Inula japonica Thunb. Aiming for discovery of a novel anti-inflammatory compound, we isolated spinacetin from the extract and investigated its in vitro and in vivo anti-inflammatory effect and the related mechanism. Effect of spinacetin on the Syk signaling pathway was studied in bone marrow-derived mast cells (BMMCs), and that on the nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) was investigated in Rat basophilic leukemia (RBL)-2H3 cells and human mast cell line (HMC-1). The in vivo anti-inflammatory activity was assessed with passive cutaneous anaphylaxis (PCA) reaction assay. Spinacetin significantly inhibited the release of histamine, and production of inflammatory mediators such as leukotriene C4 (LTC4) and interlukin-6 (IL-6) in IgE/Ag stimulated BMMCs. Analysis of the signaling pathways demonstrated that spinacetin inhibited activation of Syk, linker of activated T cells (LAT), phospholipase Cγ (PLCγ), cytosolic phospholipase A2 (cPLA2), MAPKs, Akt/NF-κB, and intracellular Ca2+ mobilization but with no effect on Fyn and Lyn. On the other hand, spinacetin suppressed IgE/Ag-induced activation of RBL-2H3 cells with inhibition against phosphorylation of extracellular signal regulated-protein kinase (ERK), c-Jun-NH2-terminal kinase (JNK), p38 MAPKs, PLCγ, translocation of cPLA2, and Akt/IκBα/NF-κB signal. However, spinacetin had no effect on PMA and A23187-induced activation of HMC-1. Furthermore, oral administration of spinacetin dose-dependently attenuated IgE/Ag-mediated PCA reaction in mouse model. Taken together, spinacetin showed the activities in preventing inflammatory processes, which might be at least partially attributed to the abolishment of Syk-dependent activation of IgE/Ag-mediated mast cells.We previously reported the anti-inflammatory and anti-asthmatic activities of the extract of the  Mast cell is known to contribute to allergic diseases such as asthma, rhinitis, urticaria, allergic conjunctivitis, and anaphylaxis . Mast ce2 to inositol trisphosphate (IP3) and diacylglycerol (DAG)-second messengers that liberate intracellular Ca2+ from internal stores and activate protein kinase C (PKC), respectively  of Fc𝜀RI and Syk following ITAM binding . Aggregaectively . The aboectively . Grb2-boectively , regulat and LTs . PI3K pr and LTs .Figure 1A) is a flavonoid isolated from Inula japonica Thunb . The LTC4 enzyme linked immunoassay (EIA) kit, and the antibody for COX-2 were from Cayman Chemical . Histamine ELISA kit was purchased from Demeditec Diagnostics GmbH .RPMI1640, fetal bovine serum (FBS) and the enhanced chemiluminescence (ECL) Western blot detection reagent were purchased from Thermo Fisher Scientific Inc. . Mouse anti-dinitrophenyl (DNP) IgE was purchased from Sigma Chemicals . DNP-human serum albumin (HSA) was from Biosearch Technologies . The antibodies specific for phospho-ERK1/2, ERK1/2, phospho-p38, p38, phospho-JNK1/2, JNK1/2, phospho-PLCγ, phospho-IκBα, IκBα, phospho-IKKα/β, β-actin, and the horseradish peroxidase-conjugated goat anti-rabbit secondary antibody were purchased from Cell Signaling Technology, Inc. . The antibodies specific for phospho-cPLAI. japonica . The plants of I. japonica were collected from Henan Province, China, and identified by Professor Y. Zhou . A voucher specimen (IJ201105) was deposited at School of Pharmacy, Tianjin Medical University, China. The flower part of I. japonica was used to isolate spinacetin. Prior to use, spinacetin was dissolved in dimethyl sulfoxide (DMSO).Spinacetin was isolated from the ethanol extract of the Bone marrow-derived mast cells were isolated from bone marrow of Balb/c mice and differentiated as described by us previously . Briefly4, histamine and IL-6 were determined using an immunoassay kit according to the manufacturer’s protocol. The HMC-1 cells were incubated with spinacetin in the presence or absence of phorbol 12-myristate 13-acetate (PMA) and calcium ionophore (A23187).Bone marrow-derived mast cells and RBL-2H3 were sensitized with 500 ng/ml of anti-DNP IgE overnight, and then treated with spinacetin or Bay 61-3606 for 1 h. After stimulated with 100 ng/ml of DNP-HSA (Ag) for 15 min or 6 h, the supernatants were collected for further analysis, respectively. The levels of LTC2+ level was determined with the FluoForte Calcium Assay Kit  as described previously .Intracellular Caeviously . BrieflyNuclear extracts were prepared as described before. Briefly, IgE-sensitized BMMCs and RBL-2H3 cells were pretreated with spinacetin for 1 h, followed by incubation with DNP-HSA for 30 min. Then the nuclear proteins were prepared by using Nuclear Extraction Kit according to the manufacturer’s protocol .IgE-sensitized BMMCs or RBL-2H3 were pretreated with spinacetin or Bay 61-3606 for 1 h and stimulated with DNP-HSA. The preparation of cell lysates and Western blot were performed as described previously . To asseTotal cell lysates were incubated with anti-Syk, anti-LAT, anti-Fyn or anti-Lyn antibody overnight, and immune-complexes were precipitated with protein A/G plus agarose. Precipitates were subjected to SDS-PAGE and immunoblotted with the respective antibodies.ICR mice  were intradermally injected with 80 ng of mouse anti-dinitrophenyl (DNP) IgE (Sigma) into one ear. After 24 h, spinacetin or dexamethasone (Dexa) was orally administered. One hour later, the mice were intravenously challenged with 60 μg of DNP-HSA in 200 μl of PBS containing 1% (w/v) Evans blue. The mice were euthanized 1 h after treatment with DNP-HSA. The ears were removed, and then dissolved with 400 μl of formamide at 63°C overnight to measure the amount of dye extravasated by Ag. The absorbance of dye was measured at 630 nm using a microplate reader . The ears were fixed with 4% formaldehyde and embedded in paraffin. Four micrometer sections of the tissues were prepared and stained with toluidine blue to count the number of mast cells. All experiments using animals were approved on Oct 27, 2017, by the Institutional Animal Care and Use Committee of Tianjin Medical University .P < 0.05.All values are expressed as means ± SEM of triplicate values. One-way ANOVA was utilized to determine the statistical significance with GraphPad Prism 7 . Differences were considered statistically significant when Figure 1B, spinacetin was not found to affect cell viability at concentration of less than 5 μM. Thus, concentrations of 1, 2, and 5 μM were chosen for further study to assess the anti-inflammatory mechanism.The MTT assay was employed to determine the cell viability of spinacetin against BMMCs. As shown in Figure 1C, spinacetin strongly inhibited release of histamine. Increased cytosolic Ca2+ mobilization leads to mast cell degranulation and production of various pro-inflammatory mediators . The enzymatic activity of cPLA2 is known to be increased by phosphorylation of MAPKs . NF-κB is known to be activated by PI3K/Akt pathway . Our results suggested that inhibition of Akt by spinacetin might be involved in suppression of IL-6 and COX-2 expression.Nuclear factor-κB positively regulates the production of IL-6 and COX-2 expression . Activat pathway . IgE/Ag in vitro studies of mast cell-mediated allergic inflammation. Stimulation of RBL-2H3 with IgE/Ag increased ERK1/2, JNK1/2, and p38 phosphorylation as compared to non-treated cells . Spinacetin reduced the activation of the MAPKs. Meanwhile, spinacetin markedly reduced translocation of the phosphorylated cPLA2 from cytosol into the nucleus . Moreover, spinacetin suppressed IκBα phosphorylation and degradation, and NF-κB activation, and also inhibited Akt which is known to activate IκBα and NF-κB . These results are consistant with that obtained in IgE/Ag-stimulated BMMCs. Furthermore, the phosphorylation of PLCγ was inhibited by spinacetin . These results demonstrate that spinacetin ameliorated the allergic reaction in IgE/Ag stimulated mast cells.RBL-2H3 cells are widely used for 2, Akt and IKKα/β in HMC-1 cells .HMC-1 cells are characterized to lack expression of Fc𝜀RI. Therefore, many researchers use calcium ionophores and phorblo esters to measure mast cell activity by using HMC-1 cells. HMC-1 can be activated by PMA and A23187, and the activated HMC-1 leads to the phosphorylation of tyrosine kinase followed by the activation of MAPKs, NF-κB, and release of pro-inflammatory cytokines . In our These results suggest that spinacetin only inhibits IgE/Ag-induced mast cells activation, and that target protein of spinacetin might be upstream of Akt.2+ and activates PKC, respectively . These observations indicated that spinacetin inhibited the IgE-mediated mast cell activation through regulating Syk dependent pathways.Lyn phosphorylates and activates Syk and LAT. Phosphorylation of LAT results in the recruitment of molecules such as Grb2, Gads, SOS, and PLCγ. These interactions with LAT lead to the formation of molecular signaling complex that is required for the release and generation of pro-inflammatory mediators in mast cells . Fyn is ectively . As showFigure 6A). Dexa was used as positive drug. As shown in Figure 6, IgE/Ag significantly induced PCA reaction, and treatment with spinacetin markedly decreased PCA reaction which is reflected by amount of diffused dye  and ear thickness , compared to the Ag challenged group. These attenuated allergic reactions might be due to the inhibitory effect of spinacetin on activation of mast cells. To confirm whether the decreased vascular permeability was related to amount of mast cells, the number of mast cells was counted at the ear reaction site. There was no variation between Ag treated group and spinacetin treated group .The Fc𝜀RI/mast cell axis is involved in triggering several intracellular signaling molecules to release preformed granules, and to generate pro-inflammatory mediators, resulting in the induction of allergy and anaphylaxis . As we aInula japonica Thunb. In the present study, we report its anti-inflammatory effect on IgE/Ag-induced mast cells and PCA mouse model as well as the related molecular mechanism.Flavonoids are abundant in plants, with a variety of biological functions such as anticancer, antiviral, antibacterial, anti-oxidative, and anti-inflammatory activities . Particu4, IL-6, TNFα, and enzyme COX-2 .Mast cells can function as effector cells in certain innate and adaptive immune responses . These cme COX-2 . Releaseme COX-2 . Histamime COX-2 . IL-6 leme COX-2 . In the 3, which binds to its receptor on the endoplasmic reticulum leading to liberation of Ca2+ from intracellular store. We found that spinacetin suppressed mobilization of Ca2+ and phosphorylation of PLCγ . The activated Vav, Grb2, and SOS lead to phosphorylation of ERK, JNK, and p38, and activation of cPLA2 to release arachidonic acid .Aggregation of the high-affinity IgE receptor Fc𝜀RI results in exocytosis of granules and production of LTs and cytokines. After binding of antigen to IgE/Fc𝜀RI complexes, Lyn transphosphorylates the ITAMs of β- and γ- chains of Fc𝜀RI, which are associated with the Src family tyrosine kinases Lyn and Fyn and with the non-Src tyrosine kinase Syk . Fyn, Lynic acid . In the Figures H). These findings suggest that spinacetin effectively inhibits IgE/Ag-induced NF-κB activation, in which suppression of Akt phosphorylation might be involved.Crosslinking of Fc𝜀RI-bound IgE-molecules with multivalent antigen or allergen induces NF-κB activation . NF-κB iFigures F). These findings are consistent with down-regulation of ERK1/2, JNK1/2, p38 MAPKs, and Akt/IκB/NF-κB signal by spinacetin in IgE/Ag-stimulated BMMCs. However, spinacetin did not affect the corresponding molecules in PMA and A23187-induced HMC-1 cells, including ERK1/2, JNK1/2, p38 MAPKs, and Akt/IKKα/β . It is known that there is no expression of Fc𝜀RI on HMC-1 surface. These results suggest that spinacetin does not affect Akt, MAPKs, and their downstream proteins, and target protein of spinacetin may be in the upstream of Akt.We also observed spinacetin potently inhibits IgE/Ag-induced activation of RBL-2H3 cells . However, it did not affect Lyn and Fyn . These results indicate that spinacetin inhibits Syk-dependent pathway in IgE/Ag-induced mast cells.In order to know whether spinacetin targeted any protein upstream of Akt, we investigated the effect of spinacetin on phosphorylation of PLCγ, LAT, Syk, Lyn, and Fyn. As a result, spinacetin inhibited phosphorylation of PLCγ, LAT, and Syk , suggesting the in vivo anti-anaphylaxis efficacy of spinacetin.Anaphylaxis is the most serious allergic reaction, and typically occurs from the activation of mast cells and basophils through crosslinking of IgE and aggregation of Fc𝜀RI . In orde2, and Ca2+ mobilization, finally suppressed histamine release, LTC4 synthesis, and IL-6 production in IgE/Ag-stimulated BMMCs . Spinacetin also inhibited the corresponding signals in IgE/Ag-induced RBL-2H3 cells, but did not affect those in PMA and A23187-induced HMC-1 cells. Furthermore, spinacetin attenuated IgE/Ag-mediated PCA reaction in mouse model. This study suggests that spinacetin could be used as a lead compound for development of novel anti-inflammatory drugs.In the present study, we demonstrated that spinacetin inhibited Syk, LAT, PLCγ activation, subsequently down-regulated Akt, IκBα, NF-κB, MAPKs, cPLANJ, SP, CS, and YC performed the experiments. ZZ, RW, and YQ provided technical assistances. NJ and MJ wrote the manuscript. MJ and DK designed the experiments and acquired funding for the study. DK edited the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Explosive volcanic eruptions are the largest non-anthropogenic perturbations for Earth’s climate, because of the injection of sulfate aerosols into the stratosphere. This causes significant radiation imbalances, resulting in surface cooling for most of the globe. However, despite its crucial importance for Antarctic ice sheet mass balance, the response of the Southern Ocean to eruptions has yet to be understood. After the eruption of Mt. Pinatubo in 1991, much of the Southern Ocean cooled; however, off the Antarctic Peninsula a warming of up to 0.8 °C is found in the observations. To understand the physical mechanisms associated with this counter-intuitive response, we combine observational analysis from the Mt. Pinatubo eruption with the Last Millennium Ensemble (850–1850) conducted with the Community Earth System Model. These results show not only that the observed warming off the Peninsula following the Mt. Pinatubo eruption is consistent with the forced response to low-latitude eruptions but further, that this warming is a response to roughly 16% weakening of subsurface Weddell Gyre outflow. These changes are triggered by a southward shift of the Southern Hemisphere polar westerlies (∼2°latitude). Our results suggest that warming induced by future volcanic eruptions may further enhance the vulnerability of the ice shelves off the Antarctic Peninsula. Large explosive eruptions are capable of injecting sulfate aerosols into the stratosphere, which are responsible for reducing incoming shortwave radiation3. The global response is an overall cooling of Earth’s surface7. The eruption of Mt. Pinatubo in 1991 was the largest eruption of the 20th century13. It caused global sea surface cooling of 0.3–0.4 °C5 with impacts on weather and hydroclimate throughout the world14. Nonetheless, its size is comparably small relative to the eruptions of the past millennium, before 1850 where anthropogenically driven warming was insignificant15. Thus, understanding the range of regional impacts associated with past large volcanic eruptions is crucial for assessing the impacts of potential future eruptions. This is particularly relevant given the potential for future anthropogenic warming to modify the impacts of volcanic eruptions17.Volcanic eruptions are important elements of the climate system, driving transient changes to global climate on interannual to decadal timescales18. In addition, it is the site of dense water formation (i.e. Antarctic Bottom Water) and unique ecosystem communities19. The Antarctic Peninsula ice shelves and ocean near it deserve special attention because of ongoing changes to ecosystems and ocean-atmosphere-cryosphere dynamics due to climate change20. The Weddell Sea region off the Antarctic Peninsula has been widely impacted by recent permanent ice-shelve collapse22 and thinning (i.e. Larsen Ice Shelf)25. Antarctic Peninsula ice shelves are highly susceptible to changes in ocean circulation, as observed in the Larsen C ice loss through basal melting26 and intrusion of warm ocean waters27. After a large eruption sea-ice extension responds more to ocean temperature and circulation changes than to the direct radiative forcing28. Climate simulations have shown that the eruption aftermath in Antarctica is an initial sea-ice expansion followed by its contraction29 linked primarily to the strengthening of the Southern Hemispheres stratospheric polar vortex and a chain of regional feedbacks. The connectivity between large-scale dynamics and local processes after an eruption in the Southern Ocean highlights several relevant features that lack of studies29, as ocean temperature exchange and regional polar interaction between ocean-atmosphere and sea-ice.The Southern Ocean is a vulnerable region to climate change, in part because of the presence of floating ice shelves around most of Antarctica’s coastline35. Here we make use of the Community Earth System Model Last Millennium Ensemble (CESM-LME33), which contains multiple realizations of the climate over the 850–2005 period. This allows us to examine both the effects of the 1991 Mt. Pinatubo eruption individually, and the potential impacts of larger Last Millennium  eruptions more broadly, to identify common physical mechanisms in the Southern Ocean response to eruptions.Climate models can provide valuable insight into the physical mechanisms driving responses to volcanic eruptions. The use of ensembles is particularly valuable, due to the large extent of internal climate variability which can mask the forced response to eruptions30 and Hadley Centre Sea Ice and Sea Surface Temperature - HadISST31) for sea surface temperature (SST) and oceanic reanalysis  for SST and wind stress. We combine the observational results with the output from CESM-LME33, which contains 15 realizations  of the 850–2005 period including the effects of volcanism. The use of 15 ensemble members allows an improved estimate of the uncertainty due to internal variability37. All results shown here are restricted to the austral summer , since the volcanic response was identified to be larger during this season4 and there is less presence of ice in the Weddell Sea. For the Mt. Pinatubo analysis the anomalies of SST and related fields are calculated relative to the 1980–2005 climatological mean, which contains the anthropogenic signal39. The time series are de-trended, over the same period, to minimize the effects of long-term positive trend of increased greenhouse gases and ozone depletion forcing. The evolution of the observed SST spatial pattern for the Mt. Pinatubo eruption from the year of the eruption  to Yr + 3 (1994) is shown in Fig. The impact of the Mt. Pinatubo eruption on the Atlantic sector of the Southern Ocean is investigated using observations  the SST anomaly presents similar pattern in all observational data sets Fig. . There iAt Yr + 2  and extends to Yr + 2 (1993), while in CESM-LME it appears in the year of the eruption (1991) and persists until 1993 (Yr0 to Yr + 2). The average of the CESM-LME volcanic-only ensemble members  the southward shift is of ~0.54° latitude in CESM-LME  and ~1.30° latitude in SODA 44. A shift of the westerlies can draw more water from deeper layers to the surface around Antarctica45 and modify subsurface temperature47.Zonal wind stress changes obtained with the results of SODA and CESM-LME reveal similar behavior Fig. . After t7, which acts to offset the anthropogenic warming48. The anomalous warming observed here has the opposite response with the potential to impact sea-ice production and melt, given the high sea-ice sensitivity to the stability of the seawater column28. This indicates a potential contribution to enhance thinning of the Larsen Ice Shelf in the Antarctic Peninsula25.The aftermath of a volcanic event is an overall decrease in the SST7, since historical eruptions are known to be much larger than the more recent ones34. The 1815 Tambora eruption, for example, loaded 109Tg of SO2 into the stratosphere49, which caused 1 °C decrease in global SST2. In contrast, the Mt. Pinatubo eruption in 1991 decreased the SST by 0.4 °C2, with a stratospheric input of sulfur-containing gases of 30Tg49. We therefore use CESM-LME results from 850 to 1850, to understand the physical mechanisms behind the Weddell Sea response to all major eruptions over the Last Millennium. Here major eruptions34 are those with an aerosol mass mixing ratio above 10−8; this leads to a set of seven eruptions , which increases the signal-to-noise ratio beyond what would be possible with either event type alone34. All anomalies shown for the LM are relative to the 850–1850 climatological mean34 only for the austral summer (DJF).The ocean response to LM eruptions is more evident than that for modern eruptions51. The surface ocean response to LM eruptions may also be faster than for the Mt. Pinatubo one, because of their larger size49.In the composite evolution from Yr0 to Yr + 3 for SST and salinity . The same pattern of meridional shift and westerlies intensification was observed after the Mt. Pinatubo eruption , which is explained by the enhanced flow in the southern limit of the ACC . We believe that the dynamical response is as follows and schematically represented in Fig. Our results show that Weddell Sea dynamics are the primary driver of warming off the northern tip of the Antarctic Peninsula following major volcanic eruptions. This warming is a response of the WG to the southward shift and strengthening of the Antarctic westerlies, together with the southward wind anomaly at the WG outflow region. In this region, warm and salty WDW waters, through convective mixing, reaches surface layers. Because of the anomalous wind response, the outflow is weakened and trapped leading to the positive SST anomaly detected.7. The physical mechanism responsible for this surface warming was investigated considering the LM (850–1850) largest eruptions. Even though the LM eruptions are known to be much stronger than Mt. Pinatubo and have different intrinsic characteristics (i.e. intensity), we observed the same robust anomalous warming near the Antarctic Peninsula in the Weddell Sea. In addition, any eruptions occurring in the future could be as strong as the LM ones, and given the robust warming response in the Weddell Sea, we anticipate that such future large eruptions would once again generate significant warming in the region.We quantified the SST perturbation caused by the Mt. Pinatubo 1991 eruption, which showed an anomalous warming response near the Antarctic Peninsula in the Weddell Sea at Yr + 2. Such response was not expected given that the known impact of volcanic eruptions is an overall cooling34, therefore the ocean response is faster.Composite ensemble average for LM eruptions showed ∼0.8 °C SST increase accompanied by positive salinity anomaly (~0.16) developed near the Antarctic Peninsula in the Weddell Sea during the first year after the eruption (Yr + 1). The composite analysis for the simulated LM eruptions presented the same sea surface temperature pattern as after the Mt. Pinatubo eruption (averages from 0.2 to 0.85 °C for different data sets), however it is the largest at Yr + 1, while the Mt. Pinatubo largest response is at Yr + 2. The different year of the largest sea surface response is related to the size of the analyzed eruptions; LM eruptions are much larger than more recent ones55 that the anomalous warm SST response to large volcanic eruptions in the Weddell Sea is due to changes in the ocean dynamics, rather than to the direct radiative forcing. During the first year after the eruption (Yr + 1), a strengthening of 0.03 N m−2 of the prevailing westerlies was observed, which was accompanied by a southward shift of ∼2° of its southernmost extension. At the same time, the meridional component of the wind stress near the northern Antarctic Peninsula changes direction from northward to southward. As a response to the wind changes, the ACC intensifies at its southern limit in about 10%, enhancing mixing off the northern Antarctic Peninsula, together with a ∼16% weakening of the WG subsurface outflow down to ~400 m deep. Consequently, some of the water that was normally exported out of the WG is trapped at the subsurface region near the Antarctic Peninsula in the Weddell Sea. The subsurface water , that is warmer and saltier than the surface water, is brought up to the surface by the enhanced mixing. Because of the anomalous wind this water gets trapped in the Weddell Sea surface outflow region near the Antarctic Peninsula.We suggest within the confines of the CESM-LME model26. The warming response of the Weddell Sea off the Antarctic Peninsula to large volcanic eruptions could impact the ongoing thinning of the Larsen Ice Shelf, through basal melting26 and the intrusion of warm ocean waters18. Considering that Earth System Models suggest that the climate response to eruptions will be amplified in the future17 due to projected increases in ocean stratification, further warming in the Weddell Sea due to volcanic eruptions could enhance the probability of sudden ice shelf collapse. Therefore, despite the global cooling associated with volcanic eruptions, the dynamical response actually enhances the regional response to anthropogenic warming in the Antarctic Peninsula, which highlights the Southern Ocean’s vulnerability25.The results shown here underscore the concern about ocean-induced thinning of ice shelves, that has been accelerating over the past years33 . For the Mt. Pinatubo response all anomalies are calculated relative to the 1980–2005 mean. The anomaly time series is de-trended over the same period, as a way to minimize CO2-induced long-term trend that could skew the results. For the same period (1980–2005), we also examined the average of the CESM-LME ensemble containing only the volcanic forcing, that allows us to isolate the volcanic signal on the SST.Volcanically induced changes in the Southern Ocean Atlantic sector are examined after the 1991 Mt. Pinatubo eruption. For that we restrict the analysis to 1980–2005 period comparing available observational data sets and output from the Last Millennium Ensemble experiment from the Community Earth System Model4. This means that in the Mt. Pinatubo analysis the austral summer of the year of the eruption (1991) consider December from this year and January and February from the next year (1992). For all the other years we used the same approach.For all the analysis we considered only the austral summer (DJF), because of the smaller sea-ice extension in the Weddell Sea during this season. In addition, the maximum response to volcanic activity is in the summer for both hemispheres, which is probably a result of shallow mixed layers at this time of the year, that respond more rapidly to changes in the heat flux30 from the National Oceanic and Atmospheric Administration (NOAA). The product has a spatial resolution of 2° and covers January 1854 up to 2015. Also, the Hadley Centre Sea Ice and Sea Surface Temperature (HadISST) data set31, with 1° latitude-longitude grid from 1870 to 2017. Other data sets used are oceanic reanalysis such as the Simple Ocean Data Assimilation (SODA) reanalysis version 3.4.136, that uses an ocean general circulation model to assimilate available in situ temperature and salinity profiles, also data from satellite, forced by European Centre for Medium-Range Weather Forecasts (ECMWF) ERA-Interim56. The product is a gridded data set of monthly values from 1980 to 2015, at 0.5° horizontal resolution32. The Ocean Reanalysis System 4 (ORAS4) from ECMWF is also compared, with spatial resolution of 1° and monthly data from 1958 to 201711.We utilized SST data from the Extended Reconstructed Sea Surface Temperature version 5 (ERSSTv5)33. The model configuration used the Community Earth System Model version 1.1 with the Community Atmosphere Model version 558. The spatial resolution of the atmosphere and land components is ∼2° and the ocean and sea-ice components use ∼1° spatial resolution and 60 layers as ocean vertical resolution33. There is a total of 30 ensemble members of forced runs for the period between 850 and 2005, which are a combination of full forcing runs evolving in time and runs considering of each forcing individually with all other forcings fixed at values from the year 850. These are volcanic only, solar59, land use61, greenhouse gas, ozone-aerosol62 and orbital only63. The ensemble approach allows improved estimate of the uncertainty from internal variability and modeling process37. The volcanic eruptions are one of the most important forcings from the LM64, CESM-LME uses eruptions from reconstruction version 149. This reconstruction49 accounts a total of 53 ice cores (32 from the Arctic and 22 from Antarctica), composing a comprehensive stratospheric volcanic sulfate mass loading for the past 1500 years49.All results from the Last Millennium  are obtained from the CESM-LME2-induced trends in the full forcing runs. The ensemble average is used to reduce the internal variability65. All anomalies presented in the LM eruptions analysis are calculated relative to the 850–1850 mean. The eruptions were selected according to a previous study that used the same model outputs34. The authors only selected eruptions with a peak aerosol mass mixing ratio greater than 10−8 and classified them in regions34. Here we selected only the tropical and southern eruptions, since both regions have shown to influence the southern extra-tropics ocean34.For the LM eruptions analysis, we consider the output from 10 full forcing and 5 volcanic-only ensemble members. This analysis has been restricted to the period 850–1850, to exclude any COAll seven selected eruptions were averaged together. The year of the eruption is referred as Yr0 in the composite analysis, which means that all 7 eruptions from the 15 ensemble members were averaged. The composite evolution starts in the year before the eruption (Yr-1) and includes the 6 subsequent years (Yr + 1 to Yr + 6). As in the Mt. Pinatubo section, we only analyze austral summer (DJF) results, for that we considered the average of December, from the year of the eruption, January and February, from the subsequent year.34. The set of the composite years  are compared with the full time series from all ensemble members. This is the nonparametric version of the t-test, which only makes the assumption of independence between the time series and equal variance, but not that the data have a known distribution66. We use the closest model grid points from the original historical stations from the SR4/WOCE (SR4 transect from World Ocean Circulation Experiment)54, which captures the subsurface inflow and outflow of Weddell circulation.The significance levels presented are determined according to the Wilcoxon Rank Sum test at 90%Supplementary Information


Sclerotinia sclerotiorum, the pandemic agent of white mold disease, can attack >400 plant species including economically important crops. Monitoring airborne inoculum of S. sclerotiorum in several French cropping areas has shown that viable ascospores are present in the air almost all the time, even when no susceptible crops are nearby. This raises the hypothesis of a distant origin of airborne inoculum. The objective of the present study was to determine the interconnectivity of reservoirs of S. sclerotiorum from distant regions based on networks of air mass movement. Viable airborne inoculum of S. sclerotiorum was collected in four distinct regions of France and 498 strains were genotyped with 16 specific microsatellite markers and compared among the regions. Air mass movements were inferred using the HYSPLIT model and archived meteorological data from the global data assimilation system (GDAS). The results show that up to 700 km could separate collection sites that shared the same haplotypes. There was low or no genetic differentiation between strains collected from the four sites. The rate of aerial connectivity between two sites varied according to the direction considered. The results also show that the aerial connectivity between sites is a better indicator of the probability of the incoming component (PIC) of inoculum at a given site from another one than is geographic distance. We identified the links between specific sites in the trajectories of air masses and we quantified the frequencies at which the directional links occurred as a proof-of-concept for an operational method to assess the arrival of airborne inoculum in a given area from distant origins.Many phytopathogenic fungi are disseminated as spores via the atmosphere from short to long distances. The distance of dissemination determines the extent to which plant diseases can spread and novel genotypes of pathogens can invade new territories. Predictive tools including models that forecast the arrival of spores in areas where susceptible crops are grown can help to more efficiently manage crop health. However, such models are difficult to establish for fungi with broad host ranges because sources of inoculum cannot be readily identified.  Puccinia pathway" in the United States wheat belt, or for wheat yellow rust in China  was assessed by using archived meteorological data providing information about air mass movements.The objective of this study was to establish a method to determine the relationships of reservoirs of Table 1). Airborne propagules were collected using a volumetric sampler called Portable Air Sampler for Agar Plates  with a flow rate of 20 L min−1. Petri plates were filled with a semi-selective medium amended with bromophenol blue (Cichorium intybus L.), in the Center-West region (CW) in a cantaloupe field (Cucumis melo L.) and in the South-West (SW) and North-West regions (NW) in carrot fields (Daucus carota L.).Air sampling was carried out at four distinct regions of France between 2014 and 2016 . For this, single pieces of hyphal tip were excised from the growing margin of a colony after 2 days of incubation on PDA and transferred to fresh medium as described by After exposure, the plates were incubated in the lab at room temperature (ca 22°C) and the presence of lic acid . MyceliaS. sclerotiorum, were spread on PDA to germinate and produce mycelium. Genomic DNA was extracted in 96-well plates from aliquots of 100 mg (fresh weight) of frozen mycelium, following the Dneasy Plant extraction Kit protocol (Qiagen). Sixteen microsatellite markers designed for S. sclerotiorum by Sclerotia, typical mycelial stroma formed by RST values (Unbiased gene diversity (Hnb) and unbiased allelic richness were computed separately for the strains collected in distinct regions with the Genetix software . The numT values .To decompose the composition of aerial samples into strains of local and distant origins we used the following logic:iN =  denote the counts of isolates from strains 1, ..., n collected from site i. Then iN was split into two parts: the part issued from site j  and a part not issued from site j  . Here, we make the crude assumption that for any strain s that was collected in both sites i and j, the corresponding count isN is issued from site j and, reversely, for any strain s that was collected in site i but not in site j, the corresponding count j is not issued from site j. Mathematically, this translates into: iN = jNif − (1 − jf) iN where if is a vector of dimension n whose component s is equal to 1 if strain s was collected in site j; otherwise it equals 0. Thus, if plays the role of a filter and iNif stands for the part in iN issued from site j whereas (1 − jf)iN stands for the part in iN not issued from site j. The specification used here for if implies that all strains in j have an equal propensity to be dispersed from j to i and an equal propensity to establish in i.We let jNif issues from site j can be measured by assuming that this vector of counts is drawn from a multinomial distribution with the vector of probabilityjp = jN/j. We let qj→i denote the multinomial probability computed at jNif with parameter jp. Thereafter, qj→i is called the probability of the incoming component (PIC) in site i from sitej.How likely qj→i was computed by subsampling with replacement k isolates in iN and jN, where k is the minimum sample size across all sites (not only i and j), computing i from site j using 4 times and the average of the 104qj→i.To avoid inconsistencies because of varying sample sizes at different sites, probability We aim to explore the association between (a) the probability that the different components of the population sampled from the air are incoming from elsewhere (PIC) and (b) the connectivity between sites via tropospheric movements. The rationale is that the greater the connectivity, the greater the PIC. This association was assessed with a log-linear regression:logqj→i = α + βCj→i + εj→i where Cj→i measures the directional connectivity from site j to site i, α is the intercept coefficient, β is the slope coefficient and εj→i is random noise.H of the air mass arriving at location ×  and date t (12:00 GMT). We computed the connectivity from site j to site i as the fraction of trajectories arriving at site i and previously going throughout a buffer zone around site j. Here, the connectivity is considered in a multisite context, and can therefore be expressed in the graph theory framework. A graph G =  is characterized by a set V of vertices and a set E of links between vertices, i.e., the edges of the graph. In our application, the vertices correspond to the sampling sites and the edges are weighted by the possibility of pathogen diffusion between two different sites via air mass movements, namely the directional connectivity terms. For our application, we calculated different connectivities corresponding to the 12 months of the year. Thus, each monthly connectivity value was based on approximately 300 (10 years × 30 days) binary variables indicating whether or not an air mass arriving at a given site went through the vicinity of another given site. More precisely, the monthly connectivity for the m-th month of the year from site j to site i was computed as follows:Directional connectivity was inferred by exploiting archived meteorological data provided by the Global Data Assimilation System (GDAS) of NOAA and the software HYSPLIT  that allmkt : k = 1,...,mK} are all dates of the m-th month of the year between 2008-01-01 and 2017-12-31, B  is a circular buffer of radius r around site j, and 1  ∩ B ) is the indicator function equal to 1 if the intersection between the trajectory H  and the buffer B  is not empty; otherwise it equals to 0. In our application, buffer zones were defined as disks of radius 25 km centered on sampling sites, and allowed us to take into account the uncertainty in the reconstruction of air mass trajectories. The use of buffer zones also implies that an assumption of local stability in pathogen composition is made. Thereafter, for the sake of simplicity in the notation, we omit the index m.where {qj→i and Cj→i was fitted to data with a least-squares approach. To achieve a satisfactory statistical power, for each pair  we only used the point  or  corresponding to the maximum connectivity. The significance of the link was assessed via a randomization directional test .Each site iN in site i ∈ {1,...,I} were drawn with the following three-stage procedure.More precisely, pathogen compositions (1)i of exogenous strains satisfies.The proportion τ(2)i = τmaxτ(3)Ci⋅→ = ∑j≠iCj→i denotes the overall connectivity of site i.where (4)i−1)K+1K+KK + 1,…,(i−1)K + K, are randomly generated by simulating K independent values ikU  in the uniform distribution over  and setting γi−1)K+kK+1,…,Ni,(i−1)K+K, are obtained by a multinomial draw with size [(1 − τi) M] and probabilitiesγi−1)K+1K+Ki are obtained by a multinomial draw with size [τiM] and vector of probabilities proportional to Δ, whose element (j − 1) K + k, k = 1,…,K, is equal to zero if j = i, and equal to γj−1)K+kj is sampled in i depends on the relative importance of this strain in j and the connectivity from j to i.Hence, the probability that an endemic strain of I, the number of isolates per site M and the maximum exogenous fraction of pathogen strains at the site level τmax. Then, we carried out two comparisons:Using the empirical simulation model described above, we made several series of 100 simulations with varying values of the number of sites (1)I, M) =   and the situation where  =  ; 1,000 isolates were sampled in both situations. For this comparison we set τmax = 0.2  and K = 10 .We compared the situation where ((2)max = 0.02) and compared two different sampling efforts:  =  and  = . For this comparison, K = 10.Then, we studied the case where the maximum proportion of exogenous strains at the site level is low, namely 2% (τTable 1). A total of 241 different multilocus haplotypes (MLH) were identified, of which 172 were represented by only one strain. Our analyses of dissemination and connectivity were based on the 69 MLH that were represented by at least 2 strains. The most frequent MLH was represented by 20 strains, all coming from the South-West. Furthermore, 18 of the MLH represented by multiple strains were collected from more than one region . The geographic distance between the collection sites of strains sharing the same haplotype was as far as 700 km.All the 16 markers used to characterize the 498 strains were polymorphic with a number of alleles ranging from 2 (for locus 36-4) to 21 (for locus 106-4). Strains collected from the Center-West region showed the highest gene diversity and allelic richness (P < 0.05).Hierarchical AMOVA revealed that 89.2% of the genetic variability was distributed within sampling years, compared to 1.6% within a region and 9.2% among regions. These three factors contributed significantly to total genetic variance (S. sclerotiorum strains with the RST fixation index statistic not significant (P > 0.05) for pairwise combination SW-CW, SW-NW, CW-NW and NW-N . There was evidence of low genetic differentiation between S. sclerotiorum strains with the RST highly significant (P < 0.01) for pairwise combination North/Center-West  and North/South-West . There was also a low genetic differentiation  between S. sclerotiorum strains from the North and the North-West  for the non-corrected dataset.There was no evidence of genetic differentiation between Figure 3 shows the graph of tropospheric connectivity in March that was inferred with HYSPLIT from archived meteorological data over the period 2008–2017. Our approach provides an assessment of the directional links between the sites where S. sclerotiorum has been sampled. Thus, we obtained, for each month of the year, a network with 4 nodes and 12 edges with varying intensities . Directionality in the links can be clearly seen on these graphs. Figure 4 gives a clearer assessment of the variation in the connectivity with time. Despite temporal variation in the directional connectivity between two given sites, we observe stability with respect to the most connected sites  and the less connected sites . The maximum connectivity is about 0.13 between NW and N sites in July . The connectivity NW→N is the largest for 7 months of the year but the connectivity SW→CW and CW→SW are also occasionally the highest ones. The correlation across time between the tropospheric connectivity and the Earth surface distance is displayed in Figure 5. This correlation around -0.75 indicates that the two variables are connected but additional information is included in the tropospheric connectivity, in particular the directionality.Figure 6 provides the result of the log-linear regression between the PIC and the tropospheric connectivity. The regression lines are rather stable across time, reflecting the relative stability of the tropospheric connectivity. They indicate a positive trend in the link between the PIC and tropospheric connectivity, the test of “slope = 0” leading to low p-values for June, September, and October. However, the small number of sites does not allow us to definitely state that this link is clearly founded. The regressions could be influenced by extreme points. As a benchmark, we performed the same analysis between the PIC and the Earth surface distance , which are expected to be negatively correlated. The limitation due to the number of sites also applies here, but the link seems weaker in Figure 7 than in Figure 6, suggesting that tropospheric connectivity is more appropriate to explain the probability of the incoming microbial component.I, M) =  and  = , when the maximum proportion of exogenous strains at the site level is rather high, i.e., τmax = 0.2. Figure 8 shows that the sampling scheme with more sites but less isolates per site leads more systematically to a positive estimate of the slope coefficient in the regression between the connectivity and the log-PIC. Using 10 sites instead of 4 allows, for each repetition, to have 10 × 9/2 = 45 points instead of 4 × 3/2 = 6 points to fit the regression. Obviously, there is a trade-off between the number of sites and the number of samples per site because a weak number of samples per site could lead to inaccurate estimates of PIC values and, therefore, inaccurate estimates of the slope. In addition, from a computational point of view, calculation time increases with the square of the number of sites.First, we compared the sampling schemes characterized by  =  and  = , when the maximum proportion of exogenous strains at the site level is rather low, i.e., τmax = 0.2. Figure 9 shows that using  =  with a 2% maximum proportion of exogenous strains at the site level is not enough to infer the link between connectivity and PIC. However, the power of the analysis tool significantly increases with the number of sampled isolates per site.Second, we compared the sampling schemes characterized by . This directional connectivity corresponds to air mass movements that change with the seasons – a notion that is obvious to anyone who is attentive to the weather as seasons change. The originality of this work is that we identified the links between specific sites that were in the trajectories of the air masses and we quantified the rates at which the different directions of the links occurred.The results also show that aerial connectivity is more informative than geographic distance between two sites to explain the PIC of inoculum at a given site from another one. Aerial connectivity adds information about the direction of potential exchanges via the atmosphere that geographic distance cannot provide. This information is particularly interesting since it makes it possible to identify more precisely the role of each site as a source or sink of spores. It can therefore be hypothesized that isolates with identical haplotypes in two regions are more likely to have been emitted by the one having aerial connectivity directed toward the other (such as NW toward N for example).The fact that aerial connectivity is more informative than geographic distance may be due to the movements of air masses between two sites that are not simply linked to linear distance they travel but also to topography, climate and the global movement of air masses on the terrestrial surface. Taking the example of NW and N regions of the present study: aerial connectivity reflects the general movements of air masses that often enter the French territories by the Atlantic coast and that move inland. In contrast, geographic distance (about 300 km) doesn’t give any information about frequency and direction of aerial exchanges between NW and N. In order for connectivity to be as informative as possible, we calculated it over several years to provide a global and robust vision of the prevailing links between two sites.Studying and predicting airborne routes of plant pathogens is of general interest because atmospheric highways have very few boundaries. Recently, key airborne dispersal routes of rust between several African and Asian countries were studied by The present study describes a method in its preliminary state. We used simulations to investigate how to improve this method and in particular how to optimize the balance between the number of sites considered and the number of strains collected at each site. These simulations revealed that to determine links between sites it is better to have more sites with few strains than few sites with many strains. However, a balance has to be found because an insufficient number of strains per site could lead to an inaccurate estimates of PIC values. This balance has to be considered according to the practical aspects of sampling . These simulations could be performed to establish the experimental design for future studies while taking into account the number of potential sampling sites and the effort needed to isolate and characterize strains of the targeted pathogen.if that implies that all strains in j have an equal propensity to be dispersed from j to i and an equal propensity to establish in i. To weaken this assumption, we tested an alternative specification for the filter that incorporates the possibility for some strains of j to not be dispersed or not be adapted to site i: the filter, denoted by fj→i, is a vector of dimension n whose component s is equal to 1 if strain s was collected both in site j and i; otherwise it is 0. With such a specification, we, however, assume that all the strains in j that can be dispersed to and establish in i have an equal propensity to make it to i. The use of this alternative filter led to different PIC values in the real-case study tackled in this article, but did not yield qualitatively different results. Further investigation should be carried out to consider more relevant filters and obtain more accurate inferences about the long-distance dispersal of pathogens.Moreover, the way the PIC was computed could be tuned to reflect more realistic processes. In this study, the PIC was obtained by introducing a filter 1. Placing trapping devices upstream of the North–West site described in this study, for example, along the French oceanic border could help to identify spores coming from outside the French territory. Information about airborne inoculum potentially arriving in a territory is important to obtain since the traits of exogenous strains may differ from endogenous ones and sometimes may lead to more damaging epidemics. For example, countries where the inoculum originated could have policies concerning the use of fungicides that are different from French policies. In this way, incoming fungal strains may have developed resistance to fungicides that have not yet been deployed in France or that are reserved for use in cases of extreme potential for crop damage. If such strains manage to infect and multiply in France, they may lead to the modification of the endogenous fungal population thereby rendering obsolete the use of certain fungicides. Different cropping systems or climates may also result in more aggressive or more robust strains than endogenous ones. Finally if an incoming fungal population of a given species is more diverse than the local endogenous population, the choice of cultivars might need to be modified otherwise resistance may be overcome by the first arrival of exogenous airborne inoculum. The tools and analytical methods that we have presented here can be further developed to address the impact of aerial inoculum from long distance sources and the capacity to account for this in strategies of crop protection.Knowledge about connectivity (its rate and its direction between sites) at a regional and global scale is useful for establishing strategies to set up networks of epidemio-surveillance for wind-dispersed fungi. Practically, spore monitoring devices could be placed at existing sites of meteorological networks, for example, or on elevated buildings , or at stations for measurement of flux of atmospheric particlesCL carried out microsatellite genotyping and genetic data analysis. CM and SS developed the initial idea of tropospheric interconnectivity. SS and MC developed and conducted the calculations concerning tropospheric interconnectivity and pathogen composition decomposition. CL coordinated the writing of the manuscript. CL, CM, MC, and SS wrote the manuscript. All authors reviewed the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Corydalis cava (Papaveraceae). Inhibitory activities of (+)-thalictricavine (1) and (+)-canadine (2) on human acetylcholinesterase (hAChE) and butyrylcholinesterase (hBChE) were evaluated with the Ellman’s spectrophotometric method. Molecular modeling was used to inspect the binding mode of compounds into the active site pocket of hAChE. The possible permeability of 1 and 2 through the blood–brain barrier (BBB) was predicted by the parallel artificial permeation assay (PAMPA) and logBB calculation. In vitro, 1 and 2 were found to be selective hAChE inhibitors with IC50 values of 0.38 ± 0.05 µM and 0.70 ± 0.07 µM, respectively, but against hBChE were considered inactive . Furthermore, both alkaloids demonstrated a competitive-type pattern of hAChE inhibition and bind, most probably, in the same AChE sub-site as its substrate. In silico docking experiments allowed us to confirm their binding poses into the active center of hAChE. Based on the PAMPA and logBB calculation, 2 is potentially centrally active, but for 1 BBB crossing is limited. In conclusion, 1 and 2 appear as potential lead compounds for the treatment of Alzheimer’s disease.In recent studies, several alkaloids acting as cholinesterase inhibitors were isolated from  Alzheimer’s disease (AD) is an age-related, progressive, neurodegenerative disorder, with onset in humans usually over the age of 65. The disease is characterized by cognitive impairment, a variety of behavioral symptoms, and restrictions in activities of daily living. The prevalence of AD increases exponentially between the ages of 65–85, doubling with every five-year age group . Two chaN-methyl-d-aspartate receptors) [The portfolio of ChEIs is limited; the U.S. Food and Drug Administration has approved donepezil, galanthamine  and rivastigmine to treat the symptoms of mild to moderate AD . From thceptors) ,7,12,13.Corydalis cava (L.) Schweigg. & Körte [Potent AChEIs have been found among natural products ,15,16, w & Körte ,18, whic & Körte .One of the principal approaches in designing of novel and effective AChEIs with a broad biological profile is associated with computational methods in the so-called computer-aided drug design . In siliFinally, if compounds are to be considered as drug candidates for AD treatment, it is necessary to determine their ability to cross the blood–brain barrier (BBB), which is a crucial aspect of all potential substances for the treatment of CNS-related disorders. The BBB is a protective system composed of endothelial cells with tight junctions that prevent substances going from blood into the brain ,28,29. MhAChE and hBChE) by the protoberberine alkaloids (+)-thalictricavine (1) and (+)-canadine (2), previously isolated from C. cava  was subsequently inspected by molecular modeling. The obtained data from the in silico assay were compared with Gal, the selective AChE inhibitor used in current AD therapy. Furthermore, the ability of alkaloids 1 and 2 to cross the BBB was predicted by the PAMPA to consider these compounds either as potential drugs or as lead scaffolds for the development of new potential substances for AD treatment.This study is focused on the elucidation of the inhibition of human cholinesterases  was calculated for both compounds , room temperature (25 °C), and a source of AChE (red blood cell ghosts) with unknown enzyme activity. Interestingly, the (+)-form of these tetrahydroprotoberberines is responsible for their significant hAChE inhibition: the (−)-form of 2 is considered inactive (hAChE IC50 = 637 ± 83 µM) [1 slightly increases the potency of AChE inhibition as compared with 2  against selected human carcinoma and non-carcinoma cells [2 possess low acute toxicity, their LD50 values in mice by oral administration were 940 and > 2000 mg/kg, respectively [1 and 2 may appear as potential lead compounds for the treatment of Alzheimer’s disease.Both compounds demonstrated significant and more selective inhibition of ar range . The cond with 2 . As menthibition . Additioma cells . Furtherectively . Thus, phAChE inhibition was elucidated for 1 and 2 using a kinetic study in order to gain information about the mode of inhibition and the binding site of the isolated alkaloids. Recombinant hAChE was used in this kinetic analysis of 1 and 2. The mechanism of the inhibition process was examined by recording the set of substrate concentration—enzyme velocity curves in the presence of various concentrations of compounds 1 and 2. The type of the inhibition mechanism was clarified by a nonlinear regression analysis. The results computed for each model type of inhibition  were compared with the sum-of-squares F-test. The analysis confirmed a competitive type of inhibition (p ˂ 0.05) for both compounds, consistent with the graphical representation in hAChE substrate. With increasing concentration of inhibitors, the apparent Vmax remained unchanged and Km increased. A Ki value of 174.3 ± 56.9 nM was estimated by the nonlinear regression analysis for 1 and 128.9 ± 44.0 nM for 2.The mode of interaction of the 1 and 2 was explored by molecular modeling in order to reveal the differences in their binding modes to the hAChE active site (Protein Data Bank (PDB) ID: 4EY6). 1 adopted a binding pose in the proximity of the catalytic anionic site (CAS) of hAChE near the catalytic triad residues (1 (2.6 Å). In addition, Glu202, as well as His447, are also implicated in ligand anchoring via hydrophobic van der Waals forces. The protonated nitrogen of the 1,2,3,4-tetrahydroisoquinoline heterocycle of 1 provided another hydrogen contact to the hydroxyl of Tyr341 (3.5 Å). Moreover, Tyr341 (3.8 Å), as well as Phe297 (4.4 Å) face the aromatic region of the 1,2,3,4-tetrahydroisoquinoline moiety in parallel and T-shaped π-π interactions, respectively. Several other hydrophobic interactions can be observed in the hAChE-1 complex including π-alkyl contacts with Tyr337, Tyr341, and Trp86. Specific π-sigma interactions also contribute to ligand anchoring, as established between Tyr341 and Phe338 and the methyl appendage of 1.The spatial conformation of residues A,B. Acco2 in the hAChE active site was observed  or a carbon-hydrogen bond (Glu202).On the contrary, a 180° rotated accommodation of observed C,D. ThishAChE complex [1 and 2 complexed with hAChE, Tyr337 is implicated only in hydrophobic interactions. Similarly, Glu202 anchored the Gal hydroxyl group via a hydrogen bond, whereas this catalytic triad residue enabled hydrophobic contact in the case of 1 and 2. Additionally, the limitation of the computational methodology using Autodock Vina makes it more difficult to anticipate the distribution of structural waters in hAChE complexed with either 1 or 2 [hAChE active site. Given the subtle difference in the in vitro results for 1 and 2, the structure similarities, together with the close calculated binding affinities  it cannot be established which of these two ligands better fits into the enzyme active site. Last, but not least, both ligand spatial orientations provided an interesting platform for synthetic modifications of these two ligands in order to escalate their inhibition potency.In both, the docking simulation successfully correlated well with the crystallographic data, as found for the Gal- complex . Howeverr 1 or 2 . It is w1 and 2 to penetrate into the brain, the PAMPA-BBB method was used, which employed a brain lipid porcine membrane [Pe) of 1 and 2 through the lipid membrane extract was determined, together with nine commercial drugs used in an experiment validation , while a 96-well plate UV reader  was used for the UV measurements of the PAMPA assay.The spectrophotometric readings for the hAChE; EC 3.1.1.7), human plasma butyrylcholinesterase , 5,5′-dithiobis(2-nitrobenzoic acid) , phosphate buffer , dimethyl sulfoxide (DMSO), acetylthiocholine (ATCh), and butyrylthiocholine (BTCh) were purchased from Sigma-Aldrich . Polystyrene Nunc 96-well microplates with flat bottom shape  were utilized for measuring purposes of cholinesterase assays and the kinetic inhibition study. (+)-Thalictricavine (1) and (+)-canadine (2) (≥98% by 1H-NMR spectroscopy) were previously isolated from C. cava [Human recombinant acetylcholinesterase , phosphate buffer saline solution at pH 7.4 (PBS), ethanol, and dodecane were purchased from Sigma, Acros Organics, Merck), Aldrich, or Fluka . The porcine polar brain lipid was from Avanti Polar Lipids . The donor plate was a 96-well filtrate plate (Multiscreen IP sterile plate PDVF (polyvinylidene fluoride) membrane, pore size 0.45 μM) and the acceptor plate an indented 96-well plate (Multiscreen), purchased from Millipore. Filter PVDF membrane units  were purchased from Symta .hAChE and hBChE inhibitory activity of the tested compounds was determined using a modified Ellman’s method [2PO4/K2HPO4 buffer, pH 7.4. Enzyme solutions were prepared at an activity of 2.0 units/mL in 2 mL aliquots. The assay medium (100 µL) consisted of 40 µL of 0.1 M phosphate buffer (pH 7.4), 20 µL of 0.01 M DTNB, 10 µL of enzyme, and 20 µL of 0.01 M substrate (ATCh iodide solution). Assay solutions with the inhibitor  were preincubated for 5 min . The reaction started by addition of 20 µL of the substrate . The enzyme activity was determined by measuring the increase in absorbance at 412 nm at 37 °C at 2 min intervals. Each concentration was assayed in triplicate. The obtained data were used to compute percentage of inhibition (I):iA indicates an absorbance change provided by the cholinesterase exposed to the cholinesterase inhibitor and ΔA0 indicates an absorbance change caused by the intact enzyme (the phosphate buffer was used instead of the inhibitor solution). Inhibition potency of tested compounds was expressed as an IC50 value (concentration of an inhibitor that causes 50% cholinesterase inhibition). Calculations were performed using software Microsoft Excel  and GraphPad Prism version 6.07 for Windows, GraphPad Software, San Diego, CA, USA (www.graphpad.com).The s method . All thehAChE inhibition was performed by using Ellman’s method (described above) [max and Km values, respectively, of the Michaelis-Menten kinetics and Ki were calculated by non-linear regression from the substrate velocity curves. Linear regression was used for calculation of Lineweaver-Burk plots. All calculations were performed using the GraphPad Prism software.The kinetic study of d above) . For thewww.pdb.org) the model of hAChE  was downloaded and prepared for flexible molecular docking by Molecular Graphics Laboratory (MGL) Tools utilities [hAChE. The following xyz coordinates of the grid box centers were applied: hAChE . The studied ligands were firstly drawn in HyperChem 8.0, then manually protonated as suggested by MarvinSketch 6.2.0. software (http://www.chemaxon.com), geometrically optimized by the semi-empirical quantum-chemistry PM3 method and stored as pdb files. The structures of the ligands were processed for docking in a similar way as the above mentioned flexible parts of the receptor by AutoDock Tools 1.5.4 program. Molecular docking was carried out in the AutoDock Vina 1.1.2 program utilizing the computer resources of the Czech National Grid Infrastructure MetaCentrum. The search algorithm of AutoDock Vina efficiently combines a Markov chain Monte Carlo like method for the global search and a Broyden-Fletcher-Goldfarb-Shano gradient approach for the local search [From the online Protein Data Bank (PDB) database , and, after 5 min, 180 μL of each compound solution was added. 1 to 2 mg of each test compound to be assayed for its ability to pass the brain barrier was dissolved in 1500 μL of EtOH and 3500 μL of PBS (pH 7.4) buffer, filtered, and then added to the donor 96-well plate. Then, the donor plate was carefully put on the acceptor plate to form a “sandwich”, which was left undisturbed for 2 h 45 min at 25 °C. During this time, the compounds diffused from the donor plate through the brain lipid membrane into the acceptor plate. After incubation, the donor plate was removed. The UV plate reader determined the concentration of compounds and clinically used drugs in the acceptor and donor wells.Prediction of brain penetration was evaluated using the PAMPA assay . Test co1 and 2 have promising and selective hAChE inhibition, which acts via a competitive inhibition mechanism. Docking experiments allowed us to estimate their binding poses into the active center of hAChE. Based on the PAMPA-BBB assay and logBB calculation, 2 is able to cross the BBB by passive permeation; however, for 1, BBB crossing is limited. Considering all of the aforementioned, compounds 1 and 2 appear as either potential drugs or lead structures in the development of potent drugs for the treatment of AD.Compounds


In the present work, its diphenolase activity was inhibited at pH 6.8 by a series of structurally related polyoxotungstates (POTs) of the α-Keggin archetype, exhibiting the general formula [Xn+W12O40](8−n)− in order to elucidate charge-dependent activity correlations. Kinetic data were obtained from the dopachrome assay and 183W NMR was applied to obtain crucial insights into the actual Keggin POT speciation in solution, facilitating a straightforward assignment of inhibition effects to the identified POT species. While [PW12O40]3− was completely hydrolyzed to its moderately active lacunary form Hx[PW11O39](7−x)− (Ki = 25.6 mM), [SiW12O40]4− showed the most pronounced inhibition effects with a Ki of 4.7 mM despite of partial hydrolysis to its ineffective lacunary form Hx[SiW11O39](8−x)−. More negative Keggin cluster charges of 5− and 6− generally resulted in preclusion of inhibitory efficacy as well as hydrolysis, but with the Ni-substituted cluster [PW11O39{Ni(H2O)}]5− enzymatic inhibition was clearly restored (Ki = 9.7 mM). The inhibitory capacity of the structurally intact Keggin POTs was found to be inversely correlated to their net charge. The here applied speciation strategy is of utmost importance for any biological POM application to identify the actually active POM species.Mushroom tyrosinase  The PPO subgroup of catechol oxidases exhibits only diphenolase activity , whereas the family of tyrosinases is capable of both monophenolase  and diphenolase activities4. PPOs share a common active site architecture comprising a type III dinuclear copper center in tight coordination by six conserved histidine residues. As the active site of PPOs is located on the protein surface5, it is well accessible for substrates and inhibitors. Among other substrate-related compounds, the most potent inhibitors are hydroquinone6, tropolone7 and kojic acid8  in the µM range. In this series, kojic acid is the strongest and best characterized inhibitor, which is known to act in a competitive inhibition mode e.g. for “mushroom tyrosinase”8 or a mixed-type inhibition mode e.g. for Bacillus megaterium tyrosinase6. For its negligible lag phase and generally faster reaction velocity, the diphenolase activity of tyrosinases is usually assayed in kinetic studies. Importantly, the inhibition mode still depends on the substrate9, for which L-DOPA is most often reported in PPO inhibition studies, including those with kojic acid cited above.Polyphenol oxidases (PPOs) constitute an important family of enzymes occurring throughout the eukaryotic and prokaryotic kingdomsAgaricus bisporus (abPPO) is predominantly used in PPO applications due to its high commercial availability as an enzymatic extract (“mushroom tyrosinase”) from mushroom fruiting bodies. Recently, a protocol for recombinant expression of abPPO4 in Escherichia coli and purification to homogeneity was established10, which was followed to prepare pure abPPO4 for this study. AbPPO4 has been thoroughly investigated and crystallized after purification both from the natural source13 and after recombinant expression10, and therefore it is one of the few enzymes with confirmed identity for its native and recombinant form.Tyrosinase PPO from 14. For their versatility in size, charge and shape as well as a wide range of possible modifications, POMs have been receiving considerable attention for biological application in the past decades, including enzyme inhibition16, cytotoxicity studies investigating antibacterial17 and anti-tumour activity18 and even protein crystallization20. POMs have been recognized for their versatile binding behaviour towards biomolecules, especially proteins, driven by polar and non-polar interactions.Polyoxometalates (POMs) are negatively charged metal oxide clusters built up from interconnected polyhedran+M12O40](8−n)− was chosen for this study. It comprises a relatively small spherical structure (ca. 10.4 Å in diameter) with well-established synthetic protocols, but at the same time remarkable compositional versatility. Thus, it is an ideal candidate for systematic variation of structural parameters. The Keggin archetype can be envisaged as an {M12O36} cage (addenda atom M: Mo(VI) or W(VI)) with zero net charge encapsulating an [Xn+O4](8−n)− tetrahedron 21. Therein, each addenda atom is linked to 6 oxygen atoms  resulting in a distorted octahedral geometry. The most stable Keggin isoform is the α-isomer with tetrahedral symmetry, and the {M12O36} shell can be grouped into four {M3O13} triads. Rotation of one, two, three or all four triads about 60° leads to the energetically less favored β-, γ-, δ- and ε-isomers22 3[PVW12O40], (HGly)4[SiIVW12O40]24 (Gly = glycine), or the monosubstituted Keggin derivatives with proposed formulas Na7[PVMo11CuIO40]25, Na8[SiIVW11CoIIO40]26 and Na6[PVMo11FeIIO40]27. However, each of these investigations was limited to only one or two different compounds, and parameters such as counterions, cluster charge and addenda atom substitution were varied in an arbitrary way, thereby obscuring possible trends in the structure-activity relationship of the applied POMs. In contradiction to their structural analogy, for (HGly)3[PW12O40] and (HGly)4[SiW12O40], completely different inhibition types and highly differing inhibitory capacities were determined24. The same discrepancies were reported for the two Keggin-POMs substituted with CuI 25 or FeII 27.The inhibition capacity of a few Keggin POMs against the enzymatic activity of PPOs has been addressed before in non-coherent and incommensurable studies investigating the polyanions in their acid forms H28.The Keggin clusters applied in this study were selected for the criteria of exhaustive structural verification, good synthetic accessibility and sufficient stability in aqueous solution. We entirely focussed on the α-Keggin isomer which can be prepared with a large number of different heteroatoms and shows unique structural stability even upon reversible reduction with up to 18 electrons+ or K+ counterions. W was chosen over Mo due to its remarkable redox stability. The tested Keggin POTs , K4[SiW12O40]29 ([SiW12]4−), K5[BW12O40]30 ([BW12]5−), Na5[AlW12O40]31 ([AlW12]5−), Na6[H2W12O40]32 ([H2W12]6−), Na6[BeW12O40]33 ([BeW12]6−), K5[PW11O39{Ni(H2O)}]34 ([PW11Ni]5−). The lacunary anions K7[PW11O39]35, K8[SiW11O39]36 and Na9[AlW11O39]31 were prepared as control compounds to account for the potential hydrolysis of the corresponding Keggin POTs. Metatungstate [H2W12]6− is the most common Keggin cluster with net charge 6−. As an isopolytungstate, it lacks the typical central atom with tetrahedral coordination and carries two protons instead, therefore showing a slightly distorted structure with respect to the other Keggin clusters and a questionable hydrolytic stability at pH > 6.437. For comparison, we investigated [BeW12]6− with higher hydrolytic stability up to pH 9.5. In the substituted Keggin anion  addenda atoms is exchanged by Ni(II) in order to possibly tune the POT’s protein affinity. The coordination sphere of the light transition metal is completed by one labile water ligand  results in a final cluster net charge of 5−, which is well comparable to [BW12]5− and [AlW12]5−. Anions with central atoms comprising an unusually low charge (+1 in [CuW12O40]7− 38) have been reported only in solution and characterized as a transient oxidation state highly sensitive to re-oxidation. Clusters with extraordinarily highly charged central atoms (+6 in [SW12O40]2− 39 and [SeW12O40]2− 40) have been obtained exclusively from non-aqueous media as counter anions for other metal complexes supporting their structural framework. Therefore they were excluded from our study due to doubtful synthetic accessibility and stability in aqueous solution.Seven different α-Keggin polyoxotungstates (POTs) were synthesized according to published procedures, with net charges in the range from 6− to 3−, and ensured high aqueous solubility with Nacf. Fig. , which c33  Keggin anions that most biological applications have focussed on up to now (e.g.42), without careful speciation of POM clusters under the applied physiological buffer conditions. Here, we present the first systematic investigation of abPPO4 inhibition towards its diphenolase activity through POT Keggin anions, including the careful speciation of respective POT clusters at physiological pH 6.8 by 183W-NMR and ESI-MS analysis to address cluster species responsible for interaction with the enzyme in solution. Thereby, we intended to derive a possible correlation between Keggin POT properties and the observed inhibitory effect against abPPO4 diphenolase activity, focussing on the overall surface charge conveyed by different central atoms as a systematically varied parameter.The hydrolytic stability of Keggin POT anions correlates with their net charge, and one single additional negative charge can increase the cluster stability by 2 to 3 pH units33 Fig. . TherefoAbPPO4 was prepared in its activated state according to the published procedure by Pretzler et al.10. ), which accounted for all potential inhibition types and allowed evaluation of inhibition constants Ki and α-parameters  salt . The strongly basic Na2[WO4] stock solution was adjusted for pH 6.8 with HCl. The resulting activity plot  the dopachrome assay was repeated at five different substrate concentrations and three different inhibitor concentrations, respectively (SI section 3.2).The type of enzymatic inhibition is commonly investigated by linear plots according to Lineweaver-BurkKi on the abscissa, as found for competitive inhibitors. The obtained Ki values of about 4.5 µM perfectly matched the data reported for kojic acid8, thus validating the here performed kinetic evaluations as a reliable experimental approach.For the kojic acid control, the Lineweaver-Burk lines Fig.  convergeKi, thus a revalidation of the Ki value determined from the activity plots before . The Dixon lines did not intersect in one common point when fitted to straight lines, which can be explained by the mixed-type behaviour44, but still allowed for Ki estimation as the average of all intersection points between the lines. The Ki values extracted by three different methods . For the sake of comparison, the pH of the [PW12]3− stock solution was re-adjusted to 6.8 prior to enzymatic measurements. The 183W NMR analysis of the [PW12]3− cluster at pH 1.5  for preparation of a 20 mM POT stock, Na1.5 Fig.  and 6.8 1.5 Fig.  unambigua51 Fig. . The pre]8− Fig.  and [PW1]7− Fig.  served a12O40]3−, hydrolytic stability at different pH values has been addressed before by 31P NMR spectroscopy and ESI mass spectrometry, with the clear result that the lacunary cluster [PW11O39]7− is the only prevailing species in the neutral pH range53, which is in perfect agreement with the NMR-based speciation in this study.For 6−, which was revealed by 27Al NMR  atom, this compound exhibited weak inhibition effects despite the overall high charge.Based on the observed lack of inhibitory capacity exhibited by highly charged Keggin POMs such as NMR Fig.  and 183Wses Fig. . Featuri183W NMR speciation of the [WO4]2− solution at pH 6.8 revealed the heptatungstate anion H[W7O24]5− 56 as the only present species to evoke the exhibited inhibitory effect  counterbalanced by their pronounced hydrolysis. Although the POT anions with higher charges 5− and 6− even effected an increase in enzymatic activity, inhibitory activity was restored with the cluster [PW11Ni]5− equipped with a nickel center for increased protein affinity.The charge-dependent enzymatic inhibition behaviour of the α-Keggin POT archetype against zed Fig. , revealiAll chemicals have been purchased from Sigma-Aldrich  and Carl-Roth  and were at least of analytical grade. Synthetic details and spectroscopic data along with the kinetic curves are presented in SI.et al.10. Briefly, a pGEX vector  was used to equip the abPPO4 gene with an N-terminal glutathione-S-transferase tag. The encoded fusion protein was expressed in E. coli BL21(DE3) in the auto-inducing medium ZYM-5052 in 2 L shaking flasks. Cells were cultivated at 20 °C for approximately 20 h, supplemented with 0.5 mM CuSO4 and grown for 20 more hours. The cell pellet was harvested by centrifugation, resuspended in a Tris/HCl lysis buffer with protease inhibitors and EDTA and disrupted by homogenization through a French pressure cell. After clarification, the supernatant containing the fusion protein was applied to a GSTrap FF column  and eluted in pure form with reduced glutathione, followed by digestion with recombinant HRV 3 C protease in order to cleave off the GST tag. A second purification step employing GSTrap yielded pure latent abPPO4 enzyme in the flow-through. The latent enzyme was treated with Proteinase K to specifically remove the C-terminal part for permanent activation and purified by size-exclusion chromatography using a Tricorn Superdex 200 16/60 column  in reaction buffer (50 mM Na-citrate pH 6.8). The combined protein fractions were concentrated and used for the kinetic analyses presented here 4.8Na1.2[BeW12O40]33 and NaClO4 in acetonitrile.Na5[BW12O40]·18 H2O was prepared according to57 and re-crystallized three times from water at 60 °C. Mono-crystalline needle-shaped crystals of K5[BW12O40]·18 H2O were identified by checking the unit cell parameters  on a Bruker D8 Venture.K3[PW12O40]·3 H2O was used instead of H3[PW12O40] as a starting material for the synthesis of K5[PW11NiO40H2]·11 H2O58.NaAll chemicals were purchased from Sigma-Aldrich (Austria) and used without further purification. The Keggin type polytungstates were synthesized according to the reported procedures Table , which wThe exact amount of crystal water in all Keggin POTs was determined gravimetrically by calcination at 200 °C for 10 hours.5[BW12O40] was additionally analyzed by IR measurements on a Bruker Vertex 70 IR Spectrometer equipped with a single-reflection diamond-ATR unit. The obtained spectrum shows the characteristic bands for the α-Keggin polyoxotungstate cluster57 and was used as a reference spectrum for all the other POT compounds yielding highly similar spectra  in 50 mM Na-citrate pH 6.8 if not stated otherwise. 183W NMR spectra were recorded with a Bruker FT-NMR spectrometer Avance Neo 500 MHz in 10 mm tubes for a total experiment time of ca. 60 hours, using standard pulse programs at 20.836 MHz and a 63° flip angle with 1 s of relaxation delay; the temperature was kept at 25 °C. Chemical shifts were measured relative to external 1 M Na2WO4. For [PW12]3−, [PW11]7− and [PW12Ni]5−, additional 31P NMR spectra were taken, structures of [AlW12]5−, [AlW11]9− and [BeW12]6− were confirmed by 27Al NMR or 9Be NMR spectroscopy, respectively. 27Al, 9Be and 31P NMR spectra were recorded at 130.368, 70.273 and 202.53 MHz, respectively. Chemical shifts are given relative to Al(NO3)3, BeSO4 or 85% H3PO4.[AlW12]5−, an aliquot was taken from the reaction solution and checked for the isomerisation state of the Keggin anion by 27Al NMR spectroscopy. The obtained spectrum }]6−, abbreviated as [AlW11Al]6−, which is characterized by two signals at 8.7 and 74.6 ppm31. The additional signal at 72.0 ppm hinting towards the intact anion [AlW12]5− is unexpected31, as the corresponding 183W NMR spectrum .In the NMR Fig. . The red[WO4]2−, 183W NMR was performed in reaction buffer pH 6.8, which had to be readjusted by a few drops of 5 M HCl to compensate for the marked basicity of [WO4]2−. The 183W NMR spectrum  to reach a final concentration of 20 mM. Surprisingly, reacidification to pH 6.8 led to a cluster rearrangement to the monosubstituted anion [AlW11O39{Al(H2O)}]6−, as also confirmed by 27Al NMR , the low excitation energy of many Ni2+ complexes to the next electronic state leads to a significant portion of [PW11Ni]5− being present in a pseudo-paramagnetic state. This results in additional signal splitting by electronic coupling as well as pronounced line-broadening with concomitantly reduced NMR signal intensities, sometimes below the observation limit. Therefore, to confirm the presence of intact [PW11Ni]5−, ESI-MS analysis was also carried out.In order to identify the tungstate species responsible for the inhibitory effect observed with the orthotungstate solution rum Fig.  reveals see Figs –S14 showNMR Fig. . In the cf. Fig.  and 183WNMR Fig.  spectra [PW11Ni]5− was verified by ESI-MS with an ESI–Qq–oaRTOF  calibrated with an ESI Tuning Mix , and data was evaluated by the corresponding Bruker Daltonics Data Analysis software. Spectra were taken in the m/z range from 50 to 1900 with a resolution <5 ppm, covering low m/z polytungstate anions (resulting from cluster decomposition in the harsh ESI ionisation conditions) and the specific signals for the intact Keggin cluster as adducts with various cations present in the solution. Negative-ion mode with a capillary voltage of 4500 V was applied for the measurements, and they were conducted in a 1:1 (v/v) H2O/ACN mixture.The substituted Keggin cluster v ([I] = 0] and vapp ([I] = 0.2–14 mM) were determined from UV absorption measurements at 475 nm using the dopachrome assay. Each reaction setup contained a fixed amount of enzyme (1 µg abPPO4) in 1 mL in a plastic cuvette and the reaction buffer was 50 mM Na-citrate pH 6.8. POT stock solutions were prepared in 20 mM concentration in reaction buffer and adjusted for pH 6.8 with NaOH, if necessary. The reaction rates were extracted from the obtained absorption curves as the slope of linear fits of the steepest curve sections close to the start of the reaction over a time-range of 30 s and were determined in triplicates. The relative enzymatic activity dependent on the inhibitor concentration was fitted to a suitable hyperbolic expression (see SI for details). For determination of inhibition modes, concentration-dependent activity measurements were performed at 5 different L-DOPA concentrations  for three different inhibitor concentrations, including [I] = 0 for the case of no inhibition, an [I] value selected to be close to the Ki (as determined from the activity plots) and an additional, usually higher concentration [I]. The dopachrome assay was performed exactly as described in section 3.1.1. This time, the observed reaction velocities vapp (in µmol/min) were calculated from the extracted reaction rates (in 1/s) using the extinction coefficient of dopachrome ε = 3700 M−1 cm−1 62. The so obtained data was plotted according to Lineweaver-Burk, and the slopes and ordinate intercepts of these graphs were used to extract inhibition parameters63.For the activity plots shown here, the ratios of reaction rates Supplementary Information


Ice accretion is detrimental to numerous industries, including infrastructure, power generation, and aviation applications. Currently, some of the leading de-icing technologies utilize a heating source coupled with a superhydrophobic surface. This superhydrophobic surface reduces the power consumption by the heating element. Further power consumption reduction in these systems can be achieved through an increase in passive heat generation through absorption of solar radiation. In this work, a superhydrophobic surface with increased solar radiation absorption is proposed and characterized. An existing icephobic surface based on a polytetrafluoroethylene (PTFE) microstructure was modified through the addition of graphite microparticles. The proposed surface maintains hydrophobic performance nearly identical to the original superhydrophobic coating as demonstrated by contact and roll-off angles within 2.5% of the original. The proposed graphite coating also has an absorptivity coefficient under exposure to solar radiation 35% greater than typical PTFE-based coatings. The proposed coating was subsequently tested in an icing wind tunnel, and showed an 8.5% and 50% decrease in melting time for rime and glaze ice conditions, respectively. Ice accumulation causes irreversible damage in systems ranging from ships, heat pumps, civil infrastructure, and aircraft ,2,3,4,5.The most common of these systems attempt to reduce ice adhesion and accumulation using mechanical and electrothermal techniques ,7. Such Further energy savings can be achieved through the increased absorption of solar energy. A substantial number of superhydrophobic coatings, especially those used in de-icing systems, reflect most radiation in the visible range ,12. An iIn this work, a superhydrophobic coating was created through modification of an existing PTFE coating . The comThe ingredients of the coating were mixed together and sonicated to ensure even distribution throughout the mixture. The mixture was then sprayed onto bare aluminum samples which had been sanded to remove the oxide layer. Point measurements of the coating thicknesses were taken at 5 mm increments on the aluminum samples using an Oxford Instruments thickness gauge from Abingdon, England. The average thickness for both coatings was 11.2 μm. The minimum coating thickness was 10 μm and the maximum was 13 μm.T is the temperature of the irradiation source.The absorptivity of the graphite coating was analyzed using a HORIBA FluoroMax-4 spectrometer from Kyoto, Japan. 5 cm × 5 cm aluminum samples were spray coated and allowed to dry at 25 °C for 24 h. The samples were then analyzed with a synchronous test which directly measured the reflectance of the sample from 350 nm to 1150 nm, which encompasses the entire visible range, as well as a large portion of the infrared range where solar irradiance is high. This test was repeated 10 times for 3 aluminum samples in order to reduce uncertainty in the measurement. The local material property absorptivity could be used to calculate an overall absorptivity for the coating when exposed to solar radiation using Equation (1):T depend on material properties, so the ratio of overallα is equal to the ratio of the irradiative heat flux to a give surface. This relationship is shown in Equation (3).Neither Due to various static and dynamic phenomena which impact hydrophobicity, characterization had to be done through analysis of both roll-off and contact angles . Both ofThe roll-off angle is a dynamic characterization which measures the angle to which a surface must be tilted for a droplet to roll. It is an indicator of the minimum force required to remove a droplet from the surface. In the characterization, the sample plate was rotated in increments of 1 degree until the droplet began to roll. A lower roll-off angle indicates increased hydrophobicity. A schematic of the roll-off angle is shown in The contact and roll-off angle tests were performed at 20 °C. Because both contact and roll-off angle tests were used to measure hydrophobic performance, the droplets did not need to be supercooled, as Morita et al. have demonstrated that temperature has negligible impact on the hydrophobicity of a surface . For watIcing wind tunnel tests were run in order to test the performance of the coatings in environmental icing conditions. Coated cylinders were placed in the Kanagawa Institute of Technology icing wind tunnel. These cylinders were 25 mm in diameter and 50 mm long. An example of a model with both coatings can be seen in Each coated cylinder was rotated at 15 rpm in supercooled airflow in order to create a uniform sheet of ice over the coated surface. The flow exposure lasted for 3 min, and the parameters of the flow, including temperature, flow velocity, relative humidity, Liquid Water Content (LWC), and Median Volume Diameter (MVD) were varied to generate different forms of environmental icing. LWC is a measure of the mass of water present in the airflow, and MVD is the median diameter of the liquid droplets suspended in the airflow. Both of these values were adjusted by varying the nozzle water and air pressure. The nozzle pressures were calibrated to produce specific MVD and LWC values before testing using a multiple rotating cylinders calibration method . The varTo be certain that the ice accumulation was constant across different tests, the models were weighed before and after being coated in ice. After weighing, the coated samples were returned to the wind tunnel and exposed to a 50 W MR16 halogen lamp with a color temperature of 3000 K at a distance of 10 cm to simulate solar radiation. The spectrum of the halogen irradiation as compared to solar irradiation is shown in Intensity from the halogen bulb peaks much later than solar radiation. However, the flatter shape of the halogen spectrum means that a substantial portion of the irradiative power is present in the visible range, where the greatest difference between the absorption spectra of the coatings is expected. Therefore, the halogen light source is able to demonstrate a difference in overall absorptivity, and thus heat absorption, between the two coatings. The cylinders were again rotated at 15 rpm to ensure an even exposure to radiation. The amount of time required for ice to be fully removed from the surface was measured, and then divided by the measured ice mass. The setup of both the flow and radiation portions of the test are shown in −3, while the graphite coating has a local absorptivity of 0.857. As described in Equation (1), the spectra were multiplied by the solar and halogen irradiation intensity and integrated to find an overall absorptivity coefficient. These coefficients are shown in The data collected from the spectrometer is shown in The overall absorptivity coefficient for the graphite coating was 35% greater than that of the PTFE coating under exposure to typical solar radiation, and 9.3% greater under exposure to the halogen light source. These increases in absorptivity correspond to identical increases in heat absorbed due to irradiation. The average contact and roll-off angle measurements for both coatings, compared to a perfectly hydrophobic surface are shown in The coatings show identical hydrophobic performance within uncertainty. Based on qualitative observations during the de-icing process, the ice sheet on the graphite coating melted at the base layer of the coating, and was removed in a single, uniform sheet. However, the ice sheet on the PTFE coating melted more from the outside, and in pieces, requiring substantially longer for the ice to be completely removed. The ice sheets after removal from the models are shown in The accumulated ice mass on the surface of the models after exposure to supercooled airflow is shown in The time required for complete ice removal is shown in As shown, the time required for ice removal was substantially lower for the graphite coating as compared to the PTFE coating. In glaze ice conditions, or temperatures between −12 °C and −4 °C, the ice on the graphite was removed more than 50% faster when compared to the PTFE coating. In rime ice conditions, or temperatures between −14 °C and −13 °C, the ice removal time for the graphite coating was an 8.5% improvement over the PTFE coating. Such a sharp change in improvement time is due to the different opacities of the ice, which changes the spectrum received at the coating layer, the overall absorptivity coefficient, and the heat absorbed by the coating. The improved absorptivity of the graphite coating is clearly evident in the spectrometer analysis, and the highest improvement in absorptivity over an unmodified PTFE coating occurs at a wavelength of 501 nm, near the peak intensity of solar irradiation spectrum . The incThe hydrophobic characterization through the contact and roll-off angle tests demonstrate no decrease in hydrophobic performance in the graphite coating, which maintained superhydrophobicity characteristic of a PTFE coating even after modification with graphite. These roll-off and contact angle tests indicate identical wettability and surface energies, within uncertainty. The graphite could be added to the coating without interfering with the PTFE microstructure, which is required for the hydrophobicity of the coating.The icing wind tunnel tests demonstrated the feasibility of the graphite coating in environmental icing conditions, and both a qualitative and quantitative observation of de-icing performance indicates improvement through the addition of graphite. Upon observing the ice sheets after they had been removed from the coating surface, it can be concluded that the graphite coating promoted substantially more melting at the base layer of the ice, which is what allowed the ice sheet to be removed in a single piece. This increase in base layer melting is due to higher absorption of radiation, which translates to higher heat generation at the coating surface. Analysis of the ice removal times shows that the graphite coating removed ice more than 50% faster in glaze ice conditions, and 8.5% faster in rime ice conditions. Rime ice, due to its opacity, limits the spectrum which penetrates to the base layer of the ice, which reduces the relative effectiveness of the coating. However, in every performance metric over a variety of test conditions, the graphite coating showed similar performance to, or an improvement over, the standard PTFE coating.