Atmospheric new particle formation (NPF) and growth significantly influences climate by supplying new seeds for cloud condensation and brightness. Currently, there is a lack of understanding of whether and how marine biota emissions affect aerosol-cloud-climate interactions in the Arctic. Here, the aerosol population was categorised via cluster analysis of aerosol size distributions taken at Mt Zeppelin  during a 11 year record. The daily temporal occurrence of NPF events likely caused by nucleation in the polar marine boundary layer was quantified annually as 18%, with a peak of 51% during summer months. Air mass trajectory analysis and atmospheric nitrogen and sulphur tracers link these frequent nucleation events to biogenic precursors released by open water and melting sea ice regions. The occurrence of such events across a full decade was anti-correlated with sea ice extent. New particles originating from open water and open pack ice increased the cloud condensation nuclei concentration background by at least ca. 20%, supporting a marine biosphere-climate link through sea ice melt and low altitude clouds that may have contributed to accelerate Arctic warming. Our results prompt a better representation of biogenic aerosol sources in Arctic climate models. Current climate models show large differences in projected outcomes of present and future Arctic warming3. Air quality regulations on emissions in the Northern Hemisphere, ocean and atmospheric circulation, and Arctic climate are inherently linked6. The Arctic has a pronounced aerosol annual cycle, with the haze period associated with high accumulation mode aerosol concentrations over the dark months8. In the beginning of the summer daylight period, changes in the general circulation that limit continental and anthropogenic inputs, along with the increased presence of low-level clouds  result in fast atmospheric clean-up of the haze11. Lower aerosol loadings and increased photochemistry lead to a peak in ultrafine aerosols (<100 nm) during the Arctic summer8. The Arctic environment undergoes various changes with the potential to affect local and regional aerosol properties. Wet removal by snow or rain is the main sink for accumulation-mode particles, whereas condensation, cloud processing, and transport may be the main sources of these particles12. Multifaceted - yet poorly understood - atmospheric processes by which aerosols contribute to the numbers of cloud droplet condensation nuclei strongly affect the radiative balance13.The climate of the Arctic is changing faster than almost everywhere else on Earth, a phenomenon known as the Arctic amplification14. Within the Arctic, such events are suggested to be formed via secondary aerosol formation and being of marine biological origin from the open waters between ice floes19. Other studies point to an alternative hypothesis involving fragmentation and/or dispersion of primary marine polymer gels also originated in water next to the ice21.The formation of new particles in the atmosphere occurs regionally, but makes an important contribution to the worldwide aerosol particle number concentrations25. Here we applied clustering analysis to a decade-long record of aerosol numbers and size distributions obtained at the Arctic monitoring site of Mount Zeppelin in Svalbard. The main objectives were (a) a categorization and quantification of the aerosol ultrafine population; (b) the association of such aerosol categories with air mass back trajectories and geographical and environmental span; (c) the apportionment of natural and anthropogenic aerosol sources by correlation with chemical markers, and (d) the impact of the elucidated aerosol categories to the Cloud Condensation Nuclei (CCN) population, with particular emphasis at the new particle formation events.Overall, our understanding of climate-relevant aerosol sources, formation processes and size distributions in the remote Arctic region remains incomplete, in part due to the scarcity of long term observations and appropriate methodologies for data interpretation8). Here, we refer to ultrafine as particles with diameters between 10 and 100 nm. Data were clustered at daily averaged resolution; in total, we classified five categories (Supporting Information), three of which clearly described the ultrafine aerosol population , it shows an aerosol mode in the smallest detectable sizes at 10 nm  of particle number size distributions using 73,000 hourly distributions collected over 11 years  were characterized by larger size modes that contributed little to the overall ultrafine aerosol population numbers , whereas accumulation I and II over consolidate packed sea ice (31–45%). Bursting ultrafine seems to have intermediate undefined conditions. Figure nucleation category is associated with air masses travelling over open pack ice. Even though both categories nucleation and nascent peak in summer  were detected during days classified into the two accumulation categories , which are more frequent in winter  during nucleation event days relative to all other days  but not gaseous ammonia  across the aerosol clusters  for the five different aerosol categories may help our interpretation. Indeed, the average CS for the different categories  events through photochemical reactions of gaseous precursors of biogenic origin. Thus, secondary aerosols seem to be the prevailing - but not sole26 - mechanism in governing ultrafine aerosol numbers in the Arctic. Nascent ultrafine likely result from a combination of marine processes26. Figure nascent possesses the lowest CS values and - regardless of occurring mainly during summer time  from the oceans Figure  shows thime Fig.  - the ab1, decreasing sea ice alters marine ecosystems by, e.g., increasing the rates of phytoplankton net primary production by 20–30%51. Therefore, the enhanced source strengths of primary organic biological particles and the aerosol precursors DMS and nitrogen volatiles are likely to increase the oceanic influence on atmospheric composition52. Some studies have suggested an increase in biological activity with increased temperature and decreased sea-ice cover during summer, leading to elevated DMS production and emission38. However, modelling results25 suggest that increased summertime DMS emissions will not cause a strong climate feedback due to the efficient removal processes for aerosols. Such results are highly dependent on aerosol arising from the microbial cycling of sulphur, which still is largely unresolved23.Despite the presence of the ice cover and extreme light and temperature conditions, the Arctic shelf seas rank amongst the most biologically productive in the world. As the region warmsWe wanted to test whether nucleation events observed throughout the 11-year period presented any trend with the recorded sea ice extent. Figure 53. The radiative properties of summertime liquid clouds are very sensitive to the number of cloud condensation nuclei (CCN), which in turn is very sensitive to the local formation of new particles when aerosol long-range transport is limited54. NPF strongly controls the number of particles ≤ 200 nm in all seasons, and particularly in summer12. NPF involves the construction of molecular clusters and the subsequent growth of these clusters to larger sizes. In order to see if the recorded nucleation events had an effect on the overall aerosol numbers and CCN concentrations, we hourly-resolved total particle number counts (N) and CCN data collected over the period 2007–2010 . Figure −3 to 86 ± 5 cm−3) during NPF events  will grow to CCN sizes.Clouds, and related climate feedbacks, are major sources of uncertainty in climate model projections, and the Arctic is not an exception. Even small changes in cloud cover and albedo may have a strong impact on the energy balance at the surface and hence on the summer ice-melt55. Greater cloudiness in this region could lead to warming and accelerated sea-ice melt. Subsequent changes in the moisture source likely result in changes in precipitation that further complicate the energy balance, creating notable uncertainties for climate predictions56. New sources of particles and CCN are expected from increased anthropogenic activities in the ice-freer Arctic in coming years55. In order to evaluate their potential impact on Arctic climate, it is necessary to better understand and quantify the natural phenomena behind ocean-ice-aerosol-cloud interactions, upon which anthropogenic aerosols exert radiative forcing24.The Arctic is a complex environment where, as opposed to large parts of the world, cloud properties together with bright ice and snow-covered surfaces cause clouds to actually warm the surface by more efficiently trapping a portion of the outgoing long-wave radiation. Various studies indicate that Arctic cloud cover has increased during recent decadesnucleation and growth mechanisms. In spring and autumn, conversely, ultrafine particle bursts are hardly accompanied by further growth, probably because of a lack of gas phase precursors. Despite this general scheme of temporal evolution, the Arctic ocean-sea ice-atmosphere coupled system remains largely unexplored but reveals highly nonlinear and spatially heterogeneous processes. Further integrated studies with joint multi-component observations are warranted.We conclude with a schematic illustration of the seasonal sea-ice and aerosol cycle Fig. . Our obsThe aerosol number size distribution observations that are presented in this study were collected at the Zeppelin observatory located on the top of Mt. Zeppelin, Svalbard , just outside the small community of Ny-Alesund. The station represents remote Arctic conditions.8. In order to group together the number size distributions (NSDs) into common sets which were dependent mainly on the shape of the distribution and not the magnitude, the NSDs were normalised to the vector sum and cluster analysed using k-means clustering57. K-means clustering aims to partition the observations into k clusters in which each observation belongs to the cluster with the nearest mean. The analysis works given a predefined number of clusters k and an optimum needs to be decided upon. The optimum cluster number was derived using the Dunn Index and Sihouette Width (silwidth) Figure 59. However, as the number of clusters increases there is a tendency of DI to decrease. A second useful measurement is the Silhouette width60, which is a measure of the similarity of the DMPS spectra within a cluster (cohesion) compared to other clusters (separation)61. The range of the silwidth is from 1 to −1, where 1 indicates that the elements within the cluster are have a high similarity with each other but a low similarity with the elements with the other clusters. For the DMPS data there is a tendency for the silwidth to be high for smaller cluster numbers which decreases as the cluster number is increased as the separation of clusters decreases. There will be a cluster number at which the natural clusters start to divide. This is point is judged as the cluster number where a common abrupt change in the DI and silwidth takes place, i.e. 9 to 10 clusters for the clustered daily DMPS spectra. Likewise, a similar approach was applied to cluster the hourly NSD data to determine the optimum number of clusters. However only the silwidth was used since the DI could not be calculated for the increased dataset size. Hence the largest number of clusters (13) was chosen with a value of the silwidth = 0.3 which showed the most abrupt decrease for an increase in cluster number. Both approaches ‘over-clustered’ the hourly and daily NSDs into 13 and 9 groups respectively when comparing time series of clusters. Clusters with similar time series and average NSD were thus merged yielding 6 hourly and 5 daily clusters.Detailed information on closed-loop Differential Mobility Particle Sizer (DMPS) measurements can be found elsewhereTwo different Condensation Particle Counter (CPC) were deployed to measure simultaneously particles at 3 nm and 10 nm .58, and the increasing uncertainty in the calculation the further back in time it goes. Because newly formed particles are likely to grow into cloud condensation nuclei in a day or two, we believe 5 days back-trajectories are more robust and more indicated for the present study. By making rows of 240 latitudes followed by 240 longitude values - sequenced by the half hour step calculated back from their arrival location - a 480 × 14,600 trajectory matrix was made for cluster analysis. By over clustering the trajectories into 10 groups, it was clear when plotting on a map that 5 groups could be used to describe the data.Two step process of ‘over-clustering’, followed by ‘merging’, was carried out when applying k-Means to the back trajectory calculation. Using the BADC Trajectory Service, 5 day back trajectories arriving at Zepellin over the 10 year span at 00:00, 06:00, 12:00 and 18:00 were calculated arriving at 100 m altitude. The length of the back-trajectory calculation is chosen as a balance between the typical lifetime of the aerosols in the arctic troposphere, which is up to two weeks (shorter in summer and longer in winter/spring) for the accumulation-mode particlesSimple calculations were also made for each of the 5-day back trajectories using daily Arctic maps of gridded sea ice information. For each of the position alone each of the trajectories the sea ice information was logged into a file from which the exposure to sea ice of all the air masses arriving at Zeppelin over the 10 year study period could be calculated.62. A similar calculation was repeated but using daily maps of sea ice percentage concentration measured on a 12.5 km grid. These Artic polar sterographic maps of 12.5 km resolution contained sea ice concentration from the 85 GHz channel of SSM/I on DMSP, available since 1992. The percentages assigned from these maps to each trajectory step allow a ‘spectrum’ of sea ice concentration of 5% width from 0 to 100% to be calculated for each of the trajectory clusters.The Polar Stereographic map of the Northern Hemisphere classified each of 1024 × 1024 24 km grid cells as land, sea, ice or snow ice, and from this, the percentage of time each clustered back trajectory spent over each type could be calculated. The snow and ice coverage values were produced by the NOAA/NESDIS Interactive Multisensor Snow and Ice Mapping System (IMS) developed under the direction of the Interactive Processing Branch (IPB) of the Satellite Services Division (SSD)10 Sampling and Analysis PM10 aerosol sampling was performed at Gruvebadet station located about 50 m a.s.l. and 1 km far from the scientific village of Ny Ålesund . The aerosol sampling performed by a TECORA Skypost sequential sampler equipped with a PM10 sampling head operating following the EN 12341 European rules. Aerosol samples were collected daily on Teflon (PALL Gelman) filters from March to September 2010, in total 151 samples were analysed38. MSA was determined by ion chromatography on the aqueous extract obtained from one half of each filter38. The Particle Soot Absorption Photometer (PSAP) is used to measure in near real time the optical extinction coefficient for absorption which can in also be used to estimate concentrations of black carbon. The resulting concentrations are reported as equivalent black carbon (EBC). EBC at daily resolution available at the NILU website data at daily resolution for the period 2001–2010. Gaseous NH3 and SO2 data were obtained at daily resolution at the NILU website data for the period 2001–2010.PMConcentrations of Cloud Condensation Nuclei (CCN) have been measured continuously using a commercial available Droplet Measurement Technology (DMT) CCN counter. The CCN counter measures the number of particles large enough to act as CCN by feeding the ambient air though the chamber in which supersaturation is achieved. The CCN can scan particle number distribution as a function of supersaturation. We set the CCNC to measure concentration of CCN for 0.2, 0.4, 0.6, 0.8, 1.0 Super Saturation (SS). During the study, flow and zero-counting were checked frequently, calibrations were carried out over the years, the instrument was found stable and overall resulting data satisfactory. In this study we have select CCN values when SS is 0.4%. In total, 1120 days of sampling were obtained at hourly resolution between 01-04-2007 and 31-12-2010.63. Calculation are described elsewhere64.The condensation sink (CS) describes how rapidly condensable vapour molecules will condense on the existing aerosol. Specifically this quantity describes the loss rate of molecules with diameter dp, diffusion coefficient D, and mean free path λv onto a distribution n(dp) (or Ni in the discrete case) of existing particles and as such, can be obtained from integrating over the particle size spectrumThe data that support the findings of this study are available from the corresponding author on request.supporting information


VBNC) state. Therefore, in this study, Rpf from Micrococcus luteus was added in the culture medium to evaluate its role in bacterial isolation and enhanced effects on cellulose‐degrading capability of bacterial community in the compost. It was found that Proteobacteria and Actinobacteria were two main phyla in the compost sample. The introduction of Rpf could isolate some unique bacterial species. The cellulase activity of enrichment cultures with and without Rpf treatment revealed that Rpf treatment significantly enhanced cellulase activity. Ten isolates unique in Rpf addition displayed carboxymethyl‐cellulase (CMCase) activity, while six isolates possessed filter paper cellulase (FPCase) activity. This study provides new insights into broader cellulose degraders, which could be utilized for enhancing cellulosic waste treatment.Nowadays, much of what we know regarding the isolated cellulolytic bacteria comes from the conventional plate separation techniques. However, the culturability of many bacterial species is controlled by resuscitation‐promoting factors (Rpfs) due to entering a viable but non‐culturable ( Sodium dodecyl sulphate (SDS)–polyacrylamide gel electrophoresis (PAGE) profiles of purified Rpf protein are presented in Fig. et al. . These results indicated that the recombinant Rpf protein obtained in the study was consistent with the previous reports that the muralytic activity of Rpf is probably responsible for its resuscitation and stimulation functions , followed by Sphingomonas (3.67 ± 0.10%), Parafilimonas (3.05 ± 0.12%) and Arachidicoccus (2.87 ± 0.18%). It is well known that Streptomycetes are important members of soil community and are particularly active in the biodegradation of recalcitrant organic contaminants . As listed in Table  and HWC3n et al.  which in, et al.  found thhttps://www.ezbiocloud.net/)  which was detected at low‐level waste site with cellulosic contaminants  and filter paper  from M. luteus containing the Rpf could stimulate and resuscitate uncultured bacteria belonging to phylum Actinobacteria , while no counterparts in control group (CG) were obtained. The remaining 11 strains which were existed in both TG and CG were analysed by retrieving the most closely related strains from EzbioCloud database (/) Table . Figure , et al.  observedet al.  used in this study had previously been described  according to the manufacturer's instructions and then was amplified by PCR using primers rpf‐F 5′‐ GCGCGGATCCATGGACACCATGACTCTCTTCACC‐3′ and rpf‐R 5′‐GATCAAGCTTTCAGGCCTGCGGCAGGACGAGCTCC‐3′  and the following conditions: 94°C for 3 min , 30 cycles of denaturation at 94°C for 30 s, annealing at 57°C for 30 s and 72°C for 1 min and finally 72°C for 8 min. The PCR products were purified and ligated into the NedI/BamHI‐treated pET‐28a vector and then transformed into chemically competent E. coli BL21 (DE3) cells. To make protein containing an N‐terminal histidine tag, the recombinant cells were cultured with kanamycin (50 μg/ml) and induced by isopropyl‐β‐d‐thiogalactopyranoside  as previously described  with a spectrophotometer ‐3′ and 806R (5′‐GGACTACNNGGGTATCTAAT‐3′)  were taken from composting mixture at different locations. Microbial DNA was extracted from compost samples using the EZNA Soil DNA Kit . The V3–V4 region of the bacterial 16S rRNA gene was amplified by PCR  using primers et al., The raw sequence data, as FASTQ files, were demultiplexed using the barcode sequence with the exact barcode matching parameter. Low‐quality base pairs were removed using Trimmomatic . Throughout the entire enrichment period, an equal amount of Rpf  and inactivated Rpf (sterilized at 121°C for 15 min) were added to the TG and CG respectively. Each experiment was performed in three replicates.The compost sample  was incubated in enrichment medium containing (per litre): 10 g carboxymethyl‐cellulose (CMC), 1 g K2‐fold to 108‐fold) was plated on CMC‐Congo red agar for enumeration of cellulose‐degrading bacteria using a modified Hendricks method  were cultured at 37°C at 160 rpm in an enzyme production media containing (per litre) 10 g avicel cellulose, 2 g (NH4)2SO4, 2 g NaCl, 2 g MgSO4·7H2O and 4 g CaCO3. The pH was adjusted to 7.0. After 4 days, the two cultures suspensions were centrifuged, and the supernatants were collected as crude enzyme preparation, respectively, for further enzyme assays. The extracellular cellulase activity of FPCase and CMCase was measured in the enzyme assay mixture containing CMC and What‐man no. 1 filter paper respectively. The mixture was incubated at 60°C for 15 and 30 min, respectively, for FPCase and CMCase assay using 3,5‐dinitrosalicylic acid (DNS) method  per ml per minute. All experiments were carried out in biological triplicates. Error bars represent standard deviation (SD) of the mean of triplicate samples.The enrichment cultures from TG and CG, as well as the cultures from selected isolates, were centrifuged  and washed twice with sterile NaCl . The washed pellets were resuspended in the same volume of sterilized NaCl solution. The cell suspensions (10None declared.Table S1. Analysis of top‐hit species of the remaining 11 strains by retrieving the most closely related strains from EzBioCloud database.Click here for additional data file.


The U.S. mid-Atlantic sea-level record is sensitive to the history of the Laurentide Ice Sheet as the coastline lies along the ice sheet's peripheral bulge. However, paleo sea-level markers on the present-day shoreline of Virginia and North Carolina dated to Marine Isotope Stage (MIS) 3, from 50 to 35 ka, are surprisingly high for this glacial interval, and remain unexplained by previous models of ice age adjustment or other local  effects. Here, we reconcile this sea-level record using a revised model of glacial isostatic adjustment characterized by a peak global mean sea level during MIS 3 of approximately −40 m, and far less ice volume within the eastern sector of the Laurentide Ice Sheet than traditional reconstructions for this interval. We conclude that the Laurentide Ice Sheet experienced a phase of very rapid growth in the 15 kyr leading into the Last Glacial Maximum, thus highlighting the potential of mid-field sea-level records to constrain areal extent of ice cover during glacial intervals with sparse geological observables. et al. present a revised glacial isostatic adjustment model that enables them to reconcile these differences.Surprisingly high Pleistocene sea-level markers on the Virginia and North Carolina shoreline are inconsistent with models of Laurentide ice sheet volume during Marine Isotope Stage 3. Here, Pico  Reconstructing the pace of ice growth towards the Last Glacial Maximum LGM, 26 ka) is critical to our understanding of ice age climate and ice sheet stability. Nevertheless, global ice volume, or equivalent global mean sea level (GMSL), and the corresponding geographical distribution of ice remain uncertain through Marine Isotope Stage 3  leading into the LGM16 ka is c46The geological markers of Pleistocene sea-level oscillations extending from Virginia to North Carolina in the Albemarle Embayment , on the 111213141517Here, we present a new set of GIA calculations that explore the sensitivity of the predictions to peak GMSL and LIS geometry during MIS 3. We conclude that a revised GIA model can reconcile the MIS 3 sea-level record at the Albemarle Embayment under two conditions: (1) peak GMSL reached near −40 m and (2) the eastern sector of LIS was significantly reduced during MIS 3 compared with previous reconstructions of ice extent.The Albemarle Embayment geological record includes interfluvial, estuarine, intertidal and shallow marine lithofacies arranged in depositional sequences that record repeated sea-level highstands dated primarily by optically stimulated luminescence to MIS 5e, 5c, 5a and 3 ref.  Fig. 1;. We adopFor the MIS 3 interval spanning 50–35 ka, three marine indicators constrain RSL to be above −0.9, −3 and −2 m ref. . We thuset al.232526Ice sheet growth and melt produces a complex spatio-temporal pattern of sea-level change21 Pa s and 1.5 × 1022 Pa s, respectively; this radial profile is consistent with inferences based on globally distributed ice age data sets29Our numerical predictions require models for Earth's viscoelastic structure and the history of global ice cover. We begin by adopting an Earth model with upper and lower mantle viscosities of 0.5 × 10et al.et al.PC, is shown in Many previous inferences of GMSL during the last glacial phase, particularly MIS 3, reconstruct higher peak sea level  than adopted in the ICE-5G history932PC). In this case, the predicted RSL ranges from −26.5 to −7.5 m at 44 ka  the eastern sector of the LIS is ice-free from 80 to 44 ka, consistent with the conclusions of Dalton et al.PC models, and thus we no longer assume that global ice geometry prior and subsequent to the LGM were identical whenever the GMSL values match. The simulation, based on this revised ICEPC2 ice model and the viscoelastic Earth model discussed above, predicts a RSL of −3 m at 44 ka, consistent with the sea-level record at the Albemarle Embayment  and a reduced crustal deformation , compared to the ICEPC prediction. The latter effect dominates, resulting in a net sea-level rise compared to the ICEPC ice distribution and the fit evident in PC2 ice history and consider a range of lithospheric thicknesses, and upper and lower mantle viscosities  that the eastern sector of the LIS was ice-free from 80 to 44 ka. In these simulations, the predicted peak RSL at the reference site in the Albemarle Embayment ranged from −11 to −1 m at 44 ka  from 80 to 44 ka, and shifting the distribution of this ice within the sector, can lower the RSL predictions by up to 3 m  by their GMSL history before the Last Glacial Maximum. These ice histories randomly sample GMSL values under the following constraints: at LIG, 122 ka, GMSL=0; at 110 ka, MIS 5d, GMSL=−40 m; at MIS 5c, GMSL=−10 m; at MIS 5a, GMSL=−15 m; during MIS 3 before 44 ka, GMSL varies in the range −40 to −80 m; at 44 ka, the GMSL value is pinned within the range −37.5±7 m . We alsoPC2 model was distributed uniformly over Fennoscandia and Antarctica (rather than over the western sector of the LIS) and found that the predicted sea-level highstand at our test site at 44 ka was only perturbed by 0.6 m.We note that while RSL predictions at the Albemarle Embayment are sensitive to ice geometry of the eastern LIS, these predictions will be relatively insensitive to the geographic distribution of ice cover in regions in the far field of the mid-Atlantic U.S. East Coast. As an example, we performed a simple test in which the ice removed from the eastern sector of LIS to construct the ICEThe data sets generated during and/or analysed during the current study are available from the corresponding author on request.How to cite this article: Pico, T. et al. Sea-level records from the U.S. mid-Atlantic constrain Laurentide Ice Sheet extent during Marine Isotope Stage 3. Nat. Commun.8, 15612 doi: 10.1038/ncomms15612 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Notes, Supplementary Tables, Supplementary Figures and Supplementary References


Helicobacter pylori activity and the possible inhibitory effect on its associated urease by Palmatine  from Coptis chinensis, and explored the potential underlying mechanism. Results indicated that Pal exerted inhibitory effect on four tested H. pylori strains  by the agar dilution test with minimum inhibitory concentration (MIC) values ranging from 100 to 200 μg/mL under neutral environment (pH 7.4), and from 75 to 100 μg/mL under acidic conditions (pH 5.3), respectively. Pal was observed to significantly inhibit both H. pylori urease (HPU) and jack bean urease (JBU) in a dose-dependent manner, with IC50 values of 0.53 ± 0.01 mM and 0.03 ± 0.00 mM, respectively, as compared with acetohydroxamic acid, a well-known urease inhibitor . Kinetic analyses showed that the type of urease inhibition by Pal was noncompetitive for both HPU and JBU. Higher effectiveness of thiol protectors against urease inhibition than the competitive Ni2+ binding inhibitors was observed, indicating the essential role of the active-site sulfhydryl group in the urease inhibition by Pal. DTT reactivation assay indicated that the inhibition on the two ureases was reversible, further supporting that sulfhydryl group should be obligatory for urease inhibition by Pal. Furthermore, molecular docking study indicated that Pal interacted with the important sulfhydryl groups and inhibited the active enzymatic conformation through N-H ∙ π interaction, but did not interact with the active site Ni2+. Taken together, Pal was an effective inhibitor of H. pylori and its urease targeting the sulfhydryl groups, representing a promising candidate as novel urease inhibitor. This investigation also gave additional scientific support to the use of C. chinensis to treat H. pylori-related gastrointestinal diseases in traditional Chinese medicine. Pal might be a potentially beneficial therapy for gastritis and peptic ulcers induced by H. pylori infection and other urease-related diseases.In this paper, we evaluated the anti- Helicobacter pylori is a Gram-negative spiral bacterium that colonizes the stomachs. H. pylori has been classified as the major risk factor of gastrointestinal diseases, including gastritis, gastric and duodenal ulceration and gastric carcinoma [H. pylori infection. Among them, the most frequently used eradication regimen was the triple therapy consisting of amoxicillin, clarithromycin and proton-pump inhibitors [arcinoma . In the hibitors . AlthougH. pylori. Urease initiates the hydrolysis of urea generating ammonia to neutralize stomach acid in order to create a suitable pH environment that the bacterium requires to survive and colonize [2+) and the sulfhydryl groups, at the active site of the enzyme, are essential for the catalytic effect of urease. The activity of urease has also been regarded as the influential factor of the nitrogen utilization in organisms. The urease causes the urea decomposition and re-absorption, which leads to abnormal environmental pH elevation and induces a variety of diseases in the human body [H. pylori infection.Urease  is known to be an important biological feature and major contributor to the pathologies induced by man body . Hence, Coptis chinensis, known as ‘huanglian’ in Chinese, is traditionally used in China for the treatment of diarrhea and H. pylori-related gastrointestinal diseases [C. chinensis could inhibit HPU at a comparatively low concentration [C. chinensis was more pronounced than its major active constituent berberine [21H25NO4, Pal), an active naturally occurring isoquinoline alkaloids, is another important bioactive component derived from C. chinensis besides berberine [Chinese Pharmacopoeia (2015 Edition), and is widely used in clinical practice for the treatment of inflammatory diseases, including gynecological inflammation and digestive disorders like bacillary dysentery and enteritis, etc [C. chinensis exhibited anti-H. pylori activity as well as inhibition on H. pylori-induced gastric inflammation [H. pylori activity. Taking into account the crucial role that urease plays in the survival and gastric colonization of H. pylori, in this follow-up study, we endeavored to further investigate the possible inhibition of Pal on H. pylori and its associated urease, and probe the potential underlying mechanism.diseases . Our earntration . And theerberine , implyinerberine , 10. Moderberine –18. In Ctis, etc . PreviouPalmatine .A total of four H. pylori strains were evaluated by using agar dilution method. Series of concentrations of 25, 50, 75, 100, 125, 150 and 200 μg/mL Pal were used. H. pylori strains were harvested and resuspended in PBS (1.0–2.0 McFarland were defined based on the strain properties). And 0.1 mL H. pylori suspension was inoculated and flooded in the Pal-containing or H2O (control) agar plate. After 72 h, the MIC values were determined as the lowest concentration at which there was no strain growth judged by visual examination. Metronidazole was used as the positive control. The experiments were evaluated under two different pH (7.4 and 5.3) conditions and repeated twice.The MICs of Pal against the four HPU was prepared as previously described . The sta50 was expressed as the concentration of inhibitor that decreased urease activity by 50% and calculated by plotting the percent of inhibition. The experiments were triply performed.Reaction mixtures comprising 0.25 mg/mL HPU or JBU and different concentrations of Pal or AHA at the volume ratio of 1:1, were incubated at 37°C in 20 mM HEPES buffer (pH 7.5) in the absence of urea. The reactions were initiated by mixing the urease and the inhibitor. The preincubation mixture was withdrawn at 20 min and immediately transferred into the standard assay mixtures for urease residual activity determination . InhibitKM and the maximum velocity vmax values were evaluated by means of Lineweaver—Burk plots, using the initial velocities at different urea concentrations (ranging from 0.3125 to 15 mM), in the absence or presence of 1.00, 0.50, and 0.25 mM Pal. The concentrations of HPU and JBU were both 0.25 mg/mL in the assays. The values were obtained by applying nonlinear regression to the Michaelis-Menten equation. All experiments were performed in triplicate.Michaelis constant For the protection with thiols-containing compounds, the mixture consisting of 0.25 mg/mL urease (HPU or JBU), 0.5 mM Pal (for both HPU and JBU) and 1.25 mM thiols  was incubated for 5, 10, 20, and 40 min at 37°C, respectively. The control sample instead of the protectors contained a proper volume of respective buffer.For the protection with inorganic compounds (BA and NaF), 0.25 mg/mL urease (HPU or JBU) was preincubated with 5 mM BA or 5 mM NaF for 20 min. Following preincubation, each sample of the protected urease was incubated with 0.5 mM Pal for 20 min. The samples containing a proper volume of respective buffer instead of Pal served as comparison with the abovementioned systems. The experiments were triply performed.The reaction mixture consisted of 0.25 mg/mL HPU or JBU, 0.5 mM Pal (for both HPU and JBU), and 1.25 mM thiols  in 20 mM HEPES buffer (pH 7.5). The components of the incubation mixture were mixed according to three procedures: (1) Urease was added to the mixture after a 20-min co-incubation of Pal with thiol. (2) Pal was added to the mixture after a 20-min co-incubation of urease with thiol. (3) Thiols were added to the mixture after a 20-min co-incubation of urease with Pal. The incubation mixture containing all components was incubated for further 20 min and the activity of urease was determined. The experiments were triply performed.The reactivation of Pal-inactivated urease was investigated by DTT application. The preincubation mixture consisted of 0.25 mg/mL HPU or JBU and 0.5 mM Pal in 20 mM HEPES buffer (pH 7.5). Then the mixture was further incubated with 1.25 mM DTT after 20 min. The enzymatic activity of urease was determined before and after the addition of DTT. After a while, aliquots were withdrawn and the urease residual activity was determined. The experiments were repeated three times.2+ were in the center of the grid. Other docking parameters were set to the software’s default values. The docked complexes were geometry optimized and the results of the most favorable free energy of binding were chosen as the resultant complex structures. The distance (Å) between hydrogen bond forming residues was measured.Docking studies were performed as our previous investigation . Pal wasP < 0.05.GraphPad Prism 5 (GraphPad Software Inc.) software was used for statistics and plotting. Data were expressed as means ± standard deviation (SD). The differences of the results were assessed using a one-way analysis of variance (ANOVA) followed by Dunnett's test. The analysis was performed using SPSS 13.0 software (SPSS Inc.). A significant difference was identified at H. pylori activity of Pal in the MIC assay was shown in H. pylori strains under neutral conditions (pH 7.4) were from 100 to 200 μg/mL, and the MICs under acidic environment (pH 5.3) ranged from 75 to 100 μg/mL. The result indicated that Pal exhibited inhibitory activity against H. pylori under both neutral and acidic conditions, and the inhibitory effect seemed to be more obvious in acidic condition than the neutral environment. Metronidazole, a well-known antibiotic, was used as a positive control. The MIC values of metronidazole were summarized in The anti-50 values of 0.53 ± 0.01 mM and 0.03 ± 0.00 mM toward HPU and JBU, respectively. The IC50 values of AHA, a well-known urease inactivator employed as a positive drug, were 0.07 ± 0.01 mM and 0.02 ± 0.00 mM for HPU and JBU, respectively. The result revealed that Pal markedly inhibited the enzymatic activities of both ureases, and the IC50 value of Pal for JBU was similar to that of AHA.The inhibition progress curves as a function of urease activity versus the incubation concentration of Pal with the two ureases were illustrated in KM and vmax of the ureolytic reaction were 1.20 ± 0.07 mM and 0.40 ±0.02 mM/min for HPU, and 8.30 ± 1.14 mM and 0.36 ± 0.02 mM/min for JBU, respectively. Moreover, the KM value did not vary significantly, while the vmax value decreased as the Pal concentration increased, indicating Pal was a noncompetitive inhibitor of both HPU and JBU.In the present study, the enzymatic kinetics were determined by applying nonlinear regression to the Michaelis-Menten equation, in the presence or absence of various concentrations of Pal. The inhibition pattern was determined by the analysis of Lineweaver-Burk plots for elucidating the mechanism of inhibition. Kinetic analysis revealed that HPU followed a Michaelis—Menten kinetics in the presence of Pal. As shown in In the current research, the role of thiol protectors  in urease inactivation by Pal was investigated by comparing the thiol-free system at four time points of incubation . According to To investigate whether the active site nickel ions was involved in the inactivation by Pal, two active-site competitive binding inhibitors (NaF and BA) were also employed in the urease protection experiment. As shown in As depicted in In this section, DTT was employed to examine the stability of the urease-inhibitor complex. 1.25 mM DTT without Pal was used as a control. By adding DTT after 20 min co-incubation of Pal with JBU or HPU, the urease activity was observed to recover time-dependently. Compared with the urease activity of addition of 1.25 mM DTT without Pal (The HPU or JBU activity was ca. 100%), the HPU activity was partly restored to ca. 46% , and JBU3 of Pal as the hydrogen bond donor, was found between the OH and the backbone N atom of ARG-338 (H∙ ∙ ∙N distance = 3.1 Å), which was located on the mobile flap closing the active site of the enzyme. And for JBU, 2-OCH3 and 3-OCH3 of Pal formed two strong O-H∙ ∙ ∙N hydrogen bond  to the backbone N atom of ARG-639. These observations were soundly supportive of the involvement of the active-site sulfhydryl groups in the inhibition process, as substantiated in the protective experiment performed with the active-site binding inhibitors and reactivation assay.To give an explanation and understanding of the observed appreciable activity and mechanism revealed by the protective experiment, molecular docking of Pal into the binding site of of HPU and JBU was performed based on the HPU (1E9Y) and JBU (3LA4) complex structures, and the best possible binding modes of Pal were depicted as cartoon model  and enzyCoptis chinensis has been commonly employed in traditional oriental medicine for the treatment of various diseases, including H. pylori-related gastrointestinal disorders. Pal was one of the major bioactive protoberberine-type alkaloids of Coptis chinensis. Han et al. previously reported that Pal exhibited inhibitory effect against H. pylori (KCCM 41351) [C. chinensis had significantly inhibitory activities against H. pylori and gastric damage, of which the in vivo anti-H. pylori effect of Pal was similar to that of the positive control ampicillin [H. pylori strains including three reference strains  and one clinical isolate (ICDC 111001) under neutral and acidic conditions. And the anti-H. pylori activity of Pal was superior under acidic environment as compared with that of the neutral condition. This finding suggested that Pal might exert inhibitory effect toward H. pylori in the stomach, which was in congruent with the the in vivo anti- H. pylori effect in the previous report [M 41351) . Jung etpicillin . In the s report .H. pylori. Urease allows H. pylori to survive in the low pH environment of the stomach during colonization by the hydrolysis of urea into ammonia and carbon dioxide to neutralize the bacterial microenvironment [H. pylori would be a potential anti-H. pylori agent. And exploration of safe and efficacious urease inhibitors derived from medicinal herbs is gaining increasing importance as an alternate therapy against H. pylori infections.Urease has been implicated as an essential colonization and virulent factor of ironment , 27. TheCanavalia ensiformis, JBU), the first enzyme to be crystallized and the best-characterized urease [H. pylori, Pal was evaluated for its anti-urease capacity. From the results obtained, it was found that Pal could effectively inhibit the activities of both HPU and JBU. This result indicated that the urease-inhibitory activity of Pal might be closely associated with the suppression of H. pylori growth.Urease of different origins such as plants and bacteria composes of inconsistent types of subunits, nevertheless, ureases share similar amino acid sequences and thus exhibit common catalytic characteristics. Urease from jack bean  and monothiols (e.g. L-cys and GSH), and the other is the inorganic compounds, such as BA  and NaF (a competitive slow binding urease inhibitor), which were reported to inhibit the urease by interacting with Ni2+ in the urease active site. In equilibration with the urease, both types of protectors occupy the active site of urease to restrain the accessibility of inactivators to the active-site of urease. Specifically, the thiol reagents inhibit the urease via interaction with the active-site sulfhydryl group of urease, while inorganics have been defined as competitive inhibitors, binding to its active site Ni2+ resulting in urease inhibition [The most pivotal catalytic characteristics of ureases are the cysteinyl residues bearing thiol groups and Nihibition , 30. In KM and vmax from the Lineweaver-Burk plots, it was proposed that the kinetic behavior might be noncompetitive for both HPU and JBU, implying Pal and substrate both got access to the urease non-competitively. In addition, the anti-urease activity of Pal was significantly affected by the incubation time. However, the thiol protection against urease inactivation by Pal was not relevant to the incubation time, but closely related to the addition order of the incubators. The results showed that the thiol-protectors might quickly combine with the sulfhydryl groups and restrict the accessibility of the inhibitor. In order to further prove whether the inactivation was reversible, DTT was employed in the reactivation of Pal-inactivated urease assay. More obvious protective effect exerted by DTT was observed in JBU as compared with that of HPU. Particularly, nearly 80% of JBU activity was recovered, but only 46% of total enzymatic activity of HPU was suppressed by Pal. This might be due to the specificity in the mode of interaction between HPU and JBU [Enzyme kinetic study is pivotal for the determination of the inhibition mechanism and sites of inhibitory binding. The Lineweaver-Burk plot was used to study the inhibition type in enzyme kinetics. According to the variations of  and JBU .2+ was not found to be obvious. It was shown that 2-OCH3 group affected the molecular conformation interacting with HPU, which enhanced the binding with ARG-338, a key residue reported previously as catalytic residue in the active site [3 and 3-OCH3 groups were observed to interact with ARG-639, a key residue located at the mobile flap covering the active site, hence preserving the flap in the open conformation and leading to urease inactivation. Results from the molecular docking simulation further supported our earlier conclusion from the urease protection experiments and DTT re-activation assay, which suggested the pivotal participation of the active-site sulfhydryl group in the inactivation of urease by Pal.Furthermore, it was also supported by molecular docking simulation. As the results depicted, Pal was found to make hydrogen bonding interactions with ARG-338 (HPU) and ARG-639 (JBU), which were located on the urease active-site mobile flap. However, the interaction with the active-site Niive site , resultiH. pylori. Pal derived from C. chinensis might play an important role in the protection and therapy of H. pylori-related gastrointestinal disease. However, the more detailed underlying mechanism merited further investigation.Hence, our data indicated that the interaction with the sulfhydryl group of the active site of urease was closely associated with the anti-urease activity of Pal, which might be one of the mechanisms by which Pal inhibited the growth of C. chinensis extract and its active principle berberine, and the in vivo anti-H. pylori activity of Pal and berberine was similar to that of ampicillin [C. chinensis alkaloids , while berberine was relatively more toxic in both in vitro and in vivo assays [50 of berberine against HPU and JBU was 10.20 ± 1.10 mM and above 13.00 mM, respectively [H. pylori and urease-inhibitory effect than berberine, and had a more favorable safety profile.In China, Pal has been developed as a safe and effective anti-inflammatory agent in clinic for the treatment of gynecological inflammation, digestive inflammatory disorders and urinary infection, etc. Previous study indicated that the anti-ulcerogenic activity of Pal was better than that of the picillin . It was o assays . In our ectively , indicatH. pylori treatment either focuses on the direct anti-H. pylori effect or gastroprotective property, and none of them harbor simultaneous anti-H. pylori, HPU-inhibitory and gastroprotective activities. Based on its simultaneous anti-H. pylori, HPU-inhibitory and gastroprotective properties, Pal was believed to possess good potential for further development into a safe and efficacious non-antibiotic therapeutic agent in the management of H. pylori-related gastrointestinal disorders, and the discovery of Pal as a safe and efficacious urease inhibitor and rational characterization of its underlying mechanisms will undoubtedly add new therapeutic dimensions to its current clinical application and also provide a foundation and justification for further research as a potential complementary therapeutic agent to the current conventional medications.The current first-line monotherapeutic agent for H. pylori growth, at least in part, through inhibition of its urease. The concentration-dependent, reversible, and noncompetitive inhibition against urease by palmatine might be attributed to its interaction with the sulfhydryl group of the active site of urease. Our work gave additional scientific support to the use of C. chinensis to treat H. pylori-related gastrointestinal disorders and might extend the clinical indications of palmatine. Palmatine deserves to be further exploited as a promising naturally-occuring urease inhibitor for the treatment of H. pylori infection and other urease-related diseases.It was concluded that palmatine might suppress S1 File2, 10% CO2, and 85% N2). And 0.1 mL H. pylori suspension was inoculated and flooded in the Pal or Met-containing or H2O (control) agar plate. After 72 h, the effects of Pal and Met on the growth of H. pylori were determined.All the strains were cultured on Columbia agar supplemented with bovine serum albumin for 72 h at 37°C under 98% humidity and microaerophilic conditions (5% O(DOCX)Click here for additional data file.S2 File(XLSX)Click here for additional data file.


T cells are central players in the immune defense, but if deregulated they may also mediate the development of pathological conditions and diseases. Therefore, how T-cell functions are regulated is the focus of intense research since many years. Important steps forward in our understanding of the molecular mechanisms underlying T-cell biology have been made recently. These advances have allowed the development of new strategies to reprogram or to modulate T-cell functions for the treatment of patients suffering from cancer, chronic inflammatory diseases, and autoimmunity.394 and Tyr505. Trans- auto-phosphorylation of Tyr394 in the kinase domain promotes an open/active conformation, whereas phosphorylation of Tyr505, located at the C-terminus, by the tyrosine kinase Csk results in a close/inactive enzyme.The Src-family kinase Lck is an essential regulator of T-cell functions , which is involved in the initiation of TCR signaling. Upon TCR engagement, Lck phosphorylates tyrosine residues within the immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR-associated CD3 chains. Phosphorylated ITAMs act as docking site for the tandem SH2 domains of the tyrosine kinase Zap-70, which is in turn activated by Lck. Signaling is further propagated upon phosphorylation of the transmembrane adaptor protein LAT by Zap-70. LAT links the TCR to the activation of intracellular signaling cascades leading to the transcription of target genes required for the execution of the appropriate T-cell response. As all members of the Src-family, Lck activity is regulated by two major phosphorylation sites Tyrde novo phosphorylation/activation of Tyr394 and opening of Lck or not [de novo activation/opening of Lck and that ITAM-phosphorylation is likely initiated by a pool of active Lck, which is present in resting T cells [Recently, the regulation of Lck activity has been at the center of intense debates whether TCR triggering induces k or not -5. Usingk or not  and thatk or not . Based u T cells .in vitro kinase assays) showing that TCR stimulation increased Lck activity. Therefore, we concluded that de novo activation and opening of Lck are required for the initiation of TCR signaling (“de novo activation” model).Conversely to the view described above, using an Lck biosensor and fluorescence lifetime imaging (FLIM)/Förster resonance energy transfer (FRET) analyses, our group has found that T-cell stimulation results in a conformational opening of about 20% of Lck molecules at the engaged TCR . The mic394 occurs at the plasma membrane upon TCR stimulation [394 is critical for inducing activation of Lck. In fact, an Lck-biosensor mutant in which the two regulatory tyrosine residues were mutated to phenylalanine  is cons T cells . How the T cells . This inDespite latest advances in our understanding of Lck activation and initiation of TCR signaling, a number of questions still remains open. It would be of particular interest to investigate whether the “active” TCR is involved in the regulation of opening/activation of Lck as suggested by our data. A better understanding of this process is a crucial step towards the development of pharmacological tools to modulate the interplay between the TCR and Lck and, hence, the activation of T cells.


Neurite growth is controlled by a complex molecular signaling network that regulates filamentous actin (F-actin) dynamics at the growth cone. The evolutionarily conserved ezrin, radixin, and moesin family of proteins tether F-actin to the cell membrane when phosphorylated at a conserved threonine residue and modulate neurite outgrowth. Here we show that Akt binds to and phosphorylates a threonine 573 residue on radixin. Akt-mediated phosphorylation protects radixin from ubiquitin-dependent proteasomal degradation, thereby enhancing radixin protein stability, which permits proper neurite outgrowth and growth cone formation. Conversely, the inhibition of Akt kinase or disruption of Akt-dependent phosphorylation reduces the binding affinity of radixin to F-actin as well as lowers radixin protein levels, resulting in decreased neurite outgrowth and growth cone formation. Our findings suggest that Akt signaling regulates neurite outgrowth by stabilizing radixin interactions with F-actin, thus facilitating local F-actin dynamics. ERM proteins also act as scaffolds for adaptor and signaling molecules that regulate cytoskeletal dynamics. The activity of each ERM protein is regulated by the phosphorylation of a conserved threonine residue in the actin-binding domain  that blocks the intramolecular association of the N- and C-terminal regions and allows ERM proteins to bind to F-actin and other proteins7. However, it is not known whether additional phosphorylation on C-terminus of ERM is related to its functions.Ezrin, radixin, and moesin, collectively known as ERM proteins, coordinate membrane–cytoskeletal interactions for various forms of cell motility including neuron morphogenesis. ERM proteins share a C-terminal actin-binding domain and an N-terminal FERM domain that binds to membrane proteins such as CD44 and the axon adhesion molecule L19. In sympathetic neurons, nerve growth factor (NGF) deprivation-induced growth cone collapse is accompanied by a local decrease in radixin levels. The suppressed expression of radixin and moesin, but not of ezrin, impairs growth cone morphology, cytoskeletal organization, and growth cone motility in cortical and hippocampal neurons11. In addition, the suppression of ERM phosphorylation by inhibiting phosphoinositide 3-kinase (PI-3K) in the growth cones of DRG axons results in growth cone collapse.In developing neurons, ERM proteins are expressed in growth cones and among ERM proteins, radixin is predominant at the leading edge of dorsal root ganglion (DRG) growth cones15, PI3K/Akt signaling has been implicated in dendritic morphogenesis16, neuronal polarity and growth17, synaptogenesis and spinogenesis18, plasticity19, axon establishment, and axon elongation during development by phosphorylating glycogen synthase kinase (GSK)-3β, which leads to GSK3β inactivation23. While Akt is localized at the axon tip, phosphorylated (inactive) GSK3β is restricted to the tip of growing axons in cultured hippocampal neurons and regulates neuronal polarity. Moreover, Akt links a host of upstream signaling molecules to axon development, axon growth, and dendrite elongation in the central nervous system (CNS) by activating mTORC1 and S6 kinase, which regulate cap-dependent protein translation, and by inhibiting TSC1/227. However, evidence suggests that an mTORC1-independent pathway regulates axon regrowth in phosphatase and tensin homolog-deficient neurons29, causing the aberrant activation of Akt signaling. Moreover, our recent study showed that Akt1 regulates the formation of growth cones and functions through the phosphorylation of S14 on inhibitor of DNA binding 2 (Id2), which is a negative regulator of basic helix–loop–helix transcription factors30. Akt-phosphorylated Id2 accumulates at the tip of growing axons where radixin is enriched, and the association with radixin contributes to axon growth and proper growth cone function. Thus, Akt may regulate growth cone dynamics through alternative signaling pathways including the phosphorylation of ERM proteins.The PI-3K pathway regulates diverse neuronal activities, mainly through the downstream molecule Akt/protein kinase B. In addition to a critical role in neuronal survival31. For instance, SCF-beta-TrCP recognizes phosphorylated IkBa or phosphorylated beta-catenin for their ubiquitination. In contrast, instead of promoting ubiquitination, phosphorylation inhibits E3 ligase substrate recognition. For example, Mdm2 binding to p53 is blocked by phosphorylation of S15/T18 in response to DNA damage32; S2 phosphorylation in the c-Mos kinase prevents recognition by an unknown E3 ligase33. Akt-mediated S143 phosphorylation on DNA methyltransferase-1 (DMNT1) interferes its methylation, which leads to UPS-dependent DNMT1 degradation, inhibiting DNMT1 ubiquitination and enhancing its stability34. In addition, Akt-mediated S14 phosphorylation in the Id2 prevents its association with Cdh1/APC/C E3 ligase, thereby stabilizing Id2 protein during neuron development30. Third, phosphorylation can influence ubiquitination by resulting substrate/ligase interaction controlling subcellular localization as shown by p27Kip1 nuclear export by its phosphorylation, allowing its degradation by cytoplasmic E3 ligase35. Finally, phosphorylation should also regulate deubiquitinating enzyme (DUB) that counteract with E3 ligase. Akt mediated S432 phosphorylation on ubiqutin specific protease (USP) 14, activates its deubiquiting activity36. Although it is well established how phosphorylation is important for ubiqutination by E3 ligases and subsequent proteolysis37, the regulation of spatial/temporal phosphorylation dependent ubiquitination is understudied and also to what extent phosphorylation can negatively regulate substrate recognition are required further exploration. In the current study, we demonstrated that Akt directly interacts with radixin and phosphorylates T573 rather than T564, which is known to be conserved threonine residue on radixin. Although C-terminal T564 phosphorylation has been well documented by several kinases and conserved throughout many of mammalian radixin, Akt was not the kinase for T564. Akt-mediated phosphorylation of C-terminal T573 on radixin enhances radixin protein stability by preventing proteasomal degradation of radixin. During the differentiation of hippocampal neurons, Akt-mediated radixin phosphorylation is essential for radixin function in the growth cone, including proper neurite outgrowth and F-actin binding. Thus, our data provide additional information on an Akt-dependent pathway for neuron growth regulation through radixin phosphorylation and stabilization.Phosphorylation can promote or inhibit protein ubiquitination in several ways. First, phosphorylation positively or negatively regulates E3 ligase activity through direct phosphorylation on it. For instance, Akt increased E3 ligase activity of Mdm2 by phosphorylation by preventing its autoubiquitination, thereby promotes UPS dependent degradation of p53, which is well known target of Mdm2. In contrast, Mdm2 activity is inhibited by c-Abl phosphorylation of Y394. Second, phosphorylation promotes recognition by an E3 ligase by creating a phosphodegron, short motif that mediate phosphorylation dependent recognition by E3 ligase. The majority of phosphorylation dependent ubiquitination targets are recognized by SCF (Skp1/cullin/ F-box protein) family of E3 ligases, in which the F-box protein associates with the phosphorylated targets30 −Akt associated with radixin−WT more strongly than kinase dead (KD)–Akt, indicating that Akt activation is necessary for the interaction with radixin  14 and enhances its deubiquiting activityP14 Fig.  and examP14 Fig. . TreatmeP14 Fig. .30. Upon phosphorylation of the conserved C-terminal threonine, radixin binds to F-actin41. Thus, we examined the effect of ERM phosphorylation on F-actin binding. The GST pull-down assay showed that the interaction between radixin and F-actin decreased (more than 50%) in the absence of Akt  are critical requirement for Akt mediated phosphorylation, distinguishing substrate specificity from that of two other mitogen stimulated AGC kinases, RSK (MAPKAP-K1) and S6K1(p70S6K), which can better tolerate K at these position. Although it is possible that there are unknown sequence contexts or macromolecular interactions within cells that might be phosphorylate motifs other than well-agreed motif, currently beyond this category, there are no rigorously demonstrate and independently confirmed Akt substrate. Based on this substrate specificity of Akt kinase, it is not surprising that Akt phosphorylates radixin on T573 (RQIRQGNT) but not T564 (AGRDKYKT) in our study  we cannot rule out the possibility that ezrin and/or moesin might be able to be phosphorylated by Akt. However, in the current study we observed that knockdown of Akt only disturbs radixin protein levels but not ezrin or moesin levels Fig. . As propThis effect of Akt-dependent phosphorylation on radixin protein levels but not on radixin mRNA expression, suggesting the regulation of protein stability, was striking because studies on radixin degradation have not been reported. CHX treatment for up to 8 h did not alter radixin protein levels, indicating that radixin is a relatively stable protein. However, overexpressing radixin–T573A or knocking down Akt markedly decreased radixin protein levels Figs  and 4A, 52. However, the E3 ligase has not been identified, and the spatiotemporal profile of radixin ubiquitination during neuronal development remains to be determined. It has been reported that ezrin may be ubiquitinated by the HECT E3 ubiquitin ligase WWP1/Aip5/Tiul1 in epithelial cells, but no proteasomal degradation was observed53. Alternatively, we also suggested USP14 as a possible deubiquitinating enzyme for radixin when Akt is activated, as demonstrating the tri-complex of Akt-radixin-USP14 upon growth factor stimulation and notable decrease of radixin ubiquitination with overexpression of USP14  supplemented with 10% FBS and 100 U/ml of penicillin–streptomycin. PC12 cells were maintained in DMEM with 10% FBS, 5% horse serum, and 100 U/ml of penicillin–streptomycin at 37 °C under a 5% CO2 atmosphere.The brains of E18 rat embryos were dissected, and hippocampi were removed and placed in a 15 ml tube with 14 ml of Hanks’ balanced salt solution on ice. The medium was carefully aspirated, leaving 2 ml of the medium in the tube. Papain (20 mg/ml in a dissection medium) was added, and the hippocampi were incubated for 20 min at 37 °C. Digestion was stopped by washing the hippocampi twice with 4 ml of complete medium supplemented with 10% fetal bovine serum (FBS). Then, 3 ml of neurobasal medium /B27 (Invitrogen 17504-044) was added, and tissues were dissociated by gently triturating the hippocampi through a fire-polished Pasteur pipette. The cell mixture was diluted to 10 ml with NB/B27 and was then filtered through a 40 or 70 µM strainer. Cells were spun at 1800 rpm for 5 min and resuspended in 10 ml of NB/B27. HEK293T and PC12 cells were cultured as previously describedAnti-radixin, and anti-Akt antibodies were obtained from Cell Signaling Technology . Anti-GFP, anti-GST, anti-HA, and anti-actin antibodies were acquired from Santa Cruz Biotechnology . Alexa Fluor 594-labeled goat anti-rabbit and Alexa Fluor 488-labeled goat anti-mouse secondary antibodies were obtained from Molecular Probes . All other chemicals were obtained from Sigma .in vitro site-directed mutagenesis system and was inserted into a pEGFP vector. Primers for T573A (5′-CAA GGC AAC GCC AAG CAG CGC ATC GAT GAG-3′ and 3′-GCG CTG CTT GGC GTT GCC TTG TCG AAT CTG-5′) were synthesized by Cosmo Genetech . WT and mutant radixin were subcloned into pGEX4T-1Rat WT radixin cDNA was cloned into a pEGFP vector and amplified using a forward primer beginning at the 5′ Kpn1 site (5′-AAA GGT ACC GCA TGC CGA AAC CAA TAA AT–3′) and a reverse primer containing the BamH1 site at the 3′ end of the gene (3′-CTC AAA CTT CGT TAC ACT CCT AGG AAA-5′). The radixin point mutation T573A was generated by RT-PCR amplification using an 3VO4, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 10 mM beta-glycerophosphate, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail . Cell lysates (0.5 to 1 mg of protein) were mixed with a primary antibody and protein A/G beads and were then incubated for 3 h at 4 °C with gentle agitation. Beads were then washed in a lysis buffer. The extracted proteins were analyzed by immunoblotting as previously described55. For GST pull-down assays, cells were rinsed with PBS and lysed in the buffer as previously described57. Cell lysates (0.5 to 1 mg of protein) were mixed with glutathione–sepharose beads and incubated for 3 h at 4 °C with gentle agitation. The beads were then washed in the lysis buffer, mixed with 2× sodium dodecyl sulfate (SDS) sample buffer, and boiled. Extracted proteins were analyzed by immunoblotting.For co-immunoprecipitation, cells were rinsed with phosphate-buffered saline (PBS) and lysed in a buffer containing 50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 1.5 mM Na59 with the following modifications. Cells grown on coverslips in 24-well plates were fixed in 4% paraformaldehyde for 15 min, permeabilized in PBS containing 0.25% Triton X-100 for 10 min, and blocked in 1% bovine serum albumin for 30 min. Cells were immunostained using primary antibodies and the appropriate Alexa Fluor 594-labeled goat anti-rabbit and Alexa Fluor 488-labeled goat anti-mouse secondary antibodies. Nuclei were counterstained with DAPI. Images of immunostained cells were acquired using a laser scanning confocal microscope  controlled by ZEN software.Immunostaining was performed as previously describedin vitro kinase assay was performed as previously described54. Recombinant active Akt  was incubated with 1.8 × 105 Bq γ-32P-ATP and 1 µg of recombinant GST fusion protein in 30 µl of kinase buffer . Reactions were run at 30 °C for 20 min and terminated by the addition of Laemmli SDS sample dilution buffer. Proteins were separated by 10% SDS-polyacrylamide gel electrophoresis, and phosphorylation was visualized by performing autoradiography.The *p < 0.05; **p < 0.005).Data are expressed as mean ± SEM of three independent experiments with triplicate measurements. Statistical analysis was performed using SigmaPlot . All studies were performed in a blinded manner. Statistical significance was defined by Student’s t-test  of Sungkyunkwan University School of Medicine (SUSM) (code16-21/16-22). SUSM is an Association for Assessment and Accreditation of Laboratory Animal Care International  accredited facility and abide by the Institute of Laboratory Animal Resources (ILAR) guide. All experimental procedures were carried out in accordance with the regulations of the IACUC guideline of Sungkyunkwan University.Supplementary information


In vitro phosphorylation assay showed that the M3 phosphorylation residues of PIN1 are the partial targets by PINOID kinase. Our data suggest that the M3 phosphorylation site is functionally conserved among long HL-PINs by playing roles for their subcellular trafficking and auxin-mediated developmental processes.Asymmetrically localized PIN-FORMED (PIN) auxin efflux carriers play key roles in regulating directional intercellular auxin movement, generating local auxin gradients, and diverse auxin-mediated growth and development. The polar localization of PINs is controlled by phosphorylation in the central hydrophilic loop (HL) of PINs. Although the M3 phosphorylation site, including phosphorylatable 5 Ser/Thr residues, is conserved among long HL-PINs, its native role has only been characterized in PIN3. In this study, we examined the role of M3 phosphorylation site of PIN1, PIN2, and PIN7 in intracellular trafficking, phosphorylation, and biological functions of those PINs in their native expressing tissues. Phosphorylation-defective mutations of the phosphorylatable residues in the M3 site of PIN1-HL led to alteration in subcellular polarity of PIN1 and caused defects in PIN1-mediated biological functions such as cotyledon development, phyllotaxy of vegetative leaves, and development of reproductive organs. The M3 mutations of PIN7 interfered with its polar recycling in the root columella cell in response to gravity stimulus and partially disrupted root gravitropism. On the other hand, the M3 site of PIN2 was shown to be necessary for its targeting to the plasma membrane.  Arabidopsis genome encodes eight PIN proteins that can be classified into two subgroups by the size of central hydrophilic loop (HL) domain. PIN1, PIN2, PIN3, PIN4, PIN6, and PIN7 have a long HL , whereas PIN5 and PIN8 have a relatively short HL   . While srt PINs) , 2014.Arabidopsis root stele and meristem region  or M3-mutant versions of those PINs and analyzed the mutation effects on each PIN’s own biological function.Several phosphorylation sites in the PIN-HL have been functionally characterized . Our preArabidopsis thaliana (Columbia ecotype) was used as the WT plant in this study. Arabidopsis plants were transformed using Agrobacterium tumefaciens strain C58C1 (pMP90) ( (pMP90) . All seeProPINs:PINs:GFP, ProPINs:M3PINs:GFP, and ProPINs:3m1PINs:GFP constructs were generated by replacing the ProE7 fragment of ProE7:PINs:GFP, ProE7:M3PINs:GFP, and ProE7:3m1PINs:GFP (PINs (ProPINs). ProPINs were amplified by PCR using the Arabidopsis genomic DNA as template and the primers listed in Supplementary Table The binary vector pCAMBIA 1300-NOS with modified cloning sites  was usedPINs:GFP  with theEscherichia coli (E. coli) for the in vitro phosphorylation assay, the PIN-HL part was obtained from Arabidopsis cDNA by using the primers in Supplementary Table pGEX-4T-1 vector  for fusion with glutathione S-transferase (GST) at the N-terminus. The protein kinase PID used in the kinase assay was as described in For heterologous expression of PIN-HL proteins in All constructs were confirmed by nucleotide sequencing, and at least five independent transgenic lines for each construct were analyzed.Phyllotatic analysis was conducted by numbering the 3-week-old vegetative leaves from early to late developmental stages, and the angles between leaves were measured by the Leica Application suite (v.2.8.1). The seed number per silique was measured under the Leica MZFLIII dissecting microscope. For the root gravitropism assay, 3-day-old seedlings grown vertically in the light were turned 90° for the indicated period of time (2–10 h) in the dark condition before observing root bending.PIN:GFP signals was performed using the histogram function of Adobe Photoshop CS6 (Adobe Systems).Observation of fluorescent reporters was conducted as described previously . To obseTo observe gravitropic changes of PIN7:GFP localization in root columella cells, 4-day-old seedlings were transferred to a slide glass containing half-strength MS medium and grown for 24 h vertically in the dark condition. Gravity stimulation was applied for the indicated time period by turning the slide 90°, subsequently the changes of PIN7:GFP signal were analyzed under a confocal microscope. To visualize cell boundaries, FM4-64 (2 μM) was applied for 3 min.E. coli (BL21DE3) cells was induced by 1 mM isopropyl β-D-1-thiogalactopyranoside for 3 h at 28°C, and the cells were harvested by centrifugation at 3900 g for 10 min. The harvested cells were lysed with B-PER bacterial protein extraction reagent  with the Halt protease inhibitor complex (Santa Cruz), and the GST-fused proteins were purified using the GST-bound agarose resin (Elpis biotech).Expression of GST-fused kinase (PID) and substrates (WT and M3-mutated PIN1-HL) in in vitro phosphorylation assays, l μg of kinase and substrate proteins, respectively, were mixed in 30 μl kinase buffer  and 1 μCi [γ-32P] ATP for 1 h at 30°C and, the reaction was halted by adding 5 μl of SDS-PAGE loading buffer and boiling for 10 min. The reaction samples were then separated in 10% acrylamide gels and stained with Coomassie Brilliant Blue. Autoradiography was performed using BAS imaging plate  and Bio-imaging Analyzer . The intensity of the phosphorylation bands was estimated by ImageJ software1 . To estimate the phosphorylated band intensity, the background intensity value of the same lane was subtracted from the intensity value of the phosphorylated PIN band.For Among 5 Ser/Thr residues of the M3 site of PIN3, single or double mutations of them barely showed the mutation effect on PIN3’s trafficking and biological roles, indicating their redundant function . HoweverProPIN1) in the pin1 mutant background.In the root, PIN1 is mainly localized at the basal PM of vascular cells and the basal and inner lateral sides of pericycle and endodermal cells . In ordeFigure 1A). Moreover, M3PIN1 tended to be more internalized than WT PIN1. Statistical analysis obviously revealed the defects of M3PIN1 and 3m1PIN1 in basal localization by showing considerably decreased ratio (~40%) of apical/basal to lateral localization . In endodermal cells, while WT PIN1 localized predominantly in basal and inner lateral sides, mutation of M3 or 3m1 disturbed this polarity by delivering these mutant PIN1 proteins to the outer lateral PM . These results indicate that the proper subcellular PIN1 polarity requires the M3 phosphorylation residues.In vascular cells, WT PIN1, as expected, predominantly localized to the basal PM, whereas the basal localization of M3PIN1 was disturbed so as for the mutant PIN1 to be more localized to lateral sides than WT PIN1 , suggesting that, though M3PIN1 tends to internalize, some M3PIN1 proteins still are able to target to the PM and properly recycle like WT PIN1. This result in root meristematic vasculature cells is consistent with our previous observation that M3PIN1 and M3PIN7 are partially internalized and accumulated in BFA compartments in the root hair cell . It is likely that the disruption of PIN subcellular polarity generally causes ectopic cell division probably by interfering local auxin concentration gradients.In a previous study, ectopic cell division was observed in the M3PIN3-expressing root pericycle . M3PIN1 pin1 mutant shows structural abnormalities in inflorescence axes, flowers, and leaves , most likely by affecting auxin-gradient formation during embryogenesis. For the phenotypic analysis of cotyledons, the segregated F1 progeny seedlings of the self-fertilized heterozygous pin1 mutant background were observed because homozygous M3PIN1-complemented pin1 mutants barely produced seeds for next generation. While complementation with WT PIN1 greatly restored the normal cotyledon phenotype, complementation with M3PIN1 or 3m1PIN1 only partially rescued the pin1 cotyledon defects . Interestingly, the phenotype ratio of ‘fused cotyledon’ was rather increased by M3 or 3m1 mutation in comparison with the complete loss of PIN1 (pin1) . Furthermore, some severe defects such as reduced or no cotyledons were observed by the complementation with both M3PIN1 and 3m1PIN1, indicating that these PIN1 mutants, by impairing its native polarity, lead to alteration of local auxin gradients and subsequent morphogenetic changes during embryogenesis.The loss of PIN1 caused aberrant cotyledon morphogenesis such as single, multiple, or fused cotyledons . This distribution pattern was dissipated in the homozygous pin1 mutant by considerably deviating its peak from the typical angle of 130–140°. Complementation of homozygous pin1 with WT PIN1 restored the peak at 130–140°. However, M3PIN1- or 3m1PIN1-complementation failed to restore the WT phyllotactic peak, indicating that the M3 phosphorylation site is necessary for PIN1’s function in phyllotaxis.Because PIN1 is associated with leaf initiation and phyllotaxis , we exampin1 mutant forms a pin-like inflorescence stem lacking lateral shoot organs . M3PIN1-complemented plants grew a pin-like primary inflorescence stem that, however, was soon aborted . This early abortion of the primary stem by M3PIN1 led to growth of secondary inflorescence stems . In contrast, 3m1PIN1-complemented plants kept growing the primary stem, although the size was smaller than that of WT plants . The M3PIN1 plants, though growing secondary stems, eventually produced aborted shoot apexes in these secondary stems . The homozygous pin1 mutant did not abort the primary stem early but maintained its growth for a while .The homozygous Figures 4D–F). The WT plants generally produced over 1.5 cm siliques. However, the number of siliques over 1 cm was decreased considerably by 3m1 and greatly by M3 mutation . Consistently, the seed number per silique also decreased by these mutations to 11% (M3) and 67% (3m1) of the WT level (40.9 seeds/silique) .In addition to their effects on inflorescence stem development, M3 or 3m1 mutation also affected silique development so as to produce aberrant or no silique , the partial loss of polarity or altered intracellular trafficking of M3PIN1 and 3m1PIN1 could cause spatial changes of auxin gradients, which subsequently leads to developmental defects.These results together suggest more than that the M3 phosphorylation site is necessary for normal biological functions of PIN1. M3PIN1- and 3m1PIN1-complemented transformants revealed new phenotypes that are not shown in the In the root columella cell, upon changes of gravity vector, PIN3 and PIN7 relocalize so as to redistribute auxin for gravitropic bending of the root . Under aPIN7 promoter was complemented into the homozygous pin7 mutant. We first observed the intracellular trafficking behavior of WT PIN7 and M3PIN7 upon gravity vector change. WT PIN7 accumulated into endocytotic vesicles within 15 min after gravity stimulus of the root whereas M3PIN7 did not show obvious accumulation in the endocytotic vesicle upon the gravity stimulus . Before gravity stimulus, WT PIN7 and M3PIN7 were symmetrically localized at the columella cell PM . After a 40-min gravity stimulus, WT PIN7 asymmetrically redistributed to the PM side facing to the new gravity direction . However, M3PIN7 did not change its symmetrical distribution after the same gravity stimulus . For a statistical analysis, we took the ratio of PIN7 signal intensity between lateral (L) and basal (B) sides of the columella cell  as a polarity parameter. While WT PIN7 significantly increased its lateral localization from 0.83 to 1.46 of L/B after the 40-min gravity stimulus, M3PIN7 maintained almost the same ratio of L/B (~1.0) regardless of the gravity stimulus, suggesting that phosphorylation of the M3 site is essential for gravity-stimulated transcytosis and relocalization of PIN7 in the root columella cell.Here, we tested whether the M3 phosphorylation site is implicated in PIN7’s function during root gravitropism. Similarly as in the PIN1 study, WT or M3-mutated PIN7 with the GFP tag under the pin7 mutant plants exhibited root gravitropic defects as reported previously . While complementation with WT PIN7 rescued root gravitropism of pin7, complementation with M3PIN7 failed to rescue . In the gravitropic kinetics analysis of the root, the M3PIN7-complemented mutant showed relatively decreased gravitropic responses compared to WT and WT PIN7-complemented plants . These results suggest that the M3 phosphorylation residues are necessary for PIN7-mediated root gravitropism most likely by modulating the transcytotic relocalization of PIN7 after gravity stimuli.Next we examined whether the defects of M3PIN7 in subcellular relocalization affect the gravitropic response of the M3PIN7 plant root. The pin2 mutant plants complemented with WT PIN2, M3PIN2, or 3m1PIN2 with the GFP tag under the PIN2 promoter. WT PIN2 polarly localized to the upper side of the root epidermal cell PM as previously reported . However, M3PIN2 showed almost no expression and 3m1PIN2 showed weak expression , which is consistent with our previous result showing that M3PIN2 and 3m1PIN2, when expressed under a root hair-specific promoter (ProE7), did not express in the root hair cell . Intriguingly, the treatment with wortmannin, an inhibitor for vacuolar trafficking, was reported to restore both M3- and 3m1PIN2 protein signals in the cytoplasm but not in the PM of the root hair cell, suggesting that the M3 phosphorylation site is implicated in regulation of vacuolar targeting of PIN2 RSXAS is the consensus of PIN2 orthologs. For the third Ser, PIN2 includes NKS rather than RRS. Finally for the region around forth Thr and fifth Ser, the PIN2 consensus is TPRAS whereas other long PINs have TPRPS. Although it has remained to be characterized, these syntactic variations around the M3 phosphorylatable residues might lead PIN2 to have different trafficking behavior most likely by different reader proteins against these different syntaxes around the phosphorylation residues.Although the phosphorylatable Ser/Thr residues in long PINs are mostly conserved, the flanking residues vary particularly in PIN2 . Compared with the WT PIN1-HL phosphorylation level, PID-mediated phosphorylation of M3-mutated PIN1-HL was considerably decreased . Conversely, 3m1 mutation marginally decreased the phosphorylation level . In our previous study with PIN3, M3 mutation showed a greater effect than 3m1 mutation on PID-mediated phosphorylation of PIN3-HL (In order to know whether the 5 Ser/Thr residues of the M3 site are involved in phosphorylation of PIN1, we performed  PIN3-HL . These r PIN3-HL . TherefoAlthough the native function of M3 phosphorylation site was studied with PIN3  and the H-TC designed the project. DK performed most of the experiments for plant analyses, microscopy, and biochemistry. DS made the transgenic constructs. All authors contributed to the interpretation of results and writing of the manuscript. All authors approve the manuscript publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The sedimentary record in the Guadix-Baza Basin (southern Spain) has proved to be a great source of information for the Miocene through the Pleistocene periods, due to the abundant faunal remains preserved, in some cases associated with lithic tools. The Solana del Zamborino (SZ) section has been the subject of controversy ever since a magnetostratigraphic analysis resulted in an age of 750–770 Kyr for Acheulean tools, a chronology significantly older than the ~600 Kyr established chronology for the first Acheulean record in Europe. Although recent findings at the “Barranc de la Boella” site (north-east of the Iberian Peninsula) seem to indicate that an earlier introduction of such technique in Europe around 0.96–0.781 Ma is possible, the precise age of the classical site at SZ is still controversial. The aim of this paper is to constrain the chronology of the site by developing a longer magnetostratigraphic record. For this purpose, we carried out an exhaustive sampling in a new succession at SZ. Our results provide a ~65 m magnetostratigraphic record in which 4 magnetozones of normal polarity are found. Our new magnetostratigraphic data suggest an age range between 300–480 Kyr for the lithic tools, closer to the age of traditional Acheulean sites in Europe. Specifically, sites such as Solana del Zamborino (SZ hereafter) have produced abundant lithic tools that have been claimed to be the oldest Acheulean tools in Europe13. The aim of this study is to provide a solid and more complete chronological framework for SZ. Our results are based on new magnetostratigraphy that covers approximately over 65 meters of section, complementing the previous results by Scott and Gibert13.The Guadix-Baza basin, located in the Betic Mountain Range (southern Spain) contains the most complete sedimentary record in Spain throughout the Miocene to Pleistocene epochs14. The basin was flooded by sea water during the Tortonian and marine sediments were deposited15. Subsequently, the basin was isolated from the sea, due to tectonic tilting related to the activation of a NE-SW fault system, and filled with continental sediments from the late Tortonian to the Upper Pleistocene16. During the Villafranchian stage, the basin was divided in two sub-basins separated by a topographic elevation: the Baza sub-basin to the NE and the sub-basin of Guadix to the SW16. The Guadix sub-basin holds an abundance of terrigenous sediments, including conglomerates and sands which were deposited by rivers, alluvial fans and lacustrine fan deltas, fed by the surrounding reliefs22.Solana del Zamborino is located in the Guadix-Baza Basin (one of the more prominent post-orogenic intramontane depressions of the Betic Range) and developed during the Miocene, with a NE-SW orientation25). Deposits are horizontal to sub-horizontal and include frequent lateral facies changes; these fluvial deposits are the two most recent units of the continental landfill of the basin, named as units V and VI20 , and south of the highest lacustrine intercalation in Guadix formation as described by Casas et al.24. Within the site, stratigraphic levels have been traditionally named alphabetically from A to F (from bottom to top) and their detailed description and characteristics are fully included in Casas et al.24.The site targeted for this study is located on the western sector of the Guadix-Baza Basin, where only the Guadix and Gorafe-Huélago formations are exposed .We sampled every meter and a half or less, whenever possible, in a total of 47 sampling sites from which we have obtained 367 individual samples  of the specimens and the progressive demagnetisation was carried out in two different laboratories: a preliminary analysis was carried out in the laboratory of Paleomagnetism and Rock Magnetism at the University of Oxford (England) on a pilot set of specimens. Low field magnetic susceptibility was measured with a Kappabridge model KLY2 with a CS3 oven incorporated. Further analyses were carried out using a cryogenic magnetometer 2 G enterprises DC including an online degausser for alternating field (AF) demagnetization. Subsequently, the remaining specimens were processed at the National Research Center for Human Evolution , using an TD-48SC oven (ASC Scientific) for thermal (TH) demagnetization, a cryogenic magnetometer 2 G model 755R-4K with a built in degausser system for AF demagnetization up to 170 mT, and an Impulse Magnetizer, model IM-10–30 (ASC Scientific) for isothermal remanent magnetization (IRM) acquisition curves.◦C until 420◦C and then increasing the steps to 50 ◦C up to 670 ◦C; and the last 109 specimens were analysed using AF to a maximum field of 0.1 T. Finally, after visual inspection of the Zijderveld diagrams, the characteristic remanent magnetization (ChRM) direction, the highest stability component of the NRM, was determined and computed for each specimen using Principal Component Analysis33, typically anchoring the directions to the origin. We used the Fisherian mean direction34 of the ChRM to calculate the corresponding virtual geomagnetic pole (VGP) position for each site . Latitudes of the VGP poles were used to establish the local magnetostratigraphy.Of the 367 samples obtained, 316 have been analysed, 32 of which have been used to obtain IRM acquisition curves. From the remaining 284 specimens, 175 were analysed by TH demagnetisation with progressive steps of 30The datasets generated during and/or analysed during the current study are available from the corresponding author upon request. The information about the individual samples analysed, and the statistical result are provided as a supplementary information.21. Overall, the specimens have a stable behaviour during both, AF and TH, demagnetization, and present normal and reverse polarity directions   Fig. , magnetoTS) Fig. , impliesTS) Fig. ) helps i20), which are of the same order of magnitude (average of 2 cm/kyr22)  Fig. .Figure 8et al.24 and Botella et al.27). In a previous study13 the archaeological layer was found above the Brunhes / Matuyama boundary. Based on an inferred accumulation rate, an age of ~760kyr was suggested by these authors. However, because this previous magnetostratigraphy lacks polarity reversals below and above the archaeological layer, an alternative stratigraphic section at the paleontological site of Cúllar-Baza38 (located more than 50 km away), was used to infer the accumulation rate for La Solana. We would like to emphasize that the section of Cúllar-Baza is not lithologicaly similar to that at La Solana, since the latter is characterised by siliciclastic sediments , whereas the former sequence is mainly composed of carbonate lacustrine-palustrine sediments5. Highly different sediment accumulation rates are expected to be found simply because of the contrasting depositional environment. Jimenez-Arenas et al.39 have also brought up inconsistencies with such an age assignment. According to these authors, there are several errors in the interpretation of the archaeological assemblage for the site, and also discrepancies with the original faunal lists published. In our new study, we have been able to locate three reversals below the archaeological site at La Solana, which allows a firmer interpretation of the local magnetostratigraphy and by inference the approximate chronology of the site. Assuming an average accumulation rate of 2 cm/kyr throughout La Solana section, the stratigraphic position of the archaeological site would have an estimated age range between 300 Kyr to 480 Kyr, significantly younger than the previously suggested age, and more in line with the conventional chronology proposed for the evolution of Acheulean in Europe40 and with the faunal record of the SZ site itself31.The ultimate goal of establishing the chronology at SZ resides in the presence of the lithic tools found at a stratigraphic level reported as level B, corresponding to the Guadix Formation  and based on the sedimentation rate, the stratigraphic position of the archaeological site at SZ would have an approximate age of ~300–480 Kyr, much younger than the age provided by Scott and GibbertSupplementary Information


Allergic, especially anaphylactic, reactions during immunoglobulin replacement therapy are rare, but their pathophysiology and classification remain ambiguous. Recent findings show positive results of skin tests with commercially available immunoglobulins, but target antigens and responsible compounds of the tested immunoglobulins have not been strictly identified.Four adult patients with recently diagnosed common variable immunodeficiency qualified for standard subcutaneous immunoglobulin replacement therapy regimen. They had no history of receiving immunoglobulins, blood or blood product transfusions. Edema, confluent wheals and erythema were observed at the site of subcutaneous immunoglobulin infusion: typical early and late phase reaction. A transient increase in various passively transferred IgG and IgE antibodies was responsible for misleading positive outcome of the serological testing for active humoral response such as type I allergy, anti-Rh, isohemagglutinins and rheumatoid factor (RF). Although the clinical presentation was very unusual and severe, the retrospective analysis showed no isohemagglutinins, RF and IgE in the patients’ serum before but it was positive after the infusion . Type I allergic reaction  came out at +14 days of replacement therapy when the patient visited countryside. In the second patient anaphylactic reaction was observed 5 days after ScIg administration, and only when the patient consumed peanuts. Therefore, IgE concentration was measured retrospectively in a series of commercial preparations used in the initial subcutaneous immunoglobulin replacement therapy that caused the adverse event (AE) and it was determined between 138 and 232 IU/ml (kU/l), i.e. 690–2100 IU per g of protein. Specific IgE was within a wide range from 198 (mix of food) to 2809 kUA/l (mix of grass) but many of the tested allergen-specific IgE were class 2 or 3 (i.e. 0.71–17.5 kUA/l).The case resembles passive cutaneous anaphylaxis and Prausnitz–Küstner reaction but clinical significance of the classical phenomena has not yet been described. This observation indicates that anaphylactic reactions during immunoglobulin replacement therapy may result from IgE or pathological IgG content. Such IgE presence was sporadically reported  in intravenous immunoglobulins that are used and monitored by healthcare professionals. In clinical practice the definition of adverse events is inadequate since individual batches of immunoglobulins come with different specificity therefore, they should be classified as transfusion products . Such new approach implies establishing (1) new control methods and strategies to ensure introduction of the safety regulations for subcutaneous home self-administration of immunoglobulins as well as (2) guidelines for the prevention of anaphylaxis in patients receiving immunoglobulins (for example peanut). The choice of methods of drug production, their further purification, sterilization, distribution and storage are critical to pharmacovigilance. Immunoglobulin preparations are of a paramount importance to management of the primary humoral immunodeficiency. Subcutaneous immunoglobulin (ScIg) infusion as replacement therapy is frequently chosen for patients with primary antibody deficiencies (PAD). Although a routine immunoglobulin therapy involves intramuscular injections, it has nowadays been replaced by subcutaneous infusion. Commercially available immunoglobulin preparations contain IgG antibodies, pooled from >3000 donors, with heterogeneous specificity and physicochemical properties  as well hypersensitivity to any compound (according to common WHO/NIH terminology of adverse drug reactions). Most of the immunoglobulin product descriptions do not specify frequency of anaphylaxis. At the same time, anti-IgA immune response is over-diagnosed as the cause of anaphylaxis [Despite progress in identification in the immunoglobulins preparations of the basic components believed to cause hypersensitivity, the basic mechanisms of these phenomena remain elusive. Not surprisingly, only a broad contraindication specified in manufacturer’s leaflets as well as product characteristics concerns cases of phylaxis , 3. It iphylaxis  since thThe aim of this report is to shed light on new mechanisms of hypersensitivity/anaphylactic reaction occurring during initial immunoglobulin replacement therapy.®). They had no history of receiving immunoglobulins, blood or blood product transfusions. Neither hypersensitivity reactions nor allergy  were observed in the natural history of the disease. Antibody of the IgE class was not detected even in patients with the giardiasis, that normally prompts a very high level of IgE in immunocompetent subjects [®), what corresponds to recommended dose of 400 mg/kg body weight per month according to the International Union of Immunological Societies Scientific Committee Guidelines, EMEA [Adult patients aged 30–50 years) with diagnosed common variable immunodeficiency (CVID) according to ESID criteria and clinical characteristics [0 years wsubjects . If IgE es, EMEA , 8 and t®) product without IgE content, wheal and LPR previously observed but substitution was made under medical supervision. Other two patients who withdrew the consent for further ScIg therapy were switched from home-based to hospital-based IVIg therapy. They were not enrolled for further evaluation.In spite of a good history of tolerance to peanuts, life-threatening anaphylactic symptoms (hypotension and then urticaria) appeared when the second patient ate peanuts  . InjectiSamples of the Ig preparations, to be representative of what the patients received were collected before the end of the infusion and preserved for further investigations . IgE anaTo achieve the highest level of safety and quality of drugs the regulation experts recommend a stepwise approach. Unfortunately, immunoglobulins and other blood-derived products are not typical drugs, since plasma processing into various types of products is highly specialized. Large-scale processing (fractionation) is very important for ensuring quality and safety profiles of the products .Many publications and ScIg product descriptions refer to “local tissue reaction” without more specific information about the reaction time , mechanism  and frequency –14. In cThe body’s reaction to administered blood-derived products depend on the duration of the patient’s exposure to the product Fig. , type ofThe positive ICT for peanuts was observed at the injection site, as well as in other areas, but the level of the reaction differed significantly . ScIg initially forms a local depot by non-covalent binding to structural proteins of the extracellular matrix  and reac® and Sandoglobulin® [® [® , sera of one patient were positive for Candida antigen before CVID diagnosis  [Specific immunoglobulins may coat Fc-receptors of various immune cells. Such phenomenon is well documented for IgE, primarily described as passive cutaneous anaphylaxis or the Prausnitz–Küstner (PK) reaction. The PK is a local hypersensitivity reaction induced by intracutaneous injection of serum from a hypersensitive individual into a healthy person. When after 24 h since the commencement of the exchange the antigen to which the donor is allergic, is injected for the second time, the wheal-and-flare response occurs. It is characteristic that “immediate” type allergic reaction is observed at the time when the specific antigen is injected  Fig. . The claIt is described that serum IgE levels regulate FcεRI expression on mast cells and basophils: half-life of FcεRI on cell surface is stabilized by binding IgE . The regOur observation of passive transfer of hypersensitivity indicates that rigorous blood donor screening for allergies should be considered and the blind-spot testing for IgE as part of ScIg quality control should be performed. Furthermore, until now late manifestation of immediate type hypersensitivity after ScIg (positive ICT remains detectable for weeks to months Fig.  has not