – original draft . Iahtisham Ul Haq: Formal analysis ; project administration ; supervision ; writing – original draft ; writing – review and editing . Masood Butt: Investigation ; methodology ; project administration ; supervision ; writing – original draft . Gulzar Ahmad Nayik: Conceptualization ; data curation ; methodology ; writing – review and editing . Sami Al Obaid: Formal analysis ; funding acquisition ; investigation ; project administration ; resources . Mohammad Javed Ansari: Methodology ; validation ; visualization ; writing – review and editing . Ioannis Konstantinos Karabagias: Formal analysis ; investigation ; methodology ; writing – review and editing . Nazmul Sarwar: Formal analysis ; resources . Seema Ramniwas: Data curation ; formal analysis ; investigation .The authors declare that they have no conflict of interest regarding the publication of this manuscript.


Non-canonical EphA2 activation plays a key role in tumor progression; however, its activation mechanism remains unclear. In the present study, we focused on cellular stress signaling as a novel inducer of non-canonical EphA2 activation. p38, instead of ERK in the case of epidermal growth factor signaling, activated RSK-EphA2 under cellular stress conditions, including anisomycin, cisplatin, and high osmotic stress. Notably, p38 activated the RSK-EphA2 axis via downstream MAPK-activated protein kinase 2 (MK2). Furthermore, MK2 directly phosphorylated both RSK1 Ser-380 and RSK2 Ser-386, critical residues for the activation of their N-terminal kinases, which is consistent with the result showing that the C-terminal kinase domain of RSK1 was dispensable for MK2-mediated EphA2 phosphorylation. Moreover, the p38-MK2-RSK-EphA2 axis promoted glioblastoma cell migration induced by temozolomide, a chemotherapeutic agent for the treatment of glioblastoma patients. Collectively, the present results reveal a novel molecular mechanism for non-canonical EphA2 activation under stress conditions in the tumor microenvironment.The receptor tyrosine kinase ephrin type-A receptor 2 (EphA2) is overexpressed in malignant tumors. We previously reported that non-canonical EphA2 phosphorylation at Ser-897 was catalyzed by p90 ribosomal S6 kinase (RSK)  Ephrin type-A receptor 2 (EphA2), a member of the receptor tyrosine kinase family, plays a critical role in modulating intercellular junctions by associating with its membrane-bound Ephrin ligands in normal epithelial cells , 4, 5. Lvia phosphorylation at Thr-573. CTK-mediated phosphorylation at Ser-380 in the linker region between CTKD and NTKD provides a docking site for 3-phosphoinositide-dependent kinase 1 (PDK1), which phosphorylates NTKD at Ser-221 and causes NTK-mediated downstream signaling. The phosphorylation of Thr-732 by NTK, in turn, dissociates ERK, which eventually inactivates NTK via the dephosphorylation of Ser-380 and Ser-221 (RSK consists of four isoforms (RSK1-4) and has a unique structure harboring two distinct kinase domains: the carboxyl-terminal kinase (CTK) domain (CTKD) and amino-terminal kinase (NTK) domain (NTKD) , 12, 13. Ser-221 . A largevia the canonical ERK-RSK pathway upon stimulation with epidermal growth factor (EGF) (We previously reported that EphA2 at Ser-897 was phosphorylated or (EGF) , 15; howA). Anisomycin, a protein synthesis inhibitor that potentially activates p38, also promoted EphA2 phosphorylation despite no significant activation of ERK. The RSK inhibitor BI-D1870 blocked both EGF- and anisomycin-induced EphA2 phosphorylation (B), whereas the p38 inhibitor SB203580 selectively inhibited the response to anisomycin. Other p38 inhibitors with different inhibition profiles exerted similar suppressive effects (C). In addition, the knockdown of p38α, the main isoform in HeLa cells, attenuated EphA2 phosphorylation (D). The platinum-based drug cisplatin (CDDP) or high osmotic stress with 300 mM sodium chloride also induced p38-mediated EphA2 phosphorylation (E). Collectively, our results demonstrated the critical involvement of p38 in the non-canonical phosphorylation of EphA2 under cellular stress conditions.To investigate whether p38 is involved in EphA2 phosphorylation at Ser-897, HeLa cells were stimulated with EGF or anisomycin. As previously reported A. Anisomrylation B, wherea effects C. In addrylation D. The plrylation E. CollecB); therefore, we investigated whether p38 activated RSK via phosphorylation at Ser-380. Anisomycin-induced RSK activation was clearly blocked by SB203580 or the knockdown of p38α (A and B). In HEK293 cells transfected with RSK1 and kinase-dead EphA2 (EphA2-KD), only the weak phosphorylation of EphA2 was detected, whereas the phosphorylation of both RSK1 and EphA2 was strongly enhanced by the co-expression with constitutively active p38α (p38α-CA (C). In contrast, kinase-dead p38α (p38α-KD) did not induce the phosphorylation of RSK1 or EphA2, suggesting that p38 activity was essential for RSK1-mediated EphA2 phosphorylation . The phosphorylation of EphA2-KD indicated that its tyrosine kinase activity was not required for p38-mediated Ser-897 phosphorylation. These results showed that p38α mediated the stress-induced non-canonical RSK-EphA2 signaling pathway.The anisomycin-induced phosphorylation of EphA2 was regulated by RSK B; theref of p38α , A and B(p38α-CA ) Fig. 2B; therefB, left). The conserved sequence of p38α substrates is pSer/Thr-Pro . As shown in Figure 3A, both EGF and anisomycin induced a shift-up band in the pMK2 blot (red arrow). The shift-up band of pMK2 arises from the use of alternative translation start sites in the mRNA, indicating the activation of MK2 (A). MK2 inhibitor or the knockdown of MK2 blocked the anisomycin- but not EGF-induced phosphorylation of RSK and EphA2 (A and B and S3B). Furthermore, wild-type (WT) MK2, but not kinase-dead MK2, strongly induced the phosphorylation of RSK and EphA2 in HEK293 cells (C). Moreover, MK2 inhibitor III blocked the CDDP- or osmotic stress-induced phosphorylation of RSK and EphA2 (D and S3C). The results of in vitro kinase assays showed that recombinant active MK2 catalyzed the phosphorylation of both RSK1 Ser-380 and RSK2 Ser-386 (E). These results demonstrated that MK2 activated by p38 directly catalyzed RSK phosphorylation, which resulted in the non-canonical activation of EphA2.To investigate the relationship between p38 and RSK, we compared the RSK1 amino acid sequence around Ser-380 and the consensus substrate recognition sequence of p38α B, left. /Thr-Pro ; however/Thr-Pro , 19, 20./Thr-Pro B, right.n of MK2 . Becausen of MK2 A. MK2 in93 cells C. Moreovnd EphA2 D and S3C Ser-386 E. These 2+-Phos-tag immunoblot analysis to compare phosphorylation states upon stimulation with EGF or anisomycin. The bandshift of a targeted protein reflects the state of phosphorylation: number and position . Upon the EGF stimulation, all bands completely shifted to positions (a), (b), and (c). In contrast, a modest bandshift was detected with the anisomycin stimulation, in which there were small, but distinct bands at positions (b) and (e). The main bands were still present at the original position (d); however, the lowest band had largely disappeared. These results indicated that the number of phosphorylated RSK proteins induced by anisomycin was less than that by EGF. The bandshift pattern of RSK2 was similar to that of RSK1, indicating the similar regulation of RSK1 and RSK2. In addition, the Ser-380 phosphorylation of RSK1, an indicator of NTK activation, was detected at positions (a) and (b), but not at position (c), (d), or (e) (B). On the other hand, phosphorylation at Thr-573, an indicator of CTK activation, was only detected at position (a) (B). Most importantly, MK2 inhibitor III blocked the anisomycin-induced bandshift to position (b) containing phosphorylated Ser-380 but did not affect the EGF-induced bandshift (C and D). Therefore, EGF induced RSK1 phosphorylation at the residues containing Ser-380 with/without Thr-573 (positions (a) and (b)). In contrast, anisomycin induced phosphorylation at Ser-380 without Thr-573 (position (b)). Collectively, these results suggested that CTK and NTK were both activated by EGF, whereas only NTK was activated by MK2.To further investigate the activation mechanisms of RSKs, we employed a Znposition , 23. As , or (e) B. On thetion (a) B. Most iandshift , C and DA, a constitutively active (CA) mitogen-activated protein kinase 1 (MEK1) (B), demonstrates that MK2-induced RSK1 phosphorylation at Ser-380 in a CTK-independent manner. This is consistent with the result showing that p38-MK2 signaling promoted the CTKm-mediated phosphorylation of EphA2 (C). Furthermore, CTKm with an additional Ser-380 to Ala substitution (SA) failed to promote EphA2 phosphorylation (D).To confirm that CTK was not necessary, HEK293 cells were transfected with WT RSK1 or a CTK-dead mutant (CTKm). As shown in Figure 51 (MEK1)  induced 1 (MEK1) B, demonsof EphA2 C. Furtherylation D.Figure E). EphA2 was not phosphorylated in the SA mutant expressed cells. In addition, the PDK1 inhibitor GSK2334470 inhibited EphA2 phosphorylation in both EGF- and anisomycin-treated cells (F), indicating that Ser-221 phosphorylation by PDK1 is a common event to the NTK activation via both the MK2 and ERK signaling pathways.EGF-induced NTK activation is dependent on the phosphorylation of Ser-221 which is catalyzed by PDK1. To determine if Ser-221 phosphorylation is involved in the MK2-promoted activation of RSK-EphA2 axis, HEK293 cells were transfected with the RSK1-CTKm with Ser-221-to-Ala substitution (SA) mutant E. EphA2 ed cells F, indicavia PDK1 is essential for both typical and atypical NTK activation (G).Taken together, MEK-ERK signaling typically induced the sequential activation of two kinase domains of RSK1: CTK activation by ERK-mediated phosphorylation at Thr-573 followed by NTK activation by CTK-mediated autophosphorylation at Ser-380. On the other hand, MK2 directly phosphorylated RSK1 at Ser-380 without CTK activation to induce atypical NTK activation in the stress-induced non-canonical activation of EphA2. Ser-221 phosphorylation tivation G.A and B). Moreover, the RSK inhibitor BI-D1870 abolished the non-canonical activation of EphA2 via the p38-MK2 axis to suppress cell migration (C and D). These results demonstrated the p38-MK2 signaling and the RSK-EphA2 signaling are acting in a linear pathway to promote cell migration.The RSK-EphA2 pathway is known to promote cell migration . Here, wigration , C and DA and S4A). TMZ-induced cell migration was inhibited by the knockdown of EphA2 using small interfering RNA (siRNA) (B). RSK inhibitors blocked EphA2 phosphorylation but not HSP27 phosphorylation, whereas MK2 inhibitors blocked both EphA2 and HSP27 phosphorylation (C and D and S4B). In addition, both RSK and MK2 inhibitors suppressed cell migration (E and F and C–E). These results demonstrated that the p38-MK2-RSK-EphA2 pathway regulated cell migration under stress conditions.Next, we used human glioblastoma U87-MG cells because treatment with the alkylating chemotherapeutic agent temozolomide (TMZ) provokes cellular stress to induce p38 activation as well as the migratory ability . TMZ actFigs. 7 (siRNA) B. RSK inIn the present study, we identified a novel molecular mechanism of non-canonical EphA2 activation . We and via the MEK-ERK pathway regulates the malignant progression of various tumors, including cancer cell motility, EMT, stemness properties, and drug resistance . The conserved sequence of MSK1 substrates is Arg-X-pSer; however, the corresponding amino acid of EphA2 near Ser-897 is Leu-Pro-pSer, suggesting that MSK does not catalyze EphA2 phosphorylation. Of note, it also does not match the RSK1 amino acid sequence around Ser-380 (B). Taken together, MSK is not involved in the p38-activated MK2-RSK-EphA2 signaling.Under stress conditions, p38 is known to activate not only MK2 but also mitogen- and stress-activated kinase (MSK). MSK belongs to the AGC subfamily of protein kinases along with RSK. Interestingly, other AGC subfamily protein kinases AKT and PKC have also been reported to catalyze EphA2 phosphorylation at Ser-897 A. The co Ser-380 B. Taken MK2 controls various cellular events, including inflammatory responses, cell motility, the cell cycle, and apoptosis, by regulating mRNA stability and protein expression , 29. Somet al.  in B cells, T cells, and fibroblasts; however, MK2 was dispensable for the activation of RSK. In the present study, we found that anisomycin, CDDP, and high osmotic stress selectively promoted p38-MK2-mediated atypical RSK activation in HeLa cells (D). In contrast, similar to the findings reported by Zaru et al., only the typical activation of RSK was induced by EGF and TPA, possibly due to the earlier and stronger activation of ERK than anisomycin  and phospho-specific antibodies against EphA2 , RSK , RSK Thr-573; #9346), p38 , ERK , and MK2 ) were cultured in Eagle’s Minimum Essential medium (Nissui) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5% CO2. Before drug treatment, the culture medium of HeLa cells was changed to DMEM with 0.5% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin, and then cells were incubated at 37 °C in 5% CO2 for 16 to 24 h.HeLa and HEK293 cells (ATCC) were cultured in Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum (Merck KGaA), 2 mM L-glutamine (Thermo Fisher Scientific), 100 U/ml penicillin , and 100 μg/ml streptomycin  at 37 °C in 5% COWhole-cell lysates, prepared as previously described , were re2+ Phos-tag SDS-PAGE was performed according to a previously reported procedure  or Hokkaido System Science. Target sequences were as follows: 5′-GCAUUACAACCAGACAGUUGAUAUU-3′ (p38α), 5′-CCAGUAUGAAUUUCCCAACCCAGAA-3′ (MK2), 5′-UGGAGUCCAUCAAGAUGCAGCAGUA-3′ (EphA2 #1), 5′-GCAAGGAAGUGGUACUGCUGGACUU-3′ (EphA2 #2) and 5′-UAAUGUACUGCGCGUGGAGAGGAA-3′ (negative control). HeLa cells were transfected with siRNAs at a final concentration of 20 to 100 nM using Lipofectamine RNAiMAX (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions.The expression vector for the human kinase-dead (KD) mutant EphA2 was provided by Dr Haruhiko Sugimura  , 46. The2, 0.2 mM ATP, 2 mM DTT, 20 mM β-glycerophosphate, and 0.1 mM sodium orthovanadate. After stopping the reaction with the addition of 30 μl of SDS–PAGE sample buffer, immunoblotting was performed as described above.The RSK1 and RSK2 proteins were obtained by immunoprecipitation. Whole-cell lysates were described earlier. Lysates were incubated with anti-RSK1 or RSK2 antibodies at 4 °C overnight and then rotated with Dynabeads protein G (Thermo Fisher Scientific) at 4 °C for 1.5 h. Beads were washed three times with PBS containing 0.1% Tween 20. Recombinant human GST-MK2 (50 ng) was then added and reacted at 30 °C for 30 min in 30 ml of reaction buffer containing 20 mM HEPES (pH 7.6), 20 mM MgClp < 0.05 were considered to be significant. Similar results were obtained in at least three independent experiments.Assay was performed as described previously . BrieflyAll data are contained in this article.This article contains The authors declare that they have no conflicts of interest with the contents of this article.


Pseudomonas aeruginosa with difficult-to-treat resistance has been designated as an urgent or serious threat by the CDC in the United States; therefore, novel antibacterial drugs and combination strategies are urgently needed. The sensor kinase RoxS is necessary for the aerobic growth of Pseudomonas aeruginosa. This study aimed to screen candidate RoxS inhibitors and evaluate their efficacy in treating multi-drug-resistant and extensively drug-resistant Pseudomonas aeruginosa in combination with meropenem and amikacin to identify promising combination strategies. RoxS protein structures were constructed using homology modeling and potential RoxS inhibitors, including Ezetimibe, Deferasirox, and Posaconazole, were screened from the FDA-approved ZINC drug database using molecular docking and molecular dynamics simulations. MIC and checkerboard assays were used to determine the in vitro antimicrobial efficacy of the three drugs in combination with antibiotics. The results of in vitro experiments showed an additive effect of 100 μg/mL Deferasirox or 16 μg/mL Posaconazole in combination with meropenem and a synergistic effect of 1.5 μg/mL Deferasirox and amikacin. In summary, these three drugs are potential inhibitors of RoxS, and their combination with meropenem or amikacin is expected to reverse the resistance of P. aeruginosa, providing new combination strategies for the treatment of clinically difficult-to-treat Pseudomonas aeruginosa. Pseudomonas aeruginosa (P. aeruginosa) is a rod-shaped, aerobic, Gram-negative bacterium that is an important causative agent of clinically acquired infections [P. aeruginosa infections in critically ill and immunocompromised patients is increasing [P. aeruginosa isolates from intensive care units, 32.9% were MDR and 24.9% were XDR [P. aeruginosa infections, however, the resistance of P. aeruginosa to these drugs increases every year [P. aeruginosa is a global challenge [P. aeruginosa; therefore, it is necessary to develop novel antimicrobial drugs and therapeutic strategies.fections . The inccreasing ,3,4,5. Acreasing ,7. Multicreasing . A prospery year ,11. Therhallenge . There iP. aeruginosa, known as the two-component system (TCS), consists of paired sensors and response regulators [P. aeruginosa are regulated by this mechanism, including motility, metabolic processes, and biofilm formation [P. aeruginosa has at least five terminal oxidases involved in aerobic respiration. P. aeruginosa adapts to different environmental conditions by regulating the expression of multiple oxidases and anaerobic energy metabolism [P. aeruginosa biofilm growth and virulence [P. aeruginosa, and its overexpression significantly inhibits the aerobic growth of the bacteria [P. aeruginosa and a regulator of terminal oxidases that represses the expression of Aa3 oxidase and activates the expression of the other four terminal oxidases. Abnormal expression of RoxSR may interfere with the aerobic respiration of bacteria and reduce their adaptability to unfavorable environments, thereby inhibiting bacterial growth. Azide compounds have an inhibitory effect on bacteria; Comolli JC et al. observed that P. aeruginosa strains lacking RoxS were less resistant to azide than the wild type [P. aeruginosa was unable to grow under 500 uM NaN3 conditions, unlike wild-type PAO1 [A class of regulatory proteins in gulators . Severalormation . Therefoormation . P. aerutabolism . Jo J etirulence ; howeverbacteria . RoxSR iild type . In addiype PAO1 . TherefoP. aeruginosa [Since 2008, our group has been dedicated to reversing drug resistance in ruginosa ,22,23. Jruginosa . RoxSR, ruginosa ,26. RoxSA total of 50 templates were searched using the Swiss-Model, and six models were built . The seqP. aeruginosa  and 351 (93.9%), respectively. The structure predicted by AlphaFold was reasonable. The Z-score of Model 1 predicted by I-TASSER was −3.34 and was at the edge of the graph area plotted by Z-score values. The Z-score of the protein predicted by Alphafold was −5.76, which is within the graph area plotted by the Z-score values of known proteins, and its energy was reasonable. Combining the results of the three evaluation systems, ERRAT, PROCHECK, and PROSA , Model 1P. aeruginosa and the RegBA system of Rhodobacter capsulatus are homologous proteins with high sequence identity and functional similarity [P. aeruginosa. The DeepSite of PlayMolecule software was used to predict the three possible docking pocket center axes for the RoxS protein , DSX , and PCZ  all had satisfactory binding free energies, which are better than the binding free energy of the homologous protein inhibitor coenzyme Q1  in complex with RoxS. The stability of the binding mode was further verified.P. aeruginosa were sputum culture isolates obtained from the Laboratory Department of The Third Affiliated Hospital of Southern Medical University. According to drug sensitivity reports and the Latin American consensus, one strain was MDR P. aeruginosa and the other was XDR P. aeruginosa [P. aeruginosa strains and the results are shown in P. aeruginosa was resistant to MEM, and XDR P. aeruginosa was severely resistant to AMK, according to the CLSI antibiotic threshold criteria [The two drug-resistant strains of ruginosa . The MICcriteria . None ofThe activities of MEM and AMK in combination with EB, DSX, and PCZ were evaluated using checkerboard assays. The results are shown in P. aeruginosa showed the combined effect of three drug candidates with MEM ((p = 0.03), which increased the inhibitory effect to 1.81 times that of MEM alone. In the checkerboard assays with DSX, MEM had an additive effect only in combination with 100 μg/mL DSX. The inhibition rate with 8 μg/mL of MEM alone was 19.73%, while the combined use of 100 μg/mL DSX increased the inhibition rate to 39.59% (p = 0.003), twice as effective as single-use. In a checkerboard test with PCZ, MEM in combination with 16 μg/mL PCZ showed the best inhibitory effect and reduced the MIC50. The combination of PCZ at 16 μg/mL increased the inhibition rate to 48.79% (p < 0.001), which was 1.99 times more effective than MEM alone. The trend was the same for higher concentrations of MEM in combination with PCZ.The results of MDR with MEM . In the P. aeruginosa (P. aeruginosa. This concentration of AMK in combination with 50 μg/mL EB increased the inhibition rate to 34.87% (p < 0.001). Approximately the same trend was seen in the results of 64 μg/mL and 128 μg/mL AMK in combination with EB; however, when the AMK concentration was 512 μg/mL, the effect was dramatically increased in combination with 6.25 μg/mL EB. In the checkerboard assays with DSX, our results showed that the combination of AMK with 0.75 μg/mL to 3 μg/mL of DSX significantly increased the inhibition rate, AMK combined with 1.5 μg/mL DSX has a synergistic effect; however, 6 μg/mL and 12 μg/mL of DSX in combination with AMK had an antagonistic effect. The bacterial inhibition rate of 16 μg/mL AMK alone was 7.36%, and the combination with 1.5 μg/mL DSX was optimal, increasing the inhibition rate to 35.84% (p = 0.006); this combination was 4.86 times more effective than single use. The effect of AMK at 64 μg/mL to 256 μg/mL combined with DSX showed the same trend as 16 μg/mL AMK. AMK at 32 μg/mL and 64 μg/mL in combination with DSX at 3 μg/mL had the best bacterial inhibitory effect. In the checkerboard assays of PCZ and AMK, the bacterial inhibition rate was −7.01% with 64 μg/mL AMK alone and 10.75% (p = 0.03) and 9.67% (p = 0.04) with the combination of 32 μg/mL and 128 μg/mL PCZ, respectively, however, the inhibitory effect was not significant. Only when 256 μg/mL of AMK was combined with PCZ the effect was seen. The inhibition rate of AMK alone was 17.42%, and the combination of PCZ with 32 μg/mL was the most effective, with the inhibition rate increased to 46.18% (p < 0.001), which was 2.65 times higher than that of the combination. Additionally, the combination of AMK with 64 μg/mL PCZ had an antagonistic effect.To examine the combined inhibition of the three drug candidates with AMK, the checkerboard assays were performed in XDR ruginosa . In the P. aeruginosa. Although EB did not have additive or synergistic effects with the antibiotics, it increased the rate of bacterial inhibition. In conclusion, all three compounds enhanced bacterial inhibition when combined with MEM and AMK. The combination of 100 μg/mL DSX and 16 μg/mL PCZ with MEM had additive effects, and the combination of 1.5 μg/mL DSX and AMK had synergistic effects, showing some potential for reversing resistance in P. aeruginosa presents a therapeutic challenge because of its inherently low susceptibility to a number of antibiotics and strong ability to develop antibiotic resistance [sistance . Combinasistance . Unfortusistance . Since Tsistance . RoxS isP. aeruginosa, 8 μg/mL MEM combined with 100 μg/mL EB increased the inhibition rate by 1.81 times that of antibiotics alone. In XDR P. aeruginosa, the combination of EB inhibited bacterial growth, while AMK had no inhibitory effect. There have been no reports of EB being used to treat P. aeruginosa infections; however, the combination therapy of EB with MEM or AMK may be an alternative therapeutic strategy for multi-drug-resistant P. aeruginosa infections in patients with preexisting hyperlipidemia to reduce the dose of antibiotics and side effects, while lowering blood lipid levels. EB is a potent inhibitor of cholesterol absorption and has been approved for the treatment of hypercholesterolemia ,37. EB hP. aeruginosa requires high levels of iron during infection [P. aeruginosa resistance to tobramycin and tigecycline [P. aeruginosa [DSX is an FDA-approved iron chelator for the treatment of iron overload that has been used clinically for over 40 years . P. aeruecycline . Thus, Druginosa . This stAspergillus fumigatus and P. aeruginosa [P. aeruginosa.Increased growth of fungal microbiota is a common side effect of antibiotic therapies. Prolonged use of antibiotics affects the growth of bacterial microbiota, which in turn causes secondary infections and leads to the proliferation of fungal microbiota . The airruginosa ,45. Mixeruginosa ,47. PCZ ruginosa . The resP. aeruginosa remained resistant to the drug combination treatment; however, the results of the present study are beneficial. The combination of 100 μg/mL DSX or 16 μg/mL PCZ with MEM had additive effects, whereas the combination of 1.5 μg/mL DSX and AMK had a synergistic effect. The combination therapy improved the inhibition of MDR and XDR P. aeruginosa. Even in cases where the antibiotics had no inhibitory effect, the combination showed some inhibition. This suggests that the selected combination strategy plays a role in reversing drug resistance in P. aeruginosa, and has great potential to fight infections in certain populations because any small or modest increase in antimicrobial activity with combination therapy may contribute to clinical success and recovery in actual clinical practice [The in vitro data showed that practice . P. aeruginosa causes pneumonia, AMK can be administered by nebulization to achieve high blood levels at the site of infection while avoiding the side effects of high-dose systemic administration [It is difficult to compare administered concentrations derived from in vitro studies with in vivo concentrations due to the influence of pharmacokinetics in humans; however, the results of the literature survey preliminarily indicate that the combination strategies screened in this study show some safety and clinical significance. The peak concentration in human plasma after injection of a standard dose of 1 g MEM is approximately 30 μg/mL . When MDstration . At a dostration . AMK is stration . The constration . The dosstration . The resP. aeruginosa but also in other bacteria homologous to P. aeruginosa, including Rhodobacter capsulatus, Syringae pseudomonas, and so on. The combination strategy explored in this study may also have potential efficacy in other bacterial infections, which merits further investigation.The limitations of this study were that potential RoxS inhibitors were only initially screened by computational techniques, as well as the limited number of strains selected for the experiments. This target still has a lot of room for exploration. We will increase samples of clinical strains and structurally modify three FDA drugs as lead compounds to explore compounds with stronger inhibitory effects. Through molecular dynamics simulations, we found that SER32 and ASN101 were important for the formation of stable conformations of the compounds. Although no interaction between these two amino acids and the ligand was found in the docking results, these two amino acids guided the conversion of the ligand to a stable conformation, providing a key idea for further modification of the compound. In addition, RoxS is present not only in https://swissmodel.expasy.org/, accessed on 16 July 2022), I-TASSER , and Alphafold  were utilized to design the three-dimensional structure of the proteins. The stereochemical quality, accuracy of the predicted models, and energy rationality were observed using PROCHECK , ERRAT, and PROSA in SAVES , respectively. Based on the evaluation scores, the best protein model with acceptable quality was selected.The RoxS protein sequences were retrieved from the NCBI database in the FASTA format. Swiss-Model .The predicted protein structure contained no ligand and could not generate a docking pocket from the self-containing ligand. In this study, a combination of computer predictions and homologous protein active sites was used to determine the docking pocket. The literature reported a conserved active site, GGXXNPF, in the evolution of RoxS, while further identifying the docking pocket in conjunction with the results of the prediction of the protein docking pocket in DeepSite in Playmolecule  was added. After incubation at 37 °C for 16–20 h, the OD600 nm of the 96-well plates was measured in an enzyme marker. And the inhibition rate was calculated (see 50). Three rows of experimental wells, one row of the positive control group and one row of the negative control group, were placed in each 96-well plate. FDA drugs require additional pure drug groups to exclude absorbance effects owing to their low solubility. The concentrations of DMSO and Tween 80 in the medium of the control group were the same as those in the experimental group. Each experiment was repeated at least thrice.The antimicrobial efficacy of the drugs was determined using the broth microdilution method of the Clinical and Laboratory Standards Institute (CLSI). On a sterile ultra-clean bench, 100 μL of MH broth was added to each well of the 96-well plate, followed by 100μL of drugs  were assessed using two-way analysis of variance (ANOVA), followed by Fisher’s least significant difference test for multiple comparisons.In each 96-well plate, the drug candidate and antibiotic were diluted in a multiplicative series  and combined in different concentrations to obtain 100 μL of mixed solution. Then, 100 μL of the bacterial suspension at a final concentration of 5 × 10The fractional inhibitory concentration index (FICi) was calculated as FIC of drug A (FICA) + FIC of drug B (FICB) , where FP. aeruginosa resistance and provide new options for the treatment of clinically MDR and XDR P. aeruginosa.In this study, potential RoxS inhibitors were identified by virtually screening FDA-approved drugs. Our results suggested that Ezetimibe, Deferasirox, and Posaconazole are potential inhibitors of RoxS; in addition, the results of the in vitro experiments showed that combinations of these three drugs with meropenem or amikacin are promising antibacterial strategies that are expected to reverse


We attribute the long-term acceleration near grounding line from 1963 to 2018 to basal melting likely induced by warm modified Circumpolar Deep Water. The speed up in shelf front during 1973–1989 was caused by a large calving front retreat. As the current trend continues, intensified monitoring in the TG region is recommended in the next decades.Wilkes Land and Totten Glacier (TG) in East Antarctica (EA) have been losing ice mass significantly since 1989. There is a lack of knowledge of long-term mass balance in the region which hinders the estimation of its contribution to global sea level rise. Here we show that this acceleration trend in TG has occurred since the 1960s. We reconstruct ice flow velocity fields of 1963–1989 in TG from the first-generation satellite images of ARGON and Landsat-1&4, and build a five decade-long record of ice dynamics. We find a persistent long-term ice discharge rate of 68 ± 1 Gt/y and an acceleration of 0.17 ± 0.02 Gt/y Historical velocity maps reveal over five decade-long acceleration and high-level discharge in Totten Glacier, East Antarctica, from 1963-2018, induced by warm modified Circumpolar Deep Water. This has been determined by estimates from altimetric, gravimetric, and optical and SAR imaging satellite observations, with the main mass loss from West Antarctica (WA) and the Antarctic Peninsula (AP)3. A reconciled solution shows a mass change rate with a large uncertainty (5 ± 46 Gt/y) in East Antarctica (EA)4. However, a recent finding indicates that Wilkes Land sets itself apart from this overall trend in EA by showing a high mass loss rate of 51 ± 13 Gt/y (refs. 6). Furthermore, studies7 have noted that the Totten Glacier (TG), fed by the largest drainage basin in Wilkes Land8, has undergone consistent mass loss since 1989, losing a total of ~175 Gt (or 0.5 mm SLR) at a rate of 7 ± 2 Gt/y from 1989 to 2015. Limited by the quality and availability of satellite data, there is generally a lack of mass balance estimates for the AIS and this region prior to 1989 (ref. 9). Consequently, the lack of long-term mass balance knowledge hinders our ability to understand earlier glaciological responses in EA and the entire AIS to climate change.The Antarctica Ice Sheet (AIS) has been losing ice mass and contributing to global sea level rise (SLR) at an accelerated pace over the last few decades10. Thus, the glacier is subject to marine ice sheet instability (MISI) and vulnerable to potential rapid ice sheet retreat due to the intrusion of the relatively warm modified Circumpolar Deep Water (mCDW) from the Southern Ocean15. With a catchment of ~570,000 km2 (ref. 12), the TG basin has the capacity to raise the global sea level by 3.9 m (ref. 16) if completely melted. Its ice shelf has been experiencing increased basal melting at a rate from ~9.1 m/y (1992–2007) (ref. 13) to ~18 m/y (2005–2011) (ref. 17). The grounding line retreated up to ~3 km during 1996–2013 (ref. 16). Between 1989 and 2015, the ice flow acceleration was found to be as large as 18% (refs. 18). However, a longer satellite observation record is needed to put the recent variability in a better context. Although recent satellite data of InSAR, altimetric and gravimetric sensors are not available before 1989, early optical images collected on films by the ARGON intelligence satellites during the 1960s (ref. 19) and the Landsat satellites in the 1970s and 1980s can be used to recover historical glacier topography and ice flow velocity fields21 with comparable uncertainties of earlier and recent velocity products23. Furthermore, these ice velocity data along with other supporting data will allow us to estimate mass balance during historical periods at both glacial and continental scales through the input–output (IO) method5.Totten Glacier is marine based, mostly grounded below sea level and the bedrock slopes downwards away from the oceanTo quantify the early state of ice dynamics and mass balance in the TG region, we developed and applied an innovative method of hierarchical network densification to systematically map Antarctic ice velocity from historical optical satellite images. Here, we used images in the TG region acquired by the first-generation satellites of ARGON and early Landsat missions in order to reconstruct the ice velocity fields of multiple periods from 1963 to 1989. We found ice flow acceleration and high-level ice discharge during the period that are linked to a large calving front retreat during 1973–1985 and increased basal melting of the ice shelf. Finally, using our historical estimates alongside recent results, we built a nearly 60-year record and proved that there has been a persistent, long-term ice discharge increase driven by continued ice shelf basal melting over 6 decades in this marine-based region, making TG the glacier with the highest mass loss in EA. We suggest that the current acceleration and mass loss process in the TG region had already started by the 1960s.23. Unique processing techniques for overcoming difficulties inherent in the historical images were developed and applied, including film deformation, large format lens distortion, and georeferencing errors exceeding 10 km (refs. 26). For instance, we adopted a semi-automatic algorithm for recognition and measurement of damaged fiducial marks on ARGON films; exterior orientation parameters were initially estimated from the ephemeris data and ground features, and then refined through a bundle adjustment procedure. Consequently, uncertainties of the produced velocity maps range from 4 m to 79 m  of velocity due to ice flow acceleration was found in long timespan velocity maps ont Fig. . The ave2 (12%). We further narrowed the retreat period to 1973–1985 using images of the Advanced Very High-Resolution Radiometer (AVHRR), during which the retreat may have resulted from a major calving event or a series of smaller calving activities initiated by numerous rifts32. This retreat is found to be the largest for the Totten Ice Shelf (TIS) since the region was photographed by the earliest ARGON satellite in 1963. In particular, a large portion of the lost area crossed the “passive shelf-ice” (PSI) boundary33 in the western shelf margin  caused velocity responses throughout the ice shelf34. Ice velocity on the ice shelf remained high in 1989, at least 4 years after the retreat, without significant changes from the 1973–1989 map , simulated with COREv2 forcing-39  and 17.9 ± 1.2 m/y from 2005 to 2011 (ref. 17). We reconstructed a velocity time series from that in Fig. R2 = 0.6 with the shelf front area change in Fig. 16, was induced by ice shelf basal melting caused by intrusion of mCDW from the continental shelf into the ice shelf cavity through the trough on the seafloor41. The combined long-term melt rates from 1960 to 2018 based on modeling and satellite observations suggest that ice shelf basal melting may have existed in TIS as early as 1960. Here we demonstrate that the long-term acceleration trend in the grounding region induced mainly by ice shelf basal melting  that is the long-term average SMB from 1979 to 2016 Fig. . Consequ42 and Williams et al.43 observed mCDW on the continental shelf adjacent to TIS in the austral summer of 1995–1996 and austral winter of 2007, respectively. Intrusion of the on-shelf warm mCDW water towards the TIS resulted in circulation of the cyclonic gyre and westward Antarctic Slope Current44, modulated by polynya activities near the calving front45. A major entrance, a ~5 km wide trough, which allows the mCDW water access to the ice shelf cavity, was found in the western part of the TIS calving front using inverted bathymetry from airborne gravity and magnetic data collected between 2008 and 2012 (ref. 8). The trough entrance was more precisely described, by shipboard bathymetry data acquired in January 2015, as being ~10 km wide, ~600 m deep, and at a depth of ~1097 m (maximum) from the sea surface41. Li et al.7 suggested that ice flow acceleration in the TG between 1989 and 2015 was linked to ocean temperature changes. Furthermore, Greene et al.14 and Gwyther et al.38 indicated that the troughs underneath the ice shelf provided effective pathways for the mCDW water to intrude into the cavity and grounding zone, causing increased ice shelf basal melting and thinning at a rate from ~9 m/y for 1992–2008 (refs. 46) to ~18 m/y for 2005–2011 (ref. 17).Bindoff et al.42), from the World Ocean Database (WOD) supported by the NOAA Climate and Global Change Program (https://www.ncei.noaa.gov/). Limited by fast ice and grounded icebergs, the CTD profiles were not deployed close to the shelf front. They reveal the presence of mCDW of relatively high temperature (>~0 °C) and salinity (>~34.5 PSU) at ~300–600 m depth, near the continental shelf break and in front of TIS  profiles (IDs 1–6) in an extended region Figure , mostly TIS Fig. , c. We fTIS Fig.  and showTIS Fig.  is primaTIS Fig.  where ic TG Fig. .Our ice shelf modeling results  and the bed topography, we suggest that the eastern lobe was at the beginning of the current retro-slope during our study period (1963–1989). The mCDW-induced grounding line retreat16 may have begun as early as the 1960s. We further suggest that the TG is currently experiencing a process that occurred earlier at Pine Island Glacier, another marine-based and rapidly changing glacier on the AIS55 where basal melting induced grounding line retreat and speedup occurred for several decades prior to sustained large scale shelf front retreat was observed since 201756. Analogously, since 1990, the observed TIS calving front positions have been changing modestly, mostly between the shelf fronts of 1973 and 1989  across the grounding line. The MB of the TG basin is finally estimated for each velocity map period by subtracting the discharge from the SMB  as the long-term reference SMB. We used the reference SMB to fill the data gap of 1963–1978 Fig. . SMB unc MB Fig.  are calc60 framework, which has modifications to include ice-ocean interaction (following refs. 62) using the three-equation parameterization63. The model domain was from 104.5°E–130°E with 1/15° resolution and 60°S–68°S with 1/30° resolution. Model bathymetry is based on RTopo64 but with the addition of the Totten ice shelf cavity including the western trough cavity entrance8. Lateral boundary forcing and wind forcing are from the COREv2 1949–2007 reanalysis dataset39, while surface heat and salt fluxes are composed of a combination of SSM/I sea ice formation observations65 with COREv2 evaporation minus precipitation fields. The combination allows for long-term estimates of polynya activity and sea ice formation. Further information is given in ref. 38.Basal melting 1960–2007. The TG is simulated with the Regional Ocean Modelling System66, a finite element model, to simulate the velocity response of the Totten glacier and ice shelf to calving front retreat. This model is created using velocity and geometry prior to 1973 973 Fig. . The mod973 Fig. . The fidmes Fig. . Finallymes Fig.  to test Supplementary InformationPeer Review File


Glioma is one of the most common primary brain tumors, and mortality due to this disease is second only to cardiovascular and cerebrovascular diseases. In traditional surgery, it is difficult to eradicate glioma; often recurrence increases its malignant degree, leading to a large number of patients killed by this disease. It is one of the most important subjects to study its pathogenesis and explore effective treatment methods. Research on glioma mechanisms mainly focuses on the effect of DNA methylation in epigenetics. Although there are many studies on protein phosphorylation, there is no overall regulatory mechanism. Protein phosphorylation regulates a variety of cell functions, such as cell growth, division and differentiation, and apoptosis. As a consequence, protein phosphorylation plays a leading part in various activities of glioma, and can also be used as a target to regulate the development of glioma. This review is aimed at studying the effect of protein phosphorylation on glioma, understanding the pathological mechanism, and an in‐depth analysis of it. The following is a discussion on glioma growth, migration and invasion, resistance and death in phosphorylation, and the possibility of treating glioma by phosphorylation. Glioma is a common primary brain tumor, and its mortality rate is extremely high. Protein phosphorylation regulates a number of cellular functions like cell growth, differentiation, apoptosis, and cell signaling, so it can also be used as a target to regulate the development of glioma. This review is a discussion on the growth, migration and invasion, resistance, and death of glioma in phosphorylation and the possibility of treating glioma by phosphorylation. Grade I is benign glioma. The second grade is between the benign and malignant boundary of the glioma. Grade III above belongs to malignant glioma. Malignant gliomas grow fast and lead to more severe brain edema. There is no exactly effective treatment for glioma. In spite of temozolomide (TMZ) chemotherapy, which increases long‐term survival, treatment failure, and rapid glioma recurrence remain common.1.2Phosphoric acidification of protein is very common. Protein phosphorylation is necessary for the initiation of many biological phenomena, including cell growth, proliferation, apoptosis, and so on, because phosphorylation can directly regulate various aspects of protein function.22.12.1.1KDM1A stabilization is related to the occurrence of glioma. Glycogen synthase kinase‐3β (GSK3β) phosphorylates KDM1A serine 683 (Ser683) upon priming by CK1α, leading to the stabilization of KDM1A, which, in turn, leads to cancer stem cell self‐renewal and GBM tumorigenesis.PGK1 threonine 243 (Thr243) by secreting interleukin‐6. In addition, inhibition of PGK1 T243 phosphorylation abolishes macrophage‐promoted tumorigenesis and glycolysis.PGK1 T243 may also be connected with the prognosis and malignancy of GBM. It has been confirmed that Atoh1 reduced the incidence of glioma and extended survival. Tyrosine 78 undergoes phosphorylation probably due to a Jak2‐mediated pathway. Phosphorylation of tyrosine 78 makes Atoh1 more stable and enhances the transcriptional activity of Atoh1.YTHDF2 protein by phosphorylating YTHDF2 Ser39 and Thr381 to promote tumorigenesis of GBM cells.KDM1A Ser683, PGK1 T243, Atoh1 tyrosine 78, YTHDF2 Ser39, and Thr381 can promote tumorigenesis, and if we can effectively control the phosphorylation of these proteins, we can prevent the development of glioma. Related literature is shown in Table The occurrence of glioma is the cause. If we control the occurrence, the glioma will decrease. 2.1.2SAE1 induces increased SUMOylation and Ser473 phosphorylation of AKT, which stimulates the growth of glioma cells in vitro and in a nude mouse tumor model.PFKP) S386 not only increased its expression but also promoted aerobic glycolysis and brain tumor growth.TRIP13 Y56 phosphorylation significantly reduced epidermal growth factor receptor (EGFR) signaling and GBM cell growth.TRIP13, all the above proteins were directly or indirectly related, especially ATK1, which was closely related to other proteins. Moreover, the above regulatory protein phosphorylation has positive significance for the size, morphology, and growth rate of glioma, so we can achieve the effect of limiting glioma growth by inhibiting the phosphorylation of one or more of the above proteins. Related literature is shown in Table After reading a lot of related literatures, I have listed a few briefly, from which we can find the relevance of phosphorylation and glioma growth. Phosphorylation has an influence on the size of glioma. A small‐molecule inhibitor (Lck‐I) was used to inhibit Lck phosphorylation, which blocked the phosphorylation of Paxillin and Crk‐II. Lck‐I could be used to treat human glioma stem cells, and it significantly inhibited self‐renewal, tumorsphere formation, and glioma size.2.1.3PFKP S386 phosphorylation could increase cell proliferation.PKMYT1 and WEE1 restrain cell cycle protein B‐CDK1 activity through the phosphorylation of CDK1‐Y15 and accelerate timely completion of mitosis in GBM stem‐like cells (GSCs).All cells can proliferate, hence we need to prevent the proliferation of glioma cells to slow the progression of the tumor. 2.2N‐acetylgalactosaminyltransferase 2 (GALNT2) was closely associated with the migration and invasion of glioma cells is that GALNT2 facilitated the malignant features of gliomas by affecting the O‐glycosylation, EGFR phosphorylation, and subsequently the downstream PI3K/Akt/mTOR axis.GALNT2 knockout decreases the level of phosphorylated EGFR, which could improve the malignant characteristics of glioma. Hence GALNT2 may serve as a potential target of action to treat glioma in the future. Small interfering RNA‐mediated WNK1 or OSR1 knockdown reduced glioma migration by eliminating NKCC1‐regulated phosphorylation activation.DAB1 tyrosine phosphorylation.OLIG2 induced TGF‐β2 expression and promoted the aggressive mesenchymal properties of glioma cells.Inside the brain, glioma cells migrate rapidly according to the extracellular environment and invade surrounding structures. The reason why 2.3Glioma resistance could make chemotherapy fail. Current treatments also hope to bypass the resistance of tumor cells, thereby achieving a breakthrough. ACT001, originally an ancient anti‐inflammatory drug, was found to demonstrate anti‐GBM activity in clinical trials. ACT001 treatment can modulate antitumor immunity in GBM by inhibiting phosphorylated STAT3.2.4PLK4 interacted with IKBKE and thus phosphorylated it, resulting in an increase in NF‐κB antiapoptosis in GBM cells.SAE1 silence obviously suppressed the phosphorylation of Akt SUMOylation and Ser473, induced a G2 phase block in the cell cycle, and led to apoptosis driven by a series of biochemical molecular events.The death of glioma cells includes apoptosis and necrosis. Glioma cell death often interferes with the experiment. 2.5DYRK1A and DYRK1B. In contrast, high DYRK1 expression increases ID2 Thr27 phosphorylation, leading to glioma stemness deficiency, which inhibits tumor growth and is more favorable to the prognosis of GBM patients.Now let us observe other effects of protein phosphorylation on glioma. Tumor heterogeneity is an important feature of malignancy, which could significantly affect the therapeutic effects and prognosis. Glioma cells exhibited heterogeneous patterns of TGF‐β pathway activation with altered phosphorylation of SMAD2, SMAD3, and SMAD1/5/8.3Currently, many drug studies refer to protein phosphorylation. Similarly, various treatments for glioma are associated with protein phosphorylation. We just need to figure out that phosphorylation has an influence on glioma, and find a way to break through from the aspect. We all know that GALNT2 is inextricably linked to the physiological processes of cell proliferation, migration, and invasion in gliomas. But the mechanism by which GALNT2 promotes the malignant features of gliomas is by affecting EGFR phosphorylation.4Protein phosphorylation is associated with the occurrence, growth, proliferation, migration, invasion, resistance, and death of glioma cells, while other properties of glioma cells remain to be further investigated. As is well known, phosphorylation and dephosphorylation of protein, which is a reversible process, almost regulate the cytoskeleton development and cell proliferation, differentiation, apoptosis, and all the process of life activity. Reversible protein phosphorylation is one of the main currently known ways of signal transduction. Therefore, we can effectively control glioma cells depending on protein phosphorylation. We just need to grasp the corresponding mechanism of influence to find a method to the problem. If we can make good use of protein phosphorylation, glioma will also have pretty treatment, not limited to surgery. Although at present, the targeted therapy of intracellular signaling pathways has not gained a great satisfactory effect, we believe that the protein phosphorylation targeted regulation can achieve great effect in the future; its targeted drugs can also be further developed and put into use in the clinic, which provides another way for the treatment of patients and has a bright prospect.Yu Pi wrote the manuscript. Chang‐Le Fang and Zhang‐Yu Su helped to modify the manuscript. All authors have read and approved the final manuscript.The authors declare no conflict of interest.Consent for publication was obtained from all authors.