Global sea-level rise is caused, in part, by more rapid ice discharge from Antarctica, following the removal of the restraining forces of floating ice-shelves after their break-up. A trigger of ice-shelf break-up is thought to be stress variations associated with surface meltwater ponding and drainage, causing flexure and fracture. But until now, there have been no direct measurements of these processes. Here, we present field data from the McMurdo Ice Shelf, Antarctica, showing that the filling, to ~2 m depth, and subsequent draining, by overflow and channel incision, of four surface lakes causes pronounced and immediate ice-shelf flexure over multiple-week timescales. The magnitude of the vertical ice-shelf deflection reaches maxima of ~1 m at the lake centres, declining to zero at distances of <500 m. Our results should be used to guide development of continent-wide ice-sheet models, which currently do not simulate ice-shelf break-up due to meltwater loading and unloading. Meltwater ponding on top of ice shelves is thought to play a role in ice-shelf flexure and fracture, however in-situ evidence of these mechanisms is lacking. Here, the authors provide field-based evidence showing the impact of the filling and draining of four surface lakes on ice-shelf flexure in Antarctica. Lakes are thought to be hazardous to ice-shelf stability by acting as concentrated loads that flex and weaken the floating ice12. Surface streams and rivers facilitate the movement of meltwater across ice-shelves6, and therefore have an important role in controlling lake filling and draining5. When meltwater is simply produced and then re-frozen in-situ, there is no mass change at the surface. Numerical model and laboratory simulations suggest that if meltwater is advected across the ice-shelf surface, causing lakes to fill and drain , the ice-shelf will flex, which may lead to the formation of fractures both within and outside the lake basins13. A modelling study has suggested that if these fractures intersect other nearby lake basins, a chain reaction of further lake-drainage events may be initiated, potentially contributing to large-scale ice shelf break-up10. This scenario is supported by remote-sensing observations of the break-up of the Larsen B Ice Shelf in 200214. Ice shelves that have undergone significant thinning due to surface ablation and ocean-driven ablation at the ice-shelf base18 may be particularly vulnerable to break-up19.Surface meltwater lakes, together with stream and river networks have been widespread on the floating ice shelves that surround the Antarctic continent for decades20, there are currently no field data confirming whether ice-shelf flexure  in response to meltwater movement, ponding or draining actually occurs. Therefore, the aim of the present study was to collect field measurements to examine this process. The key practical constraint on our field campaign was to find a suitable location to collect the data. Given the remoteness of most presently melting Antarctic ice shelves, we undertook our study at the best site available; on the McMurdo Ice Shelf (McMIS) near the logistical hub of McMurdo Station  portion of the Ross Ice Shelf . Surface ablation is further enhanced due to the relatively warm prevailing south-westerly winds, which warm adiabatically as they descend onto the McMIS from the nearby landmasses of Minna Bluff and Mount Discovery22. Our ~40 km2 study site is located <5 km from the calving front where the ice shelf is relatively thin   from early November 2016 to late January 2017. The high surface ablation rates and shallow ice depths gave us a good chance of measuring significant changes in vertical ice deflection rates in response to variations in surface meltwater ponding and draining.The McMIS is a small  Fig. . Within Here, we present field observations from in and around four meltwater lakes from early November 2016 to late January 2017 (Methods). These observations include data from 12 differential global positioning system (GPS) stations  and three water pressure sensors (one for three out of the four lake sites) Fig. . Combine22.Fieldwork survey corroborated by analysis of satellite-imagery from the previous 18 years suggests that active meltwater lakes on the McMIS form in topographically low areas with high debris concentrations, e.g. Peanut, Ring, Rift Tip and Wrong Trousers (hereafter WT) lakes . The data from all four GPS transects support this Figs.  and 4. D−1, equal to the mean seasonal uplift rate of Ring GPS 2 and 3. This steady uplift is the background trend observed at all 12 GPS stations during the melt season and is likely due to relatively high surface meltwater production and export rates, compared to low meltwater import and ponding rates, causing the ice shelf to uplift hydrostatically as it experiences a net loss of surface mass . This upward deflection trend, indicative of net unloading from the area, suggests that the majority of the meltwater that slowly fills Ring lake until mid-December is produced in-situ, as opposed to being transported in from the surrounding area via surface melt streams. Thus, although Ring lake fills between late November and mid-December, the dominant trend of Ring GPS 1 is uplift as more mass is being lost from the area near to Ring GPS 1 by meltwater production and export than is being transported into the area and is ponding.Data from the Ring site show the most pronounced example of the immediate vertical ice movement in response to the filling and draining of a lake Fig. . Until m−1. Between 21 and 22 December, Ring GPS 1 goes down rapidly by ~0.2 m, corresponding to the time when the lake fills most rapidly and reaches >2 m in depth. The downward deflection measured between 14 and 22 December is indicative of net loading in the vicinity of Ring GPS 1, suggesting that inflow of meltwater from the surrounding area is now contributing to the filling of Ring lake. On 22 December, Ring lake starts to drain and Ring GPS 1 immediately starts to rise rapidly by ~50 mm day−1. In total, Ring GPS 1 rises by 0.96 m between 22 December and 28 January , we assume that they also drain slowly by overflow .The data from Rift Tip GPS 1 also show a pronounced uplift response to lake drainage Fig. , but duer) 250 m centred on each of the 12 GPSs  on 24 December, coinciding with the time when Ring GPS 1 is at its lowest elevation, and smallest (−1.6 × 104 m3) on 18 January, when Ring GPS 1 is close to its highest elevation. The net removal of water from the r = 250 m circular area centred on Ring GPS 1 between those dates is 5.7 × 104 m3  supports this observation. For example, using a combination of constrained parameter values that produce the best match between the analytic solution and measured deflection at Ring lake’s centre , its centre is simulated to rise by ~1 m, whereas the ice surface >250 m away from the lake centre rises by <10 mm ; effective ice thickness (H); and Young’s Modulus (E). Sensitivity tests were run for varying combinations of values for these parameters within the following ranges: R = 50–250 m; H = 10–30 m; and E = 1–10 GPa. These ranges are guided by our field data (R); McMIS data collected by others (H)25; and, values derived through modelling and laboratory experiments by others (E)28. For the Ring lake site, calculated centre-lake deflection and maximum von-Mises stress for varying combinations of values for the parameters R, H and E are given in Supplementary Table 4 m3 is removed during the melt season, are found to be 125 m, 10 m, and 1 GPa, respectively  and E (1 GPa) established for the Ring lake site were then used within the analytic solution and applied to the other three lake sites to see if the analytic solution agreed with GPS 1 vertical elevation data measured there. The other lakes have different meltwater unloading inputs, as indicated by our seasonal net meltwater budget calculations . Such increased meltwater loading may increase the potential for fracture formation, and ultimately, ice-shelf break-up, perhaps once a given ponding density threshold is exceeded10. Surpassing a given ponding density may also enable a chain-reaction style lake drainage process to assist with ice-shelf break-up10. Currently on the McMIS, surface lakes are not sufficiently widespread for the localized meltwater-loading induced flexure  to affect more than one lake. Other ice shelves that are already experiencing more widespread melting and pond formation may be more vulnerable to break-up5.Although our results indicate that there is currently a negative net meltwater budget on the McMIS during the melt season, melt rates on many of Antarctica’s ice shelves are expected to increase two- to three-fold by 2050The results of this study, which are based on field measurements, show that surface meltwater ponding and drainage has a prominent and instantaneous effect on ice-shelf vertical deflection and flexure. The magnitude of the vertical ice-shelf deflection decreases from maxima of ~1 m at the centres of the maximum load changes to zero at distances of <500 m; observations that are supported by an exact analytic solution for flexure of a floating, thin elastic plate, in which constrained parameter values fall within sensible ranges.38 to produce more accurate predictions of future sea-level rise following ice-shelf collapse41. The process of ice-shelf flexure in response to surface meltwater load changes11, which may lead to fracture9, is not yet simulated by these models.Ultimately, the observations presented here provide an initial performance constraint to guide the development of regional- and continent-scale ice-sheet modelsTwelve differential GPS stations were deployed in the field area from mid-November 2016 to mid-January 2017  recorded by the GPS receivers were processed using the TRACK software package maintained by the Massachusetts Institute of Technology using the GPS base station located at McMurdo Station . Parameters specifying standard deviation for horizontal and vertical motion used by the Kalman filter within TRACK, 30 mm of motion between samples in each direction, were estimated conservatively, and variation of this choice was not found to change the processed data significantly. Following the application of TRACK to determine the vertical elevation time series, the data were quality checked for outliers and obvious cycle slips (10 s of mm displacement over time periods of <10 s of minutes), and these were replaced with interpolations.Tidal displacement showing the dominant diurnal tide of McMurdo Sound with a weak semidiurnal component and the familiar spring-to-neap tidal cycle was the principal vertical motion in the time series. To restrict attention to only long-period monotonic vertical displacements associated with load-driven ice-shelf flexure, the tidal signals from each of the 12 stations were removed. This was done using a least-squares estimation of pure sinusoidal variation within each time series, applied with 3 dominant diurnal and 4 dominant semidiurnal frequencies  to produce the long-term vertical displacement residual presented in this paper. Subsequently, the data were smoothed using a 24-h running mean to remove other lower and higher frequency signals, including non-sinusoidal signals that are nearly at the tidal frequencies .−1. Because the IBE is common to all stations, errors in its application have no effect on vertical displacement differences between stations used to infer ice-shelf flexure.Following de-tiding and smoothing, the long-term vertical displacement residual was corrected for the inverse barometer effect (IBE) using barometric data (logged every 10 min) from two AWSs located 10–15 km from the field area , Pegasus North and Willy Field). The vertical displacement coefficient for the IBE used was 0.9948 cm mbApplying the time-averaging and tide removal algorithms described above to the elevation time series presented in Figs. Eight HOBO® (U20L-01) water pressure sensors, with a 0–9 m range and a 10-min logging interval, were deployed in and around the lake study sites from mid-November 2016 until mid-January 2017 Fig.  and 4. Thttp://www.onsetcomp.com/products/data-loggers/u20l-01).To account for barometric pressure fluctuations, the data were corrected against air pressure data from the AMRC Pegasus North AWS. The sensors have a typical water level accuracy of ±3 mm and a resolution of ~2 mm  = 250 m (area = ~2 × 105 m2), centred on each of the 12 GPSs  did not change the meltwater budget trends for any of the GPS.  'Seasonal net meltwater budgets' at all GPS stations were also calculated, each defined as the maximum minus minimum net meltwater budget during the 2016/2017 melt season.  Details of meltwater ponding and meltwater production calculations are given below.The 'net meltwater budget', defined as the total meltwater ponding volume minus the total meltwater production volume, was calculated within circles of radius  spectrometer, suitable for lake area and depth estimationTM). We used each image’s metadata to convert digital numbers to top-of-atmosphere (TOA) reflectance and to correct for solar elevation. These TOA reflectance values represent an adequate proxy for surface reflectance43.Reflectance values were used to extract both the area and the depth of surface water using a combination of bands. Landsat 8 bands 2 , 4 , 7  and 8  were cropped to our area of interest (using Extract by Mask in ArcMapice), defined as:ice values. Pixels with NDWIice > 0.07 were assumed to be water-covered. This value is lower than that (0.12) used to detect water-covered pixels in Landsat 8 in other studies44, because we lowered this threshold by the minimum amount necessary (in increments of 0.01) to include pixels that we knew to be water-covered from our ground instrumentation (i.e. our 8 water pressure sensors).To identify water-covered pixels, we calculated the normalized difference water index adapted for ice (NDWINine images (15 November 2016–25 January 2017 inclusive) were found to contain water-covered pixels , is given byAd is the lake bottom albedo, R∞ is the reflectance of optically deep water, and the coefficient g accounts for losses in upward and downward travel through the water column and varies with the wavelength used43. Solving this equation for water depth (z) gives:Rwater is the reflectance of a water-covered pixel. Ad was calculated image-by-image on a pixel-by-pixel basis, by taking the mean reflectance of a ring dilated by one pixel around each ponded region48, an improvement on previous studies that used static values across a region49. Some images did not contain optically deep water, and for those images, the difference between using an R∞ value of 0 and a value obtained from the ocean was negligible; for these reasons, an R∞ value of 0 was used. The depth of each water-covered pixel was calculated by averaging the water depths (z) derived from Landsat 8’s bands 4 and 8 (ref. 43). The values for g (0.7507 for band 4 and 0.3817 for band 8) were taken from ref. 43. This method of calculating water depth makes several assumptions, including the lake substrate is homogenous and the impact of any dissolved matter in the water on absorption is negligible; and there is no scattering of light from the lake surface associated with roughness due to wind46. We appreciate that the first assumption may be particularly problematic in our study area due to the large amounts of fine debris that accumulates in the areas where water ponds.To calculate water depth for the pixels identified as being water-covered, we employed the physically-based, single-band, water-depth retrieval algorithm originally based on the Bouguer–Lambert–Beer law51, but we are not aware of the shortwave infrared band having previously been used for a similar purpose.Finally, after water depth was calculated, dry debris that was falsely identified as water, was removed from the images. As dry debris is known to have high band 7 and low band 2 reflectance values , this was done by masking out band 7 pixels with reflectance >0.4 and band 2 pixels with reflectance <0.2. These thresholds were determined by performing a combination of visual analysis and inspection of the reflectance values in bands 2 and 7 for water and debris. A blue-band threshold has similarly been employed in other studies to remove pixels falsely identified as waterice threshold (see above) is assumed to produce negligible errors in the calculation of water-covered pixel areas; and the average depth error across multiple water-covered pixels using bands 4 and 8 has been shown to be zero (because positive and negative errors cancel out)43.We do not have errors on our meltwater volume calculations as our method for determining a suitable NDWI−1, and a clean ice group with melt rates <4 mm w.e. day−1. Our dirty ice and clean ice PDD factors are the mean of the PDD factors for the three dirty stake locations (47.5 mm w.e. °C−1 day−1) and the 9 clean locations (6.4 mm w.e. °C−1 day−1), respectively. The Standard Errors of these means are 9.0 and 0.9 mm w.e. °C−1 day−1, respectively, i.e. 19% and 13% of the respective PDD factors.We used a PDD model to calculate meltwater production during the 2016/2017 melt season. Two PDD factors, one for dirty, low-albedo, ice, and one for clean, high-albedo, ice, were derived empirically from our 2-m air temperature data and in-situ ablation measurements. Air temperature data were logged every 15 min at our AWS, which was installed from 22 November 2016 to 27 January 2017 in the centre of Rift Tip lake  and dirty (i.e. black) pixels and, for quality control, they were visually inspected and compared to the true colour Landsat 8 images and histograms  in which disk-shaped meltwater loads, or anti-loads (associated with the drainage of lakes), are confined within a region r ≤ R using polar coordinates r, θ, and where R is the radius of the lake or drained-lake. The vertical displacement of the elastic plate, η(r), for 0 ≤ r ≤ ∞, is expressed in terms of Kelvin–Bessel functions (as derived by ref. 54), and displayed in ref. 11. An undeflected ice shelf at a large distance (i.e. r ≤ ∞) is assumed, which is appropriate because the grounding lines and ice front are ≥2 km from all lake centres.Flexure of an ice shelf subject to changing surface-meltwater loads can be represented, for the purpose of estimating stress magnitudes, using an analytic solution based on thin elastic (Kirchhoff) plate theoryR); effective ice thickness (H); and Young’s Modulus (E), were varied to produce the best match between the analytic solution and measured lake-centre deflection for Ring site  to find the best match between the analytic solution and measured lake-centre deflection for Rift Tip, WT and Peanut sites , we did not account for changes in the effective density of ice due to debris content. Input to the analytic solution is a disk-shaped meltwater load removal, taken as the calculated seasonal net meltwater budget for each site (Figs. Values for the three parameters: lake radius (ite Fig. . Having te Figs. .11. However, in reality, as it may take a year or more for a lake to fill, a viscous response of the ice shelf will be present too53. The maximum flexure stresses implied by the elastic solution therefore represent maximum upper bounds; with a viscoelastic model20, modelled deflections at all GPS stations, including the lake centre stations, would be lower, and the flexure stresses would also all be lower. Additionally, using a viscoelastic model, the ice-shelf flexural response to the change in lake load would be even more local than that observed with the current analytic solution for flexure of a thin elastic plate, which our GPS data would support.We use an elastic treatment of flexure stresses induced after lake drainage, under the assumption that the timescale of lake drainage via overflow is relatively short compared to the Maxwell timeSupplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1


Age is the main risk factor for cancer, cardiovascular disease, neurodegeneration and other diseases prevalent in the world’s aging population. These diseases increase the pain and suffering and result in billions of dollars in healthcare costs. Addressing the common risk factor may allow for simultaneous amelioration of these diseases providing personal and economic relief to people and societies around the globe. The mTOR signaling pathway has been shown to be a robust target in the aging process with its inhibition resulting in increased lifespan in model organisms from yeast to mice. Rapamycin is an FDA approved drug for use in transplant patients and is a potent and specific inhibitor of mTOR complex 1. Rapamycin’s use as an anti-aging therapeutic in otherwise healthy individuals is complicated by the occurrence of side effects. As such, groups studying inhibition of the mTOR pathway are searching for alternative inhibitors that may be able decouple the deleterious effects of rapamycin administration from the lifespan extending effects. To address this issue, we have developed a high-throughput yeast-based assay for the identification of novel mTOR inhibitors. By utilizing mutant strains with differential sensitivity to mTOR inhibition, comparative growth kinetics of microcultures exposed to an inhibitor can be used to discern the mTOR inhibitory status of a compound. Furthermore, the assay can provide mechanistic insight on a compound’s mode of inhibition providing a rich, fast readout of a compound’s potential for inhibiting mTOR. This approach allows for the screening of large libraries of compounds speeding the discovery process.


We suggest high heat producing Precambrian basement rocks and hydrothermal circulation along a major fault system cause this anomaly. We conclude that local geothermal flux anomalies could be more widespread in East Antarctica. Assessing their influence on subglacial hydrology and ice sheet dynamics requires new detailed geophysical observations, especially in candidate areas for deep ice core drilling and at the onset of major ice streams.Melting at the base of the Antarctic Ice Sheet influences ice dynamics and our ability to recover ancient climatic records from deep ice cores. Basal melt rates are affected by geothermal flux, one of the least constrained properties of the Antarctic continent. Estimates of Antarctic geothermal flux are typically regional in nature, derived from geological, magnetic or seismic data, or from sparse point measurements at ice core sites. We analyse ice-penetrating radar data upstream of South Pole revealing a ~100 km long and 50 km wide area where internal ice sheet layers converge with the bed. Ice sheet modelling shows that this englacial layer configuration requires basal melting of up to 6 ± 1 mm a More recent (300-100 Ma) tectonic rifts dissect East Antarctica but appear not to have significantly altered the underlying Precambrian lithosphere4. Geophysical techniques provide regionally averaged estimates of geothermal flux and yield low values (50–60 mW m−2)7 for the composite East Antarctic craton, in line with global averages for cratonic heat flux8. Such regional estimates form the basis for coupled climate-ice sheet models suggesting variations in geothermal flux have limited impact on ice sheet stability9. More recent modelling studies indicate, however, that locally elevated geothermal flux can affect ice flow patterns in the interior of East Antarctica10. Although predicted by some geological studies of coastal regions11 direct evidence for the required local geothermal anomalies beneath the East Antarctic Ice Sheet was lacking.The East Antarctic Ice Sheet drains to the coast through conduits of fast flowing ice, known as ice streams, from an interior dominated by slow moving ice of the high, cold, polar-desert. The underlying East Antarctic continent is generally considered to be an ancient craton, last affected by a major tectono-thermal event ~500 Ma12  and significant upstream topographic variations, which can modify along flow layer geometry20, are not observed. We therefore attribute the observed loss of ice to enhanced basal melting23. The greatest level of drawdown overlies an area where bed reflectivity is >10 dB higher than the regional mean  subglacial lakes identified in older reconnaissance radar data also point to basal melting in this area17  implies extremely low ice velocity at the ice-bedrock interface (≪1.5 m a−1), and we can therefore bound the heating due to basal shearing30 to be smaller than 1 mW m−2. In our analysis, we neglect the small contribution of basal shearing to the heat budget and hence assume that the observed basal melting is due to geothermal heat flux. The estimated peak geothermal flux values are twice those indicated by previous continental-scale estimates derived from passive seismic and magnetic methods7.By modelling the depth age profile of the internal layers  time scales. The impact of the imaged enhanced subglacial melting on East Antarctic Ice Sheet dynamics will depend on the routing of melt water. Basal and surface topography are the critical controls on subglacial water routing, but were poorly constrained in this region. Our new bed and ice surface topography provide improved boundary conditions Figs  and 2d a Ma time ide Fig. . Hence, 8. A number of geological factors could cause the observed elevated geothermal flux and basal melting. The adjacent West Antarctic region is predicted by many studies to be a region of elevated geothermal flux37, linked to the existence of a major Cretaceous to Cenozoic rift system38. Notably, recent passive seismic studies have revealed warm West Antarctic mantle impinging beneath the Transantarctic Mountains towards the South Pole region39  volcanics are present in neighbouring sectors of the Transantarctic Mountains  granitoids cause locally high geothermal heat flux and likely also underlie parts of the East Antarctic conjugate margin11. In other areas of East Antarctica Cambrian granites11 have been recognised which could create local geothermal anomalies of up to 120 mW m−2, and radiogenic Jurassic granites in the Ellsworth-Whitmore Mountains microcontinent45 are modelled to give geothermal anomalies of up to 95 mW m−2. Geothermal anomalies in the East Antarctic interior generated by radiogenic source rocks depend on such rocks being present. Provenance studies from the catchments of the Byrd and Nimrod glaciers  and layers linked to the Lake Vostok drill site in older reconnaissance radar data54 .All maps were made by the authors for this publication using Geosoft Oasis montaj mapping tools version 8.5, Supplementary material


Western North Pacific tropical cyclone (TC) activity is known to have a significant response to El Niño and Southern Oscillation (ENSO). Among the TC intensity classifications, “super typhoon”, is widely used as a symbolic word for warning people about the potential for experiencing the most severe typhoon. This study quantifies the contribution of super typhoons to TC activity in response to ENSO, where the Southern Oscillation Index (SOI) is used to indicate the internal variation of ENSO. It is found that the number and the genesis location of non-super typhoons are little influenced by ENSO, and the changes are mostly by the addition of super typhoons. Historical levels of El Niño and La Niña span from −2σ to +2σ of the SOI. Over this range of SOI values, the mean response is 5.6 super typhoons (7.1 for El Niño and 1.5 for La Niña) which nearly matches the mean response overall of 6 storms (24.6 for El Niño and 18.6 for La Niña). The spatial distribution of TC genesis locations for different ENSO conditions does not completely explain these results. In an El Niño environment, more TCs are likely to occur in the southeastern quadrant of the WNP3, which is the peripheral area of subtropical high modulated by ENSO. This suggests that longer-living and stronger TCs contribute to the larger TC acvitity6, which can be monitored by some activity indicators such as Accumulated Cyclone Energy (ACE)7 or Power Dissipation Index (PDI)8.The El Niño and Southern Oscillation (ENSO) is one of the most influential climate variability patterns of global ocean and atmosphere, and there have been a number of investigations on its connection to tropical cyclones (TCs). TC activity in the western North Pacific (WNP) has been found to show significant response to ENSO by its vorticity, wind shear, and moist static energy9. Within this framework, the contributions of intensity and frequency to TC activity can be individually identified.Since ACE and PDI represent merged factors such as storm count, intensity and lifetime and its deconvolution is not available, further understanding of each portion of the attributes has been limited. Recently, considerable progress has been made in the analysis framework for TC climate indicators. An empirical framework was designed to express a variability where intensity and frequency of TCs are combined through principal component analysisBased on these recent works, this study tries to quantify the annual TC activity and the contribution of super typhoons in response to ENSO. The word “super typhoon” is reserved for the highest level of storm warning category by the U.S. Joint Typhoon Warning Center, and the word is widely used as symbolic for the strongest typhoons. The threshold lifetime-maximum intensities (LMIs) for TC and super typhoon are defined as 34 kt and 130 kt, respectively. Here, Southern Oscillation Index (SOI) is used to indicate ENSO status, and the annual proportion of super typhoons to indicate the annual intensity of WNP TCs. The study period is the 30 years from 1986 to 2015. Values for all variables are averaged over the period June to November.10. SOI is another metric of ENSO variability but using sea level pressure differences between Darwin and Tahiti. The response of tropical SST to global SST11 suggests that the SST-based metrics are likely to reflect localized global warming pattern. Even the highly contested so called “global warming hiatus”12 is not seen in global mean SST (GMSST)13.Various indices have been employed to indicate the ENSO status in order understand its role in climate variability. For example, Niño 1 + 2, Niño 3, Niño 3.4 and Niño 4 are widely used indices, based on the sea surface temperature (SST) averaged over each various regions of the eastern and central tropical Pacific Ocean14 might become muted. Thus for a better representation of an internal variation showing no forced trend, in this study SOI is used as the ENSO index.On the other hand, SOI appears statistically and physically separate from global warming. Figure 4. Here, the warm (cold) phase of ENSO is defined by a negative (positive) value of SOI. The map confirms that the number of super typhoons during the warm phase of ENSO is larger then the number during the cold phase. The occurrence of super typhoons to the east of 140°E and to the south of 20°N, is apparently greater in the warm phase of ENSO. It is noted on the other hand that the number and genesis location of non-super typhoons seem to be little influenced by ENSO  ass returns standardized vector values, and where θ is the angle starting from INT, where the counterclockwise direction is positive. By Eq. . For comparison, the correlations of ACE and PDI with -SOI (El Niño) are found 0.68 and 0.69  is defined as the proportion of super typhoons among the total WNP TCs  have the same sign of the standardized SOI. Thus, the abscissa values in Fig. The annual variation of TCIopt, this study shows how both INT and FRQ contribute to overall TC activity in response to ENSO through changes in the density distribution of genesis locations. The strength of this approach is that modeled TCIopt interprets the INT and FRQ of TC activity together. The contributions of INT and FRQ to activity are quantified by the projections of the modeled TCIopt onto INT and FRQ, as it were, TCIopt·cosθ and TCIopt·sinθ, respectively.Using a unified analysis framework of TCIopt is seen as the straight outline to the upper side of the dark green area. The dark green area is the portion of super typhoons as defined as INT in Eq. (17.The resultant contribution of ENSO (indicated by SOI) to TC frequency (FRQ) alone is delineated in Fig. T in Eq. . The numT in Eq. . The aveT in Eq. . Other cThe significance of the activity in response to ENSO is tested and confirmed by 95% confidence limits see Fig. . The valHistorical levels of El Niño and La Niña span roughly between −2σ and +2σ of SOI.Over this SOI range, the average difference in total WNP TCs to El Niño and La Niña is 6.0 (24.6–18.6).The average difference in numbers of super typhoons is 5.6 (7.1–1.5), indicating that the response of WNP TC climate is mostly the result of more super typhoons.This study quantifies the influence of ENSO on WNP TC activity and the contribution of super typhoons. It shows that SOI is a better index for representing the internal variation of ENSO compared to SST-based Niño indices when considering the impact ENSO has on typhoon activity. An optimal TC climate indicator defines the TC activity that ENSO explains the best. Modeled TC activity over the SOI range is effectively deconvolved into the contributions of overall TCs and super typhoons as follows:The results also show that the number and genesis locations of non-super typhoons are little influenced by ENSO. The changes are mostly by the addition of super typhoons. The distribution pattern of TC genesis locations at different ENSO status fails to completely explain the results. The study is based on the assumption of a linear relationship between TC activity and ENSO. The linear perspective for this study is considered as simple and effective to catch the clear feature of ENSO’s influence on annual TC activity.www.nature.com/nclimate/journal/v5/n7/extref/nclimate2646-s1.pdf). Since all directional variabilities are continuously linked on the same space, the response of TC climate to its environment can be optimally detected . Space is made by a principal component analysis (PCA) using INT and FRQ as the two primary inputs. INT and FRQ are the vectors of annual TC intensity and frequency, respectively. The two principal components together with the two primary indicators form a continuous variability space  from June to November are used. The observational consensus of the LMI distribution by ENSO, between JTWC and JMA, is examined in the Supplementary Information  and the Japan Meteorological Agency (JMA) was suggested to be the years after 1984see Fig. , which sSupplementary Information


The third immunoglobulin-like domain of nectin-4 cis-interacted with domain IV of ErbB2. This region differs from the trastuzumab-interacting region but is included in the trastuzumab-resistant splice variants of ErbB2, p95-ErbB2 and ErbB2ΔEx16. Nectin-4 also cis-interacted with these trastuzumab-resistant splice variants and enhanced the activation of the phosphoinositide 3-kinase-AKT signalling pathway for DNA synthesis. In addition, nectin-4 enhanced the activation of the p95-ErbB2-induced JAK-STAT3 signalling pathway, but not the ErbB2- or ErbB2ΔEx16-induced JAK-STAT3 signalling pathway. These results indicate that nectin-4 cis-interacts with ErbB2 and its trastuzumab-resistant splice variants and enhances the activation of these receptors and downstream signalling pathways in a novel mechanism.Nectin-4 cell adhesion molecule and ErbB2 tyrosine kinase receptor are upregulated in many cancers, including breast cancer, and promote cancer cell proliferation and metastasis. Using human breast cancer cell lines T47D and SUM190-PT, in which both nectin-4 and ErbB2 were upregulated, we showed here that nectin-4  ErbB2 is frequently upregulated by its gene amplification in many types of cancers, including breast cancer, and plays a role as an oncogenic protein, which induces tumourigenesis, invasion, and metastasis5. The incidence of ErbB2-upregulated breast cancers is approximately 20–30% of total breast cancer cases7. ErbB2-targeting monoclonal antibody (mAb) drugs, such as trastuzumab  and pertuzumab , have been developed and used as therapeutic drugs for ErbB2-upregulated breast cancers8. However, two splice variants of ErbB2, p95-ErbB2 and ErbB2ΔEx16, have been identified as oncogenic proteins in trastuzumab-resistant breast cancers20, although trastuzumab-resistance to ErbB2ΔEx16 is controversial22. The incidence of p95-ErbB2- or ErbB2ΔEx16-upregulated breast cancers is 30% and 90%, respectively, of the total ErbB2-upregulated breast cancers24. Thus, the development of therapeutic drugs for trastuzumab-resistant breast cancers expressing such variants is awaited.ErbB2 is a receptor tyrosine kinase belonging to the epidermal growth factor (EGF) receptor family with four members, ErbB1, -2, -3, -4, which are also known as EGF receptor for ErbB1 and HER1, -2, -3, -4, respectively25. One EGF molecule binds to one ErbB1 molecule, resulting in its homodimerization. Crystal structure and NMR analyses have revealed that the ErbB1 molecule exists in a ‘tethered’ or an ‘extended’ structure in the absence of EGF26. In the tethered structure, the extracellular domains II and IV are linked, but in the extended structure of ErbB1, domain II, which is a binding site for another ErbB1 molecule, is released25. These two structures are in dynamic equilibrium, and the binding of one EGF molecule to domain I and domain III promotes the conversion of the tethered structure to the extended structure . This conversion leads to the homodimerization of ErbB1, promoting the tyrosine kinase activity of each receptor, followed by the intermolecular tyrosine phosphorylation . The Src homology 2 domain-containing protein Grb2 binds to the tyrosine-phosphorylated ErbB1 molecule and activates at least three signalling pathways, Ras-Raf-MEK-ERK, phosphoinositide 3-kinase (PI3K)-AKT, and JAK-STAT signalling pathways, for cell proliferation, survival, and migration, and their continuous abnormal activation eventually leads to cancer cell tumourigenesis, invasiveness, and metastasis32. The truncated mutant of ErbB1 is an oncogenic protein that possesses the homodimerization property even in the absence of EGF and continuously transduces cell proliferation signals34.Of the ErbB family members, the activation mechanism has most extensively been investigated for ErbB1d25Fig. . These ture Fig. . This coion Fig. . The Src5. Crystallographic studies have revealed that ErbB2 exists in a distinct structure in which it exhibits an extended structure in the absence of ligands so that the loop in domain II of one ErbB2 molecule is ready to bind to that of another ErbB2 moolecule35. However, the affinity of the loop in domain II of ErbB2 for its homodimerization is low and the homodimerization of ErbB2 requires its upregulation, which is frequently induced by amplification of ERBB2 in various cancers, including breast cancer31. The upregulated ErbB2-induced homodimerization enhances its tyrosine-phosphorylation and the activation of at least the same three signalling pathways as those downstream of ErbB1 for cell proliferation, survival, and migration, eventually leading to cancer cell tumourigenesis, invasiveness, and metastasis32. Thus, upregulated ErbB2 also serves as an oncogenic protein in this mechanism. Trastuzumab interacts with domain IV of ErbB2 and enhances its internalization, causing inhibition of the ErbB2 signalling pathway for cell proliferation, although its mode of action differs depending on cancer cell type18. Trastuzumab further targets tumour cells by antibody (Ab)-dependent cell-mediated cytotoxicity in a patient’s immune system. Pertuzumab interacts with domain II of ErbB2 and inhibits its heterodimerization with ErbB3 and activation, causing inhibition of the ErbB2 signalling pathway for cell proliferation37.ErbB2 has a kinase domain in the cytoplasmic tail, but any ligand for ErbB2 has not been identified, unlike ErbB1, ErbB3, and ErbB4, which have specific ligandse35Fig. . Howevers32Fig. . Thus, ue18Fig. . Trastuzn37Fig. .38. It belongs to the nectin-like molecule (Necl) family with five members , which comprises a superfamily with the nectin family with four members 41. These members trans-interact with each other in a homophilic or heterophilic manner and regulate diverse events mediated by cell-cell adhesion in various cells41. In addition, they cis-interact with cell surface membrane receptors, such as the platelet-derived growth factor receptor, the fibroblast growth factor receptor, the vascular endothelial growth factor receptor, the prolactin receptor, ErbB3, and ErbB4, and integrins, such as integrin αvβ3 and integrin α6β4, and regulate not only cell–cell adhesion but also cell migration, proliferation, differentiation, and survival45.Nectin-4 is a cell adhesion molecule (CAM), which was originally identified by Lopez’s group46, lung47, ovarian48, pancreatic49, gallbladder50, and gastric cancer51, and promotes cancer cell proliferation and metastasis53. The PI3K-AKT signalling pathway is implicated in these roles of nectin-4 through activation of the Wnt-β-catenin and Rac small G protein signalling pathways54. In addition, breast cancer cells cultured in soft agar express nectin-4 and nectin-1 that trans-interact each other in a heterophilic manner, and nectin-4 also trans-interacts in a homophilic manner52. These interactions enable the cancer cells to survive in an anchorage-independent manner in soft agar52. Nectin-4 further enables epithelial and cancer cells to resist ferroptosis by clustering each cell for survival under a matrix-detached condition55. The concept derived from these results for anti-cancer drugs and a diagnostic marker development by interception of nectin-4 function has now been applied58.Nectin-4 is upregulated in various cancers, such as breastcis-interacted with ErbB2 and enhanced its homodimerization and activation in a novel mechanism, followed by the activation of the PI3K-AKT signalling pathway for DNA synthesis. We further found that nectin-4 also cis-interacted with the trastuzumab-resistant splice variant of ErbB2, p95-ErbB2, and a more aggressive splice variant for tumourigenesis, ErbB2ΔEx16, and enhanced the activation of the PI3K-AKT signalling pathway for DNA synthesis. In addition, nectin-4 enhanced the activation of the p95-ErbB2-induced JAK-STAT3 signalling pathway, but not the ErbB2- or ErbB2ΔEx16-induced JAK-STAT3 signalling pathway.We found here that nectin-4 cis-interacts with ErbB2. FLAG-tagged nectin-4 (FLAG-Nectin-4) was co-expressed with GFP-tagged ErbB2 (ErbB2-GFP) in human embryonic kidney (HEK) 293E cells. The cells were cultured in suspension to enable the detection of a possible cis-interaction between nectin-4 and ErbB2 on the same  plasma membrane. When FLAG-Nectin-4 was immunoprecipitated using an anti-FLAG mAb, ErbB2-GFP was co-immunoprecipitated with FLAG-Nectin-4 . ErbB1-GFP, ErbB3-GFP, or ErbB4-GFP was not co-immunoprecipitated with FLAG-Nectin-4 under the same assay condition . When endogenous nectin-4 was immunoprecipitated using an anti-nectin-4 polyclonal Ab (pAb) in SUM190-PT breast cancer cells, endogenous ErbB2 was co-immunoprecipitated with endogenous nectin-4 . In this cell line, nectin-4 and nectin-1 were expressed, but nectin-2, nectin-3, Necl-1, Necl-2, Necl-3, Necl-4, or Necl-5, was not detected . ErbB1, ErbB2, ErbB3, and ErbB4 were also expressed. In addition, endogenous nectin-4 and ErbB2 were co-localized at cell-cell adhesion sites in T47D and SUM190-PT cells . These results indicate that nectin-4 cis-interacts specifically with ErbB2 among the ErbB family members and that this cis-interaction occurs on the same plasma membrane.We first examined whether nectin-4 n-4 Fig. . ErbB1-Gn-4 Fig. . In this. These results indicate that nectin-4 cis-interacts with ErbB2 and that this cis-interaction enhances its homodimerization.We then examined whether nectin-4 affects the homodimerization of ErbB2. HA-tagged ErbB2 (ErbB2-HA) and ErbB2-GFP were co-expressed with FLAG-Nectin-4 or with FLAG as control in HEK293E cells. When ErbB2-GFP was immunoprecipitated with an anti-GFP pAb, ErbB2-HA was co-immunoprecipitated with ErbB2-GFP and FLAG-Nectin-4, but not FLAG, and the amount of ErbB2-HA in the cells co-expressing FLAG-Nectin-4 was more than that co-immunoprecipitated with ErbB2-GFP in the cells co-expressing FLAG Fig. . These r61. Using mAbs, one of which recognizes phosphorylated tyrosine residue at 1139 and  the other of which  recognizes both phosphorylated tyrosine residues at 1221 and 1222, we examined whether the nectin-4-enhanced homodimerization of ErbB2 enhances the phosphorylation of these tyrosine residues. For this purpose, we used T47D breast cancer cells, which expressed both nectin-4 and ErbB2 at much lower levels than SUM190-PT cells . In this cell line, nectin-1 and Necl-2, but not nectin-2, nectin-3, Necl-1, Necl-3, Necl-4, or Necl-5, were detected. The phosphorylation of tyrosine residues at 1139, 1221, and 1222 was enhanced in the T47D cells stably expressing FLAG-Nectin-4 compared with that in the control cells . Conversely, the phosphorylation of these tyrosine residues was reduced by the siRNA-induced knockdown of NECTIN4 in SUM190-PT cells . The reduction of nectin-4 by the siRNA-induced knockdown was confirmed by Western blotting . These results indicate that nectin-4 enhances the homodimerization of ErbB2, which leads to the phosphorylation of its tyrosine residues at 1139, 1221, and 1222.The homodimerization of ErbB2 induces the tyrosine-phosphorylation of ErbB2 intermolecularly at several tyrosine residues including 1139, 1221, and 1222lls Fig. . Converslls Fig. . The reding Fig. . These r32. We therefore examined the effects of nectin-4 on the activation of these signalling pathways. The threonine-phosphorylation of AKT was markedly enhanced in the T47D cells stably expressing FLAG-Nectin-4 compared with that in the control cells, whereas the threonine- and tyrosine-phosphorylation of ERK1/2 or the tyrosine-phosphorylation of STAT3 was not significantly enhanced in the T47D cells stably expressing FLAG-Nectin-4 compared with that in the control cells . The threonine-phosphorylation of AKT was inhibited by the tyrosine kinase inhibitor for ErbB2, irbinitinib, in the T47D cells stably expressing FLAG-Nectin-4 and in the control cells . Conversely, the threonine-phosphorylation of AKT was reduced in the SUM190-PT cells in which endogenous nectin-4 was knocked down compared with that in the control cells . These results indicate that nectin-4 mainly enhances the ErbB2-mediated PI3K-AKT signalling pathway, but not the Ras-Raf-MEK-ERK1/2 signalling pathway or the JAK-STAT3 signalling pathway.The tyrosine-phosphorylation of ErbB2 leads to the activation of the PI3K-AKT, Ras-Raf-MEK-ERK, and JAK-STAT signalling pathwayslls Fig. . The thrlls Fig. . Converslls Fig. . These r32. We estimated cell proliferation by measuring the incorporation of the DNA base analogue 5-ethynyl-2′-deoxyuridine (EdU) into DNA. We first examined whether nectin-4 enhances the ErbB2-mediated DNA synthesis. The incorporation of EdU into the DNA of the T47D cells stably expressing FLAG-Nectin-4 was enhanced compared with that in the control cells . The incorporation of EdU into the DNA of the T47D cells stably expressing FLAG-Nectin-4 and the control cells was inhibited by irbinitinib . In addition, the incorporation of EdU into the DNA of the SUM190-PT cells in which endogenous nectin-4 was knocked down was reduced compared with that in the control cells . These results indicate that nectin-4 enhances the ErbB2-mediated DNA synthesis.The enhancement of the phosphorylation of tyrosine residues of ErbB2 induces cell proliferation through the PI3K-AKT, Ras-Raf-MEK-ERK, and JAK-STAT signalling pathwayslls Fig. . The incnib Fig. . In addills Fig. . These r. These results indicate that nectin-4 enhances the ErbB2-mediated DNA synthesis through the activation of the PI3K-AKT signalling pathway, and that the nectin-4-independent ErbB2-mediated activation of the Ras-Raf-MEK-ERK1/2 pathway, but not the JAK-STAT3 pathway, is also involved in the ErbB2-mediated DNA synthesis.We then examined which signalling pathway, PI3K-AKT, Ras-Raf-MEK-ERK1/2, and/or JAK-STAT3 signalling pathways, is involved in this nectin-4-enhanced ErbB2-mediated DNA synthesis. The incorporation of EdU into DNA in the T47D cells stably expressing FLAG-Nectin-4 and the control cells was inhibited by the PI3K inhibitors wortmannin and LY294002 and the MEK inhibitor U0126, but not by the JAK1/2 inhibitor ruxolitinib Fig. . These r, and full-length FLAG-tagged or each of these nectin-4 mutants was co-expressed with ErbB2-GFP in HEK293E cells. When each of the FLAG-tagged nectin-4 mutants was immunoprecipitated using the anti-FLAG mAb, ErbB2-GFP was co-immunoprecipitated with full-length nectin-4 (FLAG-Nectin-4-FL), FLAG-Nectin-4-Ig1/2/3-TM, FLAG-Nectin-4-Ig2/3-TM, and FLAG-Nectin-4-Ig3-TM, but not with FLAG-Nectin-4-TM-CP . These results indicate that nectin-4 cis-interacts with ErbB2 through the third Ig-like domain, although it cannot be excluded that other domains are also involved in the cis-interaction with ErbB2.We next attempted to determine the ErbB2-interacting domain of nectin-4. For this purpose, FLAG-tagged mutants of nectin-4 in which the cytoplasmic domain was deleted (FLAG-Nectin-4-immunoglobulin (Ig)1/2/3-transmembrane (TM)), the cytoplasmic domain and first Ig-like domains were deleted (FLAG-Nectin-4-Ig2/3-TM), the cytoplasmic domain and the first and second Ig-like domains were deleted (FLAG-Nectin-4-Ig3-TM), and the first, second, and third Ig-like domains were deleted (FLAG-Nectin-4-TM-cytoplasmic (CP)), were prepared  and full-length GFP-tagged or each of these ErbB2 mutants was co-expressed with FLAG-Nectin-4 in HEK293E cells. When each of the GFP-tagged ErbB2 mutants was immunoprecipitated using the anti-GFP pAb, FLAG-Nectin-4 was co-immunoprecipitated with full-length ErbB2 (ErbB2-FL-GFP), ErbB2-1/2/3/4-TM-GFP, ErbB2-2/3/4-TM-GFP, ErbB2-3/4-TM-GFP, and ErbB2-4-TM-GFP . Essentially the same results were obtained in the reciprocal immunoprecipitation assay . These results indicate that ErbB2 cis-interacts with nectin-4 through domain IV, although it cannot be excluded that other domains are also involved in the cis-interaction with nectin-4.We then examined the nectin-4-interacting domain of ErbB2. For this purpose, GFP-tagged mutants of ErbB2, in which the cytoplasmic domain was deleted (ErbB2-1/2/3/4-TM-GFP), the cytoplasmic domain and domain I were deleted (ErbB2-2/3/4-TM-GFP), the cytoplasmic domain, domain I, and domain II were deleted (ErbB2-3/4-TM-GFP), and the cytoplasmic domain, domain I, domain II, and domain III were deleted (ErbB2-4-TM-GFP), were prepared cis-interaction of full-length nectin-4 with ErbB2 as schematically shown in Supplementary Fig. . ErbB2-GFP was immunopurified from ErbB2-GFP-transfected HEK293E cells using anti-GFP-pAb-conjugated beads. When the fixed amount of rec-FLAG-Nectin-4-Ig1/2/3 or rec-FLAG-Nectin-4-Ig3 was incubated with the ErbB2-GFP-immobilized beads, both rec-FLAG-Nectin-4-Ig1/2/3 and rec-FLAG-Nectin-4-Ig3 were co-immunoprecipitated with ErbB2-GFP . When both recombinant proteins were simultaneously incubated with ErbB2-GFP, the amount of rec-FLAG-Nectin-4-Ig1/2/3 co-precipitated with ErbB2-GFP was reduced in the presence of rec-FLAG-Nectin-4-Ig3, and vice versa. This result indicates that the third Ig-like domain of nectin-4 is required for the cis-interaction of full-length nectin-4 with ErbB2. Together with the above result showing that the third Ig-like domain of nectin-4 alone interacts with ErbB2, these results indicate that the third Ig-like domain of nectin-4 is required and sufficient for its cis-interaction with ErbB2.We then examined whether a recombinant third Ig-like domain of nectin-4 shows an inhibitory effect on the . These results indicate that the cis-interaction of the third Ig-like domain of nectin-4 with ErbB2 enhances its homodimerization.We then examined whether the third Ig-like domain of nectin-4 enhances the homodimerization of ErbB2. ErbB2-HA and ErbB2-GFP were co-expressed with FLAG-Nectin-4-Ig3-TM or FLAG as control in HEK293E cells. When ErbB2-GFP was immunoprecipitated with the anti-GFP pAb, ErbB2-HA was co-immunoprecipitated with ErbB2-GFP and FLAG-Nectin-4-Ig3-TM, but not FLAG, and the amount of ErbB2-HA from the cells co-expressing FLAG-Nectin-4-Ig3-TM was more than that co-immunoprecipitated with ErbB2-GFP from the control cells Fig. . These r. The incorporation of EdU into the T47D cells stably expressing FLAG-Nectin-4-Ig3-TM was enhanced compared with that in the control cells . These results collectively indicate that the cis-interaction of the third Ig-like domain of nectin-4 with ErbB2 enhances the homodimerization, leading to the activation of ErbB2 and AKT and DNA synthesis, and that the cytoplasmic region of nectin-4 is not required for these reactions.We further examined whether the third Ig-like domain of nectin-4 enhances the tyrosine-phosphorylation of ErbB2, activation of AKT, and DNA synthesis. For this purpose, we established a cell line stably expressing FLAG-Nectin-4-Ig3-TM in T47D cells. The tyrosine-phosphorylation of ErbB2 and the threonine-phosphorylation of AKT were enhanced in the T47D cells stably expressing FLAG-Nectin-4-Ig3-TM compared with those in the control cells, whereas the threonine- and tyrosine-phosphorylation of ERK1/2 or the tyrosine-phosphorylation of STAT3 was not significantly enhanced in the T47D cells stably expressing FLAG-Nectin-4-Ig3-TM compared with those in the control cells Fig. . The inclls Fig. . These r17, although the trastuzumab-resistance to ErbB2ΔEx16 is controversial22. The schematic diagrams and amino acid sequences of these variants are shown in Fig. cis-interacts with these variants. FLAG-Nectin-4 was co-expressed with p95-ErbB2-GFP or ErbB2ΔEx16-GFP in HEK293E cells. The cells were cultured in suspension. When FLAG-Nectin-4 was immunoprecipitated using the anti-FLAG mAb, both p95-ErbB2-GFP and ErbB2ΔEx16-GFP were co-immunoprecipitated with it to a similar extent . These results indicate that nectin-4 cis-interacts with p95-ErbB2 and ErbB2ΔEx16 as well as ErbB2.The ErbB2 splice variants, p95-ErbB2 and ErbB2ΔEx16, were shown to be trastuzumab-resistant in breast cancersent Fig. . These rcis-interaction of nectin-4 with p95-ErbB2 and ErbB2ΔEx16 enhances their tyrosine phosphorylation and AKT activation. For this purpose, we established cell lines stably co-expressing FLAG-Nectin-4 and either p95-ErbB2-GFP or ErbB2ΔEx16-GFP in T47D cells. As a control, we also established cell lines stably expressing either of these variants in T47D cells. The phosphorylation of the tyrosine residues of p95-ErbB2-GFP that are equivalent to tyrosine residues at 1139, 1221, and 1222 of wild-type ErbB2, and the threonine-phosphorylation of AKT and the tyrosine-phosphorylation of STAT3 were enhanced in the T47D cells stably expressing FLAG-Nectin-4 and p95-ErbB2-GFP compared with that in the control cells, whereas the threonine- and tyrosine-phosphorylation of ERK1/2 was not significantly enhanced in the T47D cells stably expressing FLAG-Nectin-4 and p95-ErbB2-GFP compared with that in the control cells . The threonine-phosphorylation of AKT, but not the phosphorylation of tyrosine residues of ErbB2ΔEx16-GFP that are equivalent to tyrosine residues at 1139, 1221, and 1222 of wild-type ErbB2, the threonine- and tyrosine-phosphorylation of ERK1/2, or the tyrosine-phosphorylation of STAT3, was enhanced in the T47D cells stably expressing FLAG-Nectin-4 and ErbB2ΔEx16-GFP compared with those in the control cells . The incorporation of EdU into the T47D cells stably co-expressing FLAG-Nectin-4 and either p95-ErbB2-GFP or ErbB2ΔEx16-GFP was enhanced compared with that in the control cells . The incorporation of EdU into DNA in the T47D cells stably expressing FLAG-Nectin-4 and p95-ErbB2-GFP or ErbB2ΔEx16-GFP and the control cells expressing p95-ErbB2-GFP or ErbB2ΔEx16-GFP alone was inhibited by wortmannin, LY294002, and U0126, but not by ruxolitinib . These results collectively indicate that nectin-4 cis-interacts with not only ErbB2 but also p95-ErbB2 and ErbB2ΔEx16, that nectin-4 enhances the p95-ErbB2- and ErbB2ΔEx16-mediated DNA synthesis through the activation of the PI3K-AKT signalling pathway, and that the nectin-4-independent p95-ErbB- and ErbBΔEx16-mediated activation of the Ras-Raf-MEK-ERK1/2 signalling pathway, but not the JAK-STAT3 signalling pathway, is also involved in the p95-ErbB- and ErbBΔEx16-mediated DNA synthesis.We next examined whether the lls Fig. . The thrlls Fig. . The inclls Fig. . The incnib Fig. . These rcis-interacted with ErbB2 and enhanced its homodimerization and tyrosine-phosphorylation. This interaction selectively induced the activation of the PI3K-AKT signalling pathway for DNA synthesis, but not the Ras-Raf-MEK-ERK1/2 or JAK-STAT3 signalling pathway. As shown in Fig. cis-interacted with not only ErbB2 but also its trastuzumab-resistant splice variants, p95-ErbB2 and ErbB2ΔEx16, that are homodimerized through the disulfide bond of two cysteine residues17. The cis-interaction of nectin-4 with p95-ErbB2 enhanced the phosphorylation of its tyrosine residues at 1139, 1221, and 1222 as well as the threonine-phosphorylation of AKT, similar to that with ErbB2, but additionally enhanced the tyrosine-phosphorylation of STAT3 that was not augmented by the cis-interaction of nectin-4 with ErbB2 or ErbB2ΔEx16, as shown in Fig. cis-interaction of nectin-4 with ErbB2ΔEx16 did not enhance the phosphorylation of its tyrosine residues at 1139, 1221, or 1222, the threonine-phosphorylation of AKT was enhanced similar to that with ErbB2 as shown in Fig. The modes of action of nectin-4 on the signalling pathways of ErbB2 and its trastuzumab-resistant variants for DNA synthesis are schematically demonstrated in Fig. cis-interacted with ErbB2 through the third Ig-like domain of nectin-4 and domain IV of ErbB2 and enhanced its homodimerization and tyrosine-phosphorylation, leading to the selective activation of the PI3K-AKT signalling pathway for DNA synthesis, but not the Ras-Raf-MEK-ERK1/2 or JAK-STAT3 signalling pathway. It was previously shown that the homodimerization of ErbB2 requires its upregulation, although the ligand-dependent homodimerization of ErbB1 or the heterodimerization of ErbB2 with ErbB3 does not require the upregulation of these molecules5, indicating that the affinity for the interaction between the extended domain II of one ErbB2 molecule and that of another ErbB2 molecule might be lower than the affinity of the interaction between the extended domain II of ErbB1 and that of another ErbB1 molecule as well as the affinity of the interaction between the extended domain II of one ErbB2 molecule and that of another ErbB3 molecule. The cis-interaction of nectin-4 with ErbB2 is likely to increase the affinity for the interaction between the extended domain II of one ErbB2 molecule and that of another ErbB2 molecule.We showed here for the first time that nectin-4 5. Taken together these previous observations, the present results suggest that nectin-4 is an important co-receptor regulating the tyrosine-phosphorylation level of ErbB2 but may not be the only molecule showing this activity.We further showed here that the knockdown of endogenous nectin-4 reduced the tyrosine-phosphorylation of ErbB2 and its activation. However, the level of the tyrosine-phosphorylation of ErbB2 was not always correlated with the expression levels of nectin-4. The exact reason for this discrepancy is not clear, but it was previously shown that the growth factor receptors could dimerize depending on their expression levels and that this dimerization of the receptors enhances their tyrosine-phosphorylation and the activation of their downstream signalling pathwayscis-interaction of nectin-4 with ErbB2 induces the activation of the PI3K-AKT signalling pathway more efficiently than that of the Ras-Raf-MEK-ERK1/2 and JAK-STAT3 signalling pathways. It was previously shown that the Ras-Raf-MEK-ERK signalling pathway is a major mitogenic signalling pathway of ErbB2, but it was subsequently demonstrated that the PI3K-AKT signalling pathway is also a major mitogenic signalling pathway of ErbB263. It was more recently reported that upregulated ErbB2 enhances the phosphorylation of tyrosine residues at 1139, 1221, 1222, and 1248 of ErbB261. The mutation of tyrosine residue at 1139 reduces the phosphorylation of AKT, indicating that the tyrosine phosphorylation of this residue on ErbB2 at least leads to the activation of the PI3K-AKT signalling pathway61. It remains unclear whether the phosphorylation of other tyrosine residues leads to the activation of the PI3K-AKT signalling pathway. The novel stimulatory mechanism of ErbB2 for its homodimerization and activation by cis-interacting with nectin-4, which mainly leads to the selective activation of the PI3K-AKT signalling pathway, may be involved in the enhanced DNA synthesis of cancer cells in which nectin-4 is upregulated. The novel mechanism presented here was consistent with the previous observations that the PI3K-AKT signalling pathway is implicated in the carcinogenic role of nectin-4 in gallbladder, breast, and gastric cancers53.It was noted that the cis-interacted with the trastuzumab-resistant splice variants of ErbB2, p95-ErbB2 and ErbB2ΔEx16, although the trastuzumab-resistance to ErbB2ΔEx16 is controversial22. It was previously shown that trastuzumab interacts with domain IV of ErbB2, but p95-ErbB2 lacks the trastuzumab-interacting region, which is considered as a mechanism for trastuzumab resistance of p95-ErbB2-expressing breast cancers17. In contrast, the trastuzumab-interacting region is intact in ErbB2ΔEx16, but the efficiency of trastuzumab for its interaction with this region of ErbB2ΔEx16 is less than that of ErbB211. Considering from our data that nectin-4 cis-interacted with p95-ErbB2 and ErbB2ΔEx16, the nectin-4-interacting region in domain IV of ErbB2 is likely different from the trastuzumab-interacting region.We further demonstrated here that nectin-4 cis-interaction of nectin-4 with p95-ErbB2 enhanced the phosphorylation of its tyrosine residues at 1139, 1221, and 1222 as well as the threonine-phosphorylation of AKT and additionally enhanced the tyrosine-phosphorylation of STAT3. The mechanism for this action of p95-ErbB2 remains unknown, but the phosphorylation of tyrosine residues other than 1139, 1221, and 1222 may be enhanced by this interaction. It was noted that the cis-interaction of nectin-4 with ErbB2ΔEx16 enhanced the tyrosine-phosphorylation of AKT, but not the phosphorylation of tyrosine residues at 1139, 1221, and 1222 of ErbB2ΔEx16. The phosphorylation of tyrosine residues other than these residues may be enhanced by this interaction. Thus, nectin-4 cis-interacts with ErbB2, p95-ErbB2, and ErbB2ΔEx16, but this interaction may enhance the phosphorylation of different tyrosine residues and/or another unidentified molecule. In contrast to ErbB2, p95-ErbB2 and ErbB2ΔEx16 are homodimerized through the disulfide bond of two cysteine residues17. Therefore, it is likely that nectin-4 cis-interacts with the dimerized molecule, enhancing their activation, although nectin-4 cis-interacts with the monomer of ErbB2 to form a complex, enhancing its dimerization. The molecular mechanisms for the nectin-4-enhanced activation of p95-ErbB2 and ErbB2ΔEx16 remain unknown, but the binding of nectin-4 to domain IV of these molecules may affect the conformational changes of their cytoplasmic regions so that the intermolecular tyrosine-phosphorylation is enhanced.We finally showed here that the 58. This Ab recognizes the first Ig-like domain of nectin-4 and inhibits its trans-interaction with nectin-1. Targeting the third Ig-like domain of nectin-4, which cis-interacts with ErbB2, might be an alternative strategy for the treatment of trastuzumab-resistant breast cancers expressing p95-ErbB2 and ErbB2ΔEx16, although it was recently reported that trastuzumab is effective for breast cancers expressing ErbB2ΔEx1622. This novel strategy for the development of therapeutic drugs for trastuzumab-resistant breast cancers may be also effectively used for the screening of chemical compound drugs.Enfortumab vedotin, an Ab-drug conjugate targeting nectin-4, has been developed as a highly potent therapeutic agent in multiple preclinical cancer modelsThe method of cell culture was described in Supplementary Methods. Transfection reagents and supplements for cells used in this study were listed in Supplementary Table The cDNAs used in this study were described in Supplementary Methods.66.The Abs and reagents used in this study were listed in Supplementary Table The methods of co-immunoprecipitation assay and Western blotting were described in Supplementary Methods. The reagents for the assays were listed in Supplementary Table The methods of confocal image analysis were described in Supplementary Methods. The Abs for the assays were listed in Supplementary Table 4 cells per square centimetre on dishes and cultured for 2 days. The cells were serum-starved with RPMI-1640 or Ham’s F12 containing 0.5% fatty acid-free BSA in the absence or presence of the inhibitor at 37 °C for 24 h, and then the cells were washed with ice-cold PBS and lysed with the lysis buffer. The lysates were then heated at 80 °C in the SDS sample buffer for 2 min and subjected to SDS-PAGE, followed by Western blotting. The intensity of the bands in Western blotting was calculated using ImageJ 1.48 v 32-bit software.T47D or SUM190-PT cells were plated at a density of 3 × 10NECTIN4 and a silencer select siRNA negative control siRNA (AM4611) were purchased . Transfection reagent and siRNA sequences were listed in Supplementary Tables Silencer select siRNA against human iRNA AM461 were pu4 cells per square centimetre on dishes and cultured at 37 °C for 48 h. The cells were starved of serum, cultured with RPMI1640 or Ham’s F12 containing 0.5% fatty acid-free BSA at 37 °C in the absence or presence of the inhibitors for 24 h and then treated with 10 μM EdU at 37 °C for 12 h. After washout of EdU-containing media, EdU-treated cells were additionally cultured with serum-free medium in the absence or presence of the inhibitors at 37 °C for 12 h and fixed using 4% paraformaldehyde diluted in PBS. The signal for EdU and nuclei in the cells was determined by the Click-iT EdU Alexa Fluor 488 or 647 Imaging Kit (Thermo Fisher Scientific), according to the manufacturer’s protocol. The samples were counted by fluorescence microscopic examination. The images were acquired using a BZ-X710 microscope  with a CFI Plan Apo λ 20 × /0.75 numerical aperture objective lens  in 1,920 × 1,440 pixels. The displayed images were applied into maximum signal intensity projection from around 5 confocal images collected at a 2 μm step along the z-axis at room temperature under the control of BZ-X Viewer software (KEYENCE CORPORATION). The images were processed using ImageJ 1.48 v 32-bit software.T47D or SUM190-PT cells were plated at a density of 3 × 10The method of the purification of recombinant proteins was described in Supplementary Methods. Inhibition of the interaction between the extracellular region of nectin-4 and ErbB2 was carried out using recombinant FLAG-tagged nectin-4 Ig1/2/3 and ErbB2-GFP by adding recombinant FLAG-tagged nectin-4 Ig3. ErbB2-GFP was captured on anti-GFP pAb-covalently-conjugated beads by incubating with the lysates of HEK293E cells expressing ErbB2-GFP, and the beads were washed three times with the lysis buffer. The washed beads were resuspended in the lysis buffer containing 5 μM recombinant FLAG-tagged nectin-4 Ig1/2/3 in the presence or absence of 25 μM recombinant FLAG-tagged nectin-4 Ig3 and incubated in a total volume of 20 μl at 4 °C for 16 h with gentle mixing. After ErbB2-GFP were mixed with the recombinant proteins, the beads were washed three times with PBS containing 0.02% Tween-20, and the bound proteins were eluted by heating at 80 °C in the SDS sample buffer for 2 min. The samples were subjected to SDS-PAGE, followed by Western blotting using the indicated Abs.post hoc Tukey’s test for comparisons among more than three groups.Statistical significance was analysed using GraphPad Prism 6 software  for two-tailed Welch’s t-test for two groups, or one-way analysis of variance followed by a Supplementary Figures, Tables and Methods


We recently identified pathogenic soluble aggregated tau (tau oligomers) in the cerebral microvasculature of human patients with tauopathies, including Alzheimer’s disease (AD). The functional consequences of cerebrovascular tau accumulation are not yet understood. The aim of the present study was to determine whether pathogenic tau accumulation leads to cerebrovascular dysfunction. To this end, we measured neurovascular coupling (NVC), a highly regulated process that synchronizes cerebral blood flow to neuronal activation, using the PS19(P301S) mouse model of tauopathy. The change in cerebral blood flow evoked by whisker stimulation was measured using Laser Doppler flowmetry in PS19 and wildtype control mice and the functional contribution of neuronal and endothelial nitric oxide synthase  was calculated. Vascular reactivity was assessed using topical acetylcholine to evoke endothelium-dependent vasodilation. To assess the direct impact of pathogenic tau on cell-specific NOS function, we treated N2a neuroblastoma cells or mouse brain vascular endothelial cells with soluble tau aggregates and measured activity of nNOS and eNOS. Our data indicate isolated overexpression of mutant tau impairs NVC responses, and this deficit is mediated by a reduction in nNOS activity in vivo. Further, our studies suggest tauopathy also impairs endothelium-dependent vasoreactivity in the cortex. Additionally, soluble tau aggregates inhibit the phosphorylation of NOS in primary cultured cells. Therefore, inhibition of NOS phosphorylation by pathogenic soluble tau aggregates may underlie cerebrovascular dysfunction in tauopathies. Thus, therapeutic modulation of pathogenic tau may mitigate brain microvascular deficits, which occur prior to clinical onset in Alzheimer’s disease and potentially other tauopathies.


Lactoperoxidase , as a natural antibacterial agent, is a peroxidase enzyme secreted from salivary, mammary, and other mucosal glands. In the present study, the in vitro inhibitory effects of some secondary sulfonamide derivatives (4a–8h) were examined against LPO. The obtained results reveal that secondary sulfonamide derivatives (4a–8h) are effective LPO inhibitors. The Ki values of secondary sulfonamide derivatives (4a–8h) were found in the range of 1.096 × 10−3 to 1203.83 µM against LPO. However, the most effective inhibition was found for N-(sulfathiazole)-3,4,5-triacetoxybenzamide (6a), with Ki values of 1.096 × 10−3 ± 0.471 × 10−3 µM as non-competitive inhibition.Secondary sulfonamides ( Sulfonamides constitute a privileged class among pharmacological agents by possessing properties including carbonic anhydrase enzyme (CA) inhibition, as well as diuretic, hypoglycemic, anticancer, antibacterial, antiviral, and metalloprotease inhibitory effects. Although many years passed since their first discovery in the 1930s as a chemotherapeutic agent for antibacterial properties they are receiving increasing attention due to their newly-discovered pharmacological properties ,3,4,5. DIt was also reported that sulfonamides had a number of interesting functionalities. Some example can be given for secondary and tertiary and sulfonylureas represent particularly important structural motifs in several classes of drugs. Sulfonamides possessed some biological activities as antibiotics (sulfhamethoxazole), PDE5 inhibitors for the treatment of erectile dysfunction (sildenafil), protease inhibitor for treatment of HIV (darunavir), sulfonylureas for treatment of diabetes mellitus (glibenclamide), hepatitis C anti-viral RNA polymerase inhibitors, and non-steroidal anti-inflammatory COX-2 inhibitors (oxicam class) .Because secondary sulfonamides have been reported to be effective agents, in this study, new designs of secondary sulfonamide drugs containing thiazole, pyrimidine, pyridine, isoxazole and thiadiazole moieties were utilized to obtain acetoxybenzamide, triacetoxybenzamide, hydroxybenzamide and trihydroxybenzamide sulfonamide derivatives to achieve the synergistic effects of sulfonamides and polyphenols in one molecule. Since acetyl protection of hydroxyl groups is a well-known application in pharmacology to enhance oral bioavailability and to increase therapeutic concentration of the drug in the bloodstream, both acetylated and non-acetylated forms of compounds were examined together.2O2), and thiocyanate (SCN−). H2O2 is produced by a number of microorganisms such as lactobacilli, Lactococcus and streptococci through enzymatic oxidation of some biomolecules including ascorbic acid and glucose  plot was drawn. For the determination of the inhibition constant (Ki) values, three different secondary sulfonamide derivative (4a–8h) concentrations were used. Additionally, ABTS was used as a substrate at five different concentrations. Lineweaver–Burk plots (1/V-1/[ABTS]) were obtained for secondary sulfonamide; the Ki and the inhibition type were calculated from these plots , which showed parallel lines for different concentrations of secondary sulfonamide derivatives (4a–8h). The lines do not intersect at a common point as shown in 4a–8h) inhibition effects against LPO are commonly non-competitive with respect to the ABTS substrates. In this assay, it was suggested that the secondary sulfonamides (4a–8h) do not directly react with LPO. However, any variation in the concentration of ABTS does not affect the reactivity of secondary sulfonamides (4a–8h). This is also reflected in the non-competitive nature of the inhibition by the compounds with respect to ABTS. The inhibition data of secondary sulfonamides (4a–8h) reported here are summarized in 4a–8h) is dependent on the positioning of the inhibitor in the active site; i.e., the distance between the atoms in the secondary sulfonamide derivatives (4a–8h) and active site amino acids.Although sulfonamide derivatives are used in therapies, there has been no reported LPO activity for these synthesized compounds (of 75.6% . The pur for LPO . For sec4a–8h) exhibited effective inhibitory activity against LPO, one of the prominent enzymes in milk, with a Ki values in the range of 1.096 × 10−3 ± 0.471 × 10−3–1203.83 ± 616.78 µM (N-(sulfathiazole)-3,4,5-triacetoxybenzamide (6a) demonstrated the most powerful LPO inhibition effect with low nanomolar Ki value (1.096 nM). On the other hand, among the synthesized secondary sulfonamides (4a–8h) N-(sulfadiazine)-p-acetoxy-benzamide , N-(sulfamethazine)-p-acetoxybenzamide , N-(sulfisoxazole) p-acetoxybenzamide , N-(sulfamethizole)-p-acetoxybenzamide , N-(sulfanilamide)-p-hydroxybenzamide  and N-(sulfathiazole)-3,4,5-trihydroxybenzamide , showed effective inhibition profiles against bovine milk LPO. Additionally, in general, the acetylated sulfonamides are better inhibitors than the non-acetylated sulfonamides, although the acetylated acids are poorer inhibitors than the non-acetylated acids.All synthesized secondary sulfonamides derivatives  to the antibacterial hypothiocyanite (OSCN−). Secondary sulfonamides (4a–8h) have shown micromolar to nanomolar inhibition against LPO that is vital activity for the innate immune system because of removing bacteria from milk and mucosal secretions. If LPO activity is reduced, this means that the immune system is weakened. This is particularly undesirable, since this affects the immune system of infants that used sulfonamides as a drug.Secondary sulfonamides incorporating acetoxybenzamide, triacetoxybenzamide, hydroxybenzamide and trihydroxybenzamide–all possessing thiazole, pyrimidine, pyridine, isoxazole and thiadiazole groups–were synthesized, and biological activities were evaluated. Milk is a vital liquid secreted from the mammary glands of females of all mammal species. So, the inhibition of the LPO is very important in terms of the LPO system, which catalyses the oxidation of several different reactions by H


The results show that the increased values of Cd, EC, and TOC turned out to be the determinants of the negative impact of leachate on the groundwater quality below the landfill. The integrated water threat model determined the potential negative impact of a landfill site. The extent depended on local environmental conditions and the self-cleaning process. Deterioration of the chemical status in the quality of the groundwater within the landfill area was a consequence of the lack of efficiency of the existing drainage system, which may result from the 19-year period of its use. The applied correlation relationship between physicochemical elements between leachate and groundwater with a time shift due to the extended time of migration of contaminants or mass transfer in waterlogged ground can be an important tool to identify the threat of groundwater pollution in the area of landfill sites.The aim of this paper is to analyse changes in the physicochemical elements in groundwater in the vicinity of a small municipal solid waste landfill site located within the territory of the European Union on the basis of 7-year hydrochemical research. Samples of groundwater and leachate near the examined landfill were collected four times a year during two periods, between 2008 and 2012 during the use of the landfill and between 2013 and 2014 at the stage of its closure. The research results were analysed on the basis of general physicochemical properties: pH; total organic carbon (TOC); electrical conductivity (EC); inorganic elements: Cr, Zn, Cd, Cu, Pb, Hg; and one organic element—polycyclic aromatic hydrocarbon (PAH). The analysis was carried out in accordance with the EU and national legislation requirements regarding landfill monitoring. The assessment of the groundwater and analysis indicators of the leachate pollution allowed interpretation of the impact of the municipal solid waste landfill on the state of the water environment in the immediate vicinity In connection with the guidelines contained in Directive of the Council No. Waste storage is still the most common waste-management method in the world  and from the collective wells at the landfill site quarterly during 2008–2014. Groundwater samples for laboratory testing were taken after prior pumping of water out of the piezometers: P-1, situated in front of (above) the landfill site, served the purpose of monitoring the quality of the water flowing into the area of the landfill site; water from this piezometer formed the reference point of the water quality  and two piezometers, P-2 and P-3, situated in the vicinity of the landfill site on the side of the outflow of the groundwater, serving the purpose of identification of the concentration of pollutants below the landfill site , electrical conductivity (EC), and six inorganic elements: hexavalent chromium (Cr), zinc (Zn), cadmium (Cd), copper (Cu), lead (Pb), mercury (Hg) and one organic element: polycyclic aromatic hydrocarbon (PAH).+6, Pb, Zn, and Hg, were determined by the AAS method (atomic absorption spectroscopy). The results of the tests of the leachate from the landfill site were compared with the values listed in the Regulation of the Minister of Environment of 18 November 2014 concerning the Conditions that should be Met upon Introduction of Wastewaters to Water or to the Ground  APHA . Heavy mStatic parameters were determined for the contamination factors in the leachate: minimum and maximum values, arithmetic mean, and standard deviation. For the purpose of calculation of the static parameters, the measurement result at the level of one half of the specific limit of quantification was adopted if the values of the water indices in a specific sample fell below the limit of quantification determined by the multiple of the limit of detection, i.e. the output signal or the concentration limit above which a sample, with a stated level of confidence, may be pronounced as different from the blank sample. The aggregate results of the physiochemical elements of the condition of the groundwater from the piezometers were used to perform a statistical analysis and to draw conclusions aimed at the assessment of the influence of the landfill site on the quality of the groundwater in the immediate vicinity. A non-parametric Kruskal-Wallis test and multiple  comparison test of the mean ranges, which do not require a normal distribution or homogenous variances, were applied for assessment of the importance of the difference in the concentration of the tested groundwater parameters above and below the municipal landfill site. Non-parametric tests were applied because the distribution of the most of the analysed physiochemical characteristics was abnormal in accordance with the results of the Shapiro-Wilk test, and the variances were not equal, as determined using Fisher-Snedecor’s test. For specific elements which significantly differed between the points, extreme values, the median, and the interquartile range were presented in box plots.In order to determine the impact of the municipal landfill site on the physiochemical condition of the groundwater, correlations between the physiochemical elements of the leachate and the water taken from the piezometers situated below the landfill site were determined. Each correlation was calculated using Spearman’s range method, which yields a correlation index that is a non-parametric equivalent of the respective Pearson’s index. The range correlation can show any monotonic dependence (including in particular a non-linear one). As in the case of a parametric correlation, the Spearman range correlation index measures the force of the co-dependencies between the variables; however, no quantitative scale of normal distribution is required in this case. The correlations of the physiochemical characteristics between the leachate and water in the piezometers were calculated in three variations. In the first variation, results from specific test dates were compared. In two subsequent variations, the results for the leachate were compared with the water in the piezometers with a time shift of one or two calendar quarters due to the extended time of migration of the pollution or transport of the mass in a hydrated land site, since leachate constitutes a significant source of contamination of groundwater for the foreseeable future  in the mezoregion of the Iłżeckie plateau (Podgórze Iłżeckie). It is near an asphalt-paved road approximately 450 m south of a railway line and 3 km north of residential settlements. The average amount of rainfall in the analysed area is 468 mm, which level was recorded at the weather station in Łaziska located 4 km from the landfill site.The landfill site is in G (south of the Mazowieckie Voivodship in Poland) Fig.  as is si3. The average thickness of the waste deposited during the last period of operation of the landfill site is 2.5 m. The landfill site was operated in 1993–2012 using the horizontal method consisting in layering the waste in layers 1-m thick, which layers were further divided into working stripes. Waste was unloaded by daily working parties and subsequently transferred and mechanically densified using heavy-duty equipment which allowed for the creation of thin intermediate layers from 30 to 50-cm thick. The landfill site has its bottom and inside escarpments sealed using a layer of clay 0.5-m thick. The substratum of the landfill site comprises surface deposits belonging to quaternary formations developed in the form of glacial tills which provide good insulation for the deeper layers of the soil. The borings performed up to 6-m deep showed that nearly all of these formations consist of hardly permeable deposits. The aquifer in the area of the analysed facility is approx. 12-m deep and is present in the marls and Upper Cretaceous limestones. The groundwater in the area of the landfill site flows in a south-eastern to a north-western direction.There are no watercourses in the immediate vicinity of the site. The landfill site comprises a single accommodation which, due to its situation at a step in the ground level, has a sub-/supralevel form. The shape of the landfill site approximates a rectangle with dimensions 130 × 165 m, with a surface area of approx. 2.04 ha, of which the accommodation takes up 1.30 ha. The area of the landfill site provides for an opportunity to deposit waste of maximum volume 51,350 mThe landfill site in question is equipped with a peripheral drainage system made of ceramic filters with a diameter of 15 cm. It is situated along the slope at the bottom of the trough. Over the mineral sealing, a drainage system was placed with a view to collecting the precipitation waters permeating the deposited waste. The leachate were discharged via the drainage system to the collective wells. When the landfill site was in operation, a portion of the leachate was used to sprinkle the waste while the excess was taken to a waste water treatment plant.−1, with an average level of 1443 Mg. During the last 5 years of operation of the site, the amount of deposited waste was at the level of 7215 Mg, with a conspicuous drop in the amount  was close to the limit value. The concentrations of the other metals were at a very low level. The maximum concentration of hexavalent chromium was below 0.06 mg L−1, and the concentrations of copper and lead were below 0.2 mg L−1.During the tests which had been conducted for a period of 7 years, the concentration of substances in the leachate that are particularly harmful for the water environment was found to be a few times lower than the highest admissible value (Regulation ME −1 Table . Among t−1). However, in piezometer P-2, two physiochemical characteristics, i.e. electrical conductivity and total organic carbon, contributed to a deteriorated quality of the water. The conductivity is the index of non-dissolved inorganic ions  which classifies the water as being second class, as well as a more than threefold increase in the average concentration of total organic carbon, up to a value of 10.6 mg C L−1. Concentration of total organic carbon classifies the water into the non-satisfactory quality class (fourth class), where the value of the physiochemical characteristics was increased as a result of natural processes occurring in the groundwater and as a result of evident impact of the anthropogenic factor  were observed as well as lower concentrations of hexavalent chrome and copper in comparison with piezometer P-1. However, a sevenfold increase in the concentration of cadmium was observed, which resulted in deterioration in the quality of water from class I to class II, along with a more than factor 2 increase in the concentration of mercury.Statistical comparative analysis of the physiochemical components in the groundwater, which was carried out using the Kruskal-Wallis non-parameter test, showed that six of the ten tested indices, i.e.: pH, TOC, EC, Cd, Cu, and Pb, differed in the statistical terms among the piezometers Table . The difIn order to assess the impact of the landfill site on the physiochemical condition of the groundwater, the correlation dependence between the water in the piezometers below the landfill site and the leachate from the landfill side was analysed. A statistically important dependence (positive correlation) between the water in piezometer P-2 and the leachate was observed as far as the electrical conductivity Fig.  and conc−1 at a large landfill site in Egypt  contributed to the deterioration of groundwater quality below a landfill site. Higher content of very mobile leachate effluents with Cd concentrations of 30 μg L−1 could have contributed to the interaction of landfill waste with groundwater and the migration of pollutants into the aquatic environment (Christensen et al. Analysis of the leachate from the small landfill site conducted over 7 years showed variability of its composition. According to Fatta et al. , the var−1, than its value, ranging between 0.10 and 0.50 mg L−1, as specified by Rapti-Caputo and Vaccaro (Other researcher Tałałaj  showed t Vaccaro . Accordi Vaccaro , elevate Vaccaro , the lanDeterioration of the quality of the outflowing water in the area of the landfill site was attributable to the elevated value of the electrical conductivity, which was over 25 times higher than that demonstrated by Baun et al. . Karlık −1, the conductivity of the groundwater below the landfill site grew by 0.217 μS cm−1. Other researchers (McNeill According to researchers Mor et al.  and Desh McNeill  found a −1 total organic carbon in the outflow water was more than twice as high as that reported by Tałałaj and Biedka (−1, was reported by Koda et al. (−1. According to Anilkumar et al. (The average value of 10.6 mg Ld Biedka  and demoa et al. . The inca et al. . In the r et al. , the solr et al. . Other rr et al. .The presence of such impacts may correspond to an integrated threat on the aquatic environment from pollutants contained in landfill leachate. Researches (De Souza Machado et al. +6. Analysis of the correlation matrix showed that the functioning of the small landfill site had an influence on the pollution of the groundwater as a result of the possibly insufficient efficiency of the existing drainage system after 19 years of operation. Rowe (In addition, analysis of the correlation matrices, which pertained to the data falling below the measurement threshold of the apparatus and which were reduced by one half, demonstrated that the leachate had an influence on the quality of the groundwater below the landfill site with respect to such variables as the contents of Hg and Cron. Rowe  reportedon. Rowe , consideon. Rowe  concludeThe demonstrated results of the physicochemical analysis of the groundwater and leachate in the area of a small landfill site covered two periods of operation and a period after the site ceased to operate. According to Kjeldsen et al. , closed Despite the negative impact of the examined landfill on the quality of the groundwater, a decrease in the correlation between the quality of the groundwater in the piezometers and the leachate should be considered beneficial considering the time shift caused by the migration of contaminants in the land site. This confirms the validity of the recognition of the hydrogeological environment as a natural pollutant migration factor (Alslaibi et al. The study of leachate from a landfill site in the 1990s showed its interaction with the quality of groundwater in the region. Han et al.  showed tThe values of total organic carbon in the leachate and in the groundwater below the landfill site were elevated. Also, a significant increase of the total organic carbon in comparison with the value of that indicator in the inflowing waters was observed.With regard to a dependence between the leachate and the groundwater, increased conductivity of the leachate was observed with a concurrent increase in the conductivity of the groundwater.The increase in the content of cadmium as one of the microelements caused deterioration of the quality of the outflowing waters.Ttpdelhe integrated model of threat to the aquatic environment based on the analysis of PEW, TOC, and Cd indicators showed the negative impact of a small-area landfill on groundwater and indirectly on surface waters. The potential extent of this impact depended on local geological and hydrogeological conditions and processes occurring in the soil and water environment.The correlation between the physiochemical characteristics of the leachate and water with a time shift attributable to the extended time of migration of the pollution or transport of the mass in a hydrated land site, in spite of the observed drop in the value of Spearman’s correlation factor, should be deemed an important tool allowing for recognition of pollution of the groundwater.The interaction between the leachate and groundwater in the area of the landfill site in question proves that the efficiency of the existing drainage system, serving the purpose of discharging waste water to the defensive wells, is insufficient.On the basis of hydrochemical analysis of the leachate and groundwater in the area of a small landfill site carried out during the final period of operation of the site, the following conclusions can be drawn:


Objectives. To explore if sensory information is abnormally processed and integrated in focal hand dystonia (FHD) and if surround inhibition phenomena are operating during sensory-motor plasticity and somatosensory integration in normal humans and in patients with FHD. Methods. We looked at the MEP facilitation obtained after 5 Hz repetitive paired associative stimulation of median (PAS M), ulnar (PAS U), and median + ulnar nerve (PAS MU) stimulation in 8 normal subjects and 8 FHD. We evaluated the ratio MU/(M + U) ∗ 100 and the spatial and temporal somatosensory integration recording the somatosensory evoked potentials (SEPs) evoked by a dual nerve input.  Surround inhibition is a system that sharpens sensation by creating an inhibitory zone around the central core of activation. In the motor system, this mechanism probably contributes to the selection of voluntary movements, and it seems to be lost in dystonia. Conclusions. The inhibitory integration of somatosensory inputs and the somatosensory inhibition are normal in patients with focal dystonia as well as lateral surrounding inhibition phenomena during sensory-motor plasticity in FHD. FHD had two main abnormalities: first, the amount of facilitation was larger than normal subjects; second, the spatial specificity was lost. The MU/(M + U) ∗ 100 ratio was similar in healthy subjects and in FHD patients, and the somatosensory integration was normal in this subset of patients Dystonia is a motor disorder characterized by sustained involuntary muscular contractions resulting from cocontraction of antagonistic muscles and overflow into extraneous muscles . Focal hIn addition, it is well known that dystonia is characterized by a defective somatosensory processing within the somatosensory system  associatSeveral stimulation protocols can be used to test, noninvasively, plasticity within the somatosensory motor system. One of the most established protocols is the paired associative stimulation (PAS) where a magnetic stimulus is coupled with contralateral peripheral nerve stimulation . This prWe have characterized a new conditioning fast PAS protocol that requires only two minutes of induction called 5 Hz rPAS  which prThe aim of the present study was to evaluate the spatial integration of somatosensory inputs during sensory-motor plasticity phenomena, evaluated with 5 Hz rPAS, in healthy subjects and in patients with focal hand dystonia. To achieve this goal, we compared the amplitude of MEPs obtained after the 5 Hz rPAS protocol induced by stimulating the median and ulnar nerves simultaneously (MU) vs the MEP amplitude values being obtained from the arithmetic sum of the 5 Hz rPAS protocol elicited by stimulating the same nerves separately (M + U), looking at the amount of suppression induced by dual nerve simultaneously stimulation. Moreover, we evaluated the spatial and temporal somatosensory integration recording the somatosensory evoked potentials (SEPs) evoked by a dual nerve input to investigate the contribution of lateral inhibition in the somatosensory system. Indeed, previously, Tinazzi and coworkers proposed the MU/(M + U) ∗ 100 as a marker of lateral surround inhibition evoked by a dual input in the somatosensory system , but the amount of facilitation was different between the two groups, as revealed by the time × group interaction . This effect was produced by a larger increase in MEP amplitude in dystonic patients compared to controls. We found no time × group × conditioning interaction because all the three types of intervention induced an increase in MEP amplitude in both dystonic patients and controls   ∗ 100 after the 5 Hz rPAS between dystonic patients and controls in the APB muscle. In both groups, indeed, the percentage of inhibition was around 50%   can induce long-lasting plastic changes in cortical excitability of both dystonic patients and normal subjectsIn keeping with previous findings, focal hand dystonia patients had two main abnormalities. First, associative plasticity after PAS25 was enhanced compared to normal subjects; second, the spatial specificity was lost so that facilitation was observed in both median and ulnar innervated musclesThe inhibitory integration of somatosensory inputs as well as the somatosensory inhibition are normal in patients with focal dystoniaSurround inhibition phenomena are normal in focal dystonia when applying PAS-induced sensory-motor plasticity protocolFour main findings clearly emerge from this study:Our results confirm that 5 Hz rPAS at an interstimulus interval of 25 ms can promote lasting changes in cortical excitability. Considering that rPAS aftereffects are long lasting, reversible, and topographically specific , this prInhibitory integration of somatosensory inputs as well as the somatosensory inhibition phenomena evaluated in our population of dystonic patients did not show any abnormalities compared with the ones of normal subjects. These findings support the idea that the temporal and spatial integration along somatosensory pathways are normal at least in focal hand dystonia, and this could explain the normal integration of the simultaneous median-ulnar nerve stimulation after PAS. On the other hand, our data confirm again that in focal hand dystonia there is a clear abnormality in sensory-motor plasticity as indexed by the loss of spatial specificity after PAS that may account for the creation of abnormal motor engrams. PAS topographical specificity has a different mechanism since the afferent stimulation is not dual and is probably related to the alteration of inhibitory phenomena within motor cortex which are lost in dystonia. These results can be apparently in contrast with the previous findings of Tinazzi and coworkers and Frasson and coworkers , 9. IndeIn conclusion, the data of the present study suggest, in contrast with previous ones, that surround inhibition along somatosensory pathways are intact in FHD. In addition, we demonstrated that surround inhibition is also normal during the induction of sensory-motor plasticity phenomena. These results may suggest that, at least in FHD, spatial and temporal processing of sensory inputs are normal in patients with FHD despite the well-known alterations of spatial and temporal tactile discrimination, which are related to dysfunction in somatosensory cortex (S1) –29. In a