Other inhibitory mechanisms involve immunoreceptor tyrosine‐based inhibition motif‐containing receptors, intracellular receptors and receptor desensitization. Inhibitory cyclic nucleotide pathways are traditionally thought to represent a passive background system keeping platelets in a quiescent state. In contrast, cyclic nucleotides are increasingly seen to be dynamically involved in most aspects of platelet regulation. This review focuses on crosstalk between activating and cyclic nucleotide‐mediated inhibitory pathways highlighting emerging new hub structures and signaling mechanisms. In particular, interactions of plasma membrane receptors like P2Y12 and GPIb/IX/V with the cyclic nucleotide system are described. Furthermore, differential regulation of the RGS18 complex, second messengers, protein kinases, and phosphatases are presented, and control over small G‐proteins by guanine‐nucleotide exchange factors and GTPase‐activating proteins are outlined. Possible clinical implications of signaling crosstalk are discussed.Platelets are regulated by extracellular cues that impact on intracellular signaling. The endothelium releases prostacyclin and nitric oxide which stimulate the synthesis of cyclic nucleotides  Cyclic nucleotide‐dependent inhibition and platelet activation are depicted as separate pathways.Emerging evidence suggests that these pathways are intertwined at multiple levels.Platelet inhibition and activation often regulate the same signaling nodes in opposite directions.Better understanding of cross‐inhibition between these pathways will advance antiplatelet therapy.12) and nitric oxide (NO). PGI2 binds to a G‐protein coupled receptor (GPCR) which is linked to the Gs heterotrimeric G‐protein that stimulates cAMP synthesis by adenylate cyclase (AC). cAMP binds to the regulatory domains of cAMP‐dependent protein kinase (isoforms PKA I and II) leading to activation of the PKA catalytic subunits and to phosphorylation of a multitude of substrate proteins  which bind to specific GPCRs. The signaling events downstream of these receptors include the activation of diverse kinases and GPCR‐linked heterotrimeric G‐proteins as well as small G‐proteins of the Ras and Rho families. Furthermore, calcium ions (Ca2+) are released from intracellular stores leading to further signal amplification ultimately inducing membrane and cytoskeletal reorganization, granule release and integrin activation.2 and NO pathways, platelet activation is limited by negative feedback through immunoreceptor tyrosine‐based inhibition motif (ITIM)‐containing receptors G6b‐B and PECAM‐1,In contrast, exposure of the extracellular matrix protein collagen and binding of von Willebrand factor (VWF) after vascular injury initiates platelet activation. Collagen stimulates the immunoreceptor tyrosine‐based activation motif (ITAM)‐containing glycoprotein VI (GPVI)‐Fc receptor γ chain complex which signals via Src family and Syk tyrosine kinases, whereas VWF binds to the GPIb/IX/V complex. In parallel, vascular damage also triggers the clotting cascade leading to thrombin generation which directly activates platelets via thrombin receptors and converts soluble fibrinogen into a fibrin mesh. Activated platelets release ADP and thromboxane A2  work, at least in part, by inhibiting cAMP production, and thus by interfering with inhibitory pathways. Binding of ADP to the P2Y12 receptor causes a conformational change in the receptor allowing it to act as a guanine‐nucleotide exchange factor (GEF) and activate the membrane‐associated heterotrimeric G‐protein of the Gi‐family. The active GTP‐bound Gαi‐subunit dissociates from the β and γ subunits and binds to AC leading to diminished cAMP synthesis  called regulators of G‐protein signaling (RGS) which accelerate the intrinsic GTPase activity of Gα subunits. RGS18 is a highly expressed GAP of Gαi and Gαq in platelets Figure . Deletio2+‐release and aggregation which could, at least in part, be due to dysregulated RGS18 function.2, highlighting multiple functions of this scaffold protein in platelets. Studies on SHP‐1‐deficient mice have identified a main role of SHP‐1 in regulating GPVI and integrin αIIbß3 signaling but did not reveal any defect in GPCR signaling.Interestingly, spinophilin knockout platelets exhibit reduced Ca2.3The GPIb/IX/V complex is a highly and specifically expressed VWF receptor on the platelet membrane with a crucial role in platelet adhesion at high shear rates. The cytosolic region of the GPIbα subunit interacts with the actin‐binding protein filamin A and with 14‐3‐3.2.4The GPVI‐Fc receptor γ chain complex plays a key role in platelet activation by collagen. Dimerization of the GPVI receptor is essential for binding of collagen to GPVI. GPVI activity depends on Gi signaling, indicating an inhibitory role of PKA. Indeed, cAMP and cGMP pathways inhibit GPVI dimerization.2.52 receptor is a GPCR coupling to Gq and G13 and mediates platelet activation by TxA2. TxA2 is synthesized by cyclooxygenase‐1, the key target of the antiplatelet drug aspirin. The receptor can be phosphorylated by PKA and PKG leading to inhibited receptor signaling, although only limited evidence for the presence of this phosphorylation in platelets is available.2‐induced Gq signaling involves the RGS18 complex described above. PKA‐induced phosphorylation of Gα13 linked to the TxA2 receptor might contribute to RhoA inhibition by cAMP.The TxA2.62+‐elevations thus potentially contributing to platelet activation.2+ keeping in mind the methodological issues that might affect experiments on NO and cGMP pathways.The classical pathway for the activation of cGMP production involves endothelium‐derived NO stimulating platelet sGC. sGC represents the main receptor for endothelial NO in platelets and NO‐binding stimulates cGMP production up to 100‐fold.33.13) levels. IP3 binds to Ca2+ channels on the dense tubular system, called IP3‐receptors, resulting in Ca2+‐release into the cytosol followed by store‐operated calcium entry through the plasma membrane ultimately contributing to platelet activation.2+ levels have been modelled resulting in the validation of many static and kinetic parameters of the enzymes and ion channels involved.2+ levels has recently been confirmed in a systematic study comparing effects of ADP, thrombin, convulxin and U46619 in combination with activators of cAMP and cGMP pathways.2+ levels by platelet activators and inhibitors.3‐induced Ca2+‐release through phosphorylation of the IP3‐receptor and the IP3‐receptor‐associated G‐kinase substrate .3‐receptor/PKG complex includes PDE type 5 which might support spatial control of cGMP signaling.Gq‐coupled receptor and GPVI signaling trigger phospholipase C activation, leading to an increase in cytosolic inositol‐3‐phosphate  and one is cGMP‐specific (PDE5A).44.12 are generally thought to be blocked by cyclic nucleotide pathways,Opposite regulation of MLC kinase and MLC phosphorylation levels by activating and cyclic nucleotide‐mediated inhibitory pathways has been known for a long time,phox phosphorylation, NADPH oxidase 2, and the production of reactive oxygen species ultimately leading to an inhibition of PKG.2 and/or ADP treatment of platelets probably leading to complex inhibition/activation patterns.Platelet activation by oxidized low‐density lipoproteins has been suggested to involve Src kinases, PKC, p474.2Proteome studies document de‐phosphorylation of many sites confirming an important role for phosphatases in platelet control.55.1Small G‐proteins of the Ras and Rho families like Rap1, Rac1, and RhoA are critical regulators of platelet function.5.2Rac1 integrates signals from several platelet activation pathways and is involved in platelet lamellipodia formation and thrombus stability. Similar to Rap1, parallel regulation of the Rac1 regulating proteins ArhGEF6 and ArhGAP17 has been described whereby PKA and PKG simultaneously inhibit ArhGEF6 and stimulate ArhGAP17 leading to attenuated Rac1 function.5.3RhoA is activated downstream of G13 and is an essential regulator of platelet shape change. RhoA is also known to be differentially regulated by cyclic nucleotides and platelet activators.5.42‐ and ADP‐mediated platelet regulation.ADP‐ribosylation factor 6 (Arf6) belongs to the Arf family of small G‐proteins and is involved in receptor trafficking in platelets.6Inherited disorders affecting genes involved in the positive or negative regulation of platelet cyclic nucleotide levels underscore the importance of this regulatory system to hemostasis and thrombosis in humans. For example, Gs hyperfunction leads to an enhanced tendency for bleeding,2.2 and NO to platelets from volunteers receiving dual anti‐platelet therapy with aspirin and the P2Y12 blocker prasugrel strongly enhances platelet inhibition in the presence of TRAP‐6 or collagen.2 and NO and thus on endothelial function. Since endothelial dysfunction is commonly observed in patients with cardiovascular disease it could be beneficial to include endothelial function testing in the assessment of anti‐platelet therapy.The clinical relevance of crosstalk between activating and cyclic nucleotide‐dependent inhibitory pathways is further highlighted by the success of drugs targeting either the Gi‐coupled P2Y12 receptor or PDEs 3A and 5A,7Cyclic nucleotide‐mediated inhibitory signaling is emerging to be closely intertwined with activating pathways. On the other hand, effective platelet activation mandates interference with inhibitory pathways at multiple levels, including blocking cyclic nucleotide production and promoting their degradation, and dephosphorylating PKA substrates. Inhibitors and activators tend to regulate the same signaling nodes in opposite directions Table . Larger Authors have nothing to disclose.Z.N. and A.S. wrote the paper.


NFE2L1) is crucial for the activation of rescue factors including the synthesis of new proteasomal subunits. Even though TCF11/Nrf1 is an essential transcription factor, the exact mechanisms by which it is activated and stabilized are not fully understood. It was previously shown that the calcium-dependent protease calpain-1 interacts with TCF11/Nrf1 and the TCF11/Nrf1 cleavage site is a potential calpain target. Here, we tested the hypothesis that calpain-1 or -2 cleave TCF11/Nrf1. However, we did not find a role for calpain-1 or -2 in the activation of TCF11/Nrf1 after proteasome inhibition neither by using chemical inhibitors nor siRNA-mediated knockdown or overexpression of calpain subunits. Instead, we found that TCF11/Nrf1 is digested by calpain-1 in vitro and that calpain-1 inhibition slows down the degradation of membrane-bound TCF11/Nrf1 by the proteasome in cultured cells. Thus, we provide evidence that calpain-1 is involved in the degradation of TCF11/Nrf1. Furthermore, we confirmed DDI2 as an essential factor for TCF11/Nrf1 activation and demonstrate an undefined role of DDI2 and calpain-1 in TCF11/Nrf1 stability.Protein degradation is essential to compensate for the damaging effects of proteotoxic stress. To ensure protein and redox homeostasis in response to proteasome inhibition, the cleavage and nuclear translocation of the endoplasmic reticulum (ER)-bound transcription factor TCF11/Nrf1 ( Proteostasis describes the balance of biogenesis, folding, trafficking and degradation of proteins within cells and their surroundings. Loss of its maintenance is associated with different degenerative disorders such as Alzheimer’s disease and various types of cancer. Therefore, it is essential to compensate for the damaging effects of proteotoxic and redox stress by different mechanisms such as the removal of misfolded and damaged proteins. One essential component of the proteostatis network is the ubiquitin proteasome system (UPS) ,2.NFE2L1 gene encodes different isoforms of this transcription factor. The most important ones are the large form known as TCF11 and the slightly smaller form Nrf1. In humans, both isoforms are expressed whereas mice only express the smaller Nrf1 isoform. Thus, in human cells cleavage of the TCF11 and Nrf1 isoforms results in four detectable proteins of different size [Previously, the invers coupling of protein and redox homeostasis was demonstrated for endoplasmic reticulum (ER) and cytoplasmic proteins . A transent size .TCF11/Nrf1 usually resides in the ER-membrane from where it is extracted by p97, ubiquitinylated with the help of HRD1 and rapidly degraded by the proteasome as part of the ER-associated degradation system (ERAD). Its half-life is ~10 min with slight variations depending on cell type . Upon prBefore the identification of DDI2, there was a long-standing debate about the cleaving protease, with the proteasome and calpain-1 being the two candidates most often cited ,9. By noHowever, there is still evidence that calpain-1 may be involved in this process as a yeast-two-hybrid assay using a human heart cDNA library could show the interaction between calpain-1 and TCF11/Nrf1 and the transcription factor has a potential calpain-1 cleavage site in the region where it is cleaved to induce its activation [The calpain family consists of 15 different isoforms with the µ- and m-calpain – or calpain-1 and -2 – being the most common forms in mammalia. They are activated by calcium concentrations in the micromolar range and are involved in cytoskeletal remodelling, cellular differentiation and apoptosis. Calpain-1 and -2 are heterodimers composed of a shared small regulatory subunit CAPNS1 (29 kDa) associated with either calpain-1 or -2, which is essential for calpain-1 and -2 activity. The distinct large catalytic subunits are encoded by the genes CAPN1 and CAPN2 (encoding subunits of 80 kDa) .Due to the previous evidence of calpain-1 binding to TCF11/Nrf1 and the potential cleavage site, we followed the hypothesis that calpain-1 or -2 is the protease, which cleaves TCF11/Nrf1. So far, no experimental evidence has been published to proof, whether calpain-1 or -2 might be involved in activation of TCF11/Nrf1 at the ER or not. Here, we did not find any evidence of an involvement of calpain-1/2 in the activation of TCF11/Nrf1 after proteasome inhibition. In contrast, we provide evidence that calpain-1 is involved in the degradation of membrane-bound TCF11/Nrf1 under non-inducing conditions. In addition, we confirmed that DDI2 is necessary for the TCF11/Nrf1 cleavage.® CRL-2922™), a human endothelial cell line, or their lysates were used. Cells were grown in Basal Iscove Medium supplemented with 10% FCS and penicillin and streptomycin.For all experiments, Ea.Hy-926  for the indicated time points. For experiments without BTZ, cells were treated with the calpain inhibitors for the indicated time points. The efficacy of BAPTA-AM was verified using a fluorescence-based method with Fluo-4-AM to detect intracellular calcium and flow cytometry as a read-out. The calcium complexed by BAPTA is maintained in the cell using probenecid. Fluo-4-AM has a higher affinity for calcium than BAPTA-AM.Cells were pre-treated with different concentrations of PD150606  or PD151746 (Abcam) or BAPTA-AM (Abcam) for 1 h before being treated with Bortezomib , Santa Cruz Biotechnology), β-Aktin , C4, Santa Cruz Biotechnology), TCF11/Nrf1 , DDI2 , p97/VCP , PSMA7/α4 , PSMA1/α6 , Tubulin , Calnexin , Lamin B1 , CAPNS1 . Secondary antibodies used were anti-mouse HRP and anti-rabbit HRP .g, 4°C. The supernatant contained the non-nuclear proteins. The nuclei were lysed using puffer B  for 30 min on ice. Genomic DNA and cell fragments were removed by centrifugation for 10 min at 12000×g, 4°C.To separate nuclear and non-nuclear proteins, cells were resuspended in buffer A  and incubated for 20 min on ice. NP-40 to 0.25% (v/v) was added for 3 min and the nuclei pelleted by centrifugation for 10 min at 5000×For the decay experiments, the medium was removed and replaced with fresh medium containing 100 µg/ml cycloheximide. The cells were lysed directly in 2× sample buffer after 10, 20 or 30 min. For the control, cells were left untreated.in vitro, the membrane fraction was isolated from cell pellets. First, pellets were resuspended in buffer 1  and the cells were lysed by three freeze–thaw cycles in liquid nitrogen. The cytosolic fraction was removed by centrifugation for 10 min at 1000×g, 4°C. The remaining pellet was resuspended in buffer 2 (buffer 1 + 1% NP-40) and incubated on ice for 30 min, followed by centrifugation for 10 min at 6000×g, 4°C. The supernatant contained the membrane fraction, which was used for the digestion.To digest TCF11/Nrf1 For the digestion, the components of the Calpain Activity Assay Kit  were used. To the isolated membrane fraction, the Reaction Buffer and Active Calpain I as well as different inhibitors such as the impermeant Calpain Inhibitor Z-LLY-FML from the Calpain Activity Assay Kit, BTZ or NMS-873 (SelleckChem) were added. The mix was incubated for 30 min at 37°C before sample buffer was added to the sample and it was heated for 10 min to 95°C.2 and 2 µM ATP) and 0.5 µg proteasome isolated from human erythrocytes were added. The mix was also incubated for 30 min at 37°C before adding the sample buffer and heating the samples to 95°C for 5 min.For the digestion using the proteasome, the membrane fraction was mixed with proteasome test buffer ; secondary antibody: goat anti-rabbit Alexa-488 (LifeTechnologies).Ea.Hy-926 cells were transfected with plasmid DNA using Lipofectamin 2000 (Thermo Fisher Scientific), following the manufacturer’s manual. Nrf1-3xFlag (RDB-2867) and Nrf1(m1)-3xFlag (RDB-2868) were a kind gift from R.J. Deshaies  for 48 h before treatment.RNA was isolated using the High Pure RNA Isolation Kit (Roche) following the manufacturer’s instructions. cDNA synthesis was performed with the Transcriptor High Fidelity cDNA Synthesis Kit (Roche), following the manufacturer’s protocol using 500 ng RNA.® Gene Expression Assays and the TaqMan® Universal PCR Master Mix (Applied Biosystems) following the manufacturer’s protocol. The relative gene expression was calculated using the ΔΔCT method and normalised against hypoxanthine phosphoribosyltransferase1 (HPRT1).Quantitative real-time PCR (qRT-PCR) was carried out using TaqManHPRT1 (Hs99999909_m1), PSMB6 (Hs00382586_m1), PSMC4 (Hs00197826_m1) and PSMA3 (Hs00541061_m1).The following master mixes were used: DDI2 knockout clones were generated using the CRISPR–Cas technique following established protocols with the pSpCas9(BB)-2A-Puro (PX459) plasmid (Addgene #62988) . The seqKi value of 210 nM for calpain-1 and 370 nM for calpain-2 [Ki 260 nM) than for calpain-2 (5.33 µM). In fact, PD151746 cannot completely inhibit calpain-2 even at very high concentrations [TCF11/Nrf1 is cleaved before it can translocate into the nucleus where it acts as a transcription factor . It has alpain-2 . The secin vitro with recombinant active calpain-1 using a fluorescent substrate. We found that at concentrations of 100 µM PD150606 calpain-1 was completely inhibited. Even when very high calpain-1 concentrations were used, we could no longer detect any fluorescent signal above background  in the cell experiments to test the viability of cells after 4 h of inhibitor treatment. For both inhibitors  concentrations up to 50 µM were well tolerated; however, the viability of cells decreased starting from 50 µM inhibitor concentration (data not shown). This phenomenon was even more apparent when PD151746 was used together with BTZ (no cell sample could be obtained anymore for 100 µM PD151746 together with BTZ). With the intracellular calcium chelator BAPTA-AM cells started to detach from the tissue culture plate and could not be used for experiments anymore at concentrations over 20 µM (data not shown).Based on our starting experiments, we used the calpain inhibitor PD150606 in a range of 5–100 µM, PD151746 from 5 to 50 µM and the calcium-chelator BAPTA-AM in concentrations between 5 and 20 µM. The efficacy of BAPTA-AM as a calcium-chelator was demonstrated using Fluo-4-AM by flow cytometry showing an increase of complexed calcium in the presence of BAPTA-AM .Next, we analysed the TCF11/Nrf1 activating cleavage in Ea.Hy-926 cells that were pre-treated with calpain-1/2 inhibitors or BAPTA-AM before inducing TCF11/Nrf1 activation by inhibition of the proteasome using low concentrations of BTZ overnight. Using the inhibitors in combination with BTZ treatment, no differences were observed in the cleavage of TCF11/Nrf1 with any of the concentrations or inhibitors used  [After confirming that chemical inhibition had no effect on the activation of TCF11/Nrf1, next calpain-1’s effect on TCF11/Nrf1 was tested ure S4A) . Thus, tin vitro independent of the proteasome and independent of the previously determined cleavage site.This appeared to be in contrast with published data showing that TCF11/Nrf1 is degraded by the proteasome as part of the ERAD system . Howeverin vitro, we concluded that calpain-1 may be involved in the degradation and the stability of this transcription factor. Consequently, we analysed the impact of calpain-1/2 on the stability of TCF11/Nrf1 in cells. For these experiments, the proteasome was not inhibited as we ruled out in the previous experiments that calpain-1/2 have an effect on the cleavage and activation of TCF11/Nrf1. First, we analysed TCF11/Nrf1 protein levels using immunoblot. Inhibition of calpain-1/2 lead to increased levels of TCF11/Nrf1 protein, which was significant for the inhibitor PD151746 (PSMA3, PSMB6 and PSMC4; however, a slight but not significant increase was observed for PSMB6 (After observing that inhibition of calpain-1 affected the amount of uncleaved but not cleaved TCF11/Nrf1 and that calpain-1 was able to degrade TCF11/Nrf1 PD151746 A. Next, or PSMB6 B. InhibiFinally, the stability of TCF11/Nrf1 after calpain-1/2 inhibition was analysed after inhibition of translation using the translation inhibitor cycloheximide. Here, we found that TCF11/Nrf1 was degraded slower using the inhibitor PD151746, which is more specific for calpain-1 than for calpain-2, compared with untreated cells C.In summary, this indicates that calpain-1 contributes to degradation TCF11/Nrf1 or at least speed up its degradation by the proteasome under conditions, where the transcription factor is not essentially required for the maintance of proteostasis and cell survival. However, the mechanisms and the biological implications of this interaction remain to be identified.As it has been published previously that DDI2 is responsible for the activation of TCF11/Nrf1, we wanted to confirm this result in our system. We used CRISPR–Cas to knockout DDI2 which was confirmed using immunoblot analysis. In DDI2 knockout cells, proteasome inhibition using BTZ induced an accumulation of TCF11/Nrf1; however, it was not processed anymore, confirming published results A. FurtheThe transcription factor TCF11/Nrf1 is essential to compensate for loss of proteasomal function by up-regulating the expression of new proteasome subunits in an ERAD-dependent feedback loop. Under non-inducing conditions TCF11/Nrf1 is permanently ubiquitin modified by Hrd1, extracted by p97 and degraded by the proteasome. Under inducing conditions, the ER-membrane bound TCF11/Nrf1 is extracted by p97 and cleaved by DDI2 to translocate into the nucleus ,16. Althin vitro even in its mutant form, which is uncleavable for activation in cells. In contrast, we did not see any significant effect of calpain-1 in the context of proteasome inhibition using BTZ in combination with calpain-1/2 inhibitors in different concentrations. Only a modest increase in the protein amount of nuclear TCF11/Nrf1 and mRNA of proteasomal subunits were detected following calpain-1 inhibition and proteasome inhibition. As calpain-1 inhibition enhanced TCF11/Nrf1 protein levels without BTZ present, one might expect that there would also be significant effects following proteasome inhibition. However, it is possible that the increase in TCF11/Nrf1 protein due to calpain-1 inhibition is not high enough to induce significant effects when BTZ is present; especially considering that proteasome inhibition usually only induces a doubling in proteasomal subunit mRNA. Smaller changes in mRNA levels might, therefore, remain undetectable.Here, we show that the protease calpain-1 plays a role in the breakdown of membrane-bound TCF11/Nrf1 but not in the cleavage for activation of TCF11/Nrf1. We provide two lines of evidence for this conclusion. First, we observed that inhibition of calpain-1 had an effect on the stability of the membrane-bound forms of TCF11/Nrf1 as their protein levels were increased when using an inhibitor more specific for calpain-1 and its degradation was delayed. Second, calpain-1 is able to degrade membrane-bound TCF11/Nrf1 Our results partly agree with Zhang et al. , who couIn addition, our data suggest that calpain-1 does not directly affect the activity of TCF11/Nrf1 in the context of proteasome inhibition, but there are other inducers of TCF11/Nrf1 where it might play a role. For example, the inhibitor of complex I of the respiratory chain rotenone has been shown to activate TCF11/Nrf1 . Here, tWe confirmed that DDI2 is important for the activation of TCF11/Nrf1 when the proteasome is inhibited , as knocIn conclusion, calpain-1 is involved in speeding up the degradation of TCF11/Nrf1 but does not play a direct role in its activation. In this context, one can speculate that calpain-1 action contributes to the degradation pathway of TCF11/Nrf1 under conditions when TCF11/Nrf1 has to be removed quickly from the ER-membrane and/or its extraction by p97 is slow. There are other examples of membrane proteins that require additional proteolytic processing for their final degradation by ERAD . DDI2 in


Trypanosoma cruzi (TcCA), and Leishmania donovani chagasi (LdcCA). Both enzymes were inhibited by benzoxaboroles in the micromolar range. Preferential inhibitory potency against the β-CA LdcCA versus the α-CA TcCA was observed with submicromolar inhibitory activities. Some derivatives displayed excellent inhibitory and selectivity profile over the ubiquitous and physiological relevant human off-target hCA II. This study provides a convincing opportunity to study benzoxaborole scaffold for the design of antiprotozoan potential drugs targeting the pathogen’s carbonic anhydrases.A series of 6-substituted ureido- and thioureido-benzoxaboroles were investigated as inhibitors of carbonic anhydrases from  Both of these diseases are caused by parasites belonging to the kinetoplastidae family and belong to the vector-borne diseases which are responsible for more than 17% of infectious diseases, affecting 20 million people and killing more than 50,000 every year2 to bicarbonate with a proton release, the prokaryotic metalloproteins are considered as important components in the growth and virulence of pathogenic microorganisms. In 2013, carbonic anhydrases from the two unicellular protozoans Trypanosoma cruzi (TcCA) and Leishmania donovani chagasi (LdcCA) were cloned and characterized,The availability of the complete genome sequence of both protozoans has given the possibility of large-scale analysis, which lead to consequent identification of novel drug targets. Protozoan carbonic anhydrases  were thus recently identified as novel promising targets for chemotherapeutic interventionsImportant classes of carbonic anhydrase inhibitors (CAIs) have been investigated in detail for their inhibitory profile against these two protozoans carbonic anhydrases.2 hydration reaction (kcat of 1.21 × 106 s−1 and kcat/KM of 1.49 × 108 M−1 s−1), was shown to be inhibited in the nanomolar range by many types of aromatic/heterocyclic sulfonamides,,in vivo the three phases of the pathogen’s life cycle,,in vitro,The α-CA TcCA, which is characterized by very high catalytic activity for the CO2 hydration reaction (kcat of 9.35 × 105 s−1 and kcat/KM of 5.9 × 107 M−1 s−1), has been reported to be efficiently inhibited by sulfonamides and heterocyclic thiols with nanomolar inhibition constants,in vivo assays demonstrated that selected inhibitors in thiol series possessed anti-leishmania activity, being able to reduce parasites growth and causing their deathThe β-CA LdcCA which also possess an effective catalytic activity for the COConsidering the druggability of protozoans CAs, the design of new inhibitors with potent anti-trypasonomal or anti-leishmania activities is worth of investigation.via a new binding mode, and to be effective against human α-CA as well as β-CA from pathogenic fungi,Previously, we reported a new generation of CAIs based on benzoxaborole scaffold. This new family of inhibitors were shown to act ,Encouraged by these results but also by the interesting profile of the trypanocidal orally active benzoxaborole compound SCYX-71581–23 were previously reported by this groupCompounds 2 hydration activity2SO4 of 20 mM were also added to the assay system for maintaining constant the ionic strength. The initial rates of the CA-catalyzed CO2 hydration reaction were followed for a period of 10–100 s. The CO2 concentrations ranged from 1.7 to 17 mM for the determination of the kinetic parameters and inhibition constants. For each inhibitor at least six traces of the initial 5–10% of the reaction have been used for determining the initial velocity. The uncatalyzed rates were determined in the same manner and subtracted from the total observed rates. Stock solutions of inhibitor (0.1 mM) were prepared in distilled-deionized water and dilutions up to 0.01 nM were done thereafter with the assay buffer. Inhibitor and enzyme solutions were preincubated together for 15 min at room temperature prior to assay, in order to allow for the formation of the E–I complex. The inhibition constants were obtained by non-linear least-squares methods using PRISM 3 and the Cheng–Prusoff equation, as reported earlier,An applied photophysics stopped-flow instrument has been used for assaying the CA catalyzed CO1–23 against TcCA and LdcCA were measured by a stopped flow CO2 hydrase assay and are shown in Inhibition data of benzoxaborole 1–23 may be noted:1–23 exhibited micromolar to low micromolar inhibitory activities against protozoans TcCA and LdcCA, showing a pronounced selectivity against the β-CA LdcCA with inhibition constant ranging from 3 to 0.65 μM, and selectivity ratio (KI(TcCA)/KI(LdcCA)) ranging from 9 to 70, the more selective being compound 13. Against the TcCA isoform, KIs were ranging from 12.6 to 75.1 μM, the best inhibitor detected being the para-methoxy derivative 17.Ureido and thioureido benzoxaborole 1, 6-nitro-2 and 6-amino benzoxaboroles 3 exhibited low micromolar activities against LdcCA, as the same level of magnitude as against hCA I, with inhibition constants ranging from 2.37 to 4.01 μM. No activity was noticed for these three compounds against TcCA (KI>100 μM). Tavaborole, a benzoxaborole used as standard in this study, showed inhibition potency similar to compound 2, and 3, against LdcCA, and a slight selectivity against human isoforms hCA II with a 5.5 magnitude selectivity ratio (KI(LdcCA)/KI(hCA II)).It is worth noting that simple benzoxaborole 6 and phenylthioureido benzoxaborole 23, a selectivity of 2.5 magnitude for LdcCA was observed with the thioureido derivative 23 and no selectivity with the ureido compound 6. Substitution in the para position of the phenyl ring  also showed a 2.2 magnitude of selectivity against LdcCA over CA II in thioureido series. An inversion of selectivity can be noticed for these two compounds in ureido series where compounds 9 (4-F derivative) and 10 (4-CF3) were, respectively, more selective against hCA II over LdcCA with selectivity ratio of 1.6 and 5.6 (KI(LdcCA)/KI(hCA II)).From a general point of view, change of the ureido by a thioureido group did not affect significantly inhibitory potency. Inhibitory activity against LdcCA was comparable with the ones observed for the off-targets hCA I and hCA II. A slight change of selectivity may be noticed for some compounds when we compare LdcCA versus hCA II. For example, if we compare inhibition data for the phenylureido benzoxaborole para-nitrophenyl thioureido derivative 18 with a high-selectivity ratio against the pathogenic isoform LdcCA over hCA II (around 110). This compound could represent a slightly better solution compared with the standard clinically used sulfonamide acetazolamide, as acetazolamide showed selectivity for hCA II over LdcCA and TcCA with selectivity ratio, respectively, of 6 and 9.The most performing inhibitor was found to be the Regarding the data in 4–23 investigated here showed a preferential inhibitory activity against the β-CA from Leishmania donovani chagasi (LdcCA) versus the α-CA from TcCA. Some derivatives such as 18 displayed excellent inhibitory and selectivity profile for LdcCA over the human off-target hCA II.We report for the first time the activity of benzoxaborole derivatives against protozoans CAs. 6-Substituted ureido and thioureido benzoxaborole derivatives The present study demonstrates that benzoxaborole chemotype offers interesting opportunities for the inhibition of CA from pathogenic protozoans and for the development of anti-trypasonomal or anti-leishmania compounds with a new mechanism of action, warranting further development.


A significant reduction in the Atlantic Meridional Overturning Circulation and rapid northern Hemisphere cooling 8200 years ago have been linked to the final melting of the Laurentide Ice Sheet. Although many studies associated this cold event with the drainage of Lake Agassiz-Ojibway, recent model simulations have shown that the Hudson Bay Ice Saddle collapse would have had much larger effects on the Atlantic Meridional Overturning Circulation than the lake outburst itself. Based on a combination of Mg/Ca and oxygen isotope ratios of benthic foraminifera, this study presents the first direct evidence of a major Labrador shelfwater freshening at 8.5 ka BP, which we associate with the Hudson Bay Ice Saddle collapse. The freshening is preceded by a subsurface warming of the western Labrador Sea, which we link to the strengthening of the West Greenland Current that could concurrently have accelerated the ice saddle collapse in Hudson Bay. The exact freshwater scenario that caused the 8.2 ka cold event is still debated. This study presents new evidence for a severe Labrador shelfwater freshening 8500 years ago that was caused by the Hudson Bay Ice Saddle collapse following a warming in subsurface waters. The disturbance of North Atlantic Deep Water formation, in turn, has been linked to a severe freshening caused by the sudden outburst of ice-dammed proglacial Lake Agassiz-Ojibway9 (LAO), which formed along the southern margin of the Laurentide Ice Sheet (LIS) during the terminal stage of the last deglaciation over North America. Erosive channels in the southwestern Hudson Bay seabed suggest that LAO drained catastrophically and subglacially underneath a grounded ice dam11, dispersing a red-colored, hematite-rich sediment layer13 from the western to eastern reaches of Hudson Strait17. An estimated 163,000 km3 of freshwater18 would have drained into the North Atlantic in one or two successive pulses18 between 8740 and 8160 years BP9. However, many model simulations using a short-lived drainage pulse of LAO volume as a trigger alone21 fail to reproduce a climate perturbation that lasts 160–400 years, such as seen in many climate proxy records revealing the 8.2 event24. In contrast, model studies that have applied higher freshwater volumes and proposing that the initial flood event was followed by longer routing or rapid ice sheet melting were able to reproduce a climate perturbation resembling the 8.2 kilo annum (ka) event28. Moreover, energy mass balance, ice sheet, and climate modeling studies suggest that the collapse of the Hudson Bay Ice Saddle29 would have had much larger effects on AMOC strength than the lake outburst itself31.In order to improve future climate models and assess the potential impacts of modern Greenland Ice Sheet melt, it is crucial to obtain a better understanding of past meltwater-induced changes in ocean overturning circulation. A rapid northern hemisphere temperature drop of 1–3 °C32. In the early Holocene, when the Hudson Strait Ice Stream retreated, Hudson Strait and large parts of northeastern Hudson Bay became ice-free, while a remnant of the Hudson Bay Ice Saddle still formed an ice dam along the continental margin and the marine basin of southwestern Hudson Bay, where it blocked LAO33. Though major ice domes remained on land until about 6.7 ka BP34, the Hudson Bay Ice Saddle disappeared between 8.5 and 8.4 ka BP33, which—according to model studies—would have released freshwater for several centuries35.The Hudson Bay Ice Saddle connected the Keewatin and Quebec-Labrador ice domes of the LIS over southern Hudson Bay Fig.  and was 18O signature36. A negative excursion in δ18O values of calcitic benthic foraminifera has been detected in a northern Labrador Shelf core also around 8.2 ka BP37. A series of older early Holocene freshwater events was described in a central Labrador Shelf core based on peaks in detrital carbonate correlating with reductions in calcitic δ18O of planktic and benthic foraminifera38. In contrast to these findings, some sediment records along the proposed drainage route did not contain any indication of a prominent freshwater pulse39. Such inconsistent evidence combined with limited sampling and dating resolution of previous records have left the exact timing and origin of the most significant freshwater injection ambiguous.Although direct evidence of the Hudson Bay Ice Saddle collapse is still elusive, a prominent freshwater signal along the proposed drainage route was previously attributed to the LAO drainage event. A sediment core from the central Labrador Shelf recorded a surface water freshening around 8.2 ka BP based on a reduction in the Mg/Ca temperature-adjusted planktic foraminiferal δHere, we present a high-resolution and well-dated sediment record from the northern Labrador Shelf Fig.  that proIslandiella helenae (see Methods), representing subsurface/bottom water conditions at about 200 m water depth. The δ18Οc record is relatively steady, centered at 3.2 ± 0.3‰, with the exception of a large reduction to 1.6‰ at 1048–1043 cm depth and another decrease to 2.25‰ at 1013–993 cm depth  and relative content of detrital carbonate inferred from elemental Ca/Sr ratios measured by X-ray fluorescence (XRF) core scanning (see Methods). The most pronounced peak in Ca/Sr occurs between 1073 and 1068 cm Fig. , supportpth Fig. . The Mg/pth Fig. . These tord Fig. . The δ18pth Fig.  is only ord Fig. .Fig. 2De38, short-lived changes in the local reservoir effect are likely. Based on the present Labrador Shelf marine reservoir correction of ΔR = 144 ± 38 years40 and an additional early Holocene sea-ice correction of 200 years41, we applied a ΔR of 344 ± 38 years to all accelerator mass spectrometric (AMS) dates below 700 cm depth  and detrital carbonate (Ca/Sr). Although Jennings et al.38 associate the red bed with the opening of the Tyrell Sea, we interpret the first freshening and input of red sediment and detrital carbonate to represent the initial phase of the subglacial LAO outburst flood , using an updated ice sheet volume equation34, we calculate that the Hudson Bay Ice Saddle could have stored about 451,000 km3 of freshwater, a volume that may have been large enough to dilute Labrador Shelf waters down to 200 m depth. This scenario is also in agreement with an ice sheet model study that produced the largest early Holocene freshwater pulse during the separation of the Keewatin, Labrador, and Fox domes29. Thus, we propose that the severe Labrador Shelf freshening at 8.55–8.45 ka BP was caused by the melting and collapse of the Hudson Bay Ice Saddle, which, according to a General Circulation Model simulation experiment, would have been sufficient to trigger the 8.2 ka cooling event31.The timing and duration of the severe Labrador Shelf freshening seen in our record is consistent with the disappearance of the Hudson Bay Ice Saddle between 8.5 and 8.4 ka BP7  was recovered from the northernmost Labrador Shelf at 58°45.68 N, 61°56.25 W, at 202 m water depth , Germany. Radiocarbon dates were calibrated to “calendar” scale using Calib 7.153 based on the Marine13 dataset54 with reservoir corrections of ΔR = 144 ± 38 and 344 ± 38 years. Six AMS dates between 1043 and 1273 cm depth were excluded from linear interpolation  value reflects the ratio of magenta (700 nm) and green (500 nm).55. Each section was successively scanned with voltages of 10 kV , 30 kV , and 50 kV  to measure the full suite of elements between Al and Ba in 1-cm resolution. Instrumental variance was monitored by measuring a set of standards  prior to each section and at the end of daily scanning.The bulk sediment elemental composition was analyzed at Kiel University, using an Avaatech XRF core scanner. The archive half of the core was carefully scraped to create a smooth surface before covering it with a 4-µm-thin SPEXCertiPrepUltralene foil to avoid contaminationI. helenae were handpicked from the 200–315 µm fraction in 5-cm intervals downcore, weighed, and crushed between two glass plates. The crushed samples were then split for Mg/Ca and stable isotope measurements (see below). About two thirds of each crushed sample was transferred into pre-leached Eppendorf vials and cleaned following the full protocol of Martin and Lea56, including a reductive and oxidative cleaning step and a final leaching step with 0.001 N HNO3. After dissolving and diluting the samples in 0.1 N HNO3, they were measured with an inductively coupled plasma-optical emission spectrometry (ICP-OES) instrument with radial plasma observation at the Institute of Geosciences, Kiel University. The analytical error of Mg/Ca analyses was 0.1% relative standard deviation and accuracy was monitored with reference material JCP-1. Additional trace elements  were monitored to exclude possible contaminated or coated samples from the dataset. Based on 15 duplicate down-core sample measurements, we obtained a standard deviation of 0.07 mmol/mol Mg/Ca, which translates into 0.9 °C with respect to temperature estimates. For bottom water temperature reconstructions, we applied the calibration of Skirbekk et al.57. As the calibration is based on a temperature range of 1–4 °C, it may become less accurate when temperatures exceed 4 °C, which is the case for 25% of the samples reported here.About 60 specimens of I. helenae were cleaned with ethanol absolute, decanted, and dried at 40 °C. Stable oxygen isotope analyses were carried out at the Leibniz Laboratory for Radiometric Dating and Stable Isotope Research in Kiel. A Finnigan MAT 253 mass spectrometer coupled with a Kiel IV carbonate preparation device was used and calibrated to the Vienna Pee Dee Belemnite (V-PDB) scale, which is applied to all δ18OCaCO3 analyses. Based on 15 duplicate down-core sample measurements, we obtained a standard deviation of 0.09‰. To estimate the δ18Ow (seawater), the value of calcitic δ18Oc (‰ V-PDB) was first translated into the V-SMOW scale by adding 0.27‰ and, together with the Mg/Ca-derived temperature, applied in the Shackleton equation T = 16.9 − 4.0 (δ18Oc − δ18Ow)58. To correct for ice volume (δ18Ow-ivc), a sea-level correction of 0.0083‰ per m (1‰ for 120 m of sea level) was derived from the relative sea level curve by Austermann et al.59. Based on duplicates, δ18Ow-ivc values have a standard deviation of 0.31‰. The bottom water signals  are based on infaunal benthic foraminifera I. helenae, which tend to live several centimeters deep in the sediment. This may explain the apparent lead of the bottom water signals compared to the sedimentological proxies Ca/Sr and a*(D65).About one third of the crushed samples of Supplementary Information


E. coli, purified and tested with 20 sera from Ole e 15-sensitized patients with olive pollen allergy by ELISA experiments. The contribution of linear epitopes was analyzed using twelve overlapping peptides spanning the entire Ole e 15 sequence. All the patients displayed a diverse reduction of the IgE-reactivity to the chimeras, revealing a highly polyclonal and patient-specific response to Ole e 15. IgE-epitopes are distributed across the entire Ole e 15 surface. Two main surface areas containing relevant conformational epitopes have been characterized. This is the first study to identify important IgE-binding regions on the surface of an allergenic cyclophilin.Olive pollen is a major cause of immunoglobulin E (IgE)-mediated allergy in Mediterranean countries. It is expected to become a worldwide leading allergenic source because olive cultivation is increasing in many countries. Ole e 15 belongs to the cyclophilin pan-allergen family, which includes highly cross-reactive allergens from non-related plant, animal and mold species. Here, the amino acid differences between Ole e 15 and its weak cross-reactive human homolog PPIA were grafted onto Ole e 15 to assess the contribution of specific surface areas to the IgE-binding. Eight Ole e 15-PPIA chimeras were produced in  However, in some regions, a higher prevalence of sensitization to specific local sources can be reached. Cultivated olive tree (Olea europaea ssp. europaea var. europaea) pollen is one of the main causes of pollinosis in the Mediterranean basin, being the main sensitizer in those areas where it is extensively cultivated3. Moreover, due to the continuous promotion of the Mediterranean diet, there is a rising demand for oil and olive fruit production worldwide, which will highly increase the number of olive trees and as a consequence, the number of allergic patients to this pollen4. To date, a total of fifteen olive allergens (Ole e 1 to 15) have been identified7. Besides, an extensive proteomic profiling of olive pollen has been recently reported, making this important allergenic source one of the best characterized8.Allergy is an immunoglobulin E (IgE)-mediated hypersensitivity which has become an important health problem in industrialized countries, affecting around 30% of their population. House dust mites and grass pollen are the most important aeroallergen sourcescis-trans isomerase activity and are part of the protein superfamily of immunophilins10. Due to their essential role in protein folding, cyclophilins are present in the cells of all organisms, and have been described as allergens in animals11, molds14 and plants17. Importantly, the amino acid sequence similarity among cyclophilins from unrelated species is high, and they could be considered pan-allergens due to their wide range of cross-reactivity13. Allergenic mold cyclophilins Mala s 6, Asp f 11 and Asp f 27 from Malassezia sympodialis and Aspergillus fumigatus, are major allergens whose structure and immunological properties have been extensively studied, showing in vitro and in vivo cross-reactivity between them and also with their human homologs peptidyl-prolyl cis-trans isomerases A (PPIA) and B (PPIB)19. Ole e 15, as well as other plant pollen allergenic cyclophilins, like Bet v 7 from birch and Cat r 1 from periwinkle, belong to the subgroup of divergent cyclophilins, which present a conserved Glu83 residue, two invariable cysteines and an additional stretch of seven amino acids with the consensus sequence XXGKXLH called divergent loop20. These allergens have also been shown to cross react with cyclophilins from plants21, animals (including human)8 and molds21.About 13% of the olive pollen allergic patients sera present IgE against Ole e 15, an allergen belonging to the group of cyclophilins. These proteins normally exhibit peptidyl-prolyl 23. Multiple human, parasite and mold cyclophilins structures have been reported since the first PPIA crystal structure was determined25. Moreover, the structure of wheat, orange tree and periwinkle cyclophilins have been recently reported27, revealing important information about the distribution and accessibility of amino acids across the protein surface, and the regulatory function of specific structural elements, such as the divergent loop.The availability of structural information is essential to understand the cross-reactivity among different allergens, determine the nature and location of their IgE-binding epitopes and use this information to design safer allergen-specific immunotherapy strategies31. This makes the analysis of epitopes a challenge, and only a few conformational IgE epitopes have been structurally solved using nuclear magnetic resonance (NMR) or X-ray crystallography34. Nevertheless, there are other strategies to identify specific IgE-binding surface areas, such as the use of random-peptide libraries mimicking real epitopes23, the design of point amino acid mutations to generate hypoallergenic variants35, epitope grafting36 and protein engineering to create chimeric proteins of the allergen and homologous proteins38. Although three main linear B-cell epitopes responsible for the cross-reactivity between mold cyclophilins and PPIA have been predicted by in silico analysis39, there is no experimental information available about the relevant epitopes of these allergens. Here, we aimed to identify the contribution of specific areas of Ole e 15 to the IgE-binding. After in vitro assessment of Ole e 15 and PPIA cross-reactivity, and in silico structure comparison and calculation of solvent-accessible surface areas, eight chimeras carrying specific regions from PPIA were designed using Ole e 15 as scaffold. Their IgE-binding capacity was assessed by means of ELISA and inhibition ELISA experiments and thus, relevant IgE-binding regions of Ole e 15 were identified.IgE-binding epitopes of most aeroallergens are mainly conformational and strongly dependent on the native allergen conformation492nm values higher than 0.4 when measuring IgE-binding to Ole e 15 by ELISA were tested with PPIA , Patch 2 , and Patch 3 (Ole e 15 stretch A76 to G101: region connecting β-strand β4 to β5). A fourth patch corresponding to the most conserved surface region, including the active site, was also found . The main exposed amino acids of each patch  are described in Supplementary Fig. 2, with values of 1343.81 (9.4%), 3222.82 (22.6%), 2219.88 (15.85%) and 2472.77 Å2 (17.3%) for the amino acid stretches included in Patches 1, 2, 3 and 4, respectively  did not show a loss of IgE-recognition as high as other patients.Next, the different IgE-recognition profiles of each patient were examined. At least six different groups of patients could be distinguished according to their IgE recognition patterns , and also 12 and 13 , were the most IgG-immunoreactive , Patch 2  and Patch 3 (Peptides 7 and 8) was assessed by inhibition ELISA with four representative serum samples , contained three defined sequence stretches  and accumulated the highest number of amino acid changes between Ole e 15 and PPIA. Numerous studies describe that antibody-antigen interactions mainly depend on charged, polar or aromatic epitope residues with a high SASA46. Then, assuming that a high change in the SASA value or a change in the charge state of amino acids would most probably alter the potential IgE-binding to each region, only PPIA amino acids meeting a set of criteria  were grafted onto Ole e 15 to create Chimeras 1, 5 and 7  to IgE-binding was further studied using Chimeras 8 and 9. For Pattern 2 and Pattern 6 patients, the loss of seroreactivity by Chimeras 8 and 9 was similar to that caused by Chimeras 5 and 7, suggesting that the amino acid mutations of Chimeras 5 and 7 were sufficient to provoke the loss of IgE-binding. On the other hand, for Pattern 1 or Pattern 3 patients, those amino acids mutations were not sufficient, and the exclusive mutations of Chimeras 8 and 9 provoked a higher loss, which further supported the idea that these patients present a different IgE-repertoire against this region. Besides, the fact that Chimeras 8 and 9 showed a similar loss of IgE-reactivity by most patients indicated that the amino acid mutations of Chimera 8 were sufficient to lower IgE-binding.Mutations at the N- and C-terminal regions were combined in Chimeras 15 and 17 to further analyze their contribution to the IgE-recognition of Patch 3 in IgE-binding /IgE/allergen complexes on the surface of effector cells51. Then, this in vitro cross reactivity, although weak, might be clinically relevant, and further studies to address it by in vivo experiments and to confirm the role of Patch 4 as an IgE-binding region should be performed.The IgE-mediated cross-reactivity between allergens sharing high sequence identity with their human counterparts has been described for proteins like the pan-allergens profilins or the fungal allergens manganese superoxide dismutase, acidic ribosomal P2 protein and thioredoxin53. Besides, it is important for the design of hypoallergenic variants with low capacity of effector cell activation for allergen-specific immunotherapy55. Here, Chimeras 15, 17 and 4 displayed a very reduced IgE-binding capacity compared to Ole e 15 and their suitability as hypoallergenic variants should be addressed by basophil activation tests and T-cell reactivity experiments.The identification of relevant IgE epitopes on the surface of allergens gives fundamental information about the generation of the IgE-repertoire by allergic patients and allows for a better prediction of cross-reactivity between allergens by using epitope sequence similarity rather than whole allergen sequencein vitro cross-reactivity between PPIA and Ole e 15 and that the Ole e 15-specific IgE-response is highly polyclonal and patient specific. Two main IgE-binding regions on the surface of Ole e 15 have been identified after the immunological characterization of eight Ole e 15-PPIA chimeras displaying lower IgE-binding capacities than Ole e 15. Thus, we demonstrate that grafting specific areas from a low IgE-binding homolog like PPIA is useful to identify the main IgE-binding sites of an allergen.In summary, this study shows that there is a weak but relevant 492 nm, arbitrary units) by ELISA were considered positive. All patients included in this study reported an immediate IgE-mediated allergic reaction and a positive SPT with olive pollen.Twenty sera from olive pollen allergic patients obtained by standard operating procedures from two different Spanish populations (Córdoba and Madrid) were used according to their positive olive pollen skin prick test (SPT), presence of IgE antibodies against olive pollen extract as measured by ImmunoCAP 250  or ELISA, and presence of specific IgE against recombinant Ole e 15 (Ole e 15) as determined by ELISA. SPT was performed according to standard procedures. A wheal diameter lower than 3 mm was considered negative. Values higher than 0.35 kU/L by ImmunoCAP, or higher than 0.1 optical density at 492 nm  and Reina Sofia University Hospital (ref. 3033) approved this study involving allergic patients. All samples were handled anonymously according to the ethical and legal guidelines of the Complutense University of Madrid and Reina Sofia University Hospital after approval of the Ethical Review Boards of these institutions. All subjects in the study gave their written informed consent to participate and all experiments were performed in accordance with relevant guidelines and regulations. All patients presented asthma or rhinoconjunctivitis. Detailed demographic and clinical information of the olive pollen allergic patients is depicted in Supplementary Table 56 using the NMR solution ligand-free structure of the allergenic plant cyclophilin Cat r 1 as template . For PPIA, the NMR solution structure (PDB: 1OCA) was used. Three-dimensional structures were visualized using PyMOL 2.3  and amino acid sequences were aligned using Clustal Omega57. Sequence and structure comparison allowed for the identification of four main surface patches gathering most of the different  or the conserved (Patch 4) amino acids between Ole e 15 and PPIA , and Chimeras 5 and 7, by mutating four amino acids in the Patch 2 inner C-terminal region (Q138 to G155) and three amino acids in the Patch 2 outer C -terminal region (G155 to S172), respectively. Chimeras 15 and 17 were designed as a combination of the mutations in Chimeras 1 and 5, and Chimeras 1 and 7. For the rest of the chimeras, the above criteria were not applied. Chimera 4 was designed by changing all the corresponding amino acids in Patch 3; Chimera 8, all amino acids in the Patch 2 inner C-terminal region; and Chimera 9, all amino acids in the Patch 2 . A deletion mutant (Chimera 3) was also designed by removing the amino acids of the Ole e 15 divergent loop (K48 to H54 in Patch 1). All amino acid changes are summarized in Table A three-dimensional (3D)-model of Ole e 15 was obtained with the ExPASy homology-modelling server Swiss-Modelsee Figs . Then, sid Table . Thus, CattB-site containing oligonucleotides overlapping by twenty nucleotides. Then, each insert was simultaneously cloned into pDONR221 and pDEST-HisHALO as donor and destination (expression) vectors, respectively, using single-step combined BP/LR Gateway reactions 59. Oligonucleotides were purchased from Sigma-Aldrich  . Thirteen peptides of twenty four amino acids in length and with twelve amino acid overlaps covering the entire Ole e 15 amino acid sequence were also designed  E. coli cells. LB cultures of 10 mL with 30 μg/mL kanamycin containing the transformed cells were grown overnight and then, diluted ten times and grown until OD600nm reached 0.7. Finally, protein expression was induced with 0.4 mM isopropyl thio-β-D-thiogalactoside (IPTG) and grown at 37 °C 230 rpm four hours , 30 °C 230 rpm for sixteen hours , or at 16 °C 230 rpm for forty eight hours . Then, the cultures were centrifuged at 6,000 × g for twenty minutes at 4 °C. Proteins were purified by gravity-flow chromatography from the soluble fraction of cell lysates using Ni-NTA agarose  following the manufacturer instructions with minor modifications. Briefly, cell pellets corresponding to 250 or 500 mL E. coli cultures were resuspended in 10 mL lysis buffer , distributed in 1 mL aliquots and disrupted by five cycles of subsequent freezing in liquid N2 for forty seconds and thawing at 42 °C in a water bath. Then, soluble fractions obtained by centrifugation at 6,000 × g for twenty minutes at 4 °C were incubated sixteen hours at 4 °C in a 15 mL conical centrifuge tubes with 500 μL Ni-NTA agarose previously pre-incubated three times with 5 mL lysis buffer for two minutes. After that, Ni-NTA agarose was washed five times for ten minutes at 4 °C with 5 mL wash buffer , recovering each time the resin by centrifugation at 4,000 × g for ten minutes at 4 °C. Proteins were then eluted with 5 mL elution buffer  for twenty minutes at 4 °C on rotation. To avoid the presence of traces of Ni-NTA agarose in the eluted fractions, a final centrifugation step was performed at 12,000 × g for five minutes at 4 °C. Finally, purified proteins were desalted onto PD10 desalting columns  with 150 mM ammonium bicarbonate, quantified, aliquoted, lyophilized and stored at −20 °C until use.Ole e 15, PPIA, protein chimeras and HaloTag-fused peptides were recombinantly produced in Purity of the recombinant proteins was analyzed by 15% SDS-PAGE and subsequent staining with Coomassie Blue R-250 . Theoretical molecular mass calculations were performed using unstained protein biomarkers SM0431 .8.The rabbit polyclonal antiserum against Ole e 15 (Ole e 15-specific pAb) was previously obtainedCircular dichroism (CD) spectra of Ole e 15, PPIA and protein chimeras in 20 mM phosphate buffer pH 7.0, at 0.2 mg/mL and 20 °C were recorded in the far-UV (190–260 nm) on a J-715 spectropolarimeter  using a 0.1 cm optical-path quartz cuvette. The deconvolution of the obtained spectra was performed using the CDNN software . Finally, the obtained far UV spectra were baseline-subtracted and represented as mean residue molecular ellipticity.Protein concentration of purified Ole e 15, PPIA and protein chimeras was calculated after amino acid analysis of 14 μg purified proteins in duplicates with a BioChrom 30 Amino Acid analyzer . Protein concentration of the purified HaloTag-fused peptides was calculated by measuring the absorbance at 280 nm using a DU-7 spectrometer  after theoretical extinction coefficient calculation with the ProtParam tool from ExPASy.3-fold blocking buffer-diluted horseradish-peroxidase-conjugated mouse anti-human IgE mAb  one hour at 37 °C. Color was developed for thirty minutes with o-phenylenediamine chloride and H2O2 as substrates , and the reaction was stopped with 1 N H2SO4. Absorbance was measured at 492 nm. Incubation with three sera from non-atopic individuals, and incubation with blocking buffer instead of serum (background), were used as controls of the assay. Normalized OD values (sample OD492nm-background) were considered positive when the cut-off absorbance value was higher than 0.1 . For Ole e 15-specific pAb-ELISAs, plates were coated with 100 μL/well of 1 μg/mL proteins or 5 μg/mL fusion-peptides, in PBS. For Ole e 15, PPIA and protein chimeras titration curves, each well was incubated with 100 μL serial dilutions (from 104 to 3 × 104-fold) of Ole e 15-specific pAb in blocking buffer at 37 °C for one hour. To assess the recognition of the HaloTag fused-peptides, the antibody was 2.5 × 103-fold diluted. Specific IgG-binding was detected using a horseradish peroxidase-labeled goat polyclonal antibody against rabbit IgG .To detect IgE-binding by ELISA, high-binding microplates  were coated overnight at 4 °C with 50 μL/well of 10 μg/mL Ole e 15, PPIA and protein chimeras in phosphate-buffered saline (PBS). After washing four times with 0.5% Tween 20-PBS, non-specific binding sites were blocked at 37 °C one hour with 100 μL/well 3% skimmed milk 0.1% Tween-PBS (blocking buffer). Then, 50 μL/well sera ten-fold diluted in blocking buffer were incubated in duplicate at 37 °C for two hours. After washing, bound IgEs were detected by incubating 50 μL/well 10For all inhibition ELISAs, procedures like coating, blocking and color development were performed as described above. For IgE-inhibition with Ole e 15, PPIA and chimeras, diluted sera (seven point five to twenty five-fold) were preincubated in duplicate for two hours at room temperature with ten-fold serial dilutions, from 0.005 to 5 μg/mL, or alternatively, 0.001 to 1 μg/mL, of the inhibitor proteins. For IgE-inhibition with HaloTag-fused peptides, diluted sera (fifteen to twenty five-fold) were preincubated with each peptide at 2 μM concentration.Inhibition values were calculated with the following formula:492nm values obtained by ELISA and the inhibition experiments was analyzed using Spearman correlation (95% CI).The Friedman test and the Dunn’s post test ) were performed to analyze whether the reduction of the IgE-recognition capacity of each serum against protein chimeras and PPIA in comparison to Ole e 15, was significant. Moreover, the relationship between the ODSupplementary InformationSupplementary InformationSupplementary Information


The recent thinning and retreat of Antarctic ice shelves has been attributed to both atmosphere and ocean warming. However, the lack of continuous, multi-year direct observations as well as limitations of climate and ice shelf models prevent a precise assessment on how the ocean forcing affects the fluctuations of a grounded and floating ice cap. Here we show that a +0.3–1.5 °C increase in subsurface ocean temperature (50–400 m) in the northeastern Antarctic Peninsula has driven to major collapse and recession of the regional ice shelf during both the instrumental period and the last 9000 years. Our projections following the representative concentration pathway 8.5 emission scenario from the Fifth Assessment Report of the Intergovernmental Panel on Climate Change reveal a +0.3 °C subsurface ocean temperature warming within the coming decades that will undoubtedly accelerate ice shelf melting, including the southernmost sector of the eastern Antarctic Peninsula. Ocean warming contributes to the thinning of the Antarctic ice shelves, however, lack of observations has prevented a quantification of this contribution. Here the authors use geological records to show that 0.3–1.5 °C ocean warming has played a central role on regional ice shelf instability over the last 9000 years. This retreat durably affects the stability of the regional glaciers and the ice sheet mass balance, which ultimately contributes to the eustatic sea-level rise. Specifically, in the eastern Antarctic Peninsula (EAP), the rapid warming observed since the 1970s4 have had marked consequences on regional glaciers and ice shelves. One of the first major events occurred in 2002 when the Larsen B ice shelf in eastern Antarctic Peninsula (EAP) 5, thus drawing the premise of unprecedented major collapses in the near future.The Antarctic Peninsula has been one of the most rapidly warming regions of the world during the twentieth century7. Indeed, a surface air temperature (SAT) increase of 2–3 °C has been observed8 between the 1960s and the late 1990s  along the EAP shelf15. However, the recent and past activity of this warm water mass, including its temperature change and modification along its pathway to the continental shelf by mixing with shelf waters  onto the Antarctic shelf has been shown to promote ice shelf basal melting around AntarcticaHere we combine recent and geological records spanning the past 9000 years to investigate the variability of environmental conditions of the EAP on multiannual-to-millennial timescales and its effect on the ice shelf boundaries. Our results suggest that the ocean thermal forcing, tied to the circulation of the relatively warm WDW, has played a central role on the regional ice shelf instability during both the instrumental period and Holocene; a slight ocean warming contributing to substantial ice shelf collapse and regression. When extrapolating these results to the most pessimistic Intergovernmental Panel on Climate Change (IPCC) emission scenario, we show that a slight subsurface ocean warming will further accentuate the current erosion of the EAP ice shelf by the end of the twenty-first century.16, we first computed the variations of the Ekman pumping along the EAP margin since 1950  (50–400 m water depth) on the continental shelf reaching up to +0.6 °C, thus sharing a similar pattern with the computed Ekman pumping during the same period of time. Nevertheless, a certain time lag is sometimes observed between SOT and Ekman pumping variations, especially in the early 2000s. The subsurface temperature should quickly respond to variations in wind-driven upwelling of warmer deep ocean water. However, we suggest that the response is slowed down because of heat exchange between the upwelled water and the much colder atmosphere and surface waters. Other factors like a significant change in the temperature of the deep-water masses20 may also have participated to the delay between the two records. We therefore argue that the SOT increase and associated warming of the EAP shelf resulted from two synergistic processes: (i) the enhanced penetration of warm deep waters on the shelf due to intensified wind-driven upwelling linked to a regional or global shift of the surface wind fields21  and atmosphere (through SAT and wind stress changes) forcing have closely acted together over the last decades to either strongly destabilize the EAP ice shelves or, inversely, to limit their dislocation.We also find that the most rapid and abrupt SOT warming reconstructed around the EAP ice shelf over the last centuries concurred with the Larsen B ice shelf collapse, thus strongly suggesting that a slight SOT increase, in concert with atmospheric warming, has been pivotal in controlling the Larsen B ice shelf instability. While we miss robust observation data prior the 1970s, the continuous SOT increase during phases of smaller collapses may imply that SOTs have potentially contributed to other smaller ice shelf collapses along the EAP and around Antarctica. Comparatively, cold SOTs as observed in the early 2000s, probably due to a dominant downwelling forcing, during a period of pronounced SAT cooling27 , and 3500 and 500 years BP. The Holocene cooling was interrupted by a slightly warmer period. The first main cooling episode corresponds to a phase of major EAP ice shelf retreat reported in the literature31. Indeed, marine sedimentological data indicate that the northern JRI ice shelf initiated a transition from grounded to floating ice ~8000 years BP ago30, which coincided with the early deglaciation of the Prince Gustav Channel and the Ulu Peninsula ice shelves, northwest of JRI29. This deglaciation preceded an ice free-regime that characterized the Vega drift and surrounding areas from ~7000 to 5000 years BP34. The Larsen A ice shelf was probably destabilized at least as early as ~6300 years BP35, while evidence show that the Larsen B ice shelf experienced a continuous and significant shrinkage throughout the Holocene28. Hence, the EAP ice shelves underwent a major retreat mostly between ~8000 and 6000 years BP.Beyond the observational period, there is only one available regional SAT record in the EAP27 Fig. , situate36 drilled within the Vega Drift in the northern Prince Gustav Channel ~8200 to 7000 years BP, followed by a continuous and slow ocean warming of +0.3 °C over the course of the mid-to-late Holocene , which is shorter to the early-to-mid Holocene period given the large extension of the ice shelves in the northern part of the EAP at this time. As SOT constraints, we used a +0.3 °C (Holocene trend) and +1.5 °C (early Holocene) warming.Estimating the impact of such an ocean warming on the ice shelf basal melting throughout the Holocene is not straightforward as it depends on many complex processes. Nevertheless, an order of magnitude including large uncertainties can be obtained using a simple parameterization~3 × 1011 J s−1 and a melting rate of ~3 × 1010 m3 year−1 (or ~1 mSv) (see Methods). This would correspond to a melting of at least ~10 m of ice per year on a surface covering the whole ice shelf front and penetrating 12 km below the ice shelves. If instead we take the surface of the 1970s EAP ice shelves for which we have a reasonable estimate , this would correspond to an average melt rate of the ice shelf of about 2.4 m per year. A +1.5 °C SOT warming as recorded between ~8200 and 7000 years BP in the EAP would imply a five times larger melt rate following the same parameterization. This clearly appears sufficient to have a large impact on ice shelf dynamics, especially as parameterizations using simple linear relations between ocean temperature and melting45 likely provide a lower estimate46. Therefore, although both ocean and atmosphere thermal forcing impacts on ice shelf instability, we conclude that, during the ocean warming phases, the SOT must have played a major role on controlling the regional ice shelf retreat.When considering an average SOT warming of +0.3 °C as observed in our records over the last 7400 years BP, we estimate a minimum net heat flux from the ocean to the ice shelf of 47 and representing two potential evolutions of the future greenhouse gas concentrations , differing by their radiative forcing trajectories throughout the twenty-first centuryons Fig. . We aver48. In comparison, for the RCP8.5 scenario, projections exhibit regional atmospheric and oceanic warming trends that are three-to-four times higher than in the RCP2.6 scenario   Fig. . Such su50, the warming of ocean temperature may be subdued in model projections. Consequently, the impact of changes in the wind forcing17 on the shoaling and further penetration of the warm deep waters on the continental shelf remains potentially underestimated. Thus, the future of the EAP ice shelf may be possibly more marked than projected. If the warm deep waters were to be redirected toward the continental shelf in the southernmost part of the Weddell Sea, enhanced heat content could increase by ~20% the yearly basal ice-melt underneath the Filchner-Ronne ice shelf51. Would such a predicted warming affect the whole Weddell Sea region, its impact on the surrounding ice sheets and glaciers would be marked for sea-level rise.Given the recent impact of the +0.3 °C warming on the ice shelf as well as during the Holocene, we anticipate an accelerated melting of EAP ice shelves over the next decades due to projected anthropogenic forcing. Together with the simulated +3 °C atmospheric warming, they will probably substantially increase the occurrence of huge collapses if the climatic trend continues to follow the RCP8.5 scenario. Since features such as wind-driven ocean circulation, sea-ice cover or the amount and distribution of the freshwater released to the ocean though ice-melt still need to be improved in climate models52. The Ekman pumping has been computed along the EAP continental shelf using surface wind velocities coming from the recent twentieth century reanalysis NOAA (20CR) Project version 28, consisting of an ensemble of 56 realizations with 2° x 2° gridded 6-hourly weather data from 1950 to 2010, and from the ERA-Interim version 2.016 reanalyses  suitable for reconstructing SOT in the polar oceans39. The analysis of GDGTs was conducted at the Royal Netherlands Institute for Sea Research (NIOZ) as previously described39. To convert To reconstruct subsurface ocean conditions in the coastal Antarctic regions, we applied the The SOT anomalies have been computed by subtracting 1.84 °C to the SOT values, 1.84 corresponding to the mean of the estimated SOT values using Eqs. (1) and (2) for the entire Holocene. To estimate the impact of the SOTs on the ice shelf throughout our record and calculate the corresponding ice melting rate as shown hereafter, we compute a temperature change of +1.5 and +0.3 °C over the early Holocene (8200–7000 years BP) and the Holocene trend (7000–0 years BP), respectively, based on a linear regression.55. In the Antarctic Peninsula, the only two studies focusing on GDGT producers have reported that the Thaumarchaeota species mostly reside within the 50–200 m upper ocean layer 59 or are much less effective for polar oceans37. Indeed, application of the TEX86 global calibration reveals values higher than +10 °C on average, some reaching up to almost +16 °C (data not shown), which are unrealistically warm for this region. Although the 37, we believe that the estimated SOTs represent the 50–400 m layer, i.e., the whole water mass that flows westward below the surface layer. Looking into last decade’s ocean temperature evolution of the EAP in two reanalyses, we do not detect any major temperature change between these two water depths. Indeed, the EN4 SOTs display similar absolute values and inter-annual variability since 1979 , we suggest that the downward transport is probably relatively fast and the GDGTs compounds are attached to particles, which are produced from the melting of sea ice or ice shelf, fecal pellets or marine snow. We exclude any terrestrial organic matter that could influence the mentclass2pt{minimmentclass2pt{minim45, used for instance in ice sheet modeling60, assuming a basic linear relationship between ice shelf melting and SOT45.Sub-ice shelf melting should ideally be estimated using an ocean model coupled to an ice shelf model in order to represent the ocean circulation in the cavity beneath the shelf, the processes controlling the melting and the feedback between this melt and circulation. This is unfortunately impossible at the scales investigated here, first of all because the precise topography of the ice shelves is largely unknown. We thus rely on a very simple parameterization45:ρ is the density of seawater (1000 kg m−3); cpw is the specific heat capacity of seawater (4000 J kg−1 C−1); γt is the exchange coefficient (10–4 ms−1); Tf is the freezing point temperature at the ice shelf base; Aeff is the effective area for melting.The net heat flux ; Li is the latent heat of fusion . We assume45 that Aeff can be estimated from the length of the ice shelf front before the major collapses  and a constant cross-shelf length taken equal to 12 km.where 46, this means that the values provided here, already large in terms of contribution, represents a lower bound. The Holocene warming and the simulated one by the end of the century are of the same order (+0.3 °C). Despite the need to improve such estimation, we strongly believe that the +10 m of ice melting computed for the Holocene could give a good overview for the near future if the current climatic trend continues to follow the RCP8.5 scenario. Moreover, although the Larsen C seems to not be primarily impacted by the summer SOT as recently evidenced61, due to the limited impact of the wind forcing , we cannot exclude that a combined effect associating its recent collapse and the continuous southward migration of the Westerlies would not promote the WDW shoaling in this area in the future and therefore amplify its disintegration.Given that this parameterization tends to underestimate the calculated melting47: RCP2.6 and RCP8.5. Our selection includes the models providing for both scenarios the physical parameters that we analyzed here, i.e., SAT and SOT. The models fulfilling this criteria are the following: bcc-csm1–1-m, bcc-csm1–1, BNU-ESM, CanESM2, CCSM4, CESM1-CAM5, CNRM-CM5, CSIRO-Mk3–6–0, FGOALS-g2, FGOALS-s2, FIO-ESM, GFDL-CM3, GFDL-ESM2G, GISS-E2-H, GISS-E2-R, HadGEM2-AO, HadGEM2-ES, IPSL-CM5A-LR, IPSL-CM5A-MR, MIROC5, MIROC-ESM, MPI-ESM-LR, MPI-ESM-MR, MRI-CGCM3, NorESM1-ME, NorESM1-M (Fig. We investigate projections from 26 climate models Fig.  particip1-M Fig. .The code used to characterize the Ekman Pumping can be found as supplementary file.Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Software


The pathophysiology of idiopathic generalized epilepsy (IGE) is still unclear, but graph theory may help to understand it. Here, we examined the graph-theoretical findings of the gray matter network in IGE using anatomical covariance methods.We recruited 33 patients with IGE and 35 age- and sex-matched healthy controls. Gray matter images were obtained by 3.0-T 3D T1-weighted MRI and were normalized using the voxel-based morphometry tools of Statistical Parametric Mapping 12. The normalized images were subjected to graph-theoretical group comparison using the Graph Analysis Toolbox with two different parcellation schemes. Initially, we used the Automated Anatomical Labeling template, whereas the Hammers Adult atlas was used for the second analysis.The resilience analyses revealed significantly reduced resilience of the IGE gray matter networks to both random failure and targeted attack. No significant between-group differences were found in global network measures, including the clustering coefficient and characteristic path length. The IGE group showed several changes in regional clustering, including an increase mainly in wide areas of the bilateral frontal lobes. The second analysis with another region of interest (ROI) parcellation generated the same results in resilience and global network measures, but the regional clustering results differed between the two parcellation schemes.These results may reflect the potentially weak network organization in IGE. Our findings contribute to the accumulation of knowledge on IGE. Idiopathic generalized epilepsy (IGE) is a common subgroup of epilepsy that encompasses several well-established epilepsy syndromes with common features such as primarily generalized seizures and epileptic discharges on electroencephalography (EEG) , 2. AlthGraph theory was originally a mathematical method to analyze various networks that are regarded as consisting of nodes and edges . In the Anatomical covariance analysis is one of the important and useful methods that draws on graph theory , and a gFor IGE specifically, one previous graph-theoretical study revealed reduced resilience of the cerebral blood flow (CBF) networks and thalamic hypoperfusion . AdditioIn this study, we applied graph-theoretical techniques to the whole gray matter network in IGE and compared the results with those of previous studies.We recruited 33 patients with IGE  at our institute between March and August 2016. IGE was diagnosed if the following criteria were met: (1) the presence of primarily generalized seizures with no focal symptoms, (2) diffuse (poly)spike-wave complex on interictal conventional scalp EEG, and (3) no focal abnormality on conventional MRI. Of the 33 patients, 16 had generalized tonic-clonic seizures alone, 12 had myoclonic seizures, and 5 had typical absence seizures in addition to generalized tonic-clonic seizures. The mean ± SD seizure onset age was 15.6 ± 4.8 years.2).We also recruited 35 age- and sex-matched healthy volunteers as controls . Between the IGE and control groups, there were no significant differences in age (p = 0.64 by unpaired t-test) or sex .MRI scans for all participants were performed using a 3.0-T MR system with a 32-channel coil . We obtained three-dimensional sagittal T1-weighted MP-RAGE (magnetization-prepared rapid acquisition with gradient echo) images with the following protocol: repetition time/echo time, 7.2/3.4 ms; field of view, 26 × 26 cm; matrix, 384 × 384; effective slice thickness, 0.6 mm with no gap; number of slices, 300; and number of excitations, 1.http://www.fil.ion.ucl.ac.uk/spm/) running in MATLAB 2014a . The T1-weighted images were segmented into gray matter, white matter, cerebrospinal fluid, and other non-brain tissues by a unified tissue segmentation procedure after correction for nonuniformity of image intensity. These segmented gray and white matter images were then spatially normalized to a customized template in the standardized anatomic space  by using the DARTEL  toolbox [The imaging data were analyzed in Statistical Parametric Mapping 12 software  was used for the graph-theoretical analysis in this study . GAT is C), a measure of the number of edges that exist between a node and its nearest neighbors; characteristic path length (L), the average shortest path length between all pairs of nodes as a measure of network integration; and Crand and Lrand, where Crand and Lrand are the normalized clustering coefficient and the characteristic path length of 20 random networks, respectively. Network resilience to random failure and to targeted attack was also evaluated. Random failure was assessed by randomly removing one node from the network and measuring changes with each iteration, whereas targeted attack was assessed by removing the nodes in rank order of decreasing nodal betweenness centrality. Moreover, GAT performed nonparametric permutation tests and assessed the regional difference in clustering between the two groups.Subsequently, the following network metrics were calculated: clustering coefficient  [Furthermore, to enhance validity and reproducibility, we repeated the above graph-theoretical analyses using a second parcellation scheme, as with previous graph-theoretical studies , 22. Fornt.org/) , 24, excGAT was used to compare the areas under the curve (AUCs) of each network measure of the two groups, in which the curves extracted from thresholding across a range of densities are used. The AUC analyses were also performed for the curves of random or targeted attacks. To test the significance, either a nonparametric permutation test or a parametric t-test was performed . GAT wasTo assess morphological differences between the two groups, we tested the normalized gray matter images using the two-sample t-test analysis in SPM12, with age, sex, and intracranial volumes calculated by SPM12 as nuisance covariates. Additionally, we also investigated the correlations between gray matter volumes and disease duration in the IGE group, using the multiple regression model in SPM12, with age, sex, and intracranial volumes as nuisance covariates. Statistically, a height threshold of p < 0.05  was deemed significant. However, to consider the effect of subtle morphological changes on graph-theoretical results, we also reported all clusters thresholded by a height of p < 0.005 (uncorrected) in SPM12 analyses.The network measures and the comparison results are shown in The resilience analyses revealed significantly reduced resilience of the IGE gray matter networks to both random failure and targeted attack . This meAs for the regional clustering, the IGE group showed an increase in the bilateral frontal lobes, temporal lobes, left hippocampus, and right marginal gyrus, and a decrease in the right occipital lobe . Given tAt the conservative level of p < 0.05 , there were no significant morphological differences between the groups and no significant correlations with disease duration. At the exploratory level of p < 0.005 (uncorrected), we found mild gray matter loss mainly in fronto-temporal lobes , left anIn this study, we used anatomical covariance methods to perform graph-theoretical analyses of gray matter structural networks in IGE. We found significantly reduced resilience to attacks, no significant changes in network measures, and several regional changes in clustering. Additionally, the reduced resilience and insignificant changes in global network metrics were reproduced by repeated analysis with another parcellation. To our knowledge, this is the first study to investigate the whole-brain gray matter network in adults with IGE. The reduced resilience is particularly meaningful because the results are in line with those of a previous study of CBF networks in IGE . ReducedAdditionally, we found no significant changes in network metrics in IGE. These findings were consistent in parcellations of both the AAL and Hammers Adult atlas. The gray matter networks in IGE may have generally comparable integrations and segregations to healthy subjects, although a lack of significant differences is not necessarily evidence of a lack of differences in the functions of the whole network. Whereas many articles have reported increased path length in TLE or focal epilepsy , 28, 29,Regarding regional analyses, we found several significant changes in clustering. The regional graph-theoretical metrics can represent the disease-specific brain areas. For example, a previous study of TLE revealed distinctly different patterns, in which TLE with hippocampal sclerosis showed functional segregation of the sclerotic hippocampus and MRI-negative TLE was associated with impaired connectivity of the ipsilateral temporal neocortex . In our Although the most common finding of voxel-based morphometry (VBM) in IGE is thalamic atrophy , we founThis study has several limitations. Binary thresholding is a common method in this field but could be a source of bias . AccordiFurthermore, the results should be interpreted with caution in regard to the use of one-tailed permutation test, which might have reduced statistical significances. However, we confirmed most of the results by two different parcellation schemes, and several previous studies also adopted one-tailed test in GAT , 25, 53.In addition, IGE consists of several different epilepsy syndromes, and our IGE cohort combined patients with different seizure types into one group. However, the term “IGE” is still accepted , and somWe have identified several features of gray matter networks in the IGE brain, including reproducible reduced resilience to attacks and no significant metric changes in the clustering coefficient or characteristic path length. Increased regional clustering was found mainly and widely in frontal lobes, but it was not confirmed by the second analysis. In particular, these results would reflect the potentially weak network organization of IGE. Our findings contribute to the accumulation of knowledge on IGE.