Supraglacial lakes on the Greenland Ice Sheet are expanding inland, but the impact on ice flow is equivocal because interior surface conditions may preclude the transfer of surface water to the bed. Here we use a well-constrained 3D model to demonstrate that supraglacial lakes in Greenland drain when tensile-stress perturbations propagate fractures in areas where fractures are normally absent or closed. These melt-induced perturbations escalate when lakes as far as 80 km apart form expansive networks and drain in rapid succession. The result is a tensile shock that establishes new surface-to-bed hydraulic pathways in areas where crevasses transiently open. We show evidence for open crevasses 135 km inland from the ice margin, which is much farther inland than previously considered possible. We hypothesise that inland expansion of lakes will deliver water and heat to isolated regions of the ice sheet’s interior where the impact on ice flow is potentially large. Lakes on the Greenland Ice Sheet transfer water to the bed when they drain, but the impact is unknown. Here, the authors use a 3D model to show that lakes drain when fractures form, causing a chain reaction in which cascading lake drainages extend inland and deliver water to previously isolated regions of the bed. These supraglacial lakes (SGLs) typically start to form in late May and they grow in number and extend as surface melting progresses to higher elevations during the melt season1. SGLs have become larger and more numerous since 20003, while also expanding inland to elevations as high as 2000 m above sea level and 130 km inland from the ice margin12. The ability of these new inland lakes to enhance ice flow through rapid drainage is, however, contested13. Several studies suggest that lakes forming at high elevations tend to be larger and less likely to drain rapidly compared to lakes at lower elevations14 where extensional flow is capable of initiating hydro-fractures beneath lakes15. Hence, Poinar et al.15 argue that meltwater produced above 1600 m elevation predominantly drains on the surface and that the impact of inland SGL expansion therefore may be limited in terms of ice dynamical feedbacks. Yet, Doyle et al.16 argue that draining lakes may accelerate flow at 1840 m elevation and as far as 140 km inland from the margin. They also report year-on-year increases in ice flow corresponding to the expanding extent of SGLs, a response that differs fundamentally from the decadal slowdown observed closer to the margin18. With SGLs predicted to expand 200 km inland from the margin over the next 50 years19, it is critical to understand the lake drainage mechanism and its role in delivering surface water to the interior bed, where basal drainage is thought to be predominantly inefficient22 and the impacts therefore potentially sustained16.Each summer thousands of surface melt lakes form across the Greenland Ice Sheet and those that drain abruptlyHere we apply a well-constrained, three-dimensional (3D) ice-flow model of the Kangerlussuaq sector of the Greenland Ice Sheet to test the hypothesis that SGL drainage is dynamically triggered by the perturbation induced on the force balance of the ice sheet when surface meltwater is routed along the bed in summer. We show that distinct events, with up to 124 lakes draining over the course of a few days, occur when basal lubrication along subglacial drainage paths transiently induces high-magnitude tensile stresses near the surface. This ephemeral and previously overlooked alteration of the ice sheet’s force balance escalates into a tensile shock when many lakes drain collectively in a chain reaction. We use cascading lake drainage to describe the latter and show that most lakes drain in this dynamic manner.2 domain that extends 110 km inland from ice sheet margin near Kangerlussuaq  and includes five outlet glaciers   between observation and the initialised model . Although the total runoff produced by surface melting in 2010 was 6.58 km3 (ref. 27) and therefore considerably higher than the amount of water stored in SGLs, we focus on the latter because previous work found lake drainages to induce short-lived but sustained episodes of ice flow acceleration, consistent with the observed seasonal variation of ice flow in this11 and other sectors8 of the Greenland Ice Sheet. Although a larger quantity of surface meltwater is transferred to the bed through moulins forming where lakes have drained and where supraglacial streams intersect open crevasses, we exclude this water supply because its variability is insufficient to drive a sustained response in the ice flow of our model11.We apply the higher order Community Ice Sheet Model (CISM) to a 9000 kmnd) Fig. . Model s11 and Methods). From 1 June and onwards, the lake drainage record produces distinct intra-seasonal variations in our model, with flow rapidly accelerating by up to 400% compared to winter. The sustained, but short-lived perturbations generated by draining lakes are in good general agreement with daily ice-flow variations recorded by a GPS receiver installed at site SHR on Russell Glacier . The corresponding maximum surface velocity was 247 m a−1, which is 229% faster than the day before (108 m a−1). We also found surface velocity to increase by >5% over an area of 4750 km2  and across (y) ice flow direction can be calculated as H is the ice thickness and x direction and extension yields positive values of Rxx (see Methods).We find that basal traction is significantly reduced in regions of the bed where SGL drainage results in basal water fluxes in excess of 80 mdel Fig. . This lo29, which concentrates stresses towards the surface where ice is colder and more viscous than ice nearer the bed.The membrane stress is negligible in winter when basal traction alone counters the gravitational driving stress. However, it becomes critically important in summer when it compensates for the sudden loss of basal traction along well-lubricated, high-flux basal drainage pathways Fig. . This tr31. Hence, we specifically analyse Rxx and Ryy for the 50–150-m-thick top layer of our ice sheet model  or partially (28), transferring 20 × 106 m3 of surface water to the bed. Sixteen lakes remained unchanged  at which this tensile stress is countered by compression due the weight of the overlying ice, yielding 31. We find that tensile stresses do not only propagate existing fractures in areas where ice flow was extensional to begin with; they also initiate new fractures across an expansive area where ice flow was originally compressional  in areas covering 20% (6 June), 37% (7 June) and 42% (8–10 June) of the surface  over areas of 10% (6 June), 16% (7 June) and 18% (8–10 June) of the surface . The 10 lakes that did not drain during 6–10 June were all located outside the area impacted by this tensile shock  between flow in our initialised model and that observed during the winter 2009/2010  may have been stored subglacially in winter when the thinnest portion of the ice sheet’s margin becomes frozen, causing proglacial discharge to cease57. Observations from Russell and Leverett glacier show that subglacial meltwater is produced in winter, but not released until spring57.Although 95 lakes drained on 25–28 May Fig. , their cmer Fig. . To reprr2 = 0.83 (p < 0.01) from 1 June when lakes alone drive ice flow in our model. When the simulated spring speed-up event in late May is included, the strength of the correlation increases. Furthermore, when modelled surface velocities are averaged over the same 11-day periods used to obtain surface velocities from TerraSAR-X image pairs centred on 19 June, 11 July, 22 July and 11 November, we obtain significant correlation coefficients (p < 0.01) of 0.79, 0.92, 0.90 and 0.94, respectively . The latter is defined as the total stress (σij) minus the hydrostatic component ((1/3)σkkδij) and thus represents the stress that contributes to ice deformation40. The transverse gradients Rxy = τxy. The force balance in the x and y directions are thus58:ρ, g and h respectively denote ice density, gravitational acceleration and surface elevation. The left-hand side is the driving stress. The first term on the right-hand side is basal drag tied to frictional resistance at the bed, which directly opposes the driving stress. The second and third terms on the right-hand side are longitudinal and transverse stress gradients, which either oppose or act in cooperation with the driving stress. These gradient stresses make up the membrane stress.The longitudinal gradients in the 59:59.Open crevasses were detected in satellite imagery Fig.  and the 1. In 2006, we observed overall similar evolution of ER, although significant short-term elevation change also occurred south of 67°N, where crevasse may have opened in response to lakes starting to form on 11 May1 . All input datasets and model development code are available on request from the corresponding author.The model code used in this study is based off the Community Ice Sheet Model, which is freely available (Supplementary Information


We herein investigated the third subunit, Ecm7, under the same cell culture conditions. The deletion of ECM7 slightly lowered Ca2+ influx activity in the CNB1+ background, in which calcineurin potentially dephosphorylates Cch1, but markedly lowered this activity in the cnb1Δ background. The deletion of the C-terminal cytoplasmic region of Ecm7 also reduced Ca2+ influx activity. We identified a novel Cch1-interacting protein, Scs2, which is known as a cortical endoplasmic reticulum membrane protein. The deletion of SCS2 did not affect Ca2+ influx activity when calcineurin was inhibited by FK506, but enhanced this activity by 35% when the enzyme was not inhibited. However, this enhancement was canceled by the deletion of ECM7. These results suggest that Cch1/Mid1 is regulated differentially by Ecm7 and Scs2 in a manner that is dependent on the phosphorylation status of Cch1.The yeast Cch1/Mid1 Ca YPD medium contained 1% Bacto-yeast extract, 2% Bacto-peptone, and 2% glucose.Synthetic media SD.Ca100 and SD were prepared as described previously . SD.Ca102+ was measured according to a previously described method ). The final pellet was dissolved in SDS-sample buffer . After heating at 95˚C for 5 min followed by centrifugation at 15,000 rpm at 25˚C, the supernatant was subjected to SDS-PAGE and a Western blot analysis or stocked at -20˚C until used. Western blots were used to detect Cch1, FLAG-tagged Scs2, and Pma1.Cells 4-HIS3 URA3::(lexAop)8-lacZ ade2::(lexAop)8-ADE2 GAL4) carrying the plasmid pBCY-BTC-CCH1H, which expresses the Cch1-Cub-LexA-VP16 fusion protein as bait, was transformed with the S. cerevisiae cDNA library/NubG-x , which was designed to express prey proteins, and incubated on SD plates + Ade— at 30˚C. The transformants obtained were streaked on new plates with the same nutrient composition and incubated for several days. In the second screening, transformants were grown for 2–3 days on SD plates—. cDNA sequences on prey plasmids were then elucidated.The yeast screening strain NMY32  with α-factor for 2 h for Ca2+ accumulation assays and 8 h for viability assays. The results obtained showed that only slightly less Ca2+ accumulated in the ecm7Δ mutant (by 17%) than in the wild-type strain, while Ca2+ accumulation was reduced by 77% in the cch1Δ mutant  [The cytoplasmic C-termini of human γ subunit isoforms are highly divergent, except for the terminal seven amino acid residues, which are conserved . Similar(Phe448)  Fig 1)..Saccharo1-322 lacking all of the seven phosphorylation sites and Ecm71-412 lacking three C-terminal phosphorylation sites  containing the two phosphorylation sites and (2) Ecm71-432, a mutant Ecm7 protein with the deletion of the C-terminal region ranging from Glu433 to the C terminus. We then measured their Ca2+ accumulation activities. The results obtained showed that the two mutant Ecm7 proteins exhibited normal Ca2+ accumulation activities. Since Ecm71-412 showed reduced activity, the region between 413 and 428 amino acid residues appears to be important for the activity of Ecm7, although its contribution is restrictive.In order to examine the importance of these regions and some of the phosphorylation sites, we performed truncation analysis. The C-terminally truncated forms of Ecm7, Ecm7on sites , were exmeasured . After type Ecm7 . This reS. cerevisiae cDNA libraries as prey. We obtained 12 candidates, among which Scs2 was the most frequently detected. Details on this screening will be published elsewhere; we herein focused on Scs2, particularly its genetic study in terms of HACS regulation because this protein had been reported as a member of LACS [In the search for membrane proteins that potentially interact with HACS, we screened candidates with the split-ubiquitin membrane-based yeast two-hybrid system , 28, usiintegral membrane protein with a single C-terminal transmembrane segment that localizes at the ER membrane, and is a homolog of VAP [2+ influx through HACS, the scs2Δ mutant was constructed and examined for Ca2+ accumulation 2 h after exposure to α-factor in SD.Ca100 medium. The results obtained showed that 35% more Ca2+ accumulated in the scs2Δ mutant than in the wild-type strain , suggesting that Scs22 is not involved in the regulation of the Cch1/Mid1 channel under our assay conditions.g, SCS22 . The scsi.e. under low extracellular Ca2+ conditions: the former is a positive regulator, while the latter is a negative regulator. We showed that Ecm7, a yeast homolog of animal VGCC γ subunits, was hardly necessary for α-factor-induced HACS activity when calcineurin was functional in cells incubated in low Ca2+ medium. However, when the CNB1 gene encoding a subunit of this protein phosphatase was deleted or this enzyme was inhibited by FK506, Ecm7 switched to a strong positive regulator. Although Ecm7 possesses seven phosphorylation sites in the intracellular C-terminal region [et al., manuscript in preparation). The calcineurin deficiency-dependent activation of HACS was also reported for S. cerevisiae cells exposed to tunicamycin, an ER stressor; however, the involvement of Ecm7 was negligible, and this may have been because YPD medium, which is rich in nutrients and Ca2+, was used to cultivate cells [In the present study, we identified two regulators of HACS, Ecm7 and Scs2, under the experimental conditions that activate HACS, l region , these wte cells . A previ2+ influx activity of HACS, suggesting that the seven phosphorylation sites present in this region are less important. Particularly, two C-terminally truncated forms, Ecm71-322 and Ecm71-412, had the same activity each other  is not involved in Ecm7 function. In addition, Ecm7Δ429–432 lacking two phosphorylation sites (Ser429 and Ser432) and Ecm71-432 lacking the phosphorylation site Ser435 did not lose the activity at all (1 to γ8), only one member, γ2, was found to be phosphorylated in the cytoplasmic C-terminal region [A truncation analysis showed that the large cytoplasmic C-terminal region of Ecm7 was partially responsible for the full Cach other , suggesty at all . Therefol region , 31. SinSCS2 gene enhanced α-factor-induced HACS activity by 35% and this enhancement was canceled in the absence of Ecm7 or when cells were incubated with the calcineurin inhibitor FK506. Since Scs2 is shown to be phosphorylated at Ser106, Ser153, Thr200, Ser 201, Ser203, and Thr204 [2+ concentration, Ecm7 may activate a phosphorylated form of HACS or its regulator. However, this activation may not be applied to its dephosphorylated form. When calcineurin is functional due to a high cytosolic Ca2+ concentration, Scs2 may repress the activity of a dephosphorylated form of HACS or its regulator, but may not affect its phosphorylated form. This hypothesis warrants further studies looking at HACS phosphorylation and dephosphorylation.Another regulator, Scs2, was newly discovered by the screening of Cch1-interacting membrane proteins with the split-ubiquitin membrane-based yeast two-hybrid system. The deletion of the d Thr204 , 32, it 2+ influx through the plasma membrane.Scs2 is localized to the cortical ER membrane, at which it acts as an ER-to-plasma membrane-tethering protein . Cch1 iscnb1Δ cells exposed to α-factor in low-Ca2+ medium. However, this positive regulation is weak in CNB1+ cells. We identified the cortical ER resident protein Scs2 as a novel interactor of Cch1 and demonstrated that Scs2 negatively regulated Cch1 in calcineurin-functional cells.In conclusion, we demonstrated that the VGCC γ subunit homolog Ecm7 functioned as a strong positive regulator of Cch1 in calcineurin-deficient S1 FigTDH3 promoter and appeared to be localized in the ER and plasma membranes in the cnb1Δ and ecm7Δ cnb1Δ mutants as well as in the wild-type strain.Cch1-EGFP images of cells treated for 2 h with 6 μM α-factor were obtained by confocal scanning fluorescence microscopy. Cch1-EGFP was expressed from the (TIFF)Click here for additional data file.


Multiple, independent time markers are essential to correlate sediment and ice cores from the terrestrial, marine and glacial realms. These records constrain global paleoclimate reconstructions and inform future climate change scenarios. In the Northern Hemisphere, sub-visible layers of volcanic ash (cryptotephra) are valuable time markers due to their widespread dispersal and unique geochemical fingerprints. However, cryptotephra are not as widely identified in the Southern Hemisphere, leaving a gap in the climate record, particularly during the Last Glacial Maximum (LGM). Here we report the first identification of New Zealand volcanic ash in Antarctic ice. The Oruanui supereruption from Taupo volcano  provides a key time marker for the LGM in the New Zealand sector of the SW Pacific. This finding provides a high-precision chronological link to mid-latitude terrestrial and marine sites, and sheds light on the long-distance transport of tephra in the Southern Hemisphere. As occurred after identification of the Alaskan White River Ash in northern Europe, recognition of ash from the Oruanui eruption in Antarctica dramatically increases the reach and value of tephrochronology, providing links among climate records in widely different geographic areas and depositional environments. These records are recovered through deep ice core drilling and layer-by-layer analysis of the ice. In this study we focus on a high-resolution core (WDC06A), recovered from central West Antarctica  WDC06A chronological information, generated by a combination of layer counting and compared with the Hulu Cave record9, provides the opportunity to search for silicate ash, or tephra, from major volcanic eruptions that have previously been missed in the Antarctic record.Antarctic ice sheets capture and archive detailed climate records reaching as far back as ~1.5 Ma3of magma, or >1000 km3 of pyroclastic deposit10, present good detection targets in ice cores, and the Oruanui supereruption from Taupo volcano  are fine grained and exceptionally widely dispersed as a result of the highly explosive interaction between magma and water from a large lake12, and associated groundwater system. A detailed study of 14C ages gathered from optimal material producing 8 ages from syn-eruptive material13. We have recalculated these ages using SHCal13 calibration14 , which yielded an eruption age of 25,580  ±  258 cal a BP (±2 S.D.). This age is statistically indistinguishable from the age of 25,360  ±  160 and 25,358  ±  162 cal a BP reported in previous works15, which represent integrated ages from above and below the tephra, and were calibrated using IntCal09/SHCal09. These ages place the Oruanui eruption at an important time in earth’s recent climate history – the early LGM. This time period is central to our understanding of leads and lags in the global climate system, especially between the northern and southern hemispheres. Despite the large magnitude of this event, and evidence for widespread dispersal, tephra from the Oruanui eruption has not been previously identified in any Antarctic or Arctic ice cores. Here, we report the identification of particles from this significant eruption in ice core WDC06A, and consider the implications for poleward transport of tephra from volcanic eruptions.Tephra from the largest class of volcanic eruption, termed “super-eruptions”, characterized by magma volumes of >450 km16). A large sulfate spike and presence of particles 5–30 µm in diameter were recognized at 2660.3 m depth . Two ice samples from a depth range of 2660.000–2660.335 m were obtained from the National Ice Core Laboratory , melted, filtered and prepared for geochemical analysis following techniques of Dunbar and Kurbatov17. Particles were examined using scanning electron microscopy to document their size and shape; a routine procedure when investigating ice core tephra, in order to ascertain that volcanic material is present. Quantitative geochemical analysis was then carried out using the Cameca SX100 electron microprobe at New Mexico Tech18. As most of the particles were  <20 µm across, beam sizes of <20 µm were used for some particles, but beam sizes slightly larger than the glass particle was used. This process resulted in low analytical totals (mostly between 90 and 100%) due to overlap on epoxy, requiring analyses to be normalized to 100 wt.%. Studies suggest that this technique is effective at minimizing the unavoidable Na loss associated with analyzing small particles, and does not adversely affect the analytical accuracy, although the precision for a set of points may be slightly decreased19. Duplicate samples were prepared for microbeam geochemical analysis, and these were analyzed separately during different analytical sessions. A set of secondary standards, including rhyolitic and comenditic glasses VG568 and KN18 were analyzed during both analytical sessions to monitor accuracy and precision. In addition, four known samples of tephra from the Oruanui eruption sampled from well-documented terrestrial and marine sites were analyzed by electron microprobe using the same instrument and methods as the ice core samples. All data are presented in Supplementary Information Tephra layers in the WDC06A ice core were identified visually, or by using a downhole optical logger, and/or by observation of high particle counts during continuous melting analysis at the Desert Research Institute Trace Chemistry Laboratory   is statistically indistinguishable. Most known Antarctic tephra from this age range are trachytic24, and no rhyolitic Antarctic tephra of an appropriate age are known. The striking similarity of age and geochemistry and indistinguishable age between tephra produced by the Oruanui eruption and that found in the WDC06A ice core at 2660.3 m depth strongly support this correlation.Geochemical analyses reveal that glass shards from the WDC06A core samples are rhyolitic, chemically uniform, and, for key correlation elements such as Ca, Fe, and Ti, are indistinguishable in composition from the distal onshore and offshore tephra from the Oruanui eruption21 Fig.  Table 1. 11 Fig. , which g26. Inputs are based on unit 10 of the Oruanui eruption from Taupo volcano, as established from field-based studies of the eruption deposits12. We assumed an eruption rate of 1.5  ×  109 kg s−1, similar to the 1991 eruption of Mount Pinatubo, Philippines, and duration of three days, resulting in a total erupted volume of 150 km3 . The ash transport was modeled as an expanding umbrella cloud with a maximum height of 20 km above sea level and a lower bound of 15 km. These characteristics are consistent with large-eddy simulations of water - influenced, coignimbrite-style volcanism from previous work27. To account for the physics of a gravity-driven intrusion, Ash3d adds a radial wind field within the umbrella cloud, producing an outward expansion rate proportional to the eruption rate26. We used two particle classes. The larger size class comprises 95% of the erupted mass , representing coarser particles and ash aggregates that fall out close to the volcano. The remaining 5% represents the fine particles  that escaped aggregation and underwent long-distance transport28, with characteristics similar to cryptotephra recovered from the ice core. The 3-D time-varying atmospheric structure  was initialized from the 2.5 degree NCEP/NCAR Reanalysis2 dataset (https://rda.ucar.edu/datasets/ds091.0/). We identified 80 time periods within this dataset that fit within the plausible range of LGM conditions during the time of the Oruanui eruption, with northwesterly flow over New Zealand (Kidson type ‘Blocking HE’) and strong circumpolar westerlies around Antarctica32  is less than that of background terrestrial dust, and too small for geochemical analysis36. Our identification of cryptotephra from the Oruanui eruption, with some particles up to 20–30 µm in diameter, shows that a non-Antarctic eruption is capable of delivering large particles to the Antarctic ice sheet. Model results demonstrate that it may only take a few days to weeks for such large particles to be transported thousands of kilometers in strong winds aloft, which is consistent with the European record of Icelandic cryptotephra38. Simulations of ash transport confirm that the particles could have feasibly been transported from New Zealand, and our geochemical and morphological comparisons indicate that the particles are sourced from the Oruanui eruption.Overall, volcanic ash from non-Antarctic eruptions has been challenging to locate and geochemically fingerprint in Antarctic ice13, suggesting that the background winds were favorable for poleward transport at the time of eruption. Numerical modeling shows how the tephra would be caught up in the polar jet, and continue encroaching inland to the West Antarctic ice sheet over a period of days to weeks 40. Our modeling indicates that an Oruanui-sized eruption would produce an umbrella cloud thousands of km in diameter, transporting tephra and gas radially in all directions  for moderateAlthough this cryptotephra appears to be exceptional within the WDC06A ice core, in terms of its larger grain size and possibly higher particle concentration, its presence demonstrates that other non-Antarctic tephra layers may be present and recognizable. This particular layer will serve as a valuable early LGM time-stratigraphic marker in the emerging Antarctic tephrochronological framework. Further refined particle recognition and sample preparation techniques may allow more of these well-dated tephra layers to be identified in Antarctic ice, allowing further direct correlations between different, intercontinental, sedimentary records, and providing golden spikes for the Antarctic ice chronology.Supplementary Information S1Supplementary Information S2Supplementary Video


The human salivary α-amylase was more strongly inhibited by the hydrolysable than by the condensed tannin, with the concentrations for 50% inhibition (IC50) being 47.0 and 285.4 μM, respectively. The inhibitory capacities of both tannins on the pancreatic α-amylase were also different, with IC50 values being 141.1 μM for the hydrolysable tannin and 248.1 μM for the condensed tannin. The kinetics of the inhibition presented complex patterns in that for both inhibitors more than one molecule can bind simultaneously to either the free enzyme of the substrate-complexed enzyme (parabolic mixed inhibition). Both tannins were able to inhibit the intestinal starch absorption. Inhibition by the hydrolysable tannin was concentration-dependent, with 53% inhibition at the dose of 58.8 μmol/kg and 88% inhibition at the dose of 294 μmol/kg. For the condensed tannin, inhibition was not substantially different for doses between 124.4 μmol/kg (49%) and 620 μmol/kg (57%). It can be concluded that both tannins, but especially the hydrolysable one, could be useful in controlling the postprandial glycemic levels in diabetes.The aim of the present study was to compare the in vitro inhibitory effects on the salivary and pancreatic  The salivary amylase (HSA), the most abundant enzyme in human saliva, initiates the digestion of complex carbohydrates in the human oral cavity, where especially starch is partly digested into oligosaccharides, maltose, and glucose  plots shown in α-amylase are shown in Vmax is the maximal reaction rate, KM the Michaelis-Menten constant, [S] the substrate concentration, and [I] the inhibitor concentration. The following inhibitory complexes are allowed: EI, EI2, ESI, and ESI2; Ki1, Ki2, Ki3, and Ki4 are the corresponding dissociation constants of these complexes (inhibitor constants). If one of these complexes is lacking, limiting forms of (2) accounts for the parabolic inhibition. Agreement between theory and experiment was tested by means of a least-squares fitting procedure. Fitting was done simultaneously with two independent variables ([S] and [I]), including the rate versus inhibitor concentration data shown in Ki3 and Ki4. This means that the enzyme-substrate complex (ES) forms only one type of complex with the inhibitor, which could be either ESI or ESI2. The latter implies in a simultaneous or almost simultaneous binding of two inhibitor molecules to the enzyme. After fitting the equations corresponding to 10 mechanistic possibilities, the best fit was achieved with . This means that the first binding of the hydrolysable tannin to the free enzyme occurs much more readily than the second one. Furthermore, Ki3 is 46-fold higher than Ki1, indicating that the complex ESI forms only at relatively high concentrations of the hydrolysable tannin. The legend of v versus [S] and v versus [I] curves (2 complex.When investigating the kinetic mechanism of the inhibitions caused by the hydrolysable and condensed tannins it is indispensable to take into account the form of the 1/shown in v=VmaxSKMshown in v=VmaxSKMshown in v=VmaxSKM] curves . Only atKi1 and Ki2. This could be indicating that the free enzyme (E) forms only one type of complex with the inhibitor, which could be EI or EI2. After fitting the equations corresponding to 10 mechanistic possibilities, the best fit was achieved with  and all other parameters have the meanings already specified above. In this particular case binding of two inhibitor molecules to the free enzyme occurs simultaneously or nearly so in such a way that the complex EI is practically absent. Comparison of theory and experiment in Ki1-2 is considerably smaller than Ki3 by a factor of 3.6. Singularly, the second binding to the substrate-complexed enzyme  is facilitated over the first one, as Ki4 is smaller than Ki3.Fitting of  to the dved with , which dKM and Vmax values obtained when fitting ( fitting  to the c fitting  was fittα-amylase (2: Ki3-4 is the dissociation constant for the complex ESI2 (formed by the reaction ES + 2I → ESI2) and all other symbols have the same meanings specified above. The optimized parameters are listed in the legend of Ki1 is 4.5 times smaller than Ki3-4; the free enzyme, thus, binds much more strongly the hydrolysable tannin than the substrate-complexed enzyme.The results of the experiments that were done with the hydrolysable tannin are shown in -amylase . In the -amylase . Here ag-amylase  was unsulase  exceeds Ki1 by a factor of 3.0 and Ki2 (EI2) exceeds Ki1 by a factor of 4.5.The results of the kinetic investigations on the inhibition caused by the condensed tannin on the pancreatic For testing in vivo the inhibition caused by both the hydrolysable and condensed tannin, starch was given to rats and the glycemic levels were followed during 60 minutes. The basis for these experiments is given by the well-established notion that hydrolysis of intragastrically administered starch is a prerequisite for the entrance of the derived glucosyl units into blood. μmol/kg) did not result in a pronounced enhancement of the effect. This phenomenon can be best appreciated by comparing the areas under the glycemic curves in α-amylases by both the hydrolysable and condensed tannins presents several complexities in that for both inhibitors more than one molecule can bind simultaneously to the enzymes [v versus [I] plots and confirmed by the numerical analysis in which attempts of fitting an equation describing linear inhibition (single binding) always produced unfavourable results. Even assuming some limited degree of heterogeneity for the preparations that were used, especially for the condensed tannin [α-amylases by a pinhão coat tannin [ Phaseolus protein inhibitorα-AI [ pinhão coat tannin is also of the parabolic type [α-amylase by a gallotannin was analyzed as being of the linear type even though the Dixon plots (1/v versus [I]) that were presented are clearly indicating parabolic inhibition [v versus [I] experiments. This occurred because it is difficult to measure accurately low initial reaction rates at low substrate concentrations. Even so, the description of v versus [I] relationships by the fitted equations was very good, with minimal deviations at the highest inhibitor concentrations. For all cases analyzed in the present work inhibition was of the mixed (competitive-noncompetitive) type. This is the most frequently reported mode of inhibition [α-amylase [Inhibition of the human salivary and porcine pancreatic  enzymes , 22. Thid tannin , it shoud tannin , 22. In d tannin –23. Parat tannin  and by tlic type . Furtherlic type , is a prhibition . It shouhibition . The lon-amylase .α-amylase than the tannins. The question of which tannin is more effective can be unambiguously answered in the case of the human salivary α-amylase inhibition. The hydrolysable tannin concentration for half-maximal inhibition (IC50) of the salivary enzyme is considerably smaller than that of the condensed tannin (47 μM compared to 285 μM) and the same can be said about the tendency of binding to the free enzyme, which is 14.7 times superior in the case of the hydrolysable tannin. With respect to the pancreatic enzyme the difference is not as pronounced. In terms of their masses and at the substrate concentration of 1 g/100 mL the condensed tannin was a moderately better inhibitor because 50% inhibition occurred at the concentration of 141 μM compared to the 248 μM required for the same degree of inhibition by the hydrolysable tannin. However, the inhibition degree will vary with the substrate concentration and at low substrate concentrations the hydrolysable tannin will be a better inhibitor because of the smaller value of its inhibition constant Ki1  values for the hydrolysable and the condensed tannins were always considerably smaller than Ki3 or Ki3-4 values. This suggests that binding of the substrate, which is a large molecule, to the enzyme promotes structural modifications that make binding of the inhibitor more difficult in the case of the tannins.A characteristic of the inhibitory action of both the hydrolysable and the condensed tannin is the observation that they are bound more tightly by the free enzyme (E) than by the enzyme complexed with the substrate (SE). This is revealed by the observation that for both enzymes 16 (4 → 8), form hydrogen-bonded cross-links between the protein molecules [α-amylase by both the hydrolysable and the condensed tannin occurred in the presence of relatively low tannin concentrations, implying that the inhibitory action is due to specific molecular interactions involving specific amino acid residues of the protein and well-defined structural parts of the tannins. Binding of tannins to proteins have been demonstrated by methods that do not involve kinetics and they have been investigated for several combinations of tannins and proteins, including enzymes [α-amylase. Consistent with this is the fact that hydroxylation of flavonoids improves the inhibitory effect on α-amylase exerted by these compounds [α-amylase results from the interaction of aromatic rings of the former with aromatic amino acids of the protein. The role of the aromatic amino acids of the human salivary α-amylase in the pentagalloyl glucose binding was reinforced by kinetic studies with W58L and Y151M mutants of the enzyme: replacement of the aromatic amino acids in the active site by aliphatic ones decreased inhibition dramatically, what seems to be in accordance with a participation of these residues in the interaction of tannins with the human salivary α-amylase [Both hydrolysable and condensed tannins are well-known protein precipitating agents. Two different models have been suggested for explaining the capability of tannins to precipitate proteins . It has olecules . However enzymes –32. It c enzymes . In the  enzymes . The higompounds . At highompounds . In thisompounds  it has b-amylase .α-amylase (as well as α-glucosidase) resulted in delayed carbohydrate digestion and glucose absorption with attenuation of postprandial hyperglycemic excursions. The diminution of hyperglycemia in rats to which starch was given by both the hydrolysable and condensed tannin is thus an expected phenomenon. Demonstration of the phenomenon also proofs that both tannins are able to exert α-amylase inhibition under in vivo conditions and not only in the test tube. In spite of the relatively small difference in their inhibitory activities toward the enzyme, the hydrolysable tannin was more effective in lowering hyperglycemia when compared to the condensed tannin. The response practically ceased to increase with condensed tannin doses above 124.1 μmol/kg (100 mg/kg), whereas the effects of the hydrolysable tannin increased progressively until the dose of 620 μmol/kg. The reasons for this behavior are not clear. One possible reason is that the hydrolysable tannin is active on other enzymes equally involved in starch digestion as the α-glucosidases and invertases, for example, whereas the condensed tannin is inactive or less active [Inhibition of s active , 35. Sucα-amylase exerts two additional functions, namely, binding to the tooth surface and binding to oral streptococci [Camellia sinensis (L.) Kuntze) reduce dental caries [α-amylase inhibitory activity of tea extracts, the hypothesis has been raised that this activity could be involved in the reduction of the cariogenicity of starch-containing foods [Ki1 value of 13.2 μM, can be regarded as a useful agent for oral health.Besides participating in the initial hydrolysis of starch and other carbohydrate constituents of the diet, the salivary ptococci –38. All l caries , 40. Basng foods , 42. ForIn conclusion, both tannins are potentially useful in controlling the postprandial glycemic levels in diabetic patients, with the hydrolysable one, however, being superior. To our knowledge, the present study is the first one that presents a comparison of the effects of both types of tannins under exactly the same conditions. Clinical studies are evidently indispensable for evaluating the viability and safety of the use of preparations containing the hydrolysable or even condensed tannins, especially as food additives. With reference to the latter, more studies are also necessary to evaluate the possibility of incorporating this tannin into dental products such as dentifrices, mouthwashes, dental flosses, and chewing gums that could be helpful in the prevention of dental caries.


HMGA1, a known oncogene. HMGA1 suppresses the abundance of IGF binding protein 2/IGFBP2 and Grb14, inhibitors of IGF action. IMP2 stabilization of HMGA1 mRNA plus IMP2 stimulated IGF2 production synergistically drive cancer cell proliferation and account for IMP2’s tumor promoting action. IMP2’s ability to promote proliferation and IGF action requires IMP2 phosphorylation by mTOR.The gene encoding the Insulin-like Growth Factor 2 mRNA binding protein 2/IMP2 is amplified and overexpressed in many human cancers, accompanied by a poorer prognosis. Mice lacking IMP2 exhibit a longer lifespan and a reduced tumor burden at old age. Herein we show in a diverse array of human cancer cells that IMP2 overexpression stimulates and IMP2 elimination diminishes proliferation by 50–80%. In addition to its known ability to promote the abundance of Insulin-like Growth Factor 2/IGF2, we find that IMP2 strongly promotes IGF action, by binding and stabilizing the mRNA encoding the DNA binding protein  IMP2 and therefore produce too much the IMP2 protein. Previous research has shown that mice that lack the IMP2 protein develop fewer cancers and live longer, while patients whose cancers make too much IMP2 have a poorer prognosis.Some types of cancers develop when genes known as oncogenes or tumor promoters become faulty, and are present at abnormally high levels or inappropriately turned on. For example, cancer cells often have extra copies of the gene In healthy cells, the IMP2 protein normally helps to make new gene products by stabilising certain newly produced RNA molecules – the precursors of proteins, and in some cases by promoting the translation of these RNAs into proteins. For example, IMP2 binds to the mRNA that encodes the protein IGF2, which is a protein that helps cells to grow and is commonly produced in large quantities by cancer cells. However, until now it was not clear whether IMP2 only acts by increasing the production of IGF2 or also contributes to cancer growth in other ways.Using a range of human cancer cell lines, and healthy mouse cells, Dai et al. first confirmed that without IMP2, cancer cells made less IGF2 and grew less quickly. When IGF2 was added to the cells lacking IMP2, it only partially restored their ability to grow. Further experiments revealed that cells without IMP2 had increased levels of proteins that counteract the effects of IGF2. Usually, IMP2 binds and stabilizes the mRNA that encodes the oncogenic protein HMGA1, which is known to regulate the number of ‘anti-IGF2 proteins’. However, without IMP2, the HMGA1 levels drop, which causes an increase of the anti-IGF2 proteins.This indicates that IMP2 promotes cancer cell growth both by enabling cells to produce more IGF2 and by suppressing inhibitors of IGF2 action. This suggests that cancer patients whose tumors have abnormally high levels of IMP2 may be especially sensitive to drugs that target and inhibit IGF2. The insulin-like growth factor 2 (IGF2) mRNA-binding proteins (IGF2BP1-3 or IMP1-3) are a family of RNA binding proteins that participate in post-transcriptional gene regulation ; notablyIMPs are expressed coordinately starting ~e10.5 coincident with the onset of IGF action; the expression of Igf2, Imp1 and Imp3 is largely extinguished before birth  exhibit deficient Igf2 RNA splicing and translation and greatly slowed proliferation; the latter is rescued entirely by exogenous IGF2. In contrast, Imp2 null mice are nearly normal in size through weaning, lean and slightly small as adults, highly resistant to diet-induced obesity and long lived  indicates that amplification of the IMP2 gene is a relatively common event in comparison to amplification of IMP1 and IMP3  reduction in the rate of proliferation . In each instance, IMP2 overexpression increased proliferation in a dose-dependent manner . Reciproferation . Similar/+ cells . Seekingcontrols , however−/− MEFs . This co+/+ MEFs . Thus, t+/+Imp2 and −/−Imp2 MEFs  Technology which yielded the quantitative estimation of the abundance of 7964 polypeptides  over treatment with DMSO whereas doxycycline treatment of −/−Imp2 MEFs containing shRNA against Igfbp2 increased cell number from ~30% to ~68% that of similarly-treated +/+Imp2 MEFs. ; although the abundance of the IGF1R and InsR beta subunits does not differ, the IGF1-induced increase in IGF1R beta subunit tyrosine phosphorylation in −/−Imp2 MEFs is substantially reduced as compared with +/+Imp2 MEFs, as is the overall tyrosine phosphorylation of IRS1 +IRS2 and activation of Akt assessed by phosphorylation at Ser473 . The RNAseq from +/+Imp2 and −/−Imp2 MEFs , many more than did the proteomic analysis (275/1457); among the 636 RNAs enriched ≥1.5X in the IMP2 IP are 64 that encode TFs, of which 14 show a three-fold or greater decreased abundance in −/−Imp2 MEFs as compared with +/+Imp2 MEFs . Thus HMGA1 binds and regulates the Igfbp2 gene, suppressing its transcription, so that HMGA1 depletion increases, and overexpression decreases IGFBP2 polypeptide abundance in MEFs; surprisingly, HMGA1 affects the abundance of the Grb14 polypeptide in a virtually identical manner  and an empty Flag vector in the parental −/−Imp2 MEFs. Polyclonal populations were selected and a doxycycline dose was determined that induced polypeptide expression of each Flag-tagged IMP2 variant to a level similar to endogenous IMP2 in +/+Imp2 MEFs , the two endogenous polypeptides with the highest ratio in the entire proteome are IGFBP2 (5.7) and Grb14 (3.7)  stimulated the translation of an IGF2-leader 3-luciferase reporter in RD rhabdomyosarcoma cells to an extent similar to Flag-IMP2 wildtype, whereas Flag-IMP2(Ser162Ala/Ser164Ala) was no more effective than empty vector ; this trsix days . Imp2−/−s before . If the  IMP2-DA , right. The regulation of IMP2 phosphorylation was examined in 293E cells. Overnight withdrawal of serum, brief incubation in amino acid and serum-free medium or torin1  as well IMP2 is amplified and overexpressed in many cancers and recent reports , Hela (RRID:CVCL_0058), MB-231 (RRID:CVCL_0062), Hep3b (RRID:CVCL_0326) and 293E (RRID:CVCL_6974) cell lines were maintained in DMEM supplemented with 10% FBS. HCC-1359 (RRID:CVCL_5128), NCI-H2029 (RRID:CVCL_1516), SNU-423 (RRID:CVCL_0366), HCC-1419 (RRID:CVCL_1251) cells were cultured in RPMI-1640 supplemented with 10% FBS. The corresponding data derived from human cancer cell lines .DMSO, doxycycline, cycloheximide, rapamycin, insulin, human IGF1, human IGF2, RPL26 and actin antibodies were from Sigma-Aldrich ; Torin 1 from TOCRIS. Antibodies: P-AKT(Ser473), AKT, pS6K(Thr389), S6K, P-4E-BP1(Thr37/460), 4E-BP1, P-AMPK, AMPK, IGF1R, IRS1, IRS2, P21, Phos-Tyrosine from Cell Signaling Technology; Grb14 and IGFBP2 antibodies from abcam. IMP antibodies were described previously . The indhttp://crispr.mit.edu); . Plasmids co-expressing the sgRNA specific to the IMP2 and the Streptococcus pyogenes Cas9 nuclease were transfected into cells by Nucleofector (Lonza) according to manufacturer’s instruction.CRISPR-Cas reagents were obtained from Addgene (U6-Chimeric_BB-CBh-hSpCas9). Small guide RNAs (sgRNAs), selected for minimal predicted off-target mutagenesis, were designed using CRISPR Design software  was used to organize and process the TCGA data.The data used to construct the comparison of y number  and mRNAy number  in humanSamples were processed and analyzed through the Thermo Fisher Scientific Center for Multiplexed Proteomics at Harvard Medical School . PeptideRNA was isolated using QIAGEN RNase kit. RNA samples were examined for the integrity by Agilent bioanalyzer prior to further processing. Sequencing was carried out on Illumina HiSeq 2500, resulting in approximately 21 million of 50 bp paired-end reads per sample. STAR aligner (Pubmed ID: 23104886) was used to map sequencing reads to the Mus musculus genome version 10 (mm 10) reference genomes. Read counts for individual transcripts were produced with HTSeq-count (Pubmed ID:25260700), followed by the estimation of expression values and detection of differentially expressed transcripts using EdgeR (Pubmed ID:19910308). The RNA sequence data have been deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO series accession number GSE101311.Grb14 sense (actatgtggacgacaacagc), antisense (cataattctttctaagatacag); murine Igfbp2 sense (ccagcaggagttggaccaggt), antisense (cttaaggttgtaccggccatgc); murine P21sense (cgagaacggtggaactttgac), antisense (cagggctcaggtagaccttg).Cells were treated with 1 μM Actinomycin D (Sigma) for 12 hr to block transcription. After the indicated times, the total RNAs were extracted, followed by DNase digestion for eliminating DNA contamination and cDNA syntheses. The concentration of mRNAs were quantified by Real-Time RT-PCR using the SYBR Green. The primers for the real-time PCRs were as follow: murine Protein was cross-linked to DNA by treatment of PBS washed cells with formaldehyde. Cells were pelleted and resuspended in cell lysis buffer containing a protease inhibitor cocktail (Roche). After 10 min on ice, the nuclei were collected and chromatin was sheared by sonication. ChIP was performed using the Simple ChIP Enzymatic Chromatin IP kit  according to the manufacturers’ instructions. The sheared DNA-protein complexes were immune-precipitated using antibodies to HMGA1. A nonimmune IgG was the negative control. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by two peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Charles Sawyers as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "IGF2 mRNA binding protein-2 is a tumor promoter that drives cancer proliferation through its client mRNAs IGF2 and HMGA1" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The report addresses potential mechanisms whereby the IGF-2 mRNA binding protein IMP2 promotes tumor growth and progression. The findings reveal new information regarding the role of IMP2 in regulating HMGA1 intracellular concentrations to modulate IGFBP-2, IGF-II and Grb14. The study also elucidates the role of these factors in regulating tumor cell proliferation. Extensive genomic and proteomic analyses clearly support the conclusion that these are the predominant factors that act together to regulate this signaling system. Overall, the findings are clearly presented and conclusions are well justified by the experimental data.Essential revisions:1) One might expect there to be a synergy between IGFBP2 and Grb14, but +/+ cells, which does not appear to be significantly altered (judging by the blots) in response to IGF1. Moreover, whereas the robust decrease in IGF-1R and IRS phosphorylation in IMP2-/- cells is partially rescued upon addition of IGF1, rescue of the corresponding decrease in Akt phosphorylation appears to be minimal. This discrepancy should be explained.2) 3) The mechanism of Grb14 protein stabilization by IMP2 is unexplained. Given that Grb14 plays a role that is shown to be as important as that of IGFBP2 in disrupting IGF2-mediated cell growth, some insight into the mechanism whereby IMP2 stabilizes Grb14 ought to be provided. For example, does IGFBP2 exert an effect, direct or indirect, on Grb14 expression? Does depletion of IGFBP2 alter Grb14 expression? And if HMGA1 does not affect Grb14 stability, what are the candidate factors that may do so?4) The mechanism of HMGA1 mRNA protection by IMP2 is unexplored. Without going into excessive depth here, the investigators could easily determine whether IMP2 binds to let-7 binding sites in the 3' UTR of HMGA1. This would at least suggest whether or not HMGA-1 mRNA stability is mediated by the ability of IMP2 to block or displace let-7 family miRNA binding to their targets.5) The investigators begin their discussion by stating that their findings define the functional significance of IMP2 in cancer cells. However, other observations have shown that IMP2, in addition to other IMPs, promotes stemness in cancer cells. There are therefore probably several, possibly synergistic, mechanisms whereby IMP2 promotes tumor growth and this notion should be included in the Discussion. Furthermore, the investigators cite several studies that show a correlation between the poor prognosis of a variety of cancers and IMP2 expression. Recent work  has revealed precise mechanisms whereby IMP2 may be implicated in tumor maintenance and growth and should be cited.6) The results regarding P21 are interesting however it is not clear why lowering P21 levels had no effect on proliferation. The assumption would be that even after deliberately lowering the levels they are still relatively high and therefore even a 5.9 fold change in levels is not adequate to alter proliferation. Some comment should be made on these findings. The authors comment in two places in the manuscript that the changes in proliferation could be in part mediated through insulin receptor A. Since Foti et al. (Nature Medicine 2005) reported that knocking out HMG A1 markedly downregulated insulin receptor expression and that transfection of the cells with HMG A1 restored insulin receptor expression one would expect the manipulations herein to alter insulin receptor number. If these manipulations of HMGA1 do change insulin receptor expression then tumor proliferation could be driven through this receptor. Therefore, showing the change in insulin receptor number would strongly support their hypothesis that this is the mechanism mediating tumor cell proliferation under these conditions. It seems reasonable to measure insulin receptor expression under conditions of high and low HMGA1 expression.7) The authors should discuss the data of Foti et al. and how they apply to their system. Additionally, those authors  have published data indicating that with HMG A1 downregulation there is enhanced insulin/IGF-I sensitivity which, if discussed, would also contribute to a more complete consideration of their hypothesis. Essential revisions:1) One might expect there to be a synergy between IGFBP2 and Grb14, but -/- MEFs each restores the proliferative rate from ~25% to ~70-80% of similarly treated Imp2+/+ MEFs and dual depletion is non-additive. We agree with the reviewer that this non-additivity suggests that both interventions act through the same pathway, i.e., by restoring signaling though the InsR and/or IGF-1R. We have added a sentence to that effect .Depleting Grb14 or IGFBP2 from Imp2-/- MEFs is to sequester autocrine IGF, but doesn’t rule out other possible sites of IGFBP2 action on that pathway.The non-additivity and known ability of IGFBP2 to bind IGFs certainly supports the view that the predominant action of overabundant IGFBP2 in the Imp2-/- MEFs proliferation and that together they are not additive may indicate that each intervention is sufficient to regain the full effect of the InsR/IGF1R pathway on the proliferation of Imp2-/- MEFs; i.e., impaired signaling by the InsR/IGF1R pathway perhaps accounts for ~2/3 of the ~75% reduced proliferation of Imp2-/- MEFs and other, as yet unidentified IMP2-dependent factors, that act through other proproliferative pathways or processes, account for the remaining deficit.The finding that neither intervention enables complete restoration of Imp2+/+ cells, which does not appear to be significantly altered (judging by the blots) in response to IGF1. Moreover, whereas the robust decrease in IGF-1R and IRS phosphorylation in IMP2-/- cells is partially rescued upon addition of IGF1, rescue of the corresponding decrease in Akt phosphorylation appears to be minimal. This discrepancy should be explained.2) +/+ MEFs, basal Akt(Ser473P) is high and IGF1 causes a somewhat variable increase in Akt(Ser473P). The striking and consistent finding is the markedly lower basal and IGF1-stimulated Akt(Ser473P) signal in the Imp2-/- MEFs. Nevertheless, IGF1 addition does increase the Akt(Ser473P) signal in the Imp2-/- MEFs in all three experiments. Regardless of the difference in the basal activity of Akt, we think that the lesser Tyr-P of the IGF1R and IRS polypeptides in the Imp2-/- MEFs is strongly indicative of impaired IGF signaling.We’ve carried out this experiment three times and the other 2 experiments are shown in +/+ vs. Imp2-/- MEFs. HMGA1 has been reported to suppress the activity of PP2A, by driving expression of miR222, which promotes degradation of the mRNA encoding PPP2R2A (PP2A subunit Bα), reducing PP2A activity and activating Akt. Our proteomic analysis  The mechanism of Grb14 protein stabilization by IMP2 is unexplained. Given that Grb14 plays a role that is shown to be as important as that of IGFBP2 in disrupting IGF2-mediated cell growth, some insight into the mechanism whereby IMP2 stabilizes Grb14 ought to be provided. For example, does IGFBP2 exert an effect, direct or indirect, on Grb14 expression? Does depletion of IGFBP2 alter Grb14 expression? And if HMGA1 does not affect Grb14 stability, what are the candidate factors that may do so?We have been unable to identify the proximate mechanism by which IMP2 regulates the degradation of Grb14. Clearly, HMGA1 , like IMWe have therefore hypothesized that IMP2 acts through HMGA1 which regulates the abundance of a E2 or E3 component of the ubiquitination pathway, a deubiquitinase, a protease or protease inhibitor, or some gene product (including miRs) that regulate the abundance of such elements. The number of steps that intervene between HMGA1 and Grb14 degradation is unknown. We have been unable to identify plausible candidates among the HMGA1 transcriptome and have not identified the elements that directly control Grb14 degradation.4) The mechanism of HMGA1 mRNA protection by IMP2 is unexplored. Without going into excessive depth here, the investigators could easily determine whether IMP2 binds to let-7 binding sites in the 3' UTR of HMGA1. This would at least suggest whether or not HMGA-1 mRNA stability is mediated by the ability of IMP2 to block or displace let-7 family miRNA binding to their targets.RNAseq of IMP2 IPs confirms that IMP2 binds extensively to the HMGA1 mRNA 3’UTR , and as 5) The investigators begin their discussion by stating that their findings define the functional significance of IMP2 in cancer cells. However, other observations have shown that IMP2, in addition to other IMPs, promotes stemness in cancer cells. There are therefore probably several, possibly synergistic, mechanisms whereby IMP2 promotes tumor growth and this notion should be included in the Discussion. Furthermore, the investigators cite several studies that show a correlation between the poor prognosis of a variety of cancers and IMP2 expression. Recent work  has revealed precise mechanisms whereby IMP2 may be implicated in tumor maintenance and growth and should be cited.This is an important point, and we have modified the opening paragraph of the Discussion, to emphasize this aspect of IMP2 biology. We stress that in adult life IMP2, in contrast to IMP1/IMP3, is widely expressed and appears to be preferentially expressed in tissue resident stem cells, citing examples where IMP2 has been shown to be important to their ability to both proliferate and differentiate. We then cite the work of Degrauwe et.al., 2016b, which demonstrates the importance of IMP2 in glioblastoma stem cells.6) The results regarding P21 are interesting however it is not clear why lowering P21 levels had no effect on proliferation. The assumption would be that even after deliberately lowering the levels they are still relatively high and therefore even a 5.9 fold change in levels is not adequate to alter proliferation. Some comment should be made on these findings.-/- MEFs than in IMP2+/+ MEFs, depletion of P21 to nearly the same level accelerates proliferation by only ~30% in both cell types. This similarity suggests to us that the impedance of the IGF1R/InsR pathway in the IMP2-/- MEFs imposes a restraint on proliferation that is largely independent of that imposed by P21.That said however, we have no compelling explanation for why depletion of P21 accelerates proliferation to a similar extent in WT and IMP2 KO MEFs. An obvious possibility is that the restraint to cell proliferation attributable to P21 in WT MEFs is modest and already near maximal, so that the 5-fold increase caused by IMP2 deletion has little further inhibitory effect. We have not however examined the effect of P21 overexpression in WT MEFs, because that would be an inappropriate model for IMP2-/- MEFs, which exhibit numerous changes in cell cycle regulators other than P21, in directions that could either speed or slow proliferation  reported that knocking out HMG A1 markedly downregulated insulin receptor expression and that transfection of the cells with HMG A1 restored insulin receptor expression one would expect the manipulations herein to alter insulin receptor number. If these manipulations of HMGA1 do change insulin receptor expression then tumor proliferation could be driven through this receptor. Therefore, showing the change in insulin receptor number would strongly support their hypothesis that this is the mechanism mediating tumor cell proliferation under these conditions. It seems reasonable to measure insulin receptor expression under conditions of high and low HMGA1 expression.As seen in Similarly, in RD cells , right, We do not know the nature of the compensatory mechanisms that offset the reduction in InsR gene expression likely to have resulted from the reduced HMGA1 abundance caused by IMP2 deletion. Nevertheless, this data indicates clearly that the slowed proliferative rate of IMP2 depleted cells cannot be attributed to a reduced InsR abundance. Similarly, as shown in 7) The authors should discuss the data of Foti et al. and how they apply to their system. Additionally, those authors  have published data indicating that with HMG A1 downregulation there is enhanced insulin/IGF-I sensitivity which, if discussed, would also contribute to a more complete consideration of their hypothesis.The metabolic phenotype of the HMGA1 KO mice as described by Foti et al., 2005 and pursued in the followup paper from this group  remains inexplicable. There is a reduction in insulin receptor abundance in the KO, but serum insulin levels are lower than in WT and glucose tolerance is minimally impaired; moreover, the hypoglycemic effect of injected insulin is actually greater in the KO. Liritano et.al, show that HMGA1 deletion reduces expression of the IGF binding proteins 1 and 3; IGFBP3 binds ~60-70% of circulating IGF1/2, IGFBP1 ~5% and most of the rest is bound by IGFBP2 (which our current paper shows is greatly increased by HMGA1 KO in cell culture). Liritano finds ~3X increase in unbound/free IGF-1in HMGA1 KO mice and reports modestly enhanced glycemic lowering to exogenous IGF-1 in the KO mice. Unfortunately, inasmuch as insulin does not bind the IGFBPs or IGF1R, these findings do not explain why there is a greater hypoglycemic effect of insulin in the HMGA1 KO despite reduced InsR expression. These complexities and unexplained phenomena discouraged us from dwelling on this data in our Discussion. Nevertheless, we have added a paragraph at the end of the Discussion that summarizes the information we consider defensible regarding the relationship of the components of the tumor promoting pathway we describe herein  to type 2 diabetes and the increased risk for cancers associated with that condition.References:1. Frasca F,1 Pandini G, Scalia, P, Sciacca, L, Mineo R, CostantinoA, Goldfine ID,Belfiore A and Vigneri R. Insulin Receptor Isoform A, a Newly Recognized, High-Affinity Insulin-Like Growth Factor II Receptor in Fetal and Cancer Cells. Mol Cell Biol.1999. 19: 3278–3288.PMCID: PMC841222. Rowzee AM, Ludwig DL, Wood TL. Insulin-like growth factor type 1 receptor andinsulin receptor isoform expression and signaling in mammary epithelial cells.Endocrinology. 2009.15:3611-9. doi: 10.1210/en.2008-1473.


Setophaga chrysoparia; hereafter warbler) in urban Texas. We mapped and monitored warbler territories before (2009–2011), during (2012–2013), and after (2014) highway construction at three study sites: a treatment site exposed to highway construction and traffic noise, a control site exposed only to traffic noise, and a second control site exposed to neither highway construction or traffic noise. We measured noise levels at varying distances from the highway at sites exposed to construction and traffic noise. We examined how highway construction and traffic noise influenced warbler territory density, territory placement, productivity, and song characteristics. In addition, we conducted a playback experiment within study sites to evaluate acute behavioral responses to highway construction noises. Noise decreased with increasing distance from the highways. However, noise did not differ between the construction and traffic noise sites or across time. Warbler territory density increased over time at all study sites, and we found no differences in warbler territory placement, productivity, behavior, or song characteristics that we can attribute to highway construction or traffic noise. As such, we found no evidence to suggest that highway construction or traffic noise had a negative effect on warblers during our study. Because human population growth will require recurring improvements to transportation infrastructure, understanding wildlife responses to anthropogenic noise associated with the construction and operation of roads is essential for effective management and recovery of prioritized species.Anthropogenic noise associated with highway construction and operation can have individual‐ and population‐level consequences for wildlife . We used a before–after control–impact study design to examine the potential impacts of highway construction and traffic noise on endangered golden‐cheeked warblers ( The warbler is an endangered, migratory songbird that breeds exclusively in central Texas, USA , during (2012–2013), and after (2014) highway construction at (1) a treatment site exposed to highway construction and traffic noise, (2) a control site exposed only to traffic noise, and (3) a control site exposed to neither highway construction or traffic noise. We recorded noise at varying distances from the highways at our construction and traffic noise sites. We expected that noise would be greatest at the construction site during the years of construction and that noise would decrease with increasing distance from the highway at both sites. If warblers responded negatively to highway construction noise, we predicted they would exhibit one or more of the following responses at the construction site during‐ or postconstruction relative to the preconstruction period: reduced densities, displacement from the highway, decreased pairing or fledging success, or higher minimum song frequencies. In addition, we predicted that warblers at the control site and those farther from highways would exhibit acute responses to experimentally introduced highway construction noise as individual birds located further from the sound source would be experiencing a novel disturbance. The results of our comprehensive assessment may help inform conservation efforts for warblers. In addition, our study demonstrates the use of a robust BACI study design to quantify wildlife responses to anthropogenic noise.22.1Juniperus ashei), Texas oak (Quercus buckleyi), live oak (Q. virginiana), and various other hardwoods , located on the eastern edge of the warbler's breeding range . Our second control site (hereafter control site) included 682‐ha of potential warbler habitat located >800 m from Highway 71 or other major roads. We collected data at all sites during three treatment phases: preconstruction (2009–2011), construction (2012–2013), and postconstruction (2014).We delineated one treatment site and two types of control sites at the Barton Creek Habitat Preserve, a 1,600‐ha property managed by The Nature Conservancy. We selected study sites with similar ecological characteristics that were exposed to similar environmental conditions throughout the year . The treatment site (hereafter construction site) was a 301‐ha area of potential warbler habitat located ≤800 m from a 5.2 km segment of Highway 71 that was exposed to construction noise associated with highway expansion in 2012 and 2013 and traffic noise throughout the study. Annual average daily traffic (AADT) rates for this portion of the highway were 30,000–39,000 vehicles/day TXDOT . During 2.2We established paired transects perpendicular to Highway 71 in the construction site and perpendicular to Southwest Parkway in the traffic noise site. We spaced transect pairs across the construction and traffic noise sites to account for potential within‐site variability in warbler density. As such, the number of transect pairs (two or three) depended on the total area of each study site. We placed Extech 407764 Datalogging Sound Meters  programmed to record noise from 06:00 to 12:00 on each transect at stations 16, 32, 64, 128, 256, and 512 m from the highways. We randomly sampled one transect pair every 2 days from late March to late May, rotating between the construction and traffic noise sites. This protocol allowed us to sample 2–3 transect pairs per week throughout much of the warbler breeding season during the hours when songbirds are most active. We calculated the mean and maximum noise for each sound meter for use in analyses. We did not place sound meters at the control site because pilot data collected in 2008 indicated that highway noise could not be detected at distances >800 m from highways in the study area. During the pilot year, mean noise levels were 36 dB(A) at the control site, which is consistent with noise levels recorded in libraries and quiet rural areas.2.3We established transects ≥300 m apart covering the spatial extent of each study site. Transects in the construction and traffic noise sites extended ≤1 km from the highways. Transects in the control site were ≤1.5 km and located ≥800 m from the nearest highway. Each day from late February to late March, we slowly walked (~1 km/h) along transects from 07:00 to 13:00 and recorded the locations of singing male warblers using handheld Global Positioning System (GPS) units (≤10 m accuracy). If we heard another warbler in close proximity to our initial detection, we walked to the newly identified bird and recorded a GPS point at its location. After we obtained GPS locations on all singing males in the area, we returned to the transect to search for additional warblers. We used this methodology to ensure full survey coverage of the study sites, but we did not use detections recorded during transect surveys for analyses purposes. Rather, we used the locations to aid relocation of male warblers during subsequent territory mapping and monitoring activities.We mapped and monitored each territory for up to 1 hr at least once per week for the duration of the warbler breeding season (March–June). If we detected previously unidentified individuals during our territory mapping and monitoring surveys, we recorded GPS locations for the new individuals and added the territories to our weekly survey rotation. Our intent with this methodology was to identify all territorial males within each study site.During territory mapping in 2009–2010, we recorded GPS locations of males each time they moved ≥20 m until we recorded 3–6 locations per sampling occasion. In 2011, we modified our territory mapping protocols and recorded GPS locations of males every 2 min for 1 hr or until we could no longer detect focal individuals  across all study sites to examine how highway construction and traffic noise influenced warbler song characteristics. We placed ARUs in randomly selected territories at various distances from the highways  at the construction and traffic noise sites. We placed ARUs randomly within the control site given a lack of linear features comparable to the highways at other sites. We programmed ARUs to record from ~06:45 to 11:00 daily. We allowed each ARU to record warbler songs for 3–4 weeks before moving it to another randomly selected territory.™ v1.5.8  to extract warbler songs from our recordings. The extraction process identified warbler songs using time–frequency patterns within the waveforms of the recordings but also identified songs of other species that fell within the same frequency range . Therefore, we visually inspected the sonograms of extracted songs to ensure proper species identification. We also identified and excluded any sonograms with extensive background noise that could interfere with subsequent calculations of warbler song metrics .We used SonoBird™ v1.6.5  to manually analyze warbler songs. Warblers have two primary song types, the A‐song and the B‐song  (measured at ~5 m)—a level known to be annoying to humans but that does not cause hearing damage . We recorded the presence or absence of a behavioral response to the construction or control playback.2.7For an impact assessment using the BACI framework, data analyses should include an interaction term between treatment site and phase to determine whether there is a statistically significant difference at the treatment site following the disturbance  to test the interactive effects of site, treatment phase, and distance from the highway on our continuous response variables . We used logistic regression to examine the interactive effects of site and phase on our binary response variables . If there was a significant interaction (α = 0.05), we visually examined plots with calculated means and 95% confidence intervals (hereafter 95% CI) to determine the direction, magnitude, and biological significance of the patterns. Sample sizes or other factors sometimes required that we deviate from the general approach described above. We used a nonparametric Friedman's rank‐sum test to examine territory density in relation to site and phase Zar, . Friedma33.1F5,883 = 9.81, p < .01), indicating that differences in noise levels across the distance from highway categories depended on whether the sound meters were located in the construction site or traffic noise site. As such, we examined noise across treatment phases separately by site. Mean noise levels were 54 dB(A) ± 8 SD in the construction site and 53 dB(A) ± 6 SD in the traffic noise site. Mean maximum noise levels were 67 dB(A) ± 8 SD in the construction site and 66 dB(A) ± 7 SD in the traffic noise site. Mean and maximum noise decreased by ≤8 dB(A) with each increasing distance category from the highway across all three treatment phases at both the construction and traffic noise sites  or in the traffic noise site . Similarly, we did not find a statistically significant interaction between treatment phase and distance from the highway on maximum noise in the construction site . However, we did find a statistically significant interaction at the traffic noise site . Mean and maximum noise at distances closest to the highway in the traffic noise site were ≤10 dB(A) higher during the construction and postconstruction phases when compared to the preconstruction phase. However, the substantially overlapping 95% CIs indicated that the differences in noise were not statistically different across the treatment phases for most of the distance categories at either site . Mean territory density was 1.3 to 1.8 times greater in the control site than the construction and traffic noise sites  or phases .We mapped and monitored 450 warbler territories across the years of this study Table . We foun3.342 = 7.14, p = .13). We also found no statistically significant interaction between site and phase on warbler fledging success .Pairing success and fledging success ranged from 68% to 91% and 62% to 79%, respectively, during the three phases of our study (Table 3.4n = 72 song meters) and >3,500 B‐songs (n = 48 song meters). Sample sizes were low for B‐songs recorded during the postconstruction phase, so we were unable to examine statistically significant interactions for any of the B‐song metrics. As such, we excluded these data from this study. We found no statistically significant interactions between treatment site and phase for most A‐song metrics  or site  on warbler responses, and the predicted probability of a warbler response to experimental playback recordings of construction noise did not increase with increasing distance from Highway 71 or Southwest Parkway .We conducted 321 experimental playback surveys and 96 control surveys within 172 warbler territories. Playback surveys ranged from 48 to 1,900 m from the highway. In the construction site, six warblers (10%) responded to experimental playback and one warbler (~6%) responded to control surveys. In the traffic noise site, five warblers (~6%) responded to experimental playback and one warbler (~5%) responded to control surveys. In the control site, 20 warblers (~11%) responded to experimental playback and two warblers (~3%) responded to control surveys. We found no significant main effect of playback survey type  are less likely to place their territories in close proximity to compressors used in oil and gas production, which typically operate at 75–90 dB(A) (maximum 105 dB(A)), 24 hr a day, 365 days a year  exhibit reduced pairing success when exposed to sustained oil and gas compressor noise  suggest that the noise would have to be close range and much higher than levels typically recorded at construction sites ; Kerr, Brousseau, & Johnson, The frequency of anthropogenic noise that birds are exposed to may be more influential than amplitude .The USFWS previously indicated that noise levels occurring at ≥60 dB(A) during the loudest hour of the day may negatively influence songbirds Barrett, . HoweverAnthropogenic noise associated with highway construction and operation can have individual‐ and population‐level consequences for wildlife. However, the impacts of highway noise on birds are species specific (reviewed in Reijnen & Foppen, None declared.


Phyllostachys pubescens) leaves and Japanese apricot (Mume fructus) fruit are traditionally recognized to be safe herbs broadly used for food and medicinal purposes in Southeast Asia. Our group previously explored their antiplatelet effects. This study was designed to confirm inhibition effects of PM21 (a 2:1 mixture of bamboo leaf extract and Japanese apricot fruit extract) on platelet aggregation and evaluate its potency to use as an herbal remedy to prevent and/or treat the diseases caused by platelet aggregation and thrombus formation.Bamboo  were evaluated. In ex vivo assays, PM21 was orally administered to rats daily after overnight fasting for 3 days and blood was collected 1 h after the final treatment2, and serotonin. PM21 also reduced intracellular concentration of calcium ion, fibrinogen binding to integrin αIIbβ3, and phosphorylation of ERK2, p38, PLCγ2, and PI3 K.In ex vivo assay, PM21 inhibited platelet aggregation significantly. PM21 showed a strong antithrombotic effect by reducing significantly the length of mouse tail thrombus. PM21 increased intracellular cAMP level and reduced the release of ATP, TXA2, serotonin, and Ca2+. PM21 also appears to exert its anti-platelet effect by deactivation of ERKs activation pathway as well as inhibition of fibrinogen binding to integrin αIIbβ3.PM21 showed remarkable inhibitory effects on platelet aggregation and thrombus formation. Its inhibitory function seems to influence on GPVI binding to its ligand and subsequent initiation of a signaling cascade that involves activation of effector proteins and secretion of effector molecules, such as ATP, TXA When a blood vessel is injured platelets and fibrin play a major role to form blood coagulation to prevent blood loss from the damaged vessel. However this blood coagulation can cause a various kinds of venous or arterial pathogenesis, such as congestion, ischemia, and necrosis that can subsequently result in some serious cardiovascular diseases, such as a stroke  myocardiPlatelet aggregation is an important procedure for effective thrombus formation following the adhesion of platelets to the site of injury . The pla2 (thromboxane A2), a potent platelet activator, via prostaglandin H2 and arachidonic acid i was determined with Fura-2/AM as previously described i was calculated as previously described [Ca2+]i = 224 nM x (F - Fmin) / (Fmax - F), in which 224 nM is the dissociation constant of Fura-2-Ca2+ complex, and Fmin and Fmax represent the fluorescence intensity levels at very low and very high Ca2+ concentrations, respectively. F represents the fluorescence intensity of the Fura-2-Ca2+ complex measured at 510 nm after Fura-2-loaded platelets were pre-incubated with experimental samples and stimulated for aggregation reaction as previously described. In our experiments, Fmax was observed when platelet suspensions containing 1 mM Ca2+ were solubilized with Triton X-100 (0.1%), while Fmin was observed when platelet suspensions containing 3 mM EGTA were solubilized with Trion-100 (0.1%).The intracellular concentration of calcium ion [escribed . In thisWashed platelets were pre-incubated with experimental samples and stimulated for aggregation reaction as previously described. After terminating the reaction, lysates were then prepared by solubilizing and centrifuging platelets in a sample buffer . Protein concentration was determined by BCA assay . Total cell proteins (30 μg) obtained from platelet lysates were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes in transfer buffer . The membranes were blocked in TBS-T containing 5% nonfat dry milk and incubated with primary antibody diluted in a blocking solution. The membranes were then probed with antibodies of phospho-ERK2, ERK2, phospho-p38, p38, phosphor-PLCγ2, PLCγ2, phospho-PI3 K (p85), PI3 K and β-actin. The blots were then incubated with the horseradish peroxidase-conjugated secondary antibody. Antibody binding was visualized by enhanced chemiluminescence .Male ICR mice weighing 20 ~ 23 g were purchased from Daehan Biolink Co. Ltd. . Six mice were arranged in each experimental group. Mouse tail thrombosis was induced by carrageenan according to the previously reported method . Each moMice were orally administered with experimental samples and mouse tail thrombosis was induced as previously described. Blood was collected 1 h after the last treatment, 72 h after the carrageenan injection. Washed platelets were prepared, and platelet aggregation was induced as previously described.t-test to evaluate statistical differences between the treatments and vehicle control. Dunnett’s test was utilized to evaluate statistical differences among the data involved in three or more groups. Data obtained from this experiment expressed as mean value ± SEM (standard error of mean). P < 0.05 was considered to be statistically significant.Data were analyzed by one-way ANOVA, followed by Student’s two tailed-Ex vivo Inhibition effects of PM21 on platelet aggregation after 3 days of oral administration to SD rats are shown in Fig. Ex vivo Inhibition effects of PM21 on ATP release after 3 days of oral administration to SD rats are shown in Fig. αIIbβ3. The results show that fibrinogen binding to integrin αIIbβ3 is significantly reduced in a dose-dependent manner by the treatment of PM21 compared to collagen treated vehicle. PM21 at the doses of 50, 100, and 200 μg/mL reduced fibrinogen binding to active integrin αIIbβ3 significantly by 21.1, 32.4, and 40.6%, respectively compared to collagen treated vehicle.As shown in Fig. Platelets were incubated with different concentrations of PM21 (100 and 200 μg/mL) alone or in combination with IBMX (50 μg/mL), and increase in intracellular cAMP concentration was assessed in resting platelets. The results show that IBMX treatment increases intracellular cAMP level approximately 5.4-fold compared to the untreated vehicle, and PM21 increases intracellular cAMP levels both with and without IBMX compared to each vehicle  are important signaling molecules in the regulation of platelet function. Intracellular cAMP induces the activation of PKA, results in the inhibition of platelet aggregation . PM21 in2. TXA2 formation was monitored by TXB2 formation in this study. TXB2 release was inhibited by PM21 significantly in this study. Released serotonin enhances platelet aggregation [2 and intracellular calcium concentration assays, PM21 shows inhibition potency on the release of granule contents and on the influx of calcium ions from extracellular fluid and mobilization from intracellular pools.Activated platelets release the stored granule contents such as serotonin and TXAregation , 35. In regation . In this2+ from intracellular stores and subsequent Ca2+ entry resulting in an increase in intracellular Ca2+ concentration [αIIbβ3 [The phosphorylation of signaling molecules such as ERKs is an important step for both outside-in and inside-out signaling that is closely associated with platelet activation and aggregation , 37. GPVntration . It is aIIbβ3 .We investigated collagen-induced phosphorylation of ERKs  and found that the phosphorylation of ERK2 and p38 was inhibited by PM21 in a dose dependent manner. PM21 also inhibited the phosphorylation and expression of PLCγ2 and PI3 K markedly.αIIbβ3 and platelet secretion by reducing the release of dense granule contents, such as ATP, serotonin, TXA2, and ionized calcium that are effector molecules to activate further platelet aggregation process. PM21 has also shown to downregulate effector proteins, such as ERK2, p38, PLCγ2, and PI3 K. From experimental results, we recognize that PM21 seems to exert its antiplatelet function to downregulate major events involved in the activation of GPVI receptor and, thereafter, the downstream signaling pathway of activated GPVI.PM21 has shown to affect platelet adhesion by downregulating fibrinogen binding to integrin 2, and serotonin. It also downregulated intracellular concentration of calcium ion, fibrinogen binding to integrin αIIbβ3, and activation of ERK2, p38, PLCγ2 and PI3 K.PM21 showed remarkable inhibition effects on platelet aggregation and thrombus formation in our animal studies. In antiplatelet mechanism study, PM21 upregulated intracellular cAMP level, and downregulated the release of ATP, thromboxane TXAERKs activation pathway as well as inhibition of fibrinogen binding to integrin αIIbβ3.These findings reveal that PM21 exerts its anti-platelet and antithrombotic effects by deactivation of the collagen receptor GPVI signaling pathway and