Weshow that PFAS are incorporated into bulk sea ice during ice formationand regression analyses for individual PFAS concentrations in bulksea ice were linearly related to salinity . This shows that their distribution is strongly governedby the presence and dynamics of brine  withinthe sea ice. Furthermore, long-chain PFAS (C8–C12), were enriched in bulk ice up to 3-fold more than short-chainPFAS (C4–C7) and NaCl. This suggeststhat chemical partitioning of PFAS between the different phases ofsea ice also plays a role in their uptake during its formation. Duringsea ice melt, initial meltwater fractions were highly saline and predominantlycontained short-chain PFAS, whereas the later, fresher meltwater fractionspredominantly contained long-chain PFAS. Our results demonstrate thatin highly saline parts of sea ice (near the upper and lower interfacesand in brine channels) significant chemical enrichment (ε) ofPFAS can occur with concentrations in brine channels greatly exceedingthose in seawater from which it forms . This observation has implications forbiological exposure to PFAS present in brine channels, a common featureof first-year sea ice which is the dominant ice type in a warmingArctic.Poly- and perfluoroalkylsubstances (PFAS) are contaminants ofemerging Arctic concern and are present in the marine environmentsof the polar regions. Their input to and fate within the marine cryosphereare poorly understood. We conducted a series of laboratory experimentsto investigate the uptake, distribution, and release of 10 PFAS ofvarying carbon chain length are contaminantsof emerging Arctic concern yet their fate and behavior in the marinecryosphere is poorly understood. Here, laboratory experiments showthat PFAS are incorporated into bulk sea ice during ice formationleading to significant chemical enrichment in brine. This has implicationsfor biological exposure to PFAS in first-year sea ice which is thedominant ice type in a warming Arctic. Perfluoroalkyl acids (PFAA) are one major subgroupof PFAS that have received considerable regulatory attention, with“long-chain” perfluoroalkyl carboxylic acids  and perfluoroalkyl sulfonic acids  shown to bioaccumulate more than their “short-chain”analogues and hence pose a greater risk to higher trophic level organismsand polar marine ecosystems.8Poly- and perfluoroalkyl substances (PFAS) are present in the Polarregions due to their long-range environmental transport (via atmosphereand ocean) and are considered “contaminants of emerging Arcticconcern” (CEACs)2 indicating deposition fromthe atmosphere with accumulation in the snowpack, as well as possibleentrainment into sea ice from seawater during sea-ice growth in winter.Several studies have also observed some organochlorine persistentorganic pollutants (POPs) in young or first-year sea ice.12 Pućko et al, (2010a) measured the levels of α- andγ-HCH in first year sea ice in the Canadian Arctic and demonstratedsignificantly higher concentrations of these chemicals in the seaice brine compared to under-ice seawater. Furthermore, they determinedthat their distribution and concentration in sea ice appears to bea function of circulating brine . Recently, experimental studies in artificialsea ice also showed elevated levels of organic pollutants in brineand demonstrated their distribution in newly formed bulk sea ice wasprimarily due to the movement of brine.13 Furthermore, α-HCH was released at a faster rate from meltingsea ice compared to less soluble chemicals ,suggesting that partitioning of chemicals between internal solid icesurfaces and liquid brine was also an important process. Due to theirknown surface acting properties, we anticipate PFAS to display partitioningwithin sea ice which could result in their enrichment, presentinga motivation to undertake similar experiments to investigate theirbehavior during sea ice formation and subsequent melt.PFAS have beenobserved in the sea-ice snowpack and in sea icein the Arctic14 The relationship betweensalinity and other solutes present in sea ice is indicative of theway in which chemicals are entrained and rejected from sea ice. Salinity-normalizedconcentrations have been used to study the behavior of nutrients,15 metals16 and dissolvedorganic matter (DOM)17 during sea ice formationand melt processes.Duringsea-ice growth, brine convection causes most salts and othersolutes to be rejected into the underlying seawater . This process, referred to asgravity drainage, is the dominant process causing desalination duringsea ice formation.Understanding the behavior of PFAS in growingand melting sea icewill allow better predictions of contaminant fate during winter (freeze)and spring (thaw) periods in polar marine environments, and hencethe timing and extent of PFAS exposure to ice–associated biota.Undertaking process-based studies to resolve contaminant fate in naturalsea ice is challenging. Therefore, we used an artificial sea-ice chamberto conduct controlled experiments to quantify chemical transfer betweenseawater and sea ice. We investigate the behavior of several PFASin sea ice during ice formation (freeze) and melt (thaw), testingthe hypothesis that the uptake and distribution of PFAS  in sea ice are controlledlargely by the movement of brine.22.118 The facility consists of an insulated glass-walled tank located insidea refrigerated chamber (air can be chilled). Artificial seawater wasmade by dissolving NaCl in deionized water to a concentration of ≈35g L–1 . A submerged pump was used to continuously mix the seawaterthroughout each experiment and a series of digital thermometers measuredthe in situ sea-ice temperature profile and calculate the sea icedepth. Two freeze–thaw experiments, referred to as “Freeze–1”and “Freeze–2” hereafter, were performed at airtemperatures of (−18 ± 1) °C and (−35 ±1) °C, respectively.The study was conducted in the Roland von Glasow Air-Sea-Ice Chamber(RvG-ASIC) at the University of East Anglia, U.K.5; PFHxA, C6; PFHpA, C7; PFOA, C8; PFNA, C9; PFUnDA, C11; and PFDoDA, C12), perfluoroalkyl sulfonic acids , and one n:2 fluorotelomer sulfonicacid  were utilized in this experimentalstudy (See Table S1 – S2). An ethanolicsolution containing a mix of all the PFAS  were introduced into the artificial seawaterbefore first sea ice formation to give initial concentrations in seawaterof individual PFAS between 0.06 and 0.14 nM.A range of perfluoroalkyl carboxylicacids  and bulk ice  to determininebackground PFAS contamination associated with the sea ice chamber.Once this preliminary sea ice had melted, the mixed-PFAS “spike”was added into the experimental tank seawater. After 24 hours of mixingand before any ice formation in Freeze-1, seawater samples  were collected (c0 seawater) with furtherseawater samples taken at the beginning of Freeze–2  and end of Freeze–2  as illustrated in Figure S1 of the SI). Collection of seawater samples was always undertaken when allof the ice had melted. Seawater was sampled via a preinstalled siliconehose  with an inlet set at 0.5 m above thebase of the tank to avoid interference with any forming sea ice layer.All sample containers were made of polyethylene and precleaned withmethanol.Prior to thecommencement of Freeze–1 and the introduction of the mixed-PFAS“spike” solution into the chilled seawater, a shortperiod (2 days) of sea ice growth at −35 °C was undertakento obtain preliminary procedural blanks of seawater  in Freeze–1 (depth 0.26 ± 0.01 m) and Freeze–2(depth 0.17 ± 0.01 m) from across the tank area using techniquesdeveloped by Cottier et al., (1999) to limit brine loss. Samples weresubsequently covered in polyethylene sheets and stored in a freezer(−40 °C) until further processing. Sea-ice samples weresectioned into horizontal layers  using an electric band saw in a cold room(−25 °C) and transferred to individual polyethylene bags.Following melting at room temperature, sea ice samples were combinedwith adjacent sea ice layer samples ready for PFAS analysis. Frostflowers  on the sea ice surface ofFreeze–2 were carefully collected using a polyethylene spatulaand stored in a freezer before melting for analysis.Bulk sea-ice samples were collected (2.3n = 8) sampled from across the sea ice (Freeze–1)using a titanium corer  were sectioned to givea top (T) and bottom (B) section of approximate equal length (13 ±1) cm, placed into separate polyethylene bags, and melted at (0 ±1) °C as described by Garnett et al.13 and others.22 Meltwater fractions (n = 8) were then sequentially collected over a 48 h periodin individual containers (0.1–2.5 L) and analyzed separatelyto investigate chemical dynamics.An additionalexperiment to observe chemical dynamics during sea ice melt was conductedto assess chemical interactions with sea ice during thawing. Sea icecores  with subsequentinstrumental analysis performed by HPLC-MS/MS, using an HP 1100 LCsystem  coupled to an API 4000 triplequadrupole mass spectrometer . A more detailed descriptionof the analysis can be found in the SI.As part of the quality assurance, we added 100 μL of a solution(20 pg μL–1) containing 6 mass-labeled PFCA and 3 PFSA  to each sample to assess analyticalperformance (see Table S5). In cases wheremass-labeled analogues were not available, a surrogate standard wasused (see Table S5). All PFAS concentrationsreported in this study were recovery-corrected. Method detection limits(MDL) were calculated as the mean of the procedural blanks (n = 4) concentration plus 3 times its standard deviation. Samples were notblank-corrected as PFAS levels in the blanks were low (see Table S5). Mass-balance calculations were performedas a quality control measure to assess the recovery  of individual PFAS at twokey points within this study. First, the “expected”concentrations of PFAS in the experimental tank seawater  and the “measured”seawater concentrations 24 h after the addition of the “spike”solution based on the known amount of PFAS added to the experimentalseawater  wereexpressed as a fraction (%) to give the recovery during the experimentalsetup . Second, the average“measured” concentrations of PFAS in the experimentaltank seawater before sea ice formation in Freeze–1 and Freeze–2 were comparedto the final seawater samples when all of the ice had completely meltedat the end of Freeze–2  and expressed as a fraction (%) to show the recovery throughoutthe experiments . Formore information, see Figure S1 and Table S6.All sea ice samples were meltedand the salinity of every samplewas measured using a calibrated conductivity probe (Hach HQd40 loggerwith CDC401 probe). For PFAS, sample analysis followed establishedmethods performed previously.2.5Tables S7–S16). However, bulk sea icesamples located at the bottom of the core were previously shown tosuffer from a sampling artifact and were excluded from further calculationsalthough concentration data are still reported (see Table S7). The method precision was given as the relativestandard deviation (RSD) of initial experimental seawater  samples (n = 7) and was includedin uncertainty analysis for each individual PFAS. bulk ice for NaCl in different sea ice layer samples by the initial measuredsubsurface seawater concentrations for each PFAS. We also calculated salinity-normalized enrichment factors,εs, for each PFAS using S is the salinity(g L–1) in a sample. εs = 1 representsconservative behavior, with respect to salinity. εs ≠ 1 denotes nonconservative behavior which corresponds toa specific depletion (εs < 1) or enrichment (εs > 1), respectively.t test and student paired t test. To test for significantdifferences in PFAS enrichment between sea-ice layers, a one-way ANOVAtest was applied followed by a Tukey posthoc test for multiple pairwisecomparisons.PFAS concentrationsare used for further calculations of enrichment factors (see 33.1Table S5). Initialconcentrations of PFAS in experimental seawater were comparable tothose at the end of the study (see Table S6) demonstrating that PFAS loss from the chamber during the courseof the experiments  or addition through contamination artifactswas negligible. This ensured that the experimental system and theconditions of the sea ice chamber were suitable for conducting aninvestigation on the chemical fate of PFAS in sea ice. Low recoveriesof some of the long-chain PFAS are discussed later.Low levels of PFAS were detected in proceduralblanks ensuring that the method detection limits were well below theconcentrations arising from the addition of the PFAS “spike”to the seawater  with the data lying close to the conservative mixing line. Relationships for long-chain PFAS (C8–C12) were weaker , but still significant and concentrations generallyfell above the conservative mixing line (see SI for complete data). If the data for the two surface ice layers (uppermostice) are removed, then the correlations for the C8–C12 compounds are not significant (p > 0.05).The qualitative similarities between PFAS and salinityprofiles1 combine31 little is known about the mechanisms of enrichment . The enrichment of dissolved organic matter (DOM)maybe related to differences in the diffusion rates of chemicals, whichleads to the preferential rejection of small molecules as they diffuseat relatively faster rates in ice than larger DOM molecules.32 However, rhodamine 6G (a useful dye/tracer with a molecular massakin to PFAS) did not noticeably deviate from conservative behavior.33 The presence of extracellular polymeric substances(EPS) in sea ice  can act as a sorbent for other chemicals.34 However, in this study artificial seawater (NaCldissolved in ultrapure water) was used with no EPS.PFAS, like most other dissolved solutes, are not expectedto beincorporated within the ice crystal lattice due to their relativelylarge molecular size. Instead, they are incorporated in bulk sea-icewithin interstitial brine channels. While studies have observed nonconservativebehavior of some dissolved organic components in sea ice,35 and in experimental systems exploring enrichmentof PFAS in sea spray aerosol.31 This behavior of PFASin the surface microlayer is likely driven by the presence of colloidalorganic matter, although this is expected to have been absent in ourexperimental system but was not measured. Low recoveries for the long-chainPFAS  were apparent inthe seawater samples taken initially at the start of the experiment(see Table S6). Preferential partitioningof these long-chain compounds to a sea surface microlayer may thenhave occurred, resulting in elevated concentrations at the seawatersurface where ice growth begins and thus leading to chemical enrichmentin sea ice. However, the calculated concentrations of PFAS in seaice based on the initial seawater concentrations that accounted forthe unrecovered mass of PFAS using the assumption that they were transportedconservatively with salt, were significantly higher (up to 50-fold)than measured concentrations in the sea ice (see Tables S17 and S18). This indicates that most of the massof PFAS that was not recovered in the initial seawater measurementswas probably sorbed to the chamber surfaces (mainly glass) and hencewas unlikely to play a role in the observed PFAS enrichment in seaice. Volatilization of PFAS to the overlying chamber air was anotherpossible loss process but was considered to be insignificant. Perfluoroalkylacids have relatively high aqueous solubilities, low volatilities,and exist as their anionic form in seawater at pH ≈ 8 withnegligible water to air partitioning .37Enrichmentof long-chain PFAS in sea ice may also be related toelevated concentrations in the sea surface microlayer (top 1000 μmof surface ocean), a phenomenon that has been observed in the environments, L1 > 1) with values significantlyhigher compared to lower bulk icelayers in both Freeze–1 and Freeze–2 . Alternatively, PFAS mayserve play a role in heterogeneous ice nucleation during sea ice growthPFASwere particularly enriched in the surface layer (L1) of seaice .Frost flowers are highly saline ice structures that develop underextreme cold, calm atmospheric conditions.38 Brine and other solutes are “wicked” from the surfacelayer (1–2 mm) by capillarity and concentrated on the ice structure.The presence of PFAS in frost flowers suggests they are also transportedwith brine. However, εs, frost flowers > 1 (up to 11) also indicates that other processes may contributeto the enrichment of PFAS in frost flowers  which is known to be the source of NaCl (and other solutes)in frost flowers as sea ice grows.33 Valuesof εs, frost flowers-L1 were much closer to 1 (n = 1), we highlight that these covered a large area of the sea icein Freeze–2 (approximately 0.3 m2). We thereforebelieve that high concentrations of long-chain PFAS in frost flowersis evidence that chemical sorption to ice surfaces plays a role intheir enrichment . Initial meltwater fractions were more saline and contained higherconcentrations of short-chain PFAS than later meltwater fractions.In contrast, higher concentrations of long-chain PFAS were associatedwith the later meltwater fractions that were less saline. The totalamount of NaCl and individual PFAS in the combined slow-melt sampleswere used to derive enrichment factors in each of the meltwater fractions(see Tables S13–S16). Short- andlong-chain PFAS displayed εs, meltwater values that were ≈1 and >1, respectively (4–C7) were mainly “eluted”in the first meltwater fractions and so behaved conservatively withrespect to NaCl. Conversely, long-chain PFAS (C8–C12) showed nonconservative behavior and were preferentiallyretained in the melting ice being released in later meltwater fractions.The slow-meltexperiment was designed to investigate chemical dynamicsof PFAS during controlled sea-ice melt.ectively 4. This s40 Long-chain PFAS have, in general, been found to partitionmore toward solid phases in sediments23 and in snow.41 Our results suggest PFAS behaveanalogously in sea ice and indicate that long-chain PFAS preferentiallypartition to solid ice surfaces, relative to short-chain PFAS. Thesefindings are also supported by previous studies in sea ice which displaysimilar findings for hydrophobic POPs of varying molecular mass andaqueous solubility.13 Partitioning of PFASto ice surfaces can be linked to hydrophobic interactions, which increasewith carbon chain length. Interestingly, differences were also observedbetween PFAS with the same carbon chain length  which shows that other physicochemical properties also controlthe their behavior in sea ice, such as the number of fluorine atomsand different functional groups. On the basis of the evidence we providehere, we believe it is reasonable to assume that the nonconservativebehavior of long-chain PFAS, specifically their enrichment, is relatedto their tendency to interact with and partition to ice surfaces.The similarity in enrichment behavior of some PFAS with those that have already received global regulatory attention should caution policy makers to develop better strategiesfor grouping PFAS to protect human and environmental health.42Sea ice is composed of a solid (fresh ice matrix) and liquid(brine)phases. The distribution of the PFAS between these two phases at equilibriumis expected to vary according to carbon chain length.3.3t test, p < 0.001) in the surface layer of ice during Freeze–1,which formed at a slower rate due to a warmer ambient temperature(−18 °C), compared to Freeze–2 (−35 °C).This slower ice formation will have increased the contact time betweenice cyrstals and PFAS during the initial ice growth stages of theexperiment and this “concentrating effect” has beenobserved for organic matter43 and othercomponents in seawater such as algae.44Giventhe propensity of PFAS and other organic chemicals to sorb to icesurfaces, as indicated in the slow-melt experiment, it is possiblethat chemical partitioning to frazil ice during the onset of ice formationmay be an important process controlling the uptake of dissolved chemicalsinto bulk sea ice. This may help explain why the enrichment of PFASin the uppermost ice layers was significantly higher compared to thelower ice layers. Moreover, this is also supported by significantlyhigher enrichment factors (paired student Vb) reaches 5%.45 Above 5% brine volume, ice-associated (sympagic) organisms can thrivein the brine network commonly observed on the underside of ice floes.On the basis of this, we performed a simple calculation  to estimate the enrichment ofPFAS for the in situ sea ice brine (εbrine) and so assess the likely chemical exposure presented to brine-dwellingorganisms. This approach indicates that the sympagic community maybe exposed to individual PFAS concentrations that are more than anorder of magnitude greater  than those present in seawater (see Table S19). This demonstrates that enrichment in sea ice brineis likely to be a significant exposure pathway to an array of organismssituated at the base of the marine food web thereby exacerbating bioaccumulationof toxic contaminants particularly in brine-rich single season ice.The results demonstrate that PFAS are incorporated withinsea ice during its formation and long-chain PFAS can be enriched relativeto salt (NaCl) as well as short-chain PFAS. However, PFAS are includedwithin the ice-brine network  of sea ice, which represents a muchsmaller volume fraction of the total bulk sea ice. The salinity ofice-brine is governed by thermodynamic phase-changes, fluctuatingin the environment according to diurnal and seasonal temperature changes.Sea ice is considered to be porous when the brine volume fraction(49 The very high enrichment of PFASobserved in a frost flower sample (present at the ice-atmosphere interface)is intriguing and also raises implications for ice surfaces servingas an aerosol-driven source of PFAS to the local or regional atmosphere.36 More controlled experiments, together with carefulobservational studies in the field, are now required to understandthese complex yet potentially important processes, particularly withregard to chemical exposure to organisms at the base of the marinefoodweb.In the Arctic and other regions where sea ice is prevalent, thetotal burden of PFAS in sea ice will be a function of the many differentPFAS present in surface seawater, but also that which is depositedfrom the atmosphere through snowfall and other depositional processes.Furthermore, natural sea ice is subject to various freeze–thawcycles which could transfer organic chemical pollutants between theice-rafted snowpack and sea ice in winter and during seasonal thaw.


Glioblastoma is the most common and aggressive brain cancer, and there is a desperate need for new therapeutic strategies for this incurable cancer. Inhibition of Diacylglycerol kinase α provides novel direct mechanisms against glioblastoma cells, but also offers the potential to simultaneously boost immune cell activities against glioblastoma. This review provides an updated summary of the promising potential of Diacylglycerol kinase α inhibition against glioblastoma, with potential implications for other cancers as well.Diacylglycerol kinase α (DGKα) inhibition may be particularly relevant for the treatment of glioblastoma (GBM), a relatively common brain malignancy incurable with current therapies. Prior reports have shown that DGKα inhibition has multiple direct activities against GBM cells, including suppressing the oncogenic pathways mTOR and HIF-1α. It also inhibits pathways associated with the normally treatment-resistant mesenchymal phenotype, yielding preferential activity against mesenchymal GBM; this suggests possible utility in combining DGKα inhibition with radiation and other therapies for which the mesenchymal phenotype promotes resistance. The potential for DGKα inhibition to block or reverse T cell anergy also suggests the potential of DGKα inhibition to boost immunotherapy against GBM, which is generally considered an immunologically “cold” tumor. A recent report indicates that DGKα deficiency increases responsiveness of macrophages, indicating that DGKα inhibition could also have the potential to boost macrophage and microglia activity against GBM—which could be a particularly promising approach given the heavy infiltration of these cells in GBM. DGKα inhibition may therefore offer a promising multi-pronged attack on GBM, with multiple direct anti-GBM activities and also the ability to boost both adaptive and innate immune responses against GBM. However, both the direct and indirect benefits of DGKα inhibition for GBM will likely require combinations with other therapies to achieve meaningful efficacy. Furthermore, GBM offers other challenges for the application of DGKα inhibitors, including decreased accessibility from the blood-brain barrier (BBB). The ideal DGKα inhibitor for GBM will combine potency, specificity, and BBB penetrability. No existing inhibitor is known to meet all these criteria, but the strong potential of DGKα inhibition against this lethal brain cancer should help drive development and testing of agents to bring this promising strategy to the clinic for patients with GBM. Diacylglycerol kinase alpha (DGKα) is one of ten DGK family members that convert diacylglycerols (DAGs) to phosphatidic acids (PAs). Both DAG and PA strongly regulate numerous biologic processes. DAG is important for activating Protein Kinase C enzymes. PA is typically found at low levels relative to other phospholipids, in the cell membrane and elsewhere, but despite its low concentrations it regulates a number of signaling pathways. DGKα has been found to regulate signaling pathways integral to cancer, despite being only one member of the large DGK family—suggesting a general lack of redundant function across the family. PA has been found to control activity of mTOR, Akt, and Erk, while DGKα has been associated with NF-κB, HIF-1α, c-met, ALK, and VEGF ,6,7,8,9.GBM is the most common and most lethal primary brain tumor, causing 12,000–14,000 deaths each year in the U.S. alone . Median DGKα and its product PA are known to help maintain activity of several oncogenic pathways that have been linked to GBM malignancy and viability. These include Ras and Raf, Akt and mTOR, VEGF, and HIF-1, as described above. It is therefore unsurprising that DGKα knockdown and inhibition have been shown to have direct activity against GBM cells. While DGKα inhibitors are highly unlikely to block these individual targets and pathways as effectively or potently as specific inhibitors for each, the combined inhibition of multiple targets—even to a lesser degree—may be a promising approach for a genetically heterogenous cancer such as GBM with diverse signaling drivers. This seems particularly salient given the poor track record of highly specific single agents in clinical trials for GBM, as single agents or in combinations. In a prior report, we showed that mTOR and HIF-1α blockade are particularly important mediators of the direct activity of HIF-1α inhibition in GBM cells , but theNotably, DGKα inhibition may offer benefits versus single-agent or combined specific and potent inhibitors of targets such as mTOR and HIF-1α. Dedicated inhibitors of the mTORC1 complex are known to drive feedback loops leading to activation of the upstream driver Akt, which may yield even greater malignant behavior; however, DGKα inhibition has been shown to suppress Akt as well as mTOR activity in GBM cells . DGKα alNF1 lesions, and has been associated with higher Akt, TGF-β, and NF-κB activity [DGKα inhibition may also provide traction against a challenging and common resistance mechanism and subtype found in GBM and other cancers as well—the mesenchymal subtype. The Cancer Genome Atlas (TCGA) and other profiling efforts have suggested three GBM subtypes ,25,26: pactivity ,25,26. Mactivity ,28 and dactivity , but indactivity ,30,31. Aactivity . MES GBMactivity ,34,35. Factivity . Taken tactivity . Our finactivity . DGKα inGBM is a highly vascularized tumor, and the anti-VEGF agent bevacizumab remains in wide use for recurrent GBM. Antiangiogenic agents such as bevacizumab have yet to show an overall survival benefit in trials for all patients with GBM. However, they improve quality-of-life and reduce edema, and one retrospective analysis did show an overall survival benefit of upfront bevacizumab in patients with proneural GBM lacking IDH1/2 mutation . AntiangWhile the effects of DGKα inhibition on the immune system will be covered in more detail elsewhere in this issue, it is important to review their potential specifically for GBM therapy. As described above, GBM is considered an immunologically “cold” tumor, with a strongly immunosuppressive local milieu as well as challenges such as the BBB and decreased communication between the immune system and brain/GBM microenvironment. The immunologic functions already ascribed to DGKα suggest that its inhibition may help counteract some of the challenges involved in immunotherapy for GBM. DGKα inhibition has been found to reverse T cell anergy, a prominent mechanism by which T cells become inactivated in tumors ,41. OtheA new report from our group indicates that DGKα also acts to regulate macrophages; DGKα knockout/knockdown increases macrophage responsiveness to diverse stimuli . This maA number of reports have suggested that the addition of DGKζ knockout to DGKα knockout markedly increases T cell-activating effects. In fact, specific knockout of DGKζ may have stronger T cell-promoting effects than specific DGKα knockout, and this translated to greater anti-tumor activity in one report testing three syngeneic tumor models in mice . HoweverIt is possible that DGKα inhibition could also have beneficial effects on the expression of certain immunosuppressive checkpoint proteins. Interestingly, the treatment-resistant MES GBM subtype has much higher expression of the immune checkpoints PD-L1 and TIM3 than PN GBM ,34. In aIt is increasingly being recognized that the heterogeneity and adaptability of GBM will likely necessitate the use of combination regimens to achieve meaningful efficacy. DGKα inhibitors may be particularly well-suited for such combinations, with potential to enhance the activity of numerous therapies with direct anti-cancer activity but also immunotherapies. The broad signaling effects of DGKα inhibition, as well as preferential activity against the treatment-resistant mesenchymal phenotype in GBM and other cancers, could translate to a host of promising anti-GBM combinations with a DGKα inhibitor. We previously reported in vitro synergistic activity combining a DGKα-inhibiting compound with radiation against GBM stem-like cells , and ourCombining DGKα inhibition with immunotherapies may be particularly promising for GBM and other immunologically cold cancers. As described above, DGKα inhibition may help break T cell anergy and potentially exhaustion as well, thus eliminating barriers that impede the efficacy of immunotherapies in GBM. DGKα inhibition has already been shown to boost the anti-cancer activity of CAR-T cells in preclinical studies , and othEffectively incorporating DGKα inhibitors into regimens for GBM will of course require the development of adequate inhibitor compounds with favorable pharmacological properties, including sufficient penetration of the BBB. The most widely-used tool compounds in the laboratory in past decades, R59022 and R59949, have modest potency and activity against DGKα and also inhibit other targets more potently . HoweverNewer DGKα-inhibiting compounds with different structural bases may be able to provide better potency and specificity than ritanserin and its analogs, as well as other potential advantages. Efforts are underway to further develop such compounds, likely driven in large part by the appeal of the immunotherapeutic potential of DGKα inhibitors in combination regimens for cancer. The compound CU-3 has been reported as a DGKα inhibitor with a novel structure , and BriDGKα inhibition thus offers the highly attractive potential for multifaceted direct action against GBM with simultaneous immune-boosting activities and potential antiangiogenic activity as well (summarized in


For the last years scientific community has witnessed a rapid development of novel types of biomaterials, which properties made them applicable in numerous fields of medicine. Although nanosilver, well-known for its antimicrobial, anti-angiogenic, anti-inflammatory and anticancer activities, as well as hyaluronic acid, a natural polysaccharide playing a vital role in the modulation of tissue repair, signal transduction, angiogenesis, cell motility and cancer metastasis, are both thoroughly described in the literature, their complexes are still a novel topic. In this review we introduce the most recent research about the synthesis, properties, and potential applications of HA-nanosilver composites. We also make an attempt to explain the variety of mechanisms involved in their action. Finally, we present biocompatible and biodegradable complexes with bactericidal activity and low cytotoxicity, which properties suggest their suitability for the prophylaxis and therapy of chronic wounds, as well as analgetic therapies, anticancer strategies and the detection of chemical substances and malignant cells. Cited studies reveal that the usage of hyaluronic acid-silver nanocomposites appears to be efficient and safe in clinical practice. Nanotechnology is a rapidly developing branch of science which has a potential to influence all aspects of our lives, including medicine. Nanoparticles are objects with dimensions (or at least one dimension) that are smaller than 100 nm ,3. They Nanoparticles of metals such as palladium (Pd), rhenium (Re), osmium (Os), iridium (Ir), gold (Au), platinum (Pt), and especially silver (Ag), play significant role in nanomedicine ,6. SilvePolymeric materials may be used either as matrices for biologically active nanoparticulate systems or as a coating in order to modify their physicochemical properties , biocomp8 Da). HA constitutes the major GAG component of the extracellular matrix (ECM) [Among naturally occurring polysaccharides hyaluronic acid (HA) has a special place, mainly due to its unique biological activity. HA is a negatively charged, linear, polysaccharide, composed of D-glucuronic acid and N-acetyl-D-glucosamine, which are linked by β-1,3- and β-1,4-glycosidic bonds . It beloix (ECM) . At physix (ECM) . These tix (ECM) . As a coHA is widely present in various organisms . As much as 50% of all the HA resides in the skin (the dermis and the epidermis). It can act as passive structural macromolecule  or as a signaling molecule. It was therefore confirmed to play an important role in modulating various biological processes, including tissue repair, signal transduction, angiogenesis, cell motility, and cancer metastasis . CD44 (cIllustration created using UCSF ChimeraX (version 1.2.5.), developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases .This review gives an overview of the current state of art in the field of the composites of silver nanoparticles and biopolymers based on hyaluronic acid. It presents the methods of synthesis of such composites, their physicochemical and biological properties, and discusses medical implementation of these materials.To obtain the composites of silver particles and polymers two main steps are needed: (1) the synthesis of Ag nanoparticles (Ag NPs) and (2) introduction of the polymer chains on the Ag NPs surface or incorporation of Ag NPs into the polymer matrix a.Typically, Ag NPs are obtained by reduction of silver cations. Silver salts  are used as a source of silver ions, while ascorbic acid ,20,21, tAn interesting approach, proposed in the literature, is to modify the groups already present in the polymeric chain , corn silk extract, pectin) changing them into the effective reducing agents for silver cations ,28,29 Fb. Such mDepending on the polymer used, the composite form and the technique used to produce it, the resulting material may be tailored to the requirements of the application in order to obtain the functional product. In the case of wound dressings, where such composites are applied most often, the methods such as electrospinning ,28,29,30Electrospinning is a process in which electric force is used to form fibres from polyelectrolytes. Biopolymers can be easily processed using this method, because it does not require aggressive conditions and is well-suited for macromolecules. In medicine, it is used in tissue engineering, drug delivery and to produce nanofibrous mats used as wound dressings with favourable properties .Layer-by-layer (LbL) method allows creating thin polymeric films by a subsequent deposition of oppositely charged macromolecules. The number of layers strongly influences the mechanical features of the film, so they can be changed in a simple way by adjusting the time of the process. In the case of HA and Ag NPs, the two-component LbL composite is impossible to obtain due to negative charge of HA and no charge of Ag NPs, but the addition of positively charged polymer  resulted in the formation of functional wound dressing .Considered in the article polymers were hydrophilic , E. faecalis, S. aureus, S. pyogenes, Mycobacterium tuberculosis and P. larvae [+ ions show antibacterial effect even after bacteria form a biofilm [It was found that nanoparticles may inhibit growth of several microorganisms, including strains of . larvae ,43,44,45. larvae . However. larvae ,32 even . larvae ,33. This biofilm ,21.It is speculated that silver nanoparticles lead to protein denaturation and proton pumps destruction following the interaction with sulphur and phosphoric groups present on the fungal cell wall surface. Moreover, they most probably increase the permeability of the cell membrane, leading to its disintegration, ion equilibrium loss, metabolism, and respiration blocking, finally resulting in the cell death. Additionally, the role of oxygen free radicals is also considered ,48. It iFurthermore, the anticancer properties of nanosilver can be explained by the mitochondrial damage and the release of the reactive oxygen species, leading to a DNA damage, a disruption of the cell membrane, an impairing of the normal cells development and an induction of apoptosis .It is suggested that the antimicrobial effect of nanosilver depends on Ag ions release rather than is caused by the particle itself. This sets a challenge for the researchers to find the nanocomposites which would achieve optimal functionality ,23.Bactericidal, fungicidal, antiviral, anti-inflammatory, anti-angiogenic and anticancer properties make silver nanoparticles highly appreciated in medicine. They can also be useful in drug delivery, gene therapy, and as biosensors. Hippocrates used to cure wounds and ulcers with the silver powder . Credé iS. aureus and E. coli. What’s surprising, the material containing hyaluronic acid showed stronger antibacterial activity compared to those synthesized using heparin, possibly due to rougher surfaces, providing higher surface area exposed to the environment [Staphylococcus aureus minimal inhibitory concentration determined for the combination of allicin and nanosilver was considerably lower than that for both components separately [It is widely known that silver nanoparticles can effectively inhibit the growth of both Gram-positive and Gram-negative bacteria. Their efficacy in the complexes with biopolymers seems to be determined by the metal concentration ,32,55. Nironment . In the parately . The sphparately .Moreover, nanosilver exhibits a broad spectrum of antifungal activity. In the combination with fungicides, such as amphotericin B and fluconazole, the effectiveness increases ,60.Influenza A virus [respiratory syncytial virus (RSV) [Herpes simplex virus type 1 (HSV-1) [Hepatitis B virus (HBV) [It is still discussed whether Ag NPs can be applied in suppressing viruses. According to the recent literature it is active against  A virus , respiraus (RSV) , Herpes  (HSV-1)  and Hepaus (HBV) .Various studies suggest that silver nanoparticles demonstrate anti-angiogenic properties and may be useful in the treatment of angiogenesis-related diseases, such as cancer, inflammation, and vascular diseases. Biosynthesized silver nanoparticles reduced the expression of the vascular endothelial growth factor (VEGF) and its receptor (VEGF-R) genes, and significantly reduced the number of vessels and their length, as well as the length and weight of the embryos in chick chorioallantoic membrane (CAM) assay .Numerous in vitro and in vivo studies showed anticancer activity of nanosilver in various cancer cell lines, such as leukaemia THP-1, Jurkat, and K562 cells , MCF-7  and MDA-Silver nanoparticles can be also used in synergy with hyaluronic acid and S-nitrosothiol as the tumour targeting drug delivery system combined with photothermal therapy (PTT) and chemotherapy, increasing their selectivity ,74. WhatNanosilver can be employed in construction of catheters and tracheal tubes ,81,82. ,84 AppliPotential applications and usage of AgNPs and HA compounds are shown in the Nanosilver is commonly used in many areas of the human everyday life, for instance, in textiles, deodorants, paints, packaging, and kitchen equipment. Such a wide use of silver evokes questions about its influence on human organism. In general, Ag ions are regarded to be cytocompatible at low concentrations. However, high doses of silver ions released in a short time may induce cytotoxicity .+ ions. A direct effect of nanoparticles on the mechanisms of cell regulation and gene expression can also be considered. In a recent study, Vazquez-Muñoz and co-workers [Silver can be accumulated in the skin, generating its blue-grey colour—a primary symptom of argyria, caused by long-term exposition to silver, which has already been described in XVIII century . Recent -workers  assessed+ ions, can minimize the toxic effect of such system on mammalian tissues [However, the efficient stabilization of silver nanoparticles by the polymeric coating, and resulting low release of Ag tissues . The cyt tissues . Makvand tissues  demonstr tissues  who did S. aureus. The selectivity was explained by the ability of the bacteria to secret hyaluronidase, which degraded the hyaluronic acid coating, resulting in the release of the nanosilver. What is more, upon illumination with the near-infrared light, the temperature of the graphene oxide increased locally, which led to the high mortality of bacteria.The hyaluronidase-triggered nanosilver release was investigated by Ran and co-workers , who obtS. aureus and E. coli, while, at low concentration, they were not toxic to the L929 fibroblast cells. The in vivo test with rabbits and histological studies delivered evidence that these composites accelerate wound healing. A composite of κ-carrageen, hyaluronic acid, nanosilver and lidocaine also cured chronic ulcerations [Guanqgian Lan and co-workers  preparederations  indicatiS. aureus, and after 14 days its efficiency declined to 65–90%. That efficiency depended on a concentration of released nanosilver particles, but only for the first 14 days; then this correlation was no longer observed. Cytotoxic effect of the composites on MC3T3 mice osteoblasts also depended on the concentration of nanosilver.Properties of a titanium material covered with multilayer coat made of chitosan, hyaluronic acid and nanosilver were suitable for bone and dental implants . In the S. aureus, S. epidermidis and E. coli. The molecular weight of hyaluronic acid increased considerably when Ag NPs were formed within its structure. After admixing lecithin, the material was characterized by a more uniform profile of molecular weight, which was considerably higher than that in the lecithin—free nanocomposite, pointing out to the possibility of formation of the intermolecular complexes. Lecithin containing foils were more flexible and mechanically more resistant.Foils prepared, in a free of toxic reagents manner, from silver nitrate, xylose, hyaluronic acid and lecithin are promising dressings for healing wounds . It was E. coli K12, but an admixture of nanosilver considerably enhanced that effect. It was shown in vitro on the human keratinocyte HaCaT cell line, that neither hyaluronic acid alone nor its composites with nanosilver were cytotoxic. Studies on mice indicated positive effect of those composites against chronic ulcerations.Abdel Mohsen and co-workers prepared hyaluronic acid—nanosilver nanofibers and then built them into the nanosheets . Fibres B.Subtilis, S. Aureus and E. Coli [Sponges from polygalacturonic acid, hyaluronic acid and nanosilver were shown to exhibit bactericidal properties in vitro against  E. Coli .Composites based on Ag NPs and HA were also proposed as components of the systems for detection of marker substances, thus allowing for the early recognition of neoplasms. Yang and co-workers  exploiteOther approach to implement biopolymers into the laboratory diagnostics was proposed by Li and coworkers . Their wThe properties and potential biomedical applications of selected, important nanocomposites containing nanosilver and hyaluronic acid proposed in the literature are summarised in Despite many favorable biochemical properties of Ag NPs-HA biocomposites there are a few concerns to be solved. First of all, the nanocomposites still show cytotoxicity on the adjacent healing tissue, which was discussed previously. The method of lowering the amount of released silver ions and nanoparticles would be desirable, nevertheless currently achieved profile is acceptable and safe. The second challenge is the synthesis process. Although the synthesis of membranes and even hydrogels serving as wound dressings is not complicated, production of biosensors and drug transporter systems using HA and AgNPs still remains a challenge in terms of technology and costs. Synthesis pathways simplifying the fabrication process would positively influence the availability of composites and potentially broaden their applications. The development of green-chemistry approach to the biopolymer synthesis is also a promising way of cost reduction and in our opinion might be the main target of the future research in this field.The application of hyaluronic acid-nanosilver composites in medicine has an innovative character. Their bactericidal, antiangiogenic, and anticancer properties, as well as low cytotoxicity to human cells, encourage researchers and clinicians to seek broader application, predominantly as innovative wound dressings with antimicrobial capacity against drug-resistant microorganisms, accompanied by analgesic and healing-supportive features. Moreover, the composites appear to be promising components of oncological diagnostics and treatment. In our study we also introduced the materials that demonstrated satisfactory mechanical properties as potential medical implants designed for dentistry and orthodontics. The main advantages of hyaluronic acid-containing complexes over other silver nanocomposites include their high biodegradability and green synthesis, without use of any toxic reagents.∎Hyaluronic acid-nanosilver composites exhibit antimicrobial, anti-angiogenic, anti-inflammatory and anticancer properties, accompanied by a high biocompatibility.∎There are various techniques of synthesis of nanocomposites affecting their physicochemical properties, but the crucial points of the majority of the procedures include obtainment of the AgNPs and incorporation of them into biopolymer structure.∎Their potential applications include wound dressing production, oncological, vascular and inflammatory diseases treatment, as well as medical devices and implant fabrication.Take home message:


Basin tests were performed at the Aalto Ice Tank to gather data on ice-structure action and interaction from ice failing against a vertically sided cylindrical pile. The tests were performed with a real-time hybrid test setup, which combined physical and numerical components to simulate a range of test structures in real-time. The dataset includes results from tests with offshore wind turbine structures, structural models representing a series of single- and multi-degree-of-freedom oscillators, and scaled dynamic models of the Norströmsgrund lighthouse and the Molikpaq caisson structure. In addition, forced vibration tests and rigid structure tests were performed. Ice loads and structural response were measured with accelerometers, displacement sensors, potentiometers, strain gauges and load cells and the ice-structure interaction process was filmed from three different camera angles. The resulting raw data have been categorized and stored as unfiltered time series. A total of 259 different tests are included in the dataset. The model ice formation procedure and the test temperature were aimed at creating model ice that mimics the material behavior of full-scale saline ice during crushing failure, with a specific focus on the transition from brittle to ductile behavior. The data can be used for validation of models for dynamic ice-structure interaction. The offshore wind turbine data can be used to study the effect of wind loading on the interaction with ice and the effect of the specific dynamic properties of wind turbine structures with monopile foundations on the ice-structure interaction process. The forced-oscillation data can be used to quantify the time and speed dependant aspects of ice loading. The Norströmsgrund lighthouse and the Molikpaq data can be used as a reference comparison to full-scale data on ice loads. Data from eight ice sheets are included in the dataset. data files contained in the CSVDataFiles.zip archive. The ice sheets are identified by their creation date. The runs are numbered based on the run order in each individual ice sheet. The data files are numbered consecutively based on the order of recording. The tests are classified by a unique numeric identifier, numbered consecutively based on the ingestion date of the test data into a database.In each ice sheet, between one and 12 runs were made. A run is a single passing of the carriage from the South side of the ice basin to the North side of the basin. During a single run, one or several data recordings were made. The continuous data recordings are stored in 44 test files contained in the CSVTestFiles.zip archive. Test files are subsections of data files and contain continuous time series measurements under constant or linearly varying (in the case of carriage speed) test conditions. A total of 259 unique test files are included in the dataset which are divided over six different test types as shown in Each data file may contain several tests with different test conditions. The data recorded for each test is stored separately in x, y and z direction, two columns with the target displacement of the actuators, two columns with the measured displacements of the structure, one column for synchronization of data and video files, two columns with the numerical waterline displacement without forward prediction, a column with carriage speed and a column with carriage position. Except for the seven columns with verification data from the numerical controller, all other data is time synchronized and sampled with a sampling frequency of 2000 Hz.Each data- and test file contains 36 columns of unfiltered (raw) time series data. The readme.pdf file defines the file format. The 36 columns consist of: one column where the date is recorded, one column with the time from start of the recording, seven columns with verification data from the numerical controller, two columns with load cell data, two columns with identified ice forces on the structure, two columns with potentiometer data, 12 columns with accelerometer output in The database.xlsx file gives a full overview of all tests included in the dataset, providing information on ice properties, carriage speed, and the type of test conducted in relation to each individual CSV test file. For the tests where structural models were implemented in the numerical part of the real-time hybrid test setup a reference is made to the structural files and wind load files used for this purpose. The structural files define the dynamic properties of the structures simulated by the real-time hybrid test setup and are included in the StructuralFiles.zip archive. The wind load files contain the time-domain wind loads applied at the rotor-nacelle-assembly of (some of) the offshore wind turbine structures during the tests and are included in the WindFiles.zip archive. The file format for both these file types is provided in the readme.pdf.x-view, y-view, and table-view. The video recordings are divided into one file per viewpoint per run and divided over 20 zip-archives to avoid overly large file sizes. The camera locations are shown in Video recordings in 1080p were made for each run from three different view-points: 2The experiments were designed with the main aim to develop a model-scale benchmark dataset for offshore wind turbines loaded by crushing ice. In addition to this, tests with single-degree-of-freedom (SDOF) oscillators, existing structures for which ice-structure interaction data is described in literature, forced vibration tests and rigid structure tests were included to improve the fundamental understanding of the ice-structure interaction processes and scaling of these.For scaling of the offshore wind turbine tests the choice was made to relax some of the scaling laws and not scale time and frequency, as the time-dependant behavior of the ice in the basin could not be easily controlled and was not known in the planning phase of the experiments. In doing so, it is assumed that the scaled loads in the basin merely occur due to the smaller geometry and strength compared to full-scale, something which requires validation. The ice speed (carriage speed in the experiment) and the waterline displacements of the test structures were kept scale-invariant. To achieve structural deformations similar to full scale, a mass scaling factor was applied to the structural models, affecting the mass, stiffness and damping. The scaling factor was determined based on the ratio between the full-scale and model-scale mean brittle crushing load:This assures that the deformation of the structure in model-scale is equivalent to the full-scale deformations, consistent with the assumption that the length parameter controlling the ice failure does not change (much) between full-scale and model-scale ice, provided that an appropriate method of adjusting the model ice to better represent crushing has been obtained.2.1The different test types included in the experimental campaign are briefly described in the following sections.2.1.1x- and y-direction. Conditions without external wind loading, representing an idling turbine, and conditions with external wind loading aligned with the ice and under 30° and 90° misalignment were tested. For the latter conditions a generic wind load file was created containing a time series of the thrust force on the turbine at the top of the tower. This wind load was then applied to the numerical model in the modal domain.The offshore wind turbine tests were performed with a scaled . After the ramp test, some structures were also tested at constant carriage speeds to further investigate interaction phenomena observed during the tests with a linearly varying carriage speed profile.SDOF tests were performed for the first three global bending modes of a monopile offshore wind turbine separately. The separate offshore wind turbine modes were tested to assess, by comparing the SDOF results to the multi-modal offshore wind turbine test results, to what extend the interaction of multiple modes influenced the ice-structure interaction process. In addition, using the second global bending mode of an offshore wind turbine as a base case, variations in stiffness, damping and mass were tested to investigate the influence of these parameters on the ice-structure interaction process. In addition, simulations were performed with varying mass and stiffness but with the same natural frequency. The simulated parameter combinations were chosen such that sets of three tests could be constructed in which only one parameter was varied. All SDOF structures were tested with a linearly varying carriage speed profile . An increase in ice load can be observed in the test data at this carriage position for the tests performed on and before June 17th. 2.4The tests were performed with a real-time hybrid test setup which combines physical and numerical components to simulate the test structure in real-time. 2.4.1x-direction relative to the middle table, a middle table moving in y-direction relative to the carriage, and a top table which is fixed to the carriage through a support frame. The pile was attached to the bottom table by a bolted connection. The outer diameter of the pile interacting with the ice was 200 mm. The mass of the total setup was approximately 390 kg.x-direction, and of the middle table relative to the top table in y-direction were measured by displacement sensors (5). The table movement in x- and y-direction was driven by linear actuators (6). Accelerometers were mounted to the bottom (7) middle (8) and top (9) tables. The entire setup was connected to the carriage by an aluminium support frame (10).2.4.2X-view and Y-view. The camera positions are shown in All tests were captured by GoPro Hero 9 cameras from three different viewpoints: Table-view, 2.4.3The numerical component of the real-time hybrid test setup was handled by a Teensy USB Development Board . In real-time, this microcontroller received the voltage signal from the upper ring of strain gauges, converted the signal to a load based on a calibrated conversion factor, calculated the structure response to the measured load, and sent the target position to the actuators based on the numerically calculated waterline position of the structure.2.4.3.1Structures were represented in the numerical model by single- or multi-degree-of-freedom linear models in the modal domain. Note that the equations of motion presented in 2.4.3.2The structure response was calculated in real-time using the Euler-Cromer method . A time step size of 2.4.3.3In the real-time hybrid measurement, simulation and response loop, the ice force identified from the strain gauges needs to be sent to the control system. The control system needs to calculate the structure response to the ice load. The new waterline position needs to be sent to the actuators, and the actuators must go to the updated position. Because of this process, there is an inherent delay between the measured ice load and the physical response of the structure to the measured ice load. This delay may cause inaccuracy or instability in the structural response to the ice loads. To compensate for the delay in the measurement, simulation and response loop, actuator delay compensation was applied to the positions sent to the actuators. The delay was compensated by forward predicting the structure position:The forward prediction time was based on delay measurements performed during the initial tests of the test campaign. The delay between the numerical structure waterline position and the physical actuator position was determined by finding the maximum cross-correlation between the measured displacement and the time-shifted numerical displacement. Based on these measurements, the target position that was sent to the actuators by the control system was forward predicted by 5 ms. The directly measured pile displacement (channels DisplY_mm / DisplX_mm), the numerical structure position in the control module (channels SOutYnoFP_mm / SOutXnoFP_mm), and the forward-predicted position that was sent to the actuators (channels SOutY2_mm / SOutX2_mm) are stored separately in the test output, allowing for the evaluation of the forward prediction scheme.2.5This section describes procedures that were followed during the test campaign relevant to the use of the data.2.5.1In tests with IDs 2.5.2The strain-gauges were used in the experiments to identify the ice load. This required a calibration factor linking the output of the strain gauges to the ice load to be defined as the point of ice action on the pile was not known in advance of the experiments. This factor was obtained based on a comparison to the load cell data for tests with the actuators in fixed position, minimizing the inertial contribution of the test setup in the load cell data.2.5.3−1 either the actuators or the carriage was used to move the pile. It can be derived from the test data (DisplY_mm and CarVel_mm_s data columns) which method was used to move the pile.In most tests, the carriage to which the test setup was attached was moving at a constant, linearly varying or bilinearly varying speed. The carriage movement represents the ice drift (with the ice drift direction being opposite to the carriage movement direction). The actuators were used (with the carriage stationary) to test constant speed ice action at low speeds because this enabled a more accurate speed control. In tests at speeds of 5, 10 or 20 mm s2.5.4In most tests, an initial actuator offset from the zero-position was applied in order to prevent reaching the actuator limits during the tests. If possible, the actuator offset was set such that the actuators would not cross their zero-position during the tests, as an irregularity was observed in the force and acceleration signals when the actuators crossed their zero-position. The cause of this irregularity is currently not know and it remains in the data for those tests where the zero-position is crossed.2.5.5Ice thickness measurements were performed after most test runs. The ice thickness was measured at four equidistant points along the test channel at approximately 0, 10, 20 and 30 m. The ice thickness was measured with a calliper. The slushy ice which formed at the bottom during the day was scraped off with the calliper before taking the thickness measurement.2.5.6The model ice mechanical properties were determined according to the ITTC  recommended procedures (7.5-02-04-02) The compressive strength was measured by loading a series of square protrusions cut out of a free edge of the ice sheet. The protrusions had dimensions of around The Young's modulus was determined by measuring the deflection of the intact ice plate under a predefined load of 200 g. Assuming the ice sheet to behave as a linear-elastic plate on an elastic foundation, the measured deflection was used to estimate the Young's modulus.2.6x-direction (FPileX1_N) show a drift. This drift may be corrected by comparison to the load cell data in x-direction.The ice force derived from the strain gauge measurements (FPileX1_N and FPileY1_N) was used in the real-time hybrid test setup to calculate the structure response. We recommend to use this data as the primary source of ice load data in data analysis. The loads measured by the load cells (FLoadX_N and FLoadY_N) contain significant contributions from the inertia of the tables in the setup. The loads measured by the strain gauges in x- and y-direction, respectively.The potentiometer signals Pm1X and Pm1X represent an ice load estimate derived from local pile displacements at two locations (see Section 3.4.1 for sensor locations). The measured data shows that the ice load derived from the potentiometer signals is inaccurate. This is thought to be caused by the fact that the sensors only measure the local relative displacement at two points along the waterline, and that the distributed ice load cannot be derived from the local displacements in an accurate manner. The potentiometer signals in the data files can be converted back to the measured displacement by multiplying the recorded signals with factors 6.9901E-6 and 4.5963E-6 for the 2.7The dataset described in this article covers 259 tests out of a total of 663 tests that were performed in this test campaign. The remaining tests are excluded from the dataset mainly for two reasons.The towing carriage to which the test setup was connected was moving along rails driven by two electro-motors. The electro-motors were controlled by PID controllers with a constant or linearly varying speed target. In all data files prior to data file mode_377 (thus mode files < 377), the carriage PID controller settings were suboptimal for the highly dynamic load. The carriage was unable to maintain a constant speed during the tests, and the varying carriage speed had a significant influence on the test results. As the varying carriage speed influenced the ice-structure interaction process, this effect cannot be mitigated in post-processing. All tests in which the varying carriage speed issue occurred have been excluded from the dataset.Twenty tests were performed with structural models of offshore wind turbines containing confidential data. Data from these tests is excluded from the publicly available dataset.N/A.Hayo Hendrikse: Conceptualization, Methodology, Validation, Investigation, Data curation, Writing – original draft, Visualization, Supervision, Project administration, Funding acquisition. Tim C. Hammer: Conceptualization, Methodology, Software, Validation, Investigation, Data curation, Writing – review & editing. Marnix van den Berg: Conceptualization, Software, Validation, Investigation, Data curation, Writing – original draft, Visualization. Tom Willems: Conceptualization, Methodology, Software, Investigation, Data curation, Writing – review & editing, Supervision, Project administration, Funding acquisition. Cody C. Owen: Investigation, Data curation, Writing – review & editing, Visualization. Kees van Beek: Methodology, Software, Validation, Resources, Writing – review & editing. Nick J.J. Ebben: Validation, Investigation, Data curation. Otto Puolakka: Investigation, Resources, Writing – review & editing, Project administration. Arttu Polojärvi: Writing – review & editing, Investigation, Project administration.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Pogostone possesses various pharmacological activities, which makes it widely used in the clinic. Its effect on the activity of cytochrome P450 enzymes (CYP450s) could guide its clinical combination.To investigate the effect of pogostone on the activity of human CYP450s.The effect of pogostone on the activity of CYP450s was evaluated in human liver microsomes (HLMs) compared with blank HLMs (negative control) and specific inhibitors (positive control). The corresponding parameters were obtained with 0–100 μM pogostone and various concentrations of substrates.50 values of 11.41, 12.11, and 14.90 μM, respectively. The inhibition of CYP3A4 by pogostone was revealed to be performed in a non-competitive and time-dependent manner with the iK value of 5.69 μM and the KI/Kinact value of 5.86/0.056/(μM/min). For the inhibition of CYP2C9 and 2E1, pogostone acted as a competitive inhibitor with the iK value of 6.46 and 7.67 μM and was not affected by the incubation time.Pogostone was found to inhibit the activity of CYP3A4, 2C9, and 2E1 with the ICin vivo investigations to validate the potential interaction.The inhibitory effect of pogostone on the activity of CYP3A4, 2C9, and 2E1 has been disclosed in this study, implying the potential risk during the co-administration of pogostone and drugs metabolized by these CYP450s. The study design provides a reference for further  Pogostemon cablin (Blanco) Benth. (Lamiaceae), which is known as ‘Guang-Huo-Xiang’ in Chinese. Pogostone was reported to possess various pharmacological activities, including antibacterial  is a superfamily that contains heme-thiolate proteins and functions as monooxygenases, which are involved in the metabolism of various drugs . The substrates, including phenacetin, testosterone, coumarin, chlorzoxazone, dextromethorphan, diclofenac, mephenytoin, and paclitaxel, were obtained from Sigma Chemical Co. . Pooled human liver microsomes (HLMs) were purchased from BD Biosciences . The purity of chemicals was more than 98%, and other reagents were of analytical reagent grade.+, 10 mM glucose-6-phosphate, 1 U/mL of glucose-6-phosphate dehydrogenase, and 4 mM MgCl2), corresponding probe substrates, HLM, and 100 μM pogostone or positive inhibitors. The concentrations of positive inhibitors were: 10 μM furafylline for CYP1A2, 1 μM ketoconazole for CYP3A4, 10 μM tranylcypromine for CYP2A6, 50 μM clomethiazole for CYP2E1, 10 μM quinidine for CYP2D6, 10 μM sulphaphenazole for CYP2C9, 50 μM tranylcypromine for CYP2C19, and 5 μM montelukast for CYP2C8. The microsome protein concentration and other incubation conditions were summarized in The effect of pogostone on the activity of CYP450s was investigated with probe reactions  was added to terminate the reactions and placed on ice. The mixture was centrifuged at 12,000 rpm for 10 min, and an aliquot (50 μL) of supernatant was transferred for corresponding metabolites analysis with HPLC.50 and Ki values.The effect of pogostone on the activity of CYP450s was first investigated with 100 μM pogostone in HLMs. Then, the CYPs, of which the activity was inhibited by pogostone were incubated with 0, 2.5, 5, 10, 25, 50, and 100 μM pogostone and different probe substrates  to obtained the corresponding kinetic parameters, such as ICmK. Then, further incubations were performed to measure the residual activity. After being incubated for 10 and 30 min, the reactions were terminated by adding a 100 μL acetonitrile internal standard mix and then placed on ice; the corresponding metabolites were determined by HPLC.The effect of incubation on the inhibitory effect of pogostone was evaluated by incubating for 0, 5, 10, 15, and 30 min at 37 °C. The concentration of pogostone in the time-dependent inhibition study was 20 μM with the 1 mg/mL HLMs. After incubation, an aliquot (20 μL) was transferred to another incubation tube  containing an NADPH-generating system and probe substrates whose final concentrations were approximate to Further experiments were performed to quantify the time-dependent inhibition by the KI and Kinact values. The incubation was conducted with a higher substrate concentration, which was about 4-fold to Km and 0, 2, 5, 10, 20, and 50 μM pogostone for 0, 5, 10, 15, and 30 min. A two-step incubation scheme was performed as described above.iK is the inhibition constant, S is the concentration of the substrate and mK is the substrate concentration at half the maximum velocity (maxV) of the reaction. The mechanism of the inhibition was inspected using the Lineweaver–Burk plots and the enzyme inhibition models. The data comparison was performed using the Student’s t-test and performed using IBM SPSS statistics 20 .The Lineweaver-Burk plots were used to obtain enzyme kinetic parameters by the least-squares linear regression of the inverse substrate concentration versus the inverse velocity. Inhibition data from the experiments that were conducted using multiple compound concentrations were represented by Dixon plots, and inhibition constant (Ki) values were calculated using non-linear regression according to the following equation:p < 0.05, p < 0.05, 50 values of CYP3A4, 2C9, and 2E1 were obtained as 11.41, 12.11, and 14.90 μM.Corresponding positive inhibitors dramatically inhibited the activity of all isoforms of CYPs (iK value of CYP3A4 was further obtained as 5.69 μM (The inhibition of CYP3A4 by pogostone was fitted with the Lineweaver-Burk analysis, and it was found that the inhibition of CYP3A4 was non-competitive. The  5.69 μM . Additio 5.69 μM . The KI iK values of 6.46 and 7.67 μM (Contrary to the inhibition of CYP3A4, the inhibition of CYP2C9 and 2E1 by pogostone was found to be competitive with the  7.67 μM . While tPogostemon cablin, a commonly used traditional Chinese herb for vomit, diarrhoea, and headache (Swamy and Sinniah Pogostone is the major extraction of CYP3A4 is an important member of the CYP3A family, which is responsible for the biotransformation of a variety of drugs, such as paclitaxel, vincristine, and felodipine (Henningsson et al. Additionally, the activity of CYP2C9 and 2E1 was also dramatically inhibited by pogostone. Different from the inhibition of CYP3A4, pogostone was identified as a competitive inhibitor of CYP2C9 and 2E1. CYP2C9 and 2E1 are also responsible for the metabolism of various drugs, including losartan and acetaminophen, which are commonly used clinical drugs. Moreover, CYP2E1 was involved in the metabolism of fluoride inhalation anaesthetics, of which the high plasma concentration would induce renal toxicity and nerve injury (Schindler and Hempelmann maxC of 5 mg/kg pogostone was 28.99 μg/mL, which is larger than the IC50 value of pogostone in the inhibition of CYP3A4 and close to the IC50 values of pogostone in the inhibition of CYP2C9, and 2E1 (Chen et al. in vivo validation.Previously, Chen et al.  investigin vitro inhibitory effect of pogostone on the activity of CYP3A4, 2C9, and 2E1. Pogostone was identified as a non-competitive inhibitor of CYP3A4 and a competitive inhibitor of CYP2C9 and 2E1. Meanwhile, the incubation time was proved to be a vital factor in the inhibition of CYP3A4. These results provide reference and guidance for the clinical co-administration of pogostone with other herbs or drugs.This study revealed the


IgE characterizes the humoral response of allergic sensitization but less is known about what modulates its function and why some patients present clinical symptoms for a given IgE level and others do not. An IgE response also occurs during helminth diseases, independently of allergic symptoms. This response could be a model of non-functional IgE.To study the IgE response against environmental allergens induced during natural helminth infection.Dermatophagoïdes pteronyssinus (Dpt) and Ascaris (a marker of immunization against nematodes), and determined the IgE component sensitization profile using microarray ISAC biochips. The functional ability of IgE to induce degranulation of cultured mast cells was evaluated in the presence of Dpt.In 28 non allergic subjects from the periphery of Ho Chi Minh city with  and without helminth infection , we measured IgE and IgG4 against several components of in vitro and did not recognize usual Dpt major allergens. IgE recognized other component allergens that belong to different protein families, and most were glycosylated. Depletion of IgE recognizing carbohydrate cross-reactive determinant (CCD) did not induce a reduction in Dpt IgE. The Dpt IgG4 were not significantly different.Non allergic H+ subjects exhibited higher levels of IgE against Dpt compared to H- subjects. Dpt IgE were not functional Helminth infections induced IgE against allergens such as Dpt and molecular components that belong to different sources as well as against CCD . Dpt IgE were not able to induce degranulation of mast cells and were not explained by sensitization to usual major allergens or N-glycans. The canonical type 2 (T2) immune response involves immunoglobulin (Ig) E, IgG1, and IgG4 with eosinophils, basophils, mast cells (MCs), and alternatively activated macrophages, as well as interleukin (IL)-4, IL-5, IL-9, and IL-13. In the circulation, IgE is the least abundant class of Ig and is thought to have evolved in mammals from first-line defenses against parasites, which are too large to be phagocytosed. The IgE immune response that occurs during helminth infections is characterized by the production of large amounts of specific and non-specific IgEs . IgE indin vitro.We evaluated the IgE and IgG4 response against environmental allergens  occurring during helminth infection, before and after treatment, and compared it to healthy controls. The hypersensitivity function of specific IgE was evaluated Necator americanus or Ankylostoma. duodenale) and/or Toxocara canis infection (H+ group), and 10 were healthy controls (H- group). Diagnosis of hookworm infection was made by Kato-Katz thick smear analysis and T. canis serology . No subjects had overt symptoms of toxocariasis, and all were negative for Ascaris lumbricoides (Ouchterlony immunodiffusion test with home-made Ascaris (Asc) extract, serum samples concentrated 3×) and Shistosoma stercoralis . Exclusion criteria were pregnancy, positive skin prick test (SPT) to common aeroallergens, history of allergic and autoimmune diseases or human immunodeficiency virus infection, and previous anti-helminthic treatment during the past 6 months. Subjects from the H+ group that were infected with hookworm were evaluated at an additional visit 12 months after treatment of the infection (n = 11).A total of 28 subjects (aged 18–65 years) from a rural area in the periphery of Ho Chi Minh City (Vietnam) were included in this study. Eighteen of the subjects were infected with hookworm (Sera from a well characterized house dust mite (HDM) allergic population (n = 9) were used as a positive control for the indirect MC activation test (MAT). This European population (aged 21–37 years) was characterized by a confirmed HDM induced asthma and positive Dpt IgE (median 58.7 (IQR 15.7–119.3) kU/L; ThermoFisher, Massachusetts, USA) [The study was approved by the Ethics Committees (EC) of Pham Ngoc Thach University  and CHU Brugmann . The study was conducted in accordance with all applicable regulatory requirements and Good Clinical Practice. The ethics statement was amended by the Brugmann EC (Number 2021/72). Each participant provided signed informed consent prior to enrollment in the study. The study was registered at clinical.trials.gov (NCT02262403).Dermatophagoïdes pteronyssinus (Dpt) and its major allergens  and levels of IgE and IgG4 against Asc as a marker of immunization against nematodes. The ability of specific IgE to induce degranulation of MCs  has been tested in the presence of the corresponding allergen (Dpt and Der p 1) in order to assess the functionality of IgE.We compared different markers of allergen sensitization between the H+ and H- groups: IgE and IgG4 against T. canis was not available [Blood samples (8 mL in dry tube for serum collection) were stored at -80° until assayed. Total IgE (tIgE), specific IgE, and IgG4 were measured using ImmunoCAP  according to the manufacturer’s instructions. IgE and IgG4 were tested against Dpt, Derp 1, Derp 2, and Asc as a representative of nematodes because an ImmunoCAP for hookworm or vailable . A valueWe also performed an ImmunoCAP ISAC 112 microarray test  that includes 112 allergen components from 51 sources. The results were analyzed on a semi-quantitative basis and expressed as ISAC standardized units (ISU) (range 0.3–100 ISU-E).g for 2 min and the depleted serum was collected, pooled and immediately tested [To evaluate the interference of the glycosylated epitopes  in the detection of IgE to allergens, we repeated the measurement of Dpt and Asc using ImmunoCAP, as well as component IgE using ISAC after serum depletion of Phl p 4 antibodies, in samples exhibiting at least one positive IgE. We chose the Phl p 4 allergen to deplete anti-CCD IgE because a previous study showed that glycoproteins from pollen, such as Phl p 4, a highly glycosylated protein, were recognized by antiCCD IgE . Phl p 4y tested , 12.5 cells/mL and cultured for 21 days at 37°C, humidified 5% CO2 atmosphere. We add 300 μL Iscove’s Modified Dulbecco’s Medium  containing penicillin (100 U/mL), streptomycin (100 μg/mL), 1% insulin-transferrin-selenium (Life Technologies), 0.1% bovine serum albumin (Sigma–Aldrich), SCF (20 ng/mL), and IL-3 (20 ng/mL) twice a week for 2 weeks and with IMDM containing only SCF (IMDM + SCF 10 ng/mL) the last 7 days [MCs were cultured as described previously by Cop et al. , 14. Buft 7 days .TM software .On day 21 of culture, cells were retrieved, stained for viability (7-AAD) and surface makers with monoclonal anti-human CD45-BV510, CD203c-allophycocyanin (APC), CD117-fluorescein isothiocyanate (FITC), and FcɛRI-phycoerythrin (PE) to characterize the cultured MCs (BD Biosciences). Cultured mature human MCs were defined as CD45+, CD117+, and CD203c+low cells. Flow cytometric analysis was performed on a FACSCanto II flow cytometer  and data analyzed using BD FACSDiva2 atmosphere), centrifuged , and the cell pellet resuspended in Tyrode’s buffer (Sigma-Aldrich) at a concentration of 0.5 × 106 cells/mL. A 100 μL of the MC suspension was activated with 100 μL Tyrode’s buffer as a negative control, 100 μL of 2 μg/mL monoclonal anti-IgE (BD Biosciences) as a positive control, 100 μL of Dpt/D. farinae (Df) extract (5000 AU/mL (10−3) Glycotope, BD Biosciences), or 100 μL of 2.5 μg/mL Der p 1  for 20 min at 37°C. Reactions were stopped on ice and cells stained with 7-AAD, monoclonal antihuman CD45-BV510, CD203c-APC, CD117-BV421, and CD63-PE-Cy7. The expression of CD63 (% of viable CD117+CD203c+low) was measured as an indicator of MC activation. The AR was calculated as the expression of CD63 after stimulation with anti-IgE, Dpt, or Der p 1, divided by the expression of CD63 in the unstimulated condition [g for 15 min. Passive sensitization followed by activation was repeated.The function of IgE was evaluated by passively sensitizing cultured MCs with allogenic serum from H-, H+, and HDM-allergic subjects . Cells wondition , 17. To Statistical analyses were performed using GraphPad Prism software . Data were expressed as the median with interquartile range (IQR) and analyzed by a non-parametric Mann-Whitney test to compare unpaired data from two groups. Alternatively, the Wilcoxon test was used for paired data and Kruskal-Wallis test with corrected Dunn’s test for multiple comparisons. Correlations between antibody responses were calculated using the Spearman correlation. P ≤ 0.05 was considered significant.As expected, helminth infection was associated with a higher tIgE level (median 1409 (IQR 640–4745) vs. 112.1 (IQR 38.1–227.5) kU/L, p<0.001) in the H- group. The Asc response was characterized by higher Asc IgE, Asc IgE/tIgE ratio, Asc IgG4, and Asc IgE/Asc IgG4 ratio .Dpt IgE was also significantly increased in the H+ group, whereas IgEs against components, such as Der p 1 and Der p2, were not significantly different . The speB. tropicalis in one subject, Df and B. tropicalis in the other subject). Compared to non-sensitized subjects, both subjects seemed to exhibit higher Dpt IgE, Dpt IgE/tIgE, and Dpt IgE/Dpt IgG4 ratio  developed positive SPT against HDM  because A. lumbricoides infection was excluded by feces and serological negative results.Our study confirmed that the H+ group exhibited the characteristics of a type T2 response against helminths, i.e., a high level of tIgE and parasite-specific IgE and IgG4 . We consFirst, we investigated the response against Dpt as representative of HDMs that are a major cause of allergic rhinitis and asthma  and the The discrepancies between results from ISAC and ImmunoCAP may be explained by technical differences  rather than a specific allergen sensitization induced by helminth infection.The most often glycosylated antigen recognized was Phl p 4, which was quite surprising as MuxF3 the genuine marker of CCD sensitization was negative for most of them. However, Phl p 4 can be glycosylated at four different sites and, is therefore, a highly cross-reactive CCD-bearing allergen . After dThe IgE Dpt and Asc level were not affected by the Phl p 4 depletion, confirming results from Hamid et al. who showed that IgE targeting core β-1,2-xylose and/or ⍺-1,3-fucose substituted N-glycans does not play a role in Dpt and Asc sensitization . HoweverGlycans are produced by helminths during infection, among a large panel of molecules that can induce and regulate the T2 response  and theiThere is broad agreement from experimental studies and clinical practice that IgEs against CCDs lack clinical relevance . From a A. lumbricoides, T. trichiura, or hookworm infection) in Zimbabwe induced sensitization to α-Gal. This contradictory aspect could be explained the different helminthiasis affecting both population, or by a confounding factor inducing α-Gal sensitization not related to helminth infection (ie ectoparasites). T. canis may inhibit production of IgE to α-GAL [T. canis.Another interesting finding is the absence of sensitization to carbohydrate epitope galactose-α 1,3-galactose . This contradicts the study by Arkestål et al.  suggestito α-GAL  but in oOther components recognized by IgE in H+ subjects belong to several usual protein allergen families, such as PR10, arginine kinase, tropomyosin, serum albumin, and polcalcin, suggesting that helminth infection may induce sensitization to proteins that are well known allergens from clinically relevant sources. However, none of the subjects presented any cutaneous sensitization against common respiratory allergens or clinical symptoms of allergy. In contrast to El-Faham et al.’s report , our anain vitro model of MC passive sensitization. Overall, MC incubated with Dpt IgE positive sera from H+ subjects failed to degranulate in presence of Dpt.We investigated the reactivity to Dpt allergens using an Either, Dpt IgE present in H+ individuals may not be functional, or are only weakly functional. Another explanation could be the low level of anti-Dpt specific IgE suggesting that the occupation of FcεRI on MC was too low to be efficiently cross-linked. We attempted to increase the level of IgE by concentrating sera, but the AR was not different.in vivo. However, in vitro binding to anti-CCD IgE can result in cross-linking of FcεRI and activation of MCs, but it requires much higher concentrations [The recognition of CCDs in the extract could explain the absence of activation. Indeed, sensitization to CCDs has been described during helminth infection , 30–33 atrations .An alternative hypothesis is that IgE from H+ subjects are not or less able to initiate activation compared to allergic subjects because of a different glycosylation pattern of the antibody itself. Indeed, the sialylation of IgE was recently described to be a regulator of allergic disease . To the Finally, serum from H+ subjects could contains inhibiting factors. Antigen-specific IgG4 is linked to the modified T2 response, and the allergen-driven T2 response characterized by a high IgG4/IgE ratio has been proposed to result in immunological tolerance , 38. In When looking at the subgroup of subjects infected with hookworm evaluated 12 months after treatment, two developed a positive SPT against HDMs. Despite the limited number of subjects and the absence of a control group, it is of interest to note that both subjects presented differences compared to subjects who did not develop a positive SPT. At baseline (SPT-negative), these two subjects already had a stronger response against Dpt IgE and Asc that persisted after treatment of the infection. It suggests that subjects that become HDM-sensitized already had a stronger T2 response against both Asc and Dpt. The absence of cutaneous sensitization against HDM may be due to the tolerogenic environment of the “modified” T2 response, such as regulatory T cells  induced Overall, subject to the small number of subjects, our data indicate that the IgE response occurring during helminth infection is characterized by the production of non-functional IgE directed against environmental allergens, such as HDMs, N-glycans (CCDs), and peptidic allergens from different families. Sensitization to Dpt was not explained by sensitization to N-glycans nor to major allergens, was not able to induce MC activation, and was strongly linked to the intensity of the T2 response against helminths.S1 FigCorrelation between IgE and IgG4 response against Ascaris (Asc), D. pteronyssinus (Dpt), Der p1 and Der p2 were calculated with the Spearman correlation test for all the samples (n = 28) at T0. The Spearman r value is depicted in the figure and the correspondent p value is represented by the green scale when ≤ 0.05 and in white when non-significant.(TIF)Click here for additional data file.S2 FigT0: time of inclusion, 12M: 12 months after the hookworm treatment. Among H+ subjects that were evaluated at T0 and 12M (n = 11), wo groups were defined based on results from SPT 12M after the treatment of infection: SPT+ subjects are represented with squares and SPT–subjects with circles.(TIF)Click here for additional data file.S3 FigA: Mast cells gating strategy. Mast cells are defined as CD45+ alive and CD117+CD203c+low cells. Expression of FcɛRI is shown on the histogram in blue for CD117+CD203c+low cells and green for CD117‐CD203c+low. B: The activation of mast cells was measured by expression on CD63 on CD117+CD203c+low cells. The positive threshold was placed on the unstimulated condition.(TIF)Click here for additional data file.S4 FigActivation ratio calculated between CD63 (%) after Anti-IgE, Dpt, Der p 1 stimulation and negative control. H-: non infected group (n = 3), H+: helminth infected group (n = 9) and House dust mite (HDM) group: subjects allergic to mites (n = 5). P: pur serum and C: concentrated serum. Comparison between P and C were tested with a Wilcoxon test. A p value ≤ 0.05 was considered significant .(TIF)Click here for additional data file.S5 FigResults are expressed in ISAC Standardized Units (ISU), considered positive when above 0.30 ISU and categorized according to arbitrary ranges. Only subjects with at least one positivity were tested after Phl p 4 depletion. Red framework shows the glycosylated allergens according to manufacturer.(TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.