The Kangerlussuaq area of southwest Greenland encompasses diverse ecological, geomorphic, and climate gradients that function over a range of spatial and temporal scales. Ecosystems range from the microbial communities on the ice sheet and moisture-stressed terrestrial vegetation (and their associated herbivores) to freshwater and oligosaline lakes. These ecosystems are linked by a dynamic glacio-fluvial-aeolian geomorphic system that transports water, geological material, organic carbon and nutrients from the glacier surface to adjacent terrestrial and aquatic systems. This paraglacial system is now subject to substantial change because of rapid regional warming since 2000. Here, we describe changes in the eco- and geomorphic systems at a range of timescales and explore rapid future change in the links that integrate these systems. We highlight the importance of cross-system subsidies at the landscape scale and, importantly, how these might change in the near future as the Arctic is expected to continue to warm. Arctic ecosystems have undergone major changes over the past century  encapsulates the Arctic in an approximately 150-kilometer, approximately 6000-square-kilometer corridor from the ice sheet itself to the valley and cirque glaciers at the coast. This land mass spanning from the ice-sheet margin to the coast of the Labrador Sea includes a range of landscapes and ecosystems Figures  and 3: gBox 1.Aeolian: relating to or arising from the action of windCryoconite: dust made of small rock particles, soot, and microbes that is found on the surface of a glacier, especially on the bottom of small depressions; causes darkening of ice surfacesDirty ice: ice discolored by the deposition of atmospheric dust and/or previously entrained silt rising to the surface; important because it alters the surface albedoFluvial: of or found in a riverJökulhlaup: a type of glacial outburst floodLoess: previously deposited and biologically transformed dustMoulin: a nearly vertical shaft in a glacier, formed by surface water percolating through a crack in the iceParaglacial: referring to surface processes and landscapes directly conditioned by former glaciation and deglaciationPeriglacial: the zone peripheral to glaciersSandur: outwash plain formed by meltwater from glaciersSupraglacial: anything pertaining to the surface of a glacier but often refers to lakesTalik: region of unfrozen soil or bedrock beneath a lakeThe Kangerlussuaq area is located in the continuous permafrost zone, but permafrost conditions vary considerably in the region. Recent borehole measurements and active layer probing show that active layer thickness varies from approximately 30 centimeters in an ice-wedge polygon affected peat bog at 430 meters (m) above sea level near the GrIS margin  was –5.0 degrees Celsius (°C) and –3.9°C for the periods 1974–2012 and 2001–2012, respectively , followed by rapid ice-marginal thinning and recession of outlet glaciers  and muskoxen (Ovibos moschatus), have been conducted annually throughout the reproductive seasons of both species at a long-term study site 25 kilometers east of Kangerlussuaq  concentrations (approximately 40–100 milligrams per L) because of the long retention times and evapoconcentration over centuries  and despite their proximity: It is possible to transition from Arctic steppe or tundra to glacial ice in meters figure . MoreoveSeasonal melt events and jökulhlaups that cause widespread flooding deposit fine (less than 2000 μm) sediments across the sandur. Following recession of the water, these deposits desiccate rapidly. Strong winds can entrain the sediments causing dust storms and forming localized dunefields, effectively linking the glaciofluvial and aeolian systems figures  and 5. BDust storms can reach several hundred meters above the sandur plain figure . Dust deAeolian material is also deposited on soils and directly into lakes across the region. Modern dust deposition rates in lake catchments above the floodplain are around 70 g per square meter per year , suggesting that groundwater inputs from a retreating GrIS can be traced  cycle linkages among glacial, proglacial, aquatic and terrestrial systems throughout the Kangerlussuaq region in several ways figure . IncreasIn a warming permafrost landscape, the composition of soil and lake DOC pools are likely to change in response to shifts in terrestrially derived C and internal lake dynamics. DOC is a complex mix of compounds sourced from degraded terrestrial and aquatic primary productivity and serves as a cross-ecosystem material that is dependent on direct and indirect climatic influences. Decreasing DOC concentrations in the lakes over the past decade  and the relative fraction of labile DOC  than on the ice surface, suggesting biogeochemical modification on the passage through the subglacial system , where microbial processing and the addition of glacial flour increase the bioavailable P  indicate significant N delivery as wet deposition , whereas snow with particulates has a lower ratio (11:1), indicating enrichment with P, most likely from dust. Direct atmospheric deposition of reactive N to terrestrial and aquatic ecosystems has increased over the last 50–100 years  to conclude that Eurasia was the dominant source of atmospheric pollutants (including Hg and Pb) in SW Greenland. Little is known of how delivery of other micronutrient elements  may change in the future, with implications for microbial growth on the ice sheet and lakes. As we noted above, sulfate concentrations have increased in Kangerlussuaq lakes, raising intriguing questions about the potential for a marine–terrestrial–freshwater linkage. Oceanic phytoplankton produce dimethyl sulfate (DMS), which is aerosolized, transformed into methanesulfonic acid (MSA), and transported onto landmasses. Sharma and colleagues (2012) noted significant increases in MSA over the past decade in the Arctic, indicating a possible role for biogenic sulfate delivery and deposition across the study area. Together, these examples indicate how global change processes can initiate localized influence on the biogeochemistry of the Kangerlussuaq region.Interactions between climate, dust production, atmospheric pollutants and trophic dynamics have the potential to significantly modify a number of regional biogeochemical linkages in the future. Increasing GrIS surface melt and subsequent Watson River runoff in the future  although there will be spatial and temporal variability. The future of the tundra systems north of the Brooks Range in Alaska has been reviewed by Hobbie JE and Kling , who higThe Kangerlussuaq area of southwest Greenland is changing rapidly: Regional warming is driving increased seasonal melt on the ice sheet, altering phenology and changing landscape hydrology with associated effects on lakes and ponds. Some of these changes are interacting in unpredictable ways (meltwater pulses and dust production), whereas others may have cascading effects, such as altered herbivore densities on tundra vegetation and soil C dynamics. Our understanding of the effect of altered herbivore abundance (geese as well as large herbivores) on nutrient budgets and C fluxes is still preliminary as is the reverse subsidy of terrestrial secondary production by aquatic production.Superimposed on climate-driven processes are chronic, subtle changes in atmospheric pollutants, which may have synergistic effects, such as altered N loading on soil nutrient pools and therefore soil microbiology. This preliminary synthesis has highlighted the need to better constrain atmospheric nutrient input rates  to the ice sheet, lakes, and soils, as well as their processing by the glacier and soil subsystems. The effect of the meteorological conditions at the ice-sheet margin on pollutant (and nutrient) deposition requires clarification. The cross-system linkages between geomorphic processes  and ecosystems that influence regional biogeochemical cycling may change in unpredictable ways because of the broader regional climate changes that go beyond temperature, such as altered seasonality of precipitation, wind speeds, and evapotranspiration. The effect of future changes in precipitation  and N loading represent an important but poorly constrained aspect of altered regional biogeochemistry. Changes to the groundwater system (and therefore nutrient and major ion fluxes) and its effect on lakes and streams need to be assessed.Research in the Kangerlussuaq area is ongoing and tracking changes  in real time, but there is clearly a need to link quantitatively the numerous paleoecological records in this area with contemporary measurements. This fusion of time series will provide a more rigorous definition of natural background conditions against which twenty-first-century change can be compared. The effects of these complex linkages for regional carbon dynamics and sequestration highlight the need to take a holistic view of the changing climate, geomorphic, and ecological systems as they influence both aquatic and terrestrial communities in the twenty-first century as the Arctic continues to change.


Cats are the major source of indoor inhalant allergens after house dust mites. The global incidence of cat allergies is rising sharply, posing a major public health problem. Ten cat allergens have been identified. The major allergen responsible for symptoms is Fel d 1, a secretoglobin and not a lipocalin, making the cat a special case among mammals.Given its clinical predominance, it is essential to have a good knowledge of this allergenic fraction, including its basic structure, to understand the new exciting diagnostic and therapeutic applications currently in development. The recent arrival of the component-resolved diagnosis, which uses molecular allergens, represents a unique opportunity to improve our understanding of the disease. Recombinant Fel d 1 is now available for in vitro diagnosis by the anti-Fel d 1 specific IgE assay. The first part of the review will seek to describe the recent advances related to Fel d 1 in terms of positive diagnosis and assessment of disease severity. In daily practice, anti-Fel d 1 IgE tend to replace those directed against the overall extract but is this attitude justified? We will look at the most recent arguments to try to answer this question. In parallel, a second revolution is taking place thanks to molecular engineering, which has allowed the development of various forms of recombinant Fel d 1 and which seeks to modify the immunomodulatory properties of the molecule and thus the clinical history of the disease via various modalities of anti-Fel d 1-specific immunotherapy. We will endeavor to give a clear and practical overview of all these trends. Felis domesticus, is one of the most frequently encountered pets. It is a major source of allergens in the indoor environment and is placed in second position after dust mites for its involvement in the incidence of allergic respiratory diseases. In Western countries, the prevalence of sensitization to allergens of cat has increased dramatically to 10–30% in the general population [Worldwide, the domestic cat, pulation , 3. The Cat allergens have significant allergenicity. They are also numerous and cat allergen extracts are therefore a multi-allergenic source. Historically, 10 allergens recognized by specific IgE have been identified in studies of extracts from fur, saliva, serum and urine , 5. EighThe aim of this first chapter is to review the basic knowledge of Fel d 1 and to give an update on new clinical data, particularly the most recent clinical studies on the Fel d 1-based CRD of cat allergy and the various modalities of Fel d 1-specific immunotherapy.N-oligosaccharides. It consists of 85, 90 or 92 amino acids [Escherichia coli (E. coli). However, to produce the full molecule rFel d 1, it was necessary to use a Baculovirus [E. coli) and has a similar structure to that of the natural cat allergen nFel d 1 [Fel d 1 is a glycoprotein of about 35–38 kDa , 10. It no acids . The thrno acids , 16 . However, the IgE against cat global extract was not tested. Of the latter 65 patients, 61 were sensitized against Fel d 1 , of whom 30 (46.2%) were monosensitized. Of the 65 patients, 4 were sensitized only against Fel d 2 and/or Fel d 4 (6.1%) [Finally, a synthesis of the recent literature on the biological diagnosis of cat allergy shows that the data from the studies are not always consistent. The analytical performances of the anti-Fel d 1 specific IgE assay are close to those of the specific IgE assay directed against the overall extract. However, for a small number of cases with non-typical sensitization profiles (about 5–10%), the latter could have higher sensitivity. In our center, therefore, we consider that in the current state of knowledge an anti-overall extract specific IgE assay still has its place in daily practice in the positive diagnosis of cat allergy.The correlation between the level of Fel d 1-specific IgE and the severity of symptoms was assessed in a case–control study by ImmunoCap in 140 cat-allergic children and adults from Sweden and Austria suffering from asthma and/or rhinoconjunctivitis . PositivIn addition, it has been shown that multi-sensitization towards more than three components including animal-derived lipocalin, kallikrein and Fel d 1 was associated with cases of severe asthma and among which occurred increased bronchial inflammation and a trend towards more courses of oral corticosteroid treatment . AnotherThus, when all these data are compiled, it is clear that measuring anti-Fel d 1 specific IgE levels makes it possible to better evaluate the prognosis of cat allergy. The quantitative aspect of the assay is important since the highest levels will be correlated with the most severe forms of the disease. In daily practice in our hospital, we therefore perform anti-Feld 1 specific IgE assay as soon as it is necessary to evaluate the severity of cat allergy.Allergen specific immunotherapy (AIT), consisting in progressive administration of increasing doses of allergens by different delivery routes , results in long-term allergenic desensitization , 67. DesIn cat allergy, AIT was initially tested with cat dander extract, which was effective in the treatment of cat allergy symptoms, particularly respiratory symptoms. For example, Alvarez-Cuesta et al. , tested For these reasons, and owing to its clinical predominance mentioned above, most studies now favor the use of Fel d 1 rather than global extract for cat allergy specific immunotherapy. Several molecular approaches using standardized preparations of Fel d 1 have been developed since the recombinant forms of this allergen have become available.To reduce the allergenicity of Fel d 1, and thus increase the security of AIT, various structural changes were made to Fel d 1. First, in order to modify the B cell epitopes, the disulfide bonds linking the Fel d 1 chains together were disrupted . This le®, which has been tested in clinical trials. Norman et al. [®  administered as a subcutaneous injection for 4 weeks. A high dose of Allervax CAT® improved allergy symptoms after 6 weeks of treatment [In another approach based on the pivotal role of T cells in polarizing immune responsiveness to allergen, a team selected two peptides containing multiple T-cell epitopes from the sequence of Fel d 1. Unlike Fel d 1, these two peptides caused histamine release from basophils in < 1% of cat allergic patients and are unable to crosslink allergen-specific IgE molecules on basophils in vitro . These pn et al.  conductereatment . Howeverreatment . Thereafreatment , 74. FroAnother product for cat peptide immunotherapy was then developed and tested in allergic rhinoconjunctivitis. This product, called Cat-PAD (Cat-peptide antigen desensitization), was the first in a new class of synthetic peptide immuno-regulatory epitopes (SPIREs). It consists of a mixture of seven small peptides derived from Fel d 1 . These pA third interesting approach consists in linking to Fel d 1 another molecule that may have various immunological properties in order to target both effectors of innate or adaptive immunity. For example, the fusion protein H22-Fel d 1, composed of rFel d 1 associated with a fragment of a humanized anti-CD64 antibody, has a high affinity for FcγRI, the high affinity IgG receptor, which is present on the surface of dendritic cells. In a monocyte-derived dendritic cell model, this resulted in increased uptake of Fel d 1. H22-Fel d 1 induced a semi-maturation of dendritic cells and led to a state of tolerance by promoting the secretion of cytokines such as IL-10 and IL-5 . AnotherFinally, the team of Senti produced MAT-Feld 1 (modular antigen transport-Fel d 1), fusing the recombinant allergen Fel d 1 with the TAT peptide derived from the HIV virus (MAT-Feld 1) . They obThe appearance of the recombinant forms of Fel d 1 has led to the development of a CRD for cat allergy, which is very useful for the practitioner. Compared to the cat-specific IgE (whole extract), anti-Fel d 1 specific IgE have an equivalent or slightly lower sensitivity in terms of positive diagnosis and are correlated with disease severity and the risk of asthma occurrence. Molecular engineering has contributed to the emergence of multiple forms of Fel d 1 specific immunotherapy that are still being improved to optimize the induction of a tolerogenic immune profile. They open up great therapeutic prospects for patients in the years to come. However, it is becoming clear that the multisensitized profiles correspond to particular phenotypes of the disease, of more severe evolution. It is therefore important to carry out a complete evaluation of the cat molecular allergen, including minor fractions, to correctly characterize the patient profile, including the likely course of the disease, the potential cross-reactions and, finally, the expected immunotherapeutic response. We will deal with these aspects in the second part of this review, focusing on the less known molecular allergens of the cat, such as Fel d 2 or Fel d 4.


Ice recrystallization is of great importance to both fundamental research and practical applications, however understanding and controlling ice recrystallization processes remains challenging. Here, we report the discovery of an ion-specific effect on ice recrystallization. By simply changing the initial type and concentration of ions in an aqueous solution, the size of ice grains after recrystallization can be tuned from 27.4±4.1 to 277.5±30.9 μm. Molecular dynamics simulations show that the ability of the ion to be incorporated into the ice phase plays a key role in the ultimate size of the ice grains after recrystallization. Moreover, by using recrystallized ice crystals as templates, 2D and 3D porous networks with tuneable pore sizes could be prepared from various materials, for example, NaBr, collagen, quantum dots, silver and polystyrene colloids. These porous materials are suitable for a wide range of applications, for example, in organic electronics, catalysis and bioengineering. Ice recrystallization is important for a range of research fields, both applied and fundamental, however the effect of ions on this process is not well known. Here the authors discover an ion-specific Hofmeister type effect on ice recrystallization, using this phenomenon to prepare porous materials with tuneable pore sizes. Ice recrystallization is a ubiquitous phenomenon in nature1235678151619Ion specificity, also known as the Hofmeister effect, was first discovered by Franz Hofmeister in 1888 ref. . This ef2223242526Herein, we report both experimental and theoretical evidence of ion specificity in regulating ice recrystallization. We find that the size of ice grains after recrystallization can be regulated by more than one order of magnitude by simply changing the type or concentration of ions in the aqueous solution at the initial stage. This phenomenon can be understood on the molecular level through molecular dynamics (MD) simulations. Specifically, our simulation demonstrates that the ability of ice crystals to incorporate ions and the stability of the liquid layer between crystal domains both exhibit an ion-specific effect. More remarkably, we show that ion-specific recrystallized ice crystals can be exploited as a template-based approach for the facile fabrication of various 2D and 3D porous materials, and the pore size can be controlled simply by regulating the ice grain size via changes in the type and concentration of ions. As an alternative to conventional ice templates created via a directional ice growth process, this method provides a different approach to fabricating porous materials29303132M NaF, NaBr and NaI). The mean grain size of the ice crystals obtained from pure water is 158.3±11.2 μm  why all the ions lead to this generic trend, namely, a maximum in the mean size versus ion concentration curve  and (2) −. This result is in stark contrast to the situation for Br− and I− ions, as shown in − and 87±19 water molecules for I− in liquid phase, respectively) and the other two ions were dissolved in the ice phase at the later equilibrium stage (100 ns). Similar phenomena are also observed at the lower temperature  the thickness of the remaining vicinal liquid water layer follows the trend F−>Br−>I−. Both conclusions provide an explanation as to why the mean grain size after equilibration follows the sequence F−<Br−<I−. Because the F− ions tend to remain in the vicinal liquid water layer during ice growth, further ice crystal growth is blocked, which yields smaller ice grains than grains obtained with Br− or I− ions. Note that the same qualitative conclusions can be drawn from independent test simulations with the conventional Nosé–Hoover thermostat . Snapshots of the systems with F−, Br− andperature . All theermostat .EX-ice)  of O atoms in pure bulk ice and the O atoms surrounding the halide ions  water. Ice recrystallization was performed via the splat-cooling method as described previously34 results .Collagen  was purchased from J&K Chemicals. Aqueous salt solutions of various materials  were splat-frozen and annealed at −6 °C for 45 min. Then, the samples were freeze-dried using a freeze dryer (FD-1A-50) to obtain the 2D and 3D materials. To obtain the 2D porous collagen, the polycrystalline ice needs to be as thin as possible. Hence we set the splat-freezing height at 1.8 m to form a polycrystalline ice layer <10 μm ref. . After a+ ions and 3 anions . The initial configuration was prepared by bringing together an ice slab of 958 water molecules (per supercell) with two separate liquid slabs, each having 164 water molecules (per supercell). All three slabs were separately equilibrated at −13 °C for 1 ns. Next, the two liquid slabs were placed in contact with the left and right surfaces of the ice slab   , within −4. The particle-mesh Ewald method−4). Specifically, the real space cut-off distance was 10 Å. The charge grid spacing was 1 Å, and the fourth order B-splines were used for interpolation. The leap-frog Verlet integration algorithm with a time step of 1 fs was used for the MD simulation. To rule out the latent-heat-dissipation effects, the hybrid microcanonical-canonical ensemble is employedNVT dynamics (T=−13 °C), while the molecules lie in the other half region of the simulation box (z>5 nm) are subjected to NVE dynamics NVE/T MD simulations, the temperature control was implemented by separated Nose-Hoover four-element chain thermostats for each molecule with all four time constants equal to 1 ps . The initial configuration was prepared by replacing two tagged water molecules in ice with 1 Na+ and 1 anion. These test MD simulations were performed at a constant pressure and temperature (NPT) ensemble with using the conventional Nosé–Hoover thermostat (−13 °C). The last 100 ps data (50 configurations) were collected for the averaged potential-energy calculations. Another series of test MD simulations using the same system size were performed with slightly different Lennard–Jones energy (ɛ) and particle-size (σ) parameters of the anions  were carried out to compute average interaction energy of an ion with the surrounding water molecules in the bulk ice EX-ice . For thee anions . The GroThe data that support the findings of this study are available on request from the corresponding authors (J.W or X.C.Z.).How to cite this article: Wu, S. et al. Ion-specific ice recrystallization provides a facile approach for the fabrication of porous materials. Nat. Commun.8, 15154 doi: 10.1038/ncomms15154 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Tables, Supplementary Methods and Supplementary ReferencesMolecular dynamics simulation (100 ns) for a liquid-ice interfacial system with three ions initially located in the liquid water thin film in contact with bulk ice I.Molecular dynamics simulation (100 ns) for a liquid-ice interfacial system with three ions initially located in the liquid water thin film in contact with bulk ice I.Molecular dynamics simulation (100 ns) for a liquid-ice interfacial system with three ions initially located in the liquid water thin film in contact with bulk ice I.


H2 cell generation and longevity and promote airway remodelling in mouse models of asthma, but the mechanisms by which TNFSF14 functions in this setting are incompletely understood. Here we find that mouse and human mast cells (MCs) express TNFRSF14 and that TNFSF14:TNFRSF14 interactions can enhance IgE-mediated MC signalling and mediator production. In mouse models of asthma, TNFRSF14 blockade with a neutralizing antibody administered after antigen sensitization, or genetic deletion of Tnfrsf14, diminishes plasma levels of antigen-specific IgG1 and IgE antibodies, airway hyperreactivity, airway inflammation and airway remodelling. Finally, by analysing two types of genetically MC-deficient mice after engrafting MCs that either do or do not express TNFRSF14, we show that TNFRSF14 expression on MCs significantly contributes to the development of multiple features of asthma pathology.Asthma has multiple features, including airway hyperreactivity, inflammation and remodelling. The TNF superfamily member TNFSF14 (LIGHT), via interactions with the receptor TNFRSF14 (HVEM), can support T TNFSF14 (LIGHT) contributes to airway inflammation and remodelling. Here the authors show that TNFSF14 acting on its receptor TNFRSF14 on mast cells enhances their IgE-dependent activation and that interference with this pathway attenuates features of asthma pathology in mice. H2 cell-dependent production of antigen (Ag)-specific IgE antibodies that are thought to have a key pathogenic roleAsthma is a chronic inflammatory disease affecting >235 million people worldwide and is a major cause of morbidity2in situ, increase vascular permeability and contribute to airway remodelling69In subjects with atopic or ‘allergic' asthma, mast cells (MCs) are considered critical for the development of multiple features of the pathology3678H2 responses and/or the pathology of asthmatic airway inflammation, such as TNFSF4 -33 receptors (IL-33Rs), thymic stromal lymphopoietin receptors (TSLPRs) and toll-like receptors111314SF4 ref. , TNFRSF9SF4 ref.  or TNF i1% predictedRecent data have implicated another member of the TNF superfamily, the ligand TNFSF14 ), in asthma pathology. A study of 242 asthma patients revealed a positive correlation between elevated levels of TNFSF14 in the sputum and impaired lung function ); TNFRSF3 (lymphotoxin-beta receptor (LTβR)); and TNFRSF6B (soluble decoy receptor 3 (Dcr3)) and with two receptors in mice (TNFRSF14 and TNFRSF3), suggesting complexity in the potential targets and actions of TNFSF14 in vitro can potentiate IgE-mediated signalling and can increase significantly the secretion of pre-stored and de novo synthesized MC mediators. We also showed, using both an OVA-induced mouse model of chronic airway inflammation1 antibodies, AHR, airway inflammation and airway remodelling. These findings suggest that TNFRSF14 may represent a potential therapeutic target in asthma.In the present study, we detected TNFRSF14 expression on both human and mouse MCs, and found that TNFSF14-dependent engagement of TNFRSF14 on the MC surface in vitro derived human peripheral blood cultured MCs (huPBCMCs) from CD34+ mononuclear precursors (huPBCMCs), and instead detected strong expression of TNFRSF14 on these two human MC populations  and at high time resolution, granule secretion by huPBCMCs, as assessed by measuring the fluorescence of the granule-associated marker LAMP-1 ref. , using aTo analyse precisely the number and dimension of clusters on the surface of individual huPBCMCs, we modelled both FcɛRI/anti-IgE-A650 and TNFRSF14/TNFSF14-A594 clusters 30 min after stimulation. Combining anti-IgE-A650 with TNFSF14 dramatically increased both the number and the area of individual clusters of FcɛRI/anti-IgE-A650 and TNFRSF14/TNFSF14-A594 on the plasma membrane surface .in vitro from WT Tnfrsf14+/+  mice  monoclonal mouse IgE antibody and then challenged with DNP-HSA (Ag) in the absence or presence of soluble TNFSF14.These results were confirmed in mouse MCs (mouse bone marrow-derived cultured MCs (BMCMCs)). We detected no expression of TNFRSF3 (or TNFSF14) on BMCMCs generated ng) mice . BMCMCs 117 KIT, . We thenTnfrsf14+/+ and Tnfrsf14−/− BMCMCs in the absence of TNFSF14  and TNFRSF14/TNFSF14-A594 (red) clusters after stimulation of mouse BMCMCs with TNFSF14, IgE and antigen, or both stimuli. Exposure of IgE-sensitized mouse BMCMCs to both DNP-HSA-A650 and TNFSF14 dramatically increased both the number and area of individual clusters formed by FcɛRI and TNFSF14 on the plasma membrane surface . MoreoveTNFRSF14 . Taken tIn vitro stimulation with TNFSF14 in the absence of Ag did not detectably influence MC activation  in features of asthma inflammation and remodelling has not before been specifically investigated. Doherty RL) and dynamic compliance (Cdyn)   upon metphocytes ; 3) blootibodies ; (4) levtibodies ; (5) numtibodies ; (6) BALtibodies  and TNFStibodies ; and (7)tibodies . Histolotibodies , demonst1 and IgE antibodies  airway responses to Mch ; (2) OVRL  blo lev num con with Tnfrsf14+/+ or Tnfrsf14−/− BMCMCs. OVA sensitized and challenged KitW-sh/W-sh mice that had been engrafted with Tnfrsf14−/− BMCMCs, compared with identically treated KitW-sh/W-sh mice that had been engrafted with Tnfrsf14+/+ (TNFRSF14+) BMCMCs, exhibited significantly reduced: (1) airway responses to Mch  levs to Mch ; (3) blo and IgE ; (4) lev and IgE ; (5) num and IgE ; and (6) and IgE . Histolo+ or TNFRSF14-deficient MCs into a different type of mouse which is markedly MC- (and basophil) deficient independently of genetic abnormalities affecting c-kit, namely C57BL/6-Cpa3-Cre;Mcl-1fl/fl micec-kit-dependent' and ‘c-kit-independent' MC-deficient mice thus strongly support the conclusion that even though TNFRSF14 can be expressed on multiple cell types, TNFRSF14 expression on MCs is necessary for the full development of multiple features of asthma pathology in our OVA model.Similar results were obtained when we engrafted TNFRSF14H2 responses. Indeed, we found that OVA sensitized and challenged mice that were globally TNFRSF14-deficient or that contained TNFRSF14-deficient MCs exhibited significantly lower levels of Ag-specific IgE and IgG1 than did the corresponding control mice  5 promoter. Mcpt5-Cre transgenic mice were crossed with Cre-inducible ROSA-eYFP reporter mice, as detailed in the Methods section. BMCMCs derived from such mice are thus TNFRSF14+ (since they express a WT Tnfrsf14 gene) and specifically express eYFP. We found that the Tnfrsf14−/− mice in which only the engrafted MCs expressed TNFRSF14 exhibited multiple features of the OVA-induced asthma model, including blood levels of OVA-specific IgG1 and IgE, that were not significantly different than those in the corresponding BMCMC-engrafted Tnfrsf14+/+ mice  on their surface and that TNFSF14-mediated engagement of TNFRSF14 enhances activation of such MCs when they are co-stimulated by IgE-dependent mechanisms. H2 populationsTnfrsf14−/− mice could reflect, at least in part, actions of TNFRSF14 upstream of Ag-specific IgE production. However, we showed that the amounts of Ag-specific IgE produced by OVA-immunized Tnfrsf14−/− mice were sufficient to passively sensitize naive mice for the expression of airway responses to intra-tracheal Ag challenge. Moreover, we found that antagonizing TNFRSF14:TNFSF14 interactions using an TNFRSF14 blocking antibody administered to WT mice weeks after the period of Ag sensitization significantly reduced multiple features of the asthma pathology that develop in this setting, including AHR, lung inflammation and several measures of airway remodelling. Taken together, these findings strongly suggest that TNFRSF14:TNFSF14 interactions can influence the pathology of asthma models by mechanisms in addition to effects on the production of Ag-specific IgE.Because TNFRSF14 can influence Tin vivo. Taken together with our in vitro findings indicating that TNFSF14 co-stimulation of MCs via TNFRSF14 can markedly increase both the numbers of aggregates of anti-IgE:FcɛRI or specific-antigen:FcɛRI on the surface of MCs stimulated with anti-IgE or specific antigen, respectively, as well as the IgE-dependent activation of the Akt, PKC-δ and MAPK cascade in MCs, which is accompanied by enhanced MC production of pre-formed, lipid and newly synthesized mediators, these findings indicate that TNFRSF14:TNFSF14 interactions may have a major role in fine tuning the extent of MC activation by IgE- and Ag in the asthma models we studied and perhaps in other settings in vivo.Like other members of the TNF:TNFR superfamily, TNFRSF14 can function as co-stimulatory moleculeH2 responses and IgE production831et al.482050For example, FcɛRI/TNFRSF14 co-activation of MCs enhanced activation of Akt, PKC-δ, and the MAPK cascade, which is associated with the increased production of pro-inflammatory mediators  and pro-remodelling compounds  refs , 34, 35.in vitro to stimulate mouse BMCMCs and huPBMCs was substantially greater than that we measured in BAL fluids in vivo.It is not clear to what extent the IgE-dependent activation of MCs in lung tissues is regulated by soluble versus membrane-associated forms of TNFSF14. The ELISA we used to quantify TNFSF14 in BAL fluid only measures soluble TNFSF14. The data from our analyses of TNFSF14 in BAL fluids, which are shown in in vitro experimentsIn vivo, the biologically important TNFSF14 in airway inflammation is that which is present in immediate proximity to its receptors. Unfortunately, it is not possible directly to measure interstitial amounts of soluble TNFSF14. Moreover, TNFSF14 can bind to its receptor in either its membrane-associated or soluble formin vitro studies, IHC and so on. By studying the phenotype of our asthma models in mice that did or did not express TNFRSF14, and that did or did not contain MCs that expressed TNFRSF14, we avoided problems associated with drawing conclusions from in vitro, histological or IHC analyses taken in isolation.A number of factors may have contributed to this discrepancy. It has been reported that recombinant TNFSF14 is not stable and tends to aggregate, which potentially can influence the results of in vivo. Moreover, in WT mice, blocking TNFRSF14:TNFSF14 interactions with an antibody administered weeks after Ag sensitization can diminish multiple features of asthma pathology, including blood levels of Ag-specific IgE, airway inflammation and AHR, and airway remodelling. Taken together with correlative clinical observations showing that levels of TNFSF14 in the sputum of a large group of subjects with asthma were inversely correlated with a measure of lung functionIn summary, we have identified TNFRSF14 as an important new co-receptor of mouse and human MCs, and showed that engagement of MC TNFRSF14 by TNFSF14 can significantly enhance IgE-dependent MC activation and mediator production. TNFRSF14 expression by MCs also can exacerbate several features of the pathology in two different mouse models of Ag-induced asthma Tnfrsf14−/− mice from Mitchell Kronenberg were backcrossed onto C57BL/6J mice at Stanford for 8–9 generations. Three-to-fifteen female naive mice (of the appropriate genotype and age) that were used in individual experiments were assigned randomly to the experimental groups .All animal experiments were conducted according to the National Institutes of Health (NIH) guidelines and with the approval of the Stanford University Institutional Animal Care and Use Committee. Six to 8-week-old female C57BL/6J mice were used in experiments involving solely WT mice. Transgenic mouse strains were bred and housed with the respective control mice in the local animal facilities. −1 when the manufacturer provided information about the initial concentration of the preparation of antibody purchased. For other antibodies for which this information was not provided by the manufacturer, the antibody preparation was diluted before use according to the manufacturer's instructions.All antibodies were used at 1 μg mlFluorophore conjugated anti-TNFRSF3 antibody (31G4D8) was from BioLegend and anti-TNFSF14 (7-3 7), anti-TNFRSF14 (eBioHVEM-122) and LAMP-1 (eBioH4A3) antibodies were from eBioscience.Anti-TNFRSF14 and fluorophore-conjugated anti-TNFRSF14 (both LH1), anti-TNFRSF3 (eBio3C8), anti-CD117 (2B8), anti-CD4 (GK1.5), anti-interferon (IFN)-γ (XMG1.2), anti-IL-4 (11B11), anti-IL-17 (eBio17B7) and anti-LAMP-1 (1D4B) antibodies were purchased from eBioscience. For staining of mTNFSF14, we blocked LTβ with an anti-LTβ antibody , followed by incubation with TNFRSF3-Fc (from R&D) and fluorophore-conjugated anti-mouse IgG2a antibody (eBioscience). The same results  were obtained when we performed flow cytometry using a fluorophore-conjugated rabbit polyclonal anti-mouse TNFSF14 antibody from Bioss (cat. no. bs-2462R-PE). Fluorophore-conjugated anti-FcɛRI (MAR-1) was from Biolegend. Polyclonal anti-phospho-PKC-δ (T311), anti-phospho-Akt , anti-phospho-Mek1/2 , polyclonal anti-phospho Erk1/2 (T202/Y204), anti-PKC-δ (D10E2), anti-Akt (C67E7), anti-Mek1/2 (47E6) and polyclonal anti-Erk1/2 antibodies were all from Cell Signaling Technology.6 IgE pre-sensitized BMCMCs for each condition tested were activated as described above. At indicated time points, BMCMCs were lysed and total protein extracts were resolved onto a polyacrylamide gel. Blotted extracts were probed with indicated antibodies. Detection of Ras was performed with 20 × 106 IgE pre-sensitized BMCMC for each condition, using the Active Ras Pull-down kit (Thermo Scientific), according to the manufacturer's instructions.In all, 10 × 10in vitro from peripheral blood CD34+ mononuclear cells as previously reportedThe LAD2 human MC line was kindly provided by Dr A. Kirshenbaum  and cultured as previously describedin vitro differentiation of bone marrow precursors in WEHI-3-conditioned DMEM, for at least 5 weeksTnfrsf14−/−, Tnfrsf14+/+ or Mcpt5-eYFP mice were cultured in WEHI-3-conditioned DMEM , as a source of IL-3, for 4–5 weeks to generate cell populations that contained >99% bone-marrow-derived cultured MCs (BMCMCs). BMCMCs (2 × 106) were injected into each mouse via the tail vein, and the recipients  were used for experiments 8 weeks later.BMCMCs were obtained through −1 human IgE (Millipore) overnight, washed and stimulated with 2 μg ml−1 rabbit anti-human IgE (Bethyl). For IgE pre-sensitization and activation of BMCMCs, dinitrophenyl (DNP)-specific IgE  was obtained from Sigma. BMCMCs were pre-sensitized overnight with 1 μg ml−1 IgE in DMEM 5% FBS and stimulated with DNP-HSA (Ag) at a final concentration of 10 ng ml−1.huPBCMCs were pre-sensitized with 1 μg mlɛ26 ref.  was prov−1 recombinant human TNFSF14 (R&D Systems) at the same time as anti-IgE or PBS. Human IL-8 and TNF-α ELISA kits were from eBioscience.IgE pre-sensitized huPBCMCs were stimulated with anti-IgE as described above for the time indicated in the main text. In some cases huPBCMCs received 10 μg ml−1 recombinant mouse TNFSF14 (R&D Systems) at the same time as Ag or PBS. Histamine in the BMCMC culture supernatants was measured using an Enzyme Immunoassay Kit (Beckman Coulter) according to the manufacturer's instructions. ELISA kits for TNF-α, IL-6 and IL-13 were purchased from eBioscience. ELISA kits for LTC4 and LTE4 were purchased from Cayman Chemicals.IgE pre-sensitized BMCMCs were stimulated with Ag as described above for the time indicated in the main text. In some cases BMCMCs received 10 μg mlIgE pre-sensitized BMCMCs or huPBCMCs were stimulated with PBS, Ag or anti-IgE (with or without TNFSF14), for 30 min. Cells were then incubated in ice for 5 min to block the reaction and stained for LAMP-1. Mean fluorescence intensity was measured for each BMCMC or huPBCMC and condition tested.Mcpt5-Cre+ mice were provided by Axel RoersGt(ROSA)26Sortm1(EYFP)Cos/J; Jackson) to generate Mcpt5-eYFP BMCMCs.−1, measured with LAL Endotoxin Assay kit (Genscript)]).All experiments employed OVA preparations that contained low levels of LPS  in 100 μl PBS on days 1, 4 and 7 ref. . StartinD. pteronyssinus (Greer) in 30 μl PBS on days 1, 4 and 7. Starting on day 12, mice were challenged i.n. with 20 μg HDM in 30 μl PBS weekly for 10 weeks; control mice received i.n. challenges with PBS on the same schedule.Mice were immunized by three i.n. challenges of 100 μg HDM from 2 inhalation, the lungs were ligated, removed and lavaged with ice-cold Hanks' balanced salt solution (HBSS) (BAL). Cells were harvested, stained with Hema 3 Stain Set (Fisher Diagnostic) and counted on the basis of their morphological and staining properties. After recovery of BAL fluid, lungs were fixed  and embedded in paraffin. Sections of 5 μm were mounted on Superfrost Plus glass slides (Fisher Scientific) and stained with haematoxylin and eosin, Masson's trichrome stains or toluidine blue.Twenty-four hours after the last OVA, HDM or PBS challenge mice were killed by CO1 and IgE were purchased from Cayman Chemicals.Plasma and BAL fluid were collected 24 h after the ninth OVA or PBS challenge or after the tenth HDM or PBS challenge. ELISA kits for OVA-specific IgG−1 HDM at 4 °C overnight, followed by blocking with 1% bovine serum albumin in PBS for at least 2 h at room temperature. Sera diluted in PBS containing 1% bovine serum albumin were added and incubated in the blocked wells for 2 h at 37 °C. We detected bound IgG1 and IgE antibodies using biotinylated detection antibodies  and rat anti-mouse IgE , respectively), followed by incubation with horseradish peroxidase-conjugated streptavidin (BD Pharmingen) for 30 min at room temperature and detection using supersensitive 3,3',5,5'-tetramethylbenzidine (TMB) substrate (Sigma). Antibody titers were calculated by plotting the serum dilution that gave half-maximal signal of a reference serum. Measurements of mediators from BAL fluid was performed as described above.Each incubation step in the ELISAs described below is followed by 3–5 washing steps using PBS containing 0.05% Tween-20. For detection of HDM-specific serum antibodies, MaxiSorp ELISAs plates (Nunc) were coated with 5 μg mlLung collagen was extracted using acid pepsin method and levels of collagen were measured using a Sircol Soluble Collagen Assay Kit (Biocolor Life Science Assays).Tnfrsf14+/+or Tnfrsf14−/− mice or KitW-sh/W-sh mice that had been engrafted with Tnfrsf14+/+ or Tnfrsf14−/− BMCMCs were injected i.v. per naive C57Bl/6 mouse. 24 h later, mice were anaesthetized, tracheotomized and challenged intra-tracheally with 100 μg OVA. RL was recorded over 5 min time.Two-hundred millilitres of serum collected from 4 IgE pre-sensitized huPBCMCs or BMCMCs from Tnfrsf14+/+ or Tnfrsf14−/− mice were placed into poly-D-Lysine coated micro-chambers  in 250 μl Tyrode's buffer supplemented with 1 μg ml−1 of anti-human or mouse LAMP-1  antibody coupled to Alexa 488 (LAMP-1-A488). In some experiments, 10 μg ml−1 human or mouse TNFSF14 coupled to Alexa 594 (TNFSF14-A594), 2 μg ml−1 anti human-IgE (anti-IgE A650) (for experiments with human MCs) or 10 ng ml−1 DNP-HSA coupled to Alexa 650 (DNP-HSA-A650) (for experiments with mouse MCs) and/or vehicle control were added to the medium. Fluorescence corresponding to LAMP-1-A488, TNFSF14-A594 and anti-IgE A650 or DNP-HSA-A650 was monitored simultaneously using a confocal laser-scanning microscope and in a controlled atmosphere .In all, 5 × 102) in depicting the areas of such clusters in BMCMCs or huPBCMCs were treated as described in the above Time-lapse analysis section. 30 min (for huPBCMCs) or 15 min (for BMCMCs) after stimulation with TNFSF14-A594, anti-IgE-A650 or DNP-HSA-A650, or both reagents, or vehicle alone (control), single-cell images were acquired as z stack . 3-D reconstruction and modelling/analyses of clusters were performed using the Surface function of Imaris Bitplane Software. Because the software was unable to individually identify very large clusters that were in close proximity on the plasma membrane, and, therefore, would identify them as a single large structure, we decided to omit modelled cluster area values for those few TNFSF14/anti-IgE clusters which reached the resolution limit that could be detected by the software . PCR was performed on complementary DNA prepared using the High Capacity Reverse Transcription Kit from Applied Biosystems, according to the manufacturer's instructions. Primer sequences for mouse TNFSF14 were: Fwd: ATAGTAGCTCATCTGCCAGATGGA; Rev: CGACTGACCAGCAGTTCTAACT.–1), with individual doses for 3 min, after a 2-min acclimation period, for a total for 5 min for each dose. RL and Cdyn were continuously computed by fitting flow, volume and pressure to an equation of motion for each aerosol challenge period. Reported results were interpolated calculating the average RL and Cdyn over a single time-period.Invasive measurements of airway reactivity in anaesthetized, tracheostomized, mechanically ventilated mice were performed by administering aerosolized Mch at increasing concentrations , 500 ng ml−1 ionomycin, and 2 μM monensin. Cells were then stained with an anti-CD4 antibody, fixed, permeabilized and stained for IFN-γ (TH 1), IL-4 (TH 2) and IL-17 (TH17). Gated CD4+ cells were analysed and quantified for their positivity for IFN-γ, IL-4 and/or IL-17 using a BD FACSCalibur cytofluorimeter. Data were analysed using FlowJo Data Analysis Software.Mice were killed by COELISA kits for Mucin 5AC and TNFSF14 were purchased from Uscn Life Science Inc.t-test was used for all other analyses. The unpaired Student's t-test was used for all other analyses. A P value <0.05 was considered statistically significant. Asterisks indicate statistical significance between PBS (or non-Ag) and corresponding Ag-treated groups and daggers indicate statistical significance between indicated groups. * or †P<0.05; ** or ††P<0.01; *** or †††P<0.001. All data are presented as mean+or±s.e.m.Evaluation of histology was performed in a blinded way, in that the observer was not aware of from which group of mice the slides were derived. For all the experiments, differences in airway responses between groups were tested for statistical significance using the two-way analysis of variance test. A Mann–Whitney test  was used for Data supporting the findings of this study are available in the main article, the How to cite this article: Sibilano, R. et al. A TNFRSF14-FcɛRI-mast cell pathway contributes to development of multiple features of asthma pathology in mice. Nat. Commun.7, 13696 doi: 10.1038/ncomms13696 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures.


Binding of AMP to the γ subunit allosterically activates the kinase. Additionally, many small molecules, e.g. 991, have been identified that bind between the kinase domain and the carbohydrate-binding module of the β subunit, stabilising their interaction and leading to activation. It was reported recently that non-phosphorylated Thr172 AMPK is activated by AMP and A769662. We present here the crystal structure of non-phosphorylated Thr172 AMPK in complex with AMP and 991. This structure reveals that the activation loop, as well as the complex overall, is similar to the Thr172 phosphorylated complex. We find that in the presence of AMP and 991 non-phosphorylated Thr172, AMPK is much less active than the Thr172 phosphorylated enzyme. In human cells, the basal level of Thr172 phosphorylation is very low (∼1%), but is increased 10-fold by treatment with 2-deoxyglucose. In cells lacking the major Thr172 kinases, LKB1 and CaMKKβ, Thr172 phosphorylation is almost completely abolished, and AMPK activity is virtually undetectable. Our data show that AMP and 991 binding to non-phosphorylated Thr172 AMPK can induce an ordered, active-like, conformation of the activation loop explaining how AMPK activity can be measured in vitro without Thr172 phosphorylation. However, in a cellular context, phosphorylation of Thr172 is critical for significant activation of AMPK.AMP-activated protein kinase (AMPK) plays a major role in regulating metabolism and has attracted significant attention as a therapeutic target for treating metabolic disorders. AMPK activity is stimulated more than 100-fold by phosphorylation of threonine 172 (Thr At 24 h post-transfection, cells were sorted based on GFP expression and individual colonies were analysed by western blotting to determine CaMKKβ protein expression.n-octylamine and 1,1,2-trichlorotrifluoroethane. Adenine nucleotides in the aqueous phase were analysed by capillary electrophoresis using a P/ACE MDQ plus (Sciex) in 100 mM sodium acetate buffer (pH 3.5) containing 0.01% hydroxypropylmethylcellulose on a standard fused-silica capillary. The extract was loaded by pressure injection for 5 s at 0.5 psi and separated at 25 kV. Nucleotide peaks were detected by absorbance at 254 nm and integrated peak areas were automatically calculated. Retention times for AMP, ADP and ATP were confirmed by analysing nucleotide standards.Cells were lysed in perchloric acid (5%), insoluble material was removed by centrifugation and the supernatant was extracted twice with an equal volume of 1 : 1 tri-172 and can be phosphorylated by incubation with CaMKKβ in vitro (172 complexes was less than 1% of the corresponding pThr172 complexes (1.41 ± 0.11 vs. 882.48 ± 21.43 nmol/min/mg for α1β1γ1 non-pThr172 vs. pThr172 and 1.37 ± 0.04 vs. 189.55 ± 15.89 nmol/min/mg for α2β1γ1 non-pThr172 vs. pThr172). The activity of the non-pThr172 complexes was increased markedly in the presence of the allosteric activators 991 and AMP , a slightly lower value than that reported in the previous studies [172 enzyme is ∼3% of the pThr172 form (35.44 ± 7.21 vs. 1266.77 ± 37.05). Given that 991 stimulates the activity of non-pThr172 AMPK, we examined its binding to non-pThr172 complexes. As can be seen from the results shown in 172 (see below).Recombinant AMPK complexes purified from bacteria are intrinsicially non-phosphorylated at Thrin vitro . We dete AMP see , confirm studies ,27. With172 AMPK and stimulates its activity, we were interested in determining the conformation adopted by the kinase domain in the non-pThr172–991 complex. We obtained the crystal structure of full-length human non-pThr172 α2β1γ1 ; however, it is not able to make a further stabilising interaction with αAsn162  to generate a standard curve. We titrated different amounts of both non-pThr172 AMPK and AMPK that had been phosphorylated by CaMKKβ on the same blot as the HEK293T lysates  in HEK293T cells was determined  in cells, but do not address the issue of whether pThr172 is absolutely required for AMPK signalling in cells. To determine this, we deleted CaMKKβ from A549 cells, which lack LKB1 expression  provides sufficient energy to stabilise the active-like conformation of the activation loop by driving the binding of the kinase domain, mainly via this loop, onto the regulatory fragment of AMPK.Activation loop phosphorylation promotes the stabilisation of the active conformation of this loop and the positioning of key residues in the kinase domain. Owing to the less stable conformation of kinases lacking such phosphorylation, it is perhaps not surprising that, in general, there are relatively few examples of such structures available in the Protein Databank. However, there are examples of RD kinases that do not require phosphorylation of the activation loop to achieve partial or full activity . In some172 complex, a study reported that allosteric activation of α1β1γ1 by A769662 led to significant activity without the requirement for Thr172 phosphorylation, and the addition of AMP increased this activation further [172 α1β1γ1 by AMP and 991 reached ∼10% of the activity of pThr172 AMPK in a cell-free assay, demonstrating that full activation of AMPK does require phosphorylation of Thr172. Thus, the ordering of the activation loop that we see in the crystal structure with AMP and 991 bound supports the finding of significant, but low, kinase activity in the absence of Thr172 phosphorylation.Around the same time as we solved the crystal structure of the non-pThr further . We conf172 complexes could be highly significant in terms of potential therapeutic strategies for activating AMPK, particularly those diseases where there is a lack of upstream AMPK kinase activity, such as loss of LKB1 in certain cancers [172 phosphorylation in response to either 991 or conditions which interfere with cellular ATP production, and which would therefore be anticipated to increase AMP levels. In HEK293T cells, which express both LKB1 and CaMKKβ, the two major Thr172 kinases, we were only able to detect a significant increase in AMP levels following treatment with the glycolysis inhibitor, 2DG. Neither rotenone nor phenformin treatment caused a detectable increase in AMP, although they did lead to measurable increases in ADP. Although we were unable to reliably detect AMP under these conditions, it remains possible that AMP is increased, but falls below the limit of detection using capillary electrophoresis. To resolve this issue, a more sensitive method, such as mass spectrometry, would be required to measure AMP levels. Consistent with the modest changes in adenine nucleotide levels, the increase in Thr172 phosphorylation occurred over a fairly narrow range (between 1 and 10%) following the different treatments. Thus, most of the AMPK within the cell remains in the non-phosphorylated Thr172 form. This is consistent with a model we proposed previously that we based on the nucleotide-binding properties of AMPK in relation to the physiological concentration of adenine nucleotides in mammalian cells [172 form. Small (2–3-fold) changes in AMP and/or ADP levels lead to similar fold increases in pThr172, consistent with our experimental observations in HEK293T cells. It is important to note, however, that although the proportion of AMPK in the pThr172 form is low, following treatment with 2DG the relative increase in Thr172 phosphorylation is substantial (10-fold). There was a good correlation between the increase in phosphorylation of Thr172 and ACC for rotenone, phenformin and 2DG. In contrast, 991 treatment, which caused the smallest increase in Thr172 phosphorylation out of the conditions used, resulted in a disproportionate increase in ACC phosphorylation. A potential explanation for this finding is that 991, in addition to increasing Thr172 phosphorylation, allosterically activates AMPK [172 may be misleading, and monitoring downstream substrate phosphorylation provides a more accurate reflection of AMPK activity in cells [As has been suggested previously , the abi cancers . In an aan cells . In thisin cells .172 in HEK293 cells [172 phosphorylation between HEK293 and HEK293T cells.A previous study performed a similar analysis for estimating the degree of pThr93 cells . In this93 cells , whereas93 cells , This di172 AMPK complex reported in the current study and previously by others [172 phosphorylation in human cells, we generated A549 cells lacking the two major Thr172 kinases, CaMKKβ and LKB1. A549 cells are a human lung adenocarcinoma cell line that lack endogenous expression of LKB1 [172 kinases. Similar to lung tissue [The allosteric activation of the non-pThry others ,27 has p of LKB1 ,44. By dg tissue , A549 ceg tissue .172 compared with 991 treatment (172 phosphorylation than either treatment alone. In contrast with these results, phosphorylation of ACC and Thr172 was essentially undectable in the CaMKKβ KO A549 cells following any of the treatments. We confirmed that AMP and ADP levels were increased in CaMKKβ KO A549 cells following 2DG treatment. A very low signal for ACC phosphorylation was evident following the dual treatment of the CaMKKβ KO A549 cells, and this correlated with a similarly weak signal for Thr172 phosphorylation. These findings suggest that a low level of Thr172 kinase activity remains in the CaMKKβ KO cells, and that this results in a low level of downstream substrate phosphorylation. Whatever the source of this residual activity, our results clearly show that in the absence of appreciable Thr172 phosphorylation, cellular AMPK has negligible ability to phosphorylate its downstream substrate. Taken together, these findings demonstrate that in the absence of CaMKKβ and LKB1, Thr172 phosphorylation is virtually abolished and ACC phosphorylation prevented. At first sight, this appears to be at odds with the cell-free data, which shows that non-pThr172 AMPK is capable of substrate phosphorylation in vitro. However, the cell-free assays are performed at high substrate concentration, which will be different within the cell. In addition, phosphorylation of downstream targets within a cellular context are dynamic, due to the action of protein phosphatases, which is not the case in a cell-free assay. Furthermore, additional factors are present within the cell, that are not present in the cell-free studies, that influence the activity of the non-pThr172 complex. For example, glycogen has been shown to inhibit AMPK activation in vivo [172 phosphorylation is unsubstantiated. Moreover, we are not aware of any pathology where both CaMKKβ and LKB1 are inactive and so, the requirement for activation of the non-pThr172 form has not been proved.In A549 cells, treatment with 2DG caused a modest increase in phosphorylation of ACC and Thrreatment . The mod in vivo . In addi in vivo . Cellula


Inhibitory control may play an important part in prosocial behavior, such as donating behavior. However, it is not clear at what developmental stage inhibitory control becomes associated with donating behavior and which aspects of inhibitory control are related to donating behavior during development in early to middle childhood. The present study aimed to clarify these issues with two experiments. In Experiment 1, 103 3- to 5-year-old preschoolers completed cool (Stroop-like) and hot (delay of gratification) inhibitory control tasks and a donating task. The results indicated that there were no relationships between cool or hot inhibitory control and donating behavior in the whole group and each age group of the preschoolers. In Experiment 2, 140 elementary school children in Grades 2, 4, and 6 completed cool (Stroop-like) and hot (delay of gratification) inhibitory control tasks and a donating task. The results showed that inhibitory control was positively associated with donating behavior in the whole group. Cool and hot inhibitory control respectively predicted donating behavior in the second and sixth graders. Therefore, the present study reveals that donating behavior increasingly relies on specific inhibitory control, i.e., hot inhibitory control as children grow in middle childhood. Prosocial behavior refers to voluntary behavior that benefits other people  and moral emotion attribution  were selected. Because Stroop-like tasks and delay of gratification tasks are typical cool and hot inhibitory control tasks, they were used to assess cool and hot inhibitory control, respectively. A revised day-night Stroop task . The 4-year-old group consisted of 46 preschoolers  between 4.00 and 4.92 years old . The five-year-old group comprised 31 preschoolers  between 5.00 and 5.92 years old . All the preschoolers came from middle-class families. The study was approved by the Research Ethics Board of School of Psychology of Capital Normal University. Informed written consent was obtained from the parents of all the participants.One hundred three preschoolers were recruited from a local kindergarten. The 3-year-old group comprised 26 preschoolers  between 3.00 and 3.92 years old  = 13.96, p < 0.001, η2 = 0.218. A post hoc Scheffe test indicated that the 5-year-olds performed significantly better on the Stroop-like task than did the 3-year-olds (p < 0.001) and the 4-year-olds (p < 0.001), and 3-year-olds and the 4-year-olds performed similarly (p = 0.663). For hot inhibitory control, there was also a significant effect of age group, F = 3.19, p = 0.045, η2 = 0.060. A post hoc Scheffe test indicated that the 5-year-olds waited longer in the delay of gratification task than did the 3-year-olds (p = 0.048) but not the 4-year-olds (p = 0.276). The two younger groups had similar performances on this task (p = 0.501). In the donating task, the effect of age group was not significant, F = 0.72, p = 0.490, η2 = 0.014. The numbers of stickers that were donated were similar for all three age groups.Descriptive statistics of Experiment 1 are shown in Table p < 0.001) and quadratic trend  in Stroop scores and a significant linear trend in delay of gratification scores . There were no specific developmental trends in donating behavior scores. Different development trends in inhibitory control and donating behavior imply that they may not be associated with each other in preschoolers as a whole.Polynomial trend analyses found a significant age-related linear . The fourth-grade group consisted of 43 children  between 9.08 and 10.17 years old . The sixth-grade group included 44 children  between 11.08 and 12.50 years old . All the children came from middle-class families. The study was approved by the Research Ethics Board of School of Psychology of Capital Normal University. Informed written consent was obtained from all the participants.One hundred and forty children in the second, fourth and sixth grades participated in the experiment. They came from a local elementary school. The second-grade group comprised 53 children  between 7.00 and 9.17 years old  = 20.35, p < 0.001, η2 = 0.230. A post hoc Bonferroni test showed that the sixth graders performed significantly better on the fruit Stroop task than did the second and fourth graders, p < 0.001, p = 0.018, respectively. The fourth graders outperformed the second graders, p = 0.002. For hot inhibitory control, there were no significant differences between the age groups in the delay of gratification task, F = 0.29, p = 0.750, η2 = 0.004. For donating behavior, an ANOVA yielded a significant effect of age group, F = 15.14, p < 0.001, η2 = 0.185. A post hoc Bonferroni test indicated that the sixth graders donated more than the second graders (p < 0.001) but not the fourth graders (p = 0.150). The fourth graders donated more than the second graders, p = 0.004. In addition, donating time did not differ between the age groups, F = 0.40, p = 0.668, η2 = 0.006.Descriptive statistics of Experiment 2 are shown in Table p < 0.001) and no specific developmental trends in delay of gratification scores. There was also a significant linear trend in the donating behavior scores . Similar developmental trends in cool inhibitory control and donating behavior suggest that they may be associated with each other in the elementary school children as a whole.Polynomial trend analyses indicated a significant age-related linear trend in fruit Stroop scores  = 2.43, p = 0.077. The control variables accounted for 13% of the variance in donating behavior. Donating time negatively predicted donating behavior. When inhibitory control variables were entered in the second step, the regression model was significant, F = 3.11, p = 0.017. The inhibitory control variables explained an additional 12% of the variance in donating behavior. More importantly, only cool inhibitory control (the fruit Stroop task) was a significant positive predictor of donating behavior. For the sixth graders, the regression model in the first step failed to reach significance, F = 0.74, p = 0.536. The control variables accounted for 6% of the variance in donating behavior. After the inhibitory control variables were entered in the second step, the regression model became significant, F = 2.54, p = 0.046. An additional 21% of the variance in donating behavior was explained by the inhibitory control variables. However, only hot inhibitory control (delay of gratification) significantly positively predicted donating behavior.As the relationships between specific aspects of inhibitory control and donating behavior were found for the second- and sixth graders, hierarchical multiple regression analyses were conducted for the younger and older groups. The results are shown in Table Experiment 2 demonstrated that cool inhibitory control increased during middle childhood. Archibald and Kerns  found siMoreover, inhibitory control was positively associated with donating behavior in middle childhood. Ciairano et al.  also fouThe present study explored whether inhibitory control was associated with donating behavior during development in early to middle childhood. Experiment 1 indicated that inhibitory control was not related to donating behavior in early childhood. Experiment 2 further indicated that inhibitory control was related to donating behavior in middle childhood. Moreover, cool inhibitory control specifically predicted donating behavior during the early stage of middle childhood, whereas hot inhibitory control specifically predicted donating behavior during the later stage of middle childhood.Classic and age-appropriate Stroop-like and delay of gratification tasks were used to assess cool and hot inhibitory control, respectively, in the present study. The age-related trends generally replicated those of previous studies. The donating task used in the present study is also widely adopted in previous studies (e.g., Rubin and Schneider, The main focus of the present study was the relationships between inhibitory control and donating behavior. The results demonstrated that these relationships differed considerably between early childhood and middle childhood. Inhibitory control and donating behavior were not related to each other in early childhood but were closely connected in middle childhood. Unlike preschoolers, who are characterized by egocentrism, elementary school children are less egocentric and are able to pay more attention to others' needs. Previous studies also confirm that elementary school children produce more moral justifications during moral evaluations than preschoolers do, and their moral justifications involve the welfare of others (Malti et al., The present study further revealed that during middle childhood, inhibitory control was positively associated with donating behavior, but specific aspects of inhibitory control were related to donating behavior at specific ages. First, there were positive relationships between inhibitory control and donating behavior in the whole group of elementary school children. Telzer et al.  found thSecond, cool inhibitory control predicted donating behavior in young elementary school children, whereas hot inhibitory control specifically predicted donating behavior in older elementary school children. During middle childhood, children's self-conscious emotions and emotion understanding increase with their self-awareness Berk, . StudiesDespite the new findings, the present study also has some limitations. First, the results provide an executive function account of donating behavior in middle childhood. However, it is not clear whether this executive function account can be applied to other prosocial behavior. Second, the purpose of the present study is to first clarify the developmental stage at which inhibitory control is associated with donating behavior and the specific aspects of inhibitory control associated with donating behavior at specific ages. Thus, a cross-sectional design was used. Future studies should further confirm the causal relationships between inhibitory control and donating behavior with longitudinal designs. Third, it is important to further examine whether prosocial behavior is associated with other general cognitive abilities, such as intelligence. Finally, some of the tasks provided a very narrow range of scores and small variance in the present study. This might have prevented the finding of correlations between some of the variables. In addition, the sample sizes of the age groups were relatively small and this might have influenced the statistic power. Thus, future studies should use tasks with a wide range of scores and groups of large sample sizes to further examine the relationships between inhibitory control and prosocial behavior such as donating behavior.Inhibitory control is not related to donating behavior in early childhood, but they are closely connected in middle childhood. During middle childhood, cool and hot inhibitory control, respectively, predict young and older children's donating behavior. Therefore, donating behavior increasingly relies on specific inhibitory control, i.e., hot inhibitory control as children grow in middle childhood.JH proposed the concept and designed the work, performed the acquisition of data for the work, analyzed and interpreted the data and drafted the work. JH revised the work for important intellectual content. JH finally approved the version to be published. JH agreed to be accountable for all aspects of the work in terms of ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The author declares 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 small GTPases of the Rho family comprising RhoA, Rac1 and Cdc42 function as molecular switches controlling several essential biochemical pathways in eukaryotic cells. Their activity is cycling between an active GTP-bound and an inactive GDP-bound conformation. The exchange of GDP to GTP is catalyzed by guanine nucleotide exchange factors (GEFs). Here we report a novel regulatory mechanism of Rac1 activity, which is controlled by a phosphomimetic (Ser179Glu) mutant of syndecan-4 (SDC4). SDC4 is a ubiquitously expressed transmembrane, heparan sulfate proteoglycan. In this study we show that the Ser179Glu mutant binds strongly Tiam1, a Rac1-GEF reducing Rac1-GTP by 3-fold in MCF-7 breast adenocarcinoma cells. Mutational analysis unravels the PDZ interaction between SDC4 and Tiam1 is indispensable for the suppression of the Rac1 activity. Neither of the SDC4 interactions is effective alone to block the Rac1 activity, on the contrary, lack of either of interactions can increase the activity of Rac1, therefore the Rac1 activity is the resultant of the inhibitory and stimulatory effects. In addition, SDC4 can bind and tether RhoGDI1 (GDP-dissociation inhibitor 1) to the membrane. Expression of the phosphomimetic SDC4 results in the accumulation of the Rac1–RhoGDI1 complex. Co-immunoprecipitation assays (co-IP-s) reveal that SDC4 can form complexes with RhoGDI1. Together, the regulation of the basal activity of Rac1 is fine tuned and SDC4 is implicated in multiple ways. The Rho GTPases, of which the best characterized are RhoA, Rac1 and Cdc42, are the major evolutionarily conserved regulators of the cytoskeleton, migration, cell polarity and differentiation . The homThe release of the strongly bound GTPases from the RhoGDI1 complex is regulated mostly by specific phosphorylation of RhoGDI1; e.g. Rac1 is released upon phosphorylation of the Ser174 of RhoGDI1 by Pak1 (p21-activated kinase1) .Pak1 was the first identified and the best characterized member of the PAK family, which is activated by Cdc42-GTP or Rac1-GTP . PAKs haTiam1, a typical member of the Dbl-like family of GEFs, activates the Rho-family GTPase Rac1 . It is mSyndecans constitute a family of four transmembrane heparan sulfate proteoglycans comprising a type II PDZ binding motif at their C-termini. Despite the conserved amino acid sequence of syndecans (EFYA) Tiam1 binds the different syndecans with altering affinities. It prefers SDC1 and SDC3 more than SDC2 or SDC4 . Among tHere we report that Tiam1 and SDC4 were pulled down together in SDC4 phosphorylation- and PDZ-dependent manner controlling the bound Rac1 in the Tiam1 complex. The basal level of the Rac1 activity was regulated thereby. Further, we examined the role of RhoGDI1 in the Rac1 activation and showed that RhoGDI1 was pulled down with SDC4 independently from the mutations, and RhoGDI1—Rac1 was accumulated in the cells expressing the phosphomimetic mutant SDC4.Tiam1 is a ubiquitously expressed Rac1-GEF comprising a PDZ domain ,23, and We investigated the interaction between SDC4 and Tiam1 with co-immunoprecipitation (co-IP) experiments using anti-Tiam1 antibody, at the same time monitored the connection of Tiam1 and Rac1 . SDC4 anAt the same time the interaction of Rac1 and Tiam1 was investigated in the same immunoprecipitate  followinTaken together, the endogenous Tiam1 level was not changed by the expressions of the different SDC4 constructs but the pulled down SDC4 and Rac1 were varied in the Tiam1 precipitates meaning that the association of Rac1 and Tiam1 was controlled by SDC4 in the different cell lines. Since the interaction of Ser179Glu SDC4 and Tiam1 was the strongest and the bound Rac1 was reduced in the Tiam1 complex, we concluded that the phosphomimetic SDC4 with PDZ binding site restricted the interaction between Tiam1 and Rac1. The deletion of the PDZ binding site (ΔPDZ-Ser179Glu) enhanced the Rac1binding to Tiam1. Therefore to block the enzymatic function of Tiam1 simultaneous interactions of the phosphomimetic mutation and PDZ binding site of SDC4 were needed. We suggested a model, how phosphomimetic SDC4 could restrict the binding of Rac1 to Tiam1 . Most prNext we investigated how Tiam1 contributed to the Rac1-GTP level of MCF7 cells. To do so, the level of Rac1-GTP was characterized in different conditions and compared to that of the Ser179Glu SDC4 cells. The cells were transfected transiently by Tiam1 shRNA construct, and with its scrambled sequence or the media was supplemented with NSC23766 . The NSCThe level of Rac1-GTP was characterized in cells expressing the different SDC4 mutants with a specific pull-down assay using the Cdc42/Rac1 interactive binding (CRIB) domain of Pak1 . The actWe were curious whether SDC4 could be detected in the Rac-assay, so SDC4 was tested with anti-GFP antibody. SDC4 was pulled down by CRIB domain-based assay with similar pattern that was observed in the case of Rac1 /5th slabBecause of Pak1 kinase is involved in the regulation of Rac1 activity, we investigated the interaction of SDC4 with Pak1 kinase. To do so the presence of Pak1 was tested in SDC4 co-IP . Indeed,Previous studies showed that PKC alpha could contribute to the activation of Rac1 –21 throuBlocking an intermediate step of a cyclic metabolic pathway arrests the cycle leading to accumulation of the downstream and depletion of the upstream intermediates. Therefore we investigated whether SDC4 had any effect on the accumulation of RhoGDI1-Rac1, which might influence the regulation of the activity of Rac1. The phosphorylation of the regulatory Ser174 of RhoGDI1  was moniSDC4 was suggested to tether RhoGDI1 to the membrane via PDZ interaction of synectin establishing SDC4-synectin-RhoGDI1 complex. However in that study direct interaction between SDC4 and RhoGDI1 was failed to be investigated . Thus, wSince, the phosphorylation of RhoGDI1 was even /1st slabTaken together, the phosphorylation of Ser174 of RhoGDI1 was unchanged in the cells expressing different SDC4 mutants; however the stability of the Rac1-RhoGDI1 complex was increased in the cells expressing the Ser179Glu mutation. If there is a given pool of RhoGDI and the members of the Rho-GTPase family members compete for GDI binding to avoid the degradation  the acceBecause of the activity of RhoA is antagonistic with that of Rac1  and the The activity of Rho family GTPases is highly tuned process. Any perturbation in their activity leads to the failure in polarization, migration, cell proliferation ,2,31,32.Earlier the PDZ interaction of Tiam1 and syndecans was studied and it was shown that Tiam1 binds syndecans with different strength; SDC1 or SDC3 were bound 15-fold stronger than SDC2 or SDC4 ,25. HoweStudying the Tiam1 –Rac1 interaction we found that the amount of Rac1 pulled down by Tiam1 was reduced greatly in the cells expressing the phosphomimetic SDC4. On the contrary, the deletion of PDZ binding site of SDC4 increased the pulled down Rac1 by Tiam1 and consequently the Rac1 activity by 20%. Therefore we surmise that the only interaction of the phosphomimetic SDC4 might relief the auto inhibitory conformation of Tiam1 by binding its N-terminal region. The simultaneous interactions of PDZ and the 179Glu are required to maintain the inactive conformation of Tiam1 ,29,11 F.It has already been suggested that SDC4 is necessary to suppress Rac1 activity; however the suggested regulatory mechanism was characterized in stress conditions –21. HereThe Rac1 activation circle can also be driven by the release of Rac1 from the RhoGDI1-Rac1 complex that depends on the phosphorylation of Ser174 of RhoGDI1 by Pak1 . The phoThe direct interaction of SDC4 and RhoGDI1 was investigated in co-IP experiments. They could pull down each other conversely and this interaction independent of the PDZ binding site . The intThe accumulation of RhoGDI1-Rac1 complex in the Ser179Glu cells is very interesting. It definitely means that PDZ binding is necessary to the release of Rac1 from RhoGDI1, therefore other protein(s) should be involved in the Rac1 release that might be synectin.In the cells the total amount of Rac1 was constant, at the same time the active Rac1 was reduced, and the amount of the Rac1-RhoGDI1 complex was elevated in the Ser179Glu cell line. We tested RhoA in complex of RhoGDI1, but it was not affected by the access of Rac1. However, the active RhoA was increased approximately two-fold that is most probably due to the reciprocal balance of the RhoA—Rac1 activity.We could not find any evidence for the implication of PKC alpha in the regulation of the basal activity of Rac1 in MCF7 cells, although the indispensable role of PKC alpha was emphasized in the regulation of the activity of Rac1 in some stress conditions ,20.Lβ2 integrin heterodimer). Down-regulation of Tiam1 inhibited Rac1 activity, cell adhesion, and migration [Activation of Tiam1 and consequently Rac1 was reported in T cells upon TCR-induced activation of LFA-1  blocking a Rac1-GEF complex and 2) interfering with RhoGDI1–Rac1 complex to promote the release of Rac1 .MCF-7 human breast adenocarcinoma cells were obtained from ATCC, cultured in DMEM:F12 (Sigma-Aldrich) containing penicillin-streptomycin-amphoterycin B  and supplemented with 10% fetal bovine serum (FBS) (Life Technologies/Gibco), unless otherwise stated. The stably transfected cell lines were propagated in DMEM:F12 medium supplemented with 10% FBS and 200 μg/ml G418 (Cambrex).The human SDC4 was isolated and cloned into pCMV vector (Clontech Laboratories Inc.). Green fluorescent protein (GFP) was introduced to the extracellular domain of SDC4 as it was characterized elsewhere . Briefly5’-GAGACTCCTCCGTACAGTAAT and its scrambled sequence cloned into pSIREN-RetroQ plasmid (Clontech Laboratories Inc.) according to the protocol of the manufacturer. The MCF-7 cells were transiently transfected with shTiam1 construct for 48 hours prior to harvesting and processing.Different cell lines were developed expressing the wt SDC4, Ser179Ala, Ser179Glu, and PDZ binding site deletion double mutants (ΔPDZ-Ser179Ala and ΔPDZ-Ser179Glu). Short hairpin RNA targeting human Tiam1 (shTiam1) was designed with the sequence of The next antibodies were used at appropriate dilutions: mouse monoclonal anti-GFP, Life Technologies/Zymed; polyclonal anti-SDC4: from rabbit ZMD.293, Life Technologies/Zymed; from goat D-16/sc9499 and from rabbit 5G9/sc12766, Santa Cruz Biotechnology inc.; polyclonal anti-Tiam1 C-16, Santa Cruz Biotechnology Inc.; A2484 and A2609, Sigma-Aldrich; polyclonal anti-phospho-S174-RhoGDI ab74142, Abcam Ltd; anti-RhoGDI1 : polyclonal rabbit A-20/sc-360 and monoclonal mouse B-10/sc-13120, Santa Cruz Biotechnology Inc.; polyclonal anti-Pak1 #2602, Cell Signaling Technology; anti-Rac1 monoclonal mouse #05–389, Merck Millipore; anti-Rho monoclonal mouse #05–778, Merck Millipore. HRP-conjugated goat polyclonal anti-rabbit (P0448), rabbit polyclonal anti-mouse (P0161) and rabbit polyclonal anti-goat (P0449) secondary antibodies were from DAKO Agilent Technologies.® v.8.For immunofluorescence staining the cells were seeded on fibronectin coated glass coverslips, and fixed for 5 min with 4% paraformaldehyde or methanol. After permeabilization with 0.1% Triton X-100 for 5 min, and blocking with 1% BSA in PBS cells were incubated for 30 min at room temperature with primary antibodies diluted in 1% BSA in PBS, then were washed 3 times with PBS, incubated with secondary antibodies for 20 min at room temperature. Following the nuclear staining the coverslips were mounted with Fluorescent Mounting Medium (DAKO). Cells were studied with Zeiss Axio Imager (Zeiss) and Nikon Eclipse 600 fluorescent microscopes, or Bio-Rad MRC-1024 (Bio-Rad) confocal microscope. Images were analyzed using Adobe PhotoshopRac1 and RhoA activation was estimated by applying Rac or RhoA-activation assay kit (Merck Millipore Corporate). The Rac-kit utilizes the Cdc42/Rac1 interactive binding (CRIB) domain of Pak1 kinase and the Rho-kit based on GST-tagged RhoA binding domain of Rhotekin. The cells were treated with PKC alpha inhibitor Gö6976 (Merck Millipore Corporate)  or with Tiam1 inhibitor NSC23766 (Santa Cruz Biotechnology Inc.) , when it was stated. The active Rac1 and RhoA were detected by Western blotting using mouse anti-Rac1 or RhoA antibodies (Merck Millipore Corporate /Upstate). In control experiments cell lysates were incubated with GTP-gammaS (10 mM) or GDP (100 mM), according to the manufacturer’s instructions.2+-lysis buffer  supplemented with 1 mM Na-fluoride, 1 mM Na3VO4 and protease inhibitor cocktail (Sigma-Aldrich). The lysates were centrifuged and the supernatants were pre-cleared by addition of 5 μL of protein A/G magnetic beads slurry for 2 hours, then the supernatants were incubated with the antibody of interest overnight. 10 μl of protein A/G slurry was added for 2 hours, collected by pulse centrifugation, washed 3 times with lyses buffer, and the eluted immune complex was subjected to SDS-PAGE, followed by immunoblotting with the appropriate primary antibodies. Specific binding was detected with a secondary peroxidase-conjugated antibody (DAKO), and peroxidase activity was visualized by the ECL procedure .Cells were lysed in MgData are represented as the mean from at least three independent experiments, otherwise stated. Results are expressed as means ± standard error of the mean (SEM). Differences between experimental groups were evaluated by using one-way analysis of variance (ANOVA). Values of p<0.05 were accepted as significant.S1 Fig(A) PKC alpha mediated activity of Rac1 was studied without or with Gö6976 . Quantitation of Rac1-GTP levels was normalized to nt MCF-7 cells. These results are representatives of 3 independent experiments; data are reported as mean ±SEM (n = 3). (B) Light micrographs of MCF-7 cells indicated that there was a change in the cell morphology upon Gö6976 administration.(JPG)Click here for additional data file.S2 FigBlack box indicated the cropped part included in the corresponding figure.(TIF)Click here for additional data file.


Descriptors including vsurf_DW23, E_sol, Dipole and vsurf_G were determined to be related to the inhibitory activity of flavonoids. The lack of 2,3-double bond, 3′-OH, 4′-OH and the increased number of methoxylated substitutions were shown to be beneficial for the inhibition of P-gp. These results are important for the screening of flavonoids for inhibitory activity on P-gp.P-glycoprotein (P-gp) serves as a therapeutic target for the development of inhibitors to overcome multidrug resistance (MDR) in cancer cells. In order to enhance the uptake of chemotherapy drugs, larger amounts of P-gp inhibitors are required. Besides several chemically synthesized P-gp inhibitors, flavonoids as P-gp inhibitors are being investigated, with their advantages including abundance in our daily diet and a low toxicity. The cytotoxicity of daunorubicin (as a substrate of P-gp) to KB/MDR1 cells and the parental KB cells was measured in the presence or absence of flavonoids. A two-dimensional quantitative structure–activity relationship (2D-QSAR) model was built with a high cross-validation coefficient (Q ABCB1 gene. The overexpression of P-gp has been associated with multidrug resistance (MDR) in cancer cells, a limiting factor in the success of cancer chemotherapy. Given the role of P-gp in influencing cancer chemotherapy, methods to overcome P-gp-mediated efflux have been investigated. It is generally believed that the mechanisms of P-gp inhibition mainly comprise four aspects: competitively, non-competitively or allosterically blocking the drug binding site; interfering with the ATP hydrolysis process; altering the integrity of cell membrane lipids; decreasing the P-gp expression [P-glycoprotein (P-gp) is a member of the ATP-binding cassette (ABC) family, which is encoded by the pression  and cyclpression , were fopression . The derpression . Elacridpression . Therefopression .Flavonoids are a class of compounds based on the diphenylpropane (C6–C3–C6) skeleton, which are widespread in our common diet, including in fruits and vegetables. Flavonoids have been shown to be beneficial to human health for their antioxidant, anti-inflammatory, anticarcinogenic and antiviral activities . Several50 of daunorubicin) of 31 flavonoids (The aim of this study was to investigate the quantitative structure–activity relationship for the flavonoid-mediated inhibition of P-gp in KB/MDR1 cells overexpressed with P-gp. Daunorubicin  has beenavonoids  was meas50) of flavonoids on the cytotoxicity of daunorubicin to KB and KB/MDR1 cells is shown in 50 value indicates a higher flavonoid inhibitory activity. Elacridar (10 μM) was used as a positive control. The IC50 values of daunorubicin as a negative control (without any inhibitors) in KB cells were significantly lower compared to those in KB/MDR1 cells, suggesting that daunorubicin is less sensitive to KB/MDR1 cells due to the overexpression of P-gp. Elacridar significantly enhanced the cytotoxicity of daunorubicin in KB/MDR1 cells, being more pronounced in KB/MDR1 cells (RFKB/MDR1 = 6.818) compared to KB cells (RFKB = 1.512). As shown in 17 (quercetin) and 28 (daidzein), respectively, indicating that quercetin and daidzein can increase the sensitivity of KB/MDR1 cells to anticancer drug substrates. However, the fact that the RFKB/MDR1 of quercetin and daidzein was 0.580 and 0.704, respectively, indicated an increased P-gp function, while their RFKB was as high as 10.414 and 3.531. These results may be due to the fact that although there is no P-gp expression in KB cells, expression of other proteins like multidrug resistance protein (MRP), or breast cancer resistance protein (BCRP) may occur, which also have the same function as P-gp.The reversal effect , 2 , 3 , and 4 , we found that with an increased number of methoxylated substitutions, an increased inhibitory activity on P-gp occurred. The structure–affinity relationship implicated that 3′-OH and 4′-OH are not conducive to the inhibitory activity of flavonoids by comparing 7  and 11 . It was also supported by an inhibitor galangin  and a non-inhibitor quercetin .Flavonoids 50 as the dependent variable and the calculated molecule descriptors as independent variables. The best QSAR model established using a training set consisting of 24 flavonoids and a test set of seven flavonoids is as follows:50 = 0.183vsurf_DW23 − 0.359E_sol − 3.181dipole + 10.627vsurf_G − 9 .859IC2 = 0.892, Radj2 = 0.869, Q2 = 0.829, F = 39.073, p < 0.01, RMSE = 0.492, Rpred2 = 0.905RThe model was obtained utilizing IC50 and related molecular descriptors is shown in 50, indicating that vsurf_DW23 and E_sol play an important role in the inhibitory activity of flavonoids. Also, the Pearson correlation coefficient |r| < 0.5 between each descriptor indicates that the model has not been over-fitted. The square of the correlation coefficient between the experimental and predicted IC50 values reached 0.904 (2) was as high as 0.829, suggesting that the obtained model has great prediction ability. The test set prediction correlation coefficient reached 0.905, indicating that the model has better external prediction ability.The correlation matrix between ICed 0.904 , indicatThe QSAR model contains two molecular descriptors, E_sol and dipole, which are positively correlated with the inhibitory activity of flavonoids as well as two negatively correlated descriptors, vsurf_DW23 and vsurf_G.50, indicating that this descriptor related negatively to the inhibitory activity of flavonoids on P-gp. With increasing IC50 value, the inhibitory activity of flavonoids decreased. As shown in 11 (14.221), 17 (11.597), and 28 (16.523), showed high IC50.Vsurf_DW23 and vsurf_G are the vsurf_descriptors, which are similar to the VolSurf descriptors. Vsurf_DW23 is a hydrophobic zone parameter that represents the contact distance of vsurf_EWmin (lowest hydrophilic energy) when a water probe interacts with a target molecule [50 of E_sol is illustrated by comparing the E_sol and IC50 of flavonoid 16  . Moreover, flavonoid 12, with a low vsurf_DW23 value (1.118), exhibited a high IC50 (5.501 μM) because of the lowest E_sol value (−11.881). We observed that the higher the E_sol value, the smaller the IC50.The positive contribution of E_sol is the potential energy descriptor that represents the solvation energy. The influence on IC.463 μM)  versus 227 showed a low IC50, but exhibited the highest dipole value due to its structure with four chiral centers. Dipole was positively correlated to IC50, but negatively correlated to the inhibitory activity, as shown in the model.Dipole is the conformation-dependent charge descriptor that represents the dipole moment calculated from the partial charges of the molecule. It displays the distribution of charge and the separation degree of negative charge and positive charge . Flavono12, 13, 21, and 29 have a high vsurf_G value. Flavonoid 27 also exhibits a high vsurf_G value related to its special structure. Vsurf_G plays an important role in the inhibitory activity on P-gp.Vsurf_G is a shape descriptor describing the surface globularity of the molecule . FlavonoIt is widely reported that the main cause of the low bioavailability of chemotherapy drugs is related to transport of P-gp . The mos13, 18 and 30 showed high inhibitory activity. Our results also indicated that the KB/MDR1 cells showed a 10-fold resistance to daunorubicin compared to the KB cells exposed to flavonoid 17, indicating quercetin. The sensitivity of KB/MDR1 cells to anticancer drug substrates was increased, which is consistent with previous studies reporting that quercetin is a potential modulator of P-gp [In the current study, we investigated the cytotoxicity of daunorubicin in KB/MDR1 and KB cells. The KB/MDR1 cells showed a 4.34-fold  resistan of P-gp ,12,13. A of P-gp  showed tp < 0.01) and E_sol (p < 0.05) are significantly related to IC50 and the Pearson correlation coefficient |r| < 0.5. These results indicated that the model was reliable. The 2D-QSAR showed a high prediction ability and internal stability with Q2 value (0.829) > 0.5 [pred2 value (0.905) > 0.6 [In order to determine the molecular properties associated with the inhibitory activity, the QSAR model was built. It was observed that the descriptors of vsurf_DW23  > 0.6 .As seen in the model, vsurf_DW23 was confirmed to be an important factor affecting the inhibitory activity of flavonoids. Vsurf_DW23 is a hydrophobic zone parameter that represents the contact distance of vsurf_EWmin (lowest hydrophilic energy) when a water probe interacts with a target molecule [10 and 17 were reported to be substrates [Based on previous research in the laboratory , we founbstrates  as well bstrates ,34. CompIn conclusion, flavonoids that had high inhibitory activity on P-gp should have small values of vsurf_DW23 and vsurf_G and large values of E_sol and dipole. Daunorubicin was used as the substrate of P-gp in order to investigate the inhibitory activity of flavonoids. The absence of 2,3-double bond and both 3′-OH and 4′-OH, and an increased number of methoxylated substitutions were shown to be beneficial to P-gp inhibition. Therefore, specific flavonoids meet these criteria to be modulators of P-gp.v/v) DMSO. Fetal bovine serum (FBS) was obtained from Zhejiang Tianhang Biotechnology Co.,Ltd. . Dulbecco’s modified essential medium (DMEM) was purchased from Hyclone . The 96-well plates were purchased from Corning Costar .3--2 and 5-diphenylte trazolium bromide (MTT) were purchased from Gen-View Scientific Inc. . Flavonoids were purchased from Aladdin Chemistry Co., Ltd.  (purity > 98%). Elacridar was purchased from MedChemExpress.  (purity > 98%) (CAS: 143664-11-3) and used as an inhibitor of P-gp. Daunorubicin hydrochloride was purchased from Tokyo Chemical Industry Co. Ltd.  (purity > 98%) (CAS:23541-50-6) and used as a substrate of P-gp. All the compounds were dissolved and diluted to a final concentration of 0.1% .The KB cell line was purchased from Culture Collection of the Chinese Academy of Sciences . KB/MDR1 cells were stable, transfected with cDNA of human P-gp, which was confirmed by RT-PCR and Western blotting in our previous study . Both KB3 cells/well and incubated in an atmosphere of 5% CO2 at 37 °C for 24 h. In order to test the inhibition of flavonoids, cells were treated with daunorubicin (0–20 μM) in the absence or presence of flavonoids at the non-cytotoxic concentration (40 μM)  of flavonoids was estimated by comparing the IC50 (concentration of 50% inhibition) of the daunorubicin in the absence of inhibitors to that obtained in the presence of inhibitors. RF was obtained by comparing the IC50 of daunorubicin in the KB/MDR1 cells and the parental KB cells.The cytotoxicity of flavonoids and daunorubicin was determined by MTT assay. The cells were seeded in 96-well plates at a density of 7 × 10 (40 μM)  for 24 hThe structures of the 31 flavonoids were drawn by ChemBioDraw ultra 12.0. The initial optimization of these flavonoids was performed by Sybyl X-2.0 . The molecular descriptors were calculated by Sybyl X-2.0 and Molecular Operating Environment (MOE) 2009.50 values of the 31 flavonoids in KB/MDR1 cells, in the range 0.676 to 5.894  were chosen randomly as the test set; the rest of the flavonoids were used as the training set to construct the model. The best molecular descriptors which filtered through the method of the stepwise multiple linear regression (MLR) were adopted as independent variables to build the model. In order to prevent over-fitting between descriptors, correlation analysis between descriptors was performed, and descriptors with an absolute value of correlation coefficients less than 0.5 were selected [The ICto 5.894  were useselected .2 of the resulting model is calculated in order to test the predictive ability of the model. The predicted IC50 in both the training and test sets was calculated from the QSAR model. For the model to be considered reliable, Q2 should be >0.5 [pred2) for the test set should be >0.6 [2 value was calculated by the following equation (1):The 2D-QSAR model was constructed utilizing the partial least squares (PLS) in Sybyl X-2.0. In the “leave-one-out” method, the Q be >0.5  and the  be >0.6 . The bes be >0.6 . Q2 valun is the number of the sample in the training set, 50 of the ith sample, and 50 of all samples.