Interferons (IFNs) are a group of secreted proteins that play critical roles in antiviral immunity, antitumor activity, activation of cytotoxic T cells, and modulation of host immune responses. IFNs are cytokines, and bind receptors on cell surfaces to trigger signal transduction. The major signaling pathway activated by IFNs is the JAK/STAT  pathway, a complex pathway involved in both viral and host survival strategies. On the one hand, viruses have evolved strategies to escape from antiviral host defenses evoked by IFN-activated JAK/STAT signaling. On the other hand, viruses have also evolved to exploit the JAK/STAT pathway to evoke activation of certain STATs that somehow promote viral pathogenesis. In this review, recent progress in our understanding of the virus-induced IFN-independent STAT signaling and its potential roles in viral induced inflammation and pathogenesis are summarized in detail, and perspectives are provided. Interferons (IFNs) are a group of secreted proteins that play key roles in host antiviral immunity. IFNs are typically induced by the activation of host pattern-recognition receptors (PRRs), mainly retinoic acid-inducible gene 1 (RIG-I)-like receptors (RLR) and Toll-like receptors (TLR) during viral infection . To dateInduction of IFNs typically results from activation of host PRRs during virus infection. PRRs mainly include RLRs and TLRs ,2. AfterThe JAK/STAT pathway was initially characterized based on its role in type I IFN-mediated responses . The STANotably, viruses antagonize JAK/STAT signaling pathway . TherefoThere are three key components in the JAK/STAT pathway: a membrane-bound receptor, JAKs, and STATs . So far,In mammalian species, there are seven members  of the STAT family: STAT1, 2, 3, 4, 5A, 5B, and 6 . ExistenBefore activation of the JAK/STAT pathway, JAKs non-covalently associate with the cytoplasmic tails of cytokine receptors, and are functionally inactive. Upon ligand binding, dimerization or oligomerization of specific membrane receptors leads to JAKs apposition and autophosphorylation of tyrosine residues, thereby switching on tyrosine kinase activities of JAKs. Next, the activated JAKs phosphorylate tyrosine residues located in the cytokine-receptor cytoplasmic domains to provide binding sites for STATs SH2 domains, which recruit STATs to interact with JAKs. STATs are then phosphorylated by JAKs on tyrosine residues around amino acid (aa) 700, depending on the length of a particular STAT (750–850 aa in length) . FinallySTAT1 and STAT2 are the major players involved in type I IFN-mediated signaling and form interferon-stimulated gene factor 3 (ISGF3) complex along with IRF9 . Other cIt is believed that phosphorylation of tyrosine resides (around residue 700) in STATs is an essential step for canonical activation of the JAK/STAT pathway induced by cytokines, including IFNs . HoweverThe functions of U-STATs have been extensively investigated for STAT1 and STAT2, since these STATs have been linked to IFN-mediated signaling. After stimulation of the JAK/STAT pathway by high dose IFN treatment (1000 U/mL), tyrosine phosphorylation of STAT1 and STAT2 could be observed within a half hour, but steadily decreased after two hours, and to basal levels by eight hours . This reFurther analysis has indicated that U-STAT1 and U-STAT2, along with IRF9, can support the formation of unphosphorylated ISGF3 (U-ISGF3) . U-ISGF3The function of U-STAT3 has been investigated as well, since STAT3 is linked with oncogenesis and IFN-activated responses . When a In addition to tyrosine phosphorylation, STAT serine residues within the C-terminal transactivation domain (TAD) can be phosphorylated as well . InitialStat1−/− cells demonstrated the occurrence of STAT1-independent, STAT2-dependent gene expression is a delayed event during the transcriptional response to type I IFNs [Legionella pneumophila infection than were wild-type macrophages [Stat1−/− mice upon infection with intracellular microbe Listeria monocytogenes [Stat1−/− mice. However, the NK maturation defect in the STAT1Y701F mice was similar to that observed in Stat1−/− mice [Stat1-S727A mice exhibited slightly elevated numbers of mature NK cells (mNKs) in bone marrow, spleen, and blood [Stat1-S727A mice show significantly higher cytotoxicity against a range of tumor cells [So far, the in vivo function of serine monophosphorylation within the TAD of STATs has been exclusively investigated for STAT1. Previous studies in e I IFNs . Intrigurophages . Howeverytogenes ,70. Mean−/− mice ,70. Convnd blood . Unexpecor cells ,72. AlthCurrent understanding of tyrosine phosphorylation-independent non-canonical STATs activation remains limited. Previous studies focusing on U-STATs mainly investigated phosphorylation of tyrosine, but rarely examined serine phosphorylation status within the TAD domain at the same time ,55,73. TThe kinase responsible for monophosphorylation of serine residues of STAT TAD domains in the absence of tyrosine phosphorylation remains elusive so far. Screening of specific CDK8 kinase inhibitors as targeted drugs for cancer therapy has demonstrated that inhibition of CDK8 kinase can result in decreased phosphorylation of STAT1 at S727 in a variety of cancer cells, and phosphorylation of STAT1-S727 could serve as a biomarker of CDK8 kinase activity in vitro and in vivo ,76,77. MIn addition to be activated by IFNs, STAT1 also responds to other cytokines. Studies from gain-of-function mutations suggest that increased and prolonged phosphorylation of STAT1 is observed in response to IL-6 and IL-21 . STAT2 aSTAT3 was initially identified as an IL-6-dependent transcription factor that promotes acute phase gene expression . It is nSTAT3 has also been shown to be involved in mammary gland development. Lack of STAT3 causes a severe delay in post-lactational regression of the mammary gland, as observed in epithelium-specific STAT3-knockout mice . HoweverSTAT4 was initially identified through screening for STAT homologues, and was found to share 52% identical amino acids with STAT1 . ExpressAlthough STAT4 plays important roles in driving differentiation of T helper cells, its molecular mechanism of action is largely unknown. By using chromatin immunoprecipitation and high-throughput sequencing to compare the transcriptional profiles of STAT4 and STAT6, STAT4 was found to bind over 4000 genes with distinct binding motifs . Among tSTAT5a and STAT5b [STAT5A and STAT5B were found to be encoded by two linked genes, d STAT5b . These td STAT5b . In addid STAT5b . Althougd STAT5b . Meanwhid STAT5b , as welld STAT5b . STAT5 cd STAT5b ,109. It d STAT5b . Moreoved STAT5b , and thid STAT5b . In addid STAT5b . AnalyseSTAT6 was originally identified from cellular extracts as an IL-4-stimulated STAT, and was soon shown to be activated by IL-13 as well ,113. STASince STAT proteins function as essential mediators of cytokine- or hormone-induced signaling to promote cell development, proliferation, and differentiation, activation of STATs is tightly regulated. Suppressive regulators of STAT activity include protein inhibitors of activated STAT (PIAS) family, suppressors of cytokine signaling (SOCS), and ubiquitin carboxy-terminal hydrolase 18 (USP18) ,119.In mammals, PIAS proteins are encoded by four genes: PIAS1, PIAS2 , PIAS3, and PIAS4  . Except The suppressor of cytokine signaling (SOCS) family is another group of negative regulators for the JAK/STAT pathway . The SOCUSP18, a protein of 368 aa in length and an ISG15 isopeptidase, is a negative regulator of type I and III IFN-activated JAK/STAT signaling , and is Besides the well-defined inhibitors described above, JAK/STAT signaling can also be regulated by cysteine-based protein tyrosine phosphatases (PTPs), which dephosphorylate pTyr residues in the JAK/TYK activation loop or phosphorylation sites within cytoplasmic domains of cytokine receptors . HoweverIn addition to SUMOylation of STATs by PIAS, post-translation modification (PTM) has been shown to regulate STATs activation as revealed by proteomic technologies. Modification of certain sites among STATs can result in either positive or negative effects on STATs activation . AcetylaAnother mechanism for regulating STAT activation involves both arginine- and lysine-based methylation of STATs ,151. TheBesides methylation and acetylation, ISGylation involving covalent bonding of targets to interferon-stimulated gene 15  has been shown to regulate IFN signaling as well ,158. An Collectively, PTM of STATs represents a novel mechanism for JAK/STAT regulation. However, more studies are needed to understand this regulatory mechanism, since crosstalk between methylation, SUMOylation, and acetylation is still not understood. Moreover, a recent study demonstrated that inhibition of HDAC enhances STAT acetylation but blocks NF-κB signaling during renal inflammation and fibrosis in haplotype Npr1+/− male mice . TherefoSince the discovery of virus-encoded IFN antagonists, it has been proposed that JAK/STAT pathway antagonism is a virulence factor that might be exploited to achieve virus attenuation during vaccine development. This speculation is further supported by the observation that mice lacking intact JAK/STAT signaling machinery (IFN receptor or STAT1 knockout mice) are highly susceptible to virus infection ,22,23,24As a typical example, porcine reproductive and respiratory syndrome virus (PRRSV) is known for its ability to inhibit both IFN induction and IFN-activated JAK/STAT signaling, with several PRRSV antagonists of JAK/STATs previously identified ,166,167.As discussed in the previous section, monophosphorylation of serine residues in STATs has been frequently reported as non-canonical TAD serine phosphorylation in the absence of tyrosine phosphorylation . This obAs mentioned above, HP-PRRSV infection in pigs is characterized by aberrant expression of pro-inflammatory cytokines and chemokines, leading to a robust inflammatory response ,171. OurIn addition to PRRSV, virus-induced serine monophosphorylation of STAT1 has been reported for other DNA and RNA viruses, such as Epstein–Barr virus (EBV) and human immunodeficiency virus-1 (HIV-1), as well ,173,174.Besides EBV, participation of STATs in viral pathogenic responses was also observed in human immunodeficiency virus (HIV)-1 as well. The Tat protein of HIV-1, a regulatory protein for viral transcription enhancement, contributes to the immune evasion of HIV by inducing SOCS3 expression to antagonize IFN-induced canonical STAT activation . HoweverMeanwhile, studies on Kaposi’s sarcoma-associated herpesvirus (KSHV) demonstrated that latent protein kaposin B promotes serine monophosphorylation of STAT3 at S727 in the absence of tyrosine phosphorylation at Y705 . KSHV inSo far, serine monophosphorylation induced by virus infection has been only reported for STAT1 and STAT3. Although it is still unknown if other STATs could have monophosphorylation of serine residues at TAD domains, neither tyrosine phosphorylation nor serine phosphorylation of STAT2, STAT5, and STAT6 was observed in HBMECs exposed to HIV-1 virion. . TherefoSince induction and signaling pathways of IFNs are well-defined, and great progress has been made to understand virus antagonism of the IFN-JAK/STAT axis, many questions regarding the IFN-JAK/STAT axis remain unanswered. Although IFN types and subtypes (type I and type III) appear to be redundant in their functions due to the activation of common downstream JAK/STAT pathway, the exact differences between those IFN subtypes remain unknown. Indeed, such distinctions may be an artifact of an arbitrarily simple classification of IFNs that overlooks the functionally heterogeneous nature of IFN types and subtypes. Additionally, since the antiviral functions of IFNs rely on ISGs, elucidation of ISG functions is also of interest, as most ISGs are not well-defined. Notably, some ISGs also respond to other transcription factors, such as members of the IRF family, activator protein 1 (AP-1), and NF-κB ,185. TheMeanwhile, the roles of IFN-activated JAK/STAT signaling in viral virulence and pathogenesis also require further investigation. This is especially true with regard to the in vivo role that IFN antagonists play, since most of these viral antagonists have been identified using in vitro screening methods. Moreover, crosstalk between virus-mediated antagonism of canonical JAK/STAT activation, viral-induced activation of non-canonical JAK/STAT pathway, and inflammatory responses promoted by viral infection may be also linked to viral pathogenesis. In the coming decades, additional mechanisms likely will be revealed to further our understanding of the connection between non-canonical JAK/STAT activation and inflammation-linked viral pathogenesis.


Muscle atrophy, an involuntary loss of muscle mass, is involved in various diseases and sometimes leads to mortality. However, therapeutics for muscle atrophy thus far have had limited effects. Here, we present a new approach for therapeutic target prediction using Petri net simulation of the status of phosphorylation, with a reasonable assumption that the recovery of abnormally phosphorylated proteins can be a treatment for muscle atrophy.p-value < 0.05). Furthermore, we noticed that they included several proteins that could not be characterized by the shortest path analysis. The three potential targets, i.e. BMPR1B, ROCK, and LEPR, were manually validated with the literature.The Petri net model was employed to simulate phosphorylation status in three states, i.e. reference, atrophic and each gene-inhibited state based on the myocyte-specific phosphorylation network. Here, we newly devised a phosphorylation specific Petri net that involves two types of transitions (phosphorylation or de-phosphorylation) and two types of places (activation with or without phosphorylation). Before predicting therapeutic targets, the simulation results in reference and atrophic states were validated by Western blotting experiments detecting five marker proteins, i.e. RELA, SMAD2, SMAD3, FOXO1 and FOXO3. Finally, we determined 37 potential therapeutic targets whose inhibition recovers the phosphorylation status from an atrophic state as indicated by the five validated marker proteins. In the evaluation, we confirmed that the 37 potential targets were enriched for muscle atrophy-related terms such as actin and muscle contraction processes, and they were also significantly overlapping with the genes associated with muscle atrophy reported in the Comparative Toxicogenomics Database  contains supplementary material, which is available to authorized users. Muscle atrophy is defined as the involuntary loss of muscle mass, resulting in low quality of life, morbidity and mortality . It is aFor therapeutic target prediction, the effects of gene inhibition (knock-down) are necessarily analyzed with a general assumption that most drugs inhibit their targets. shRNA knockdown, one of the in-vitro experiments, is a popular and typical tool for the purpose. However, it is time-consuming and labor-intensive to perform the experiments in genome-scale, especially with a specific disease condition (e.g. atrophic state in this case). Thus, in this study, we employed the Petri net, an in-silico method, which enables dynamic simulations on a large-scale network with incomplete kinetic parameters . The PetIn this study, we focused on phosphorylation status in an atrophic state. Phosphorylation plays one of the key regulatory roles in signal transduction by sequentially activating/deactivating various proteins and enabling cells to respond to external stimuli . Due to We predicted therapeutic targets for muscle atrophy by analyzing phosphorylation status among reference, atrophic and each gene-inhibited state, which were computed by Petri net simulations on large-scale phosphorylation network. Here, we developed a phosphorylation-specific Petri net model whose token represents the amount of phosphate and that involves two types of transitions (phosphorylation or de-phosphorylation) and two types of places (activation with or without phosphorylation). The difference between the phosphorylation status of reference and atrophic states were validated by Western blotting experiments detecting five marker proteins, i.e. RELA, SMAD2, SMAD3, FOXO1 and FOXO3. Among 331 kinases and phosphatases, all of which link to the five validated marker proteins in the integrated network, we generated 37 potential therapeutic targets whose inhibition recovers the five abnormally phosphorylated proteins in atrophic states. The predicted therapeutic targets were validated by the genes associated to muscle atrophy reported in the Comparative Toxicogenomics Database (CTD) , and theth gene-inhibited state). Then, we selected proteins whose inhibition recovers the phosphorylation status from atrophic to reference state in terms of the five validated proteins in  and 7283 edges (phosphorylation and de-phosphorylation) were constructed from four public databases, i.e., KEGG pathway , DEPOD , Phospho in Fig. .Fig. 2MeWe constructed an integrated phosphorylation network, whose nodes represent proteins and whose edges represent either phosphorylation or de-phosphorylation. To this end, we retrieved phosphorylation and de-phosphorylation interactions from three databases, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway , the DepThe kinases, phosphatases and their substrate proteins in the extracted interactions were mapped to Entrez ID. We deleted 215 duplicated interactions and removed 10 conflicted interactions with literature evidence, which resulted in 9669 interactions (7737 phosphorylation and 1932 de-phosphorylation interactions) among 2326 proteins. In order to consider muscle cell (myocyte)-specific conditions on the constructed network, we removed interactions that included non-detected proteins in myocytes reported in The Human Protein Atlas . This fiactivation and de-phosphorylation or both inhibition and phosphorylation are concurrently assigned for a certain enzyme Q to K in KEGG pathways. The other 390 kinases and 111 phosphatases were regarded to be activated with phosphorylation based on the assumption , and phosphorylation and de-phosphorylation interactions are represented by transitions (7283 transitions), and the amount of phosphates is represented by the number of tokens, an integer. The amount of phosphates in a specific protein (the phosphorylation status) is represented by the number of tokens in the corresponding place, whose value is an integer that is equal to or greater than zero. For the purpose of the study, we developed a phosphorylation-specific Petri net configuration that involves two types of transitions and two types of places, which is quite different from a typical Petri net model.There are two types of transitions, phosphorylation (5891 transitions) and de-phosphorylation (1392 transitions), whose corresponding Petri net transitions were depicted in Fig. We also presented the two types of places as introduced in the above section. It should be noted that the criteria for the two types of places is an activation condition of the enzymes, such as activation with phosphorylation or without phosphorylation, and not a function of the enzymes, such as kinase or phosphatase that has been already considered in transitions. For an input place that is activated with phosphorylation (309 kinases and 111 phosphatases), its transition is enabled when the number of tokens in the input place is greater than the enabling condition, 35. On the other hand, for an input place that is activated without phosphorylation (19 kinases and 6 phosphatases), its transition is enabled when the number of tokens in the input place is less than 35 Fig. .We performed Petri net simulations on the phosphorylation network for three states, i.e. reference, atrophic and each gene-inhibited state. To reduce computation time in the massive simulations on the large-scale network, we partially adopted the Petri net simulation procedure presented in Derek Ruths’ work . In one In state-specific perturbations, there is no perturbation for the reference state. On the other hand, for atrophic state, high activity is assigned to myostatin that is a protein inhibiting muscle cell growth and inducing muscle atrophy . This peDue to randomness in assigning the initial marking and shuffling transition list, 10 Petri net simulations were performed for the reference, atrophic and each gene-inhibited state, respectively (10 iterations). We noticed that tokens were saturated at the final block (the 100th block) for most places in all three states, and for each state we computed the phosphorylation status of 1985 proteins by averaging the final markings (the marking of 100th block) of the 10 iterations. As a result, we obtained the phosphorylation status of 1985 proteins in one reference, one atrophic, and 331 gene inhibited states Fig. . All of From three Western blotting experiment papers , 25–27, For the six marker proteins, the differences in the simulation results between reference and atrophic state were compared to those of the Western blotting experiments in Fig. The Petri net simulation results highly depend on the enabling threshold we selected. Thus, we selected the most robust threshold, i.e. 35, among the eleven enabling thresholds . To do this, for each enabling threshold, ten simulations were performed in reference and atrophic states, respectively, with consideration of randomness in assigning initial marking and shuffling transition list. Then, for each simulation, we generated a list of the ranked places by their final marking (the marking of 100th block), which describes the saturated phosphorylation status. Among the ten list of the ranked places in an enabling threshold, we calculated rank correlations in a pair-wise manner with the spearman rank correlation, which results in 45 rank correlations (except for the correlation of itself). When averaging the 45 rank correlations, the enabling threshold of 35 yields the highest rank correlation (0.791), which indicates that the threshold of 35 is the most robust among the eleven thresholds. Thus, we decided to use 35 as the enabling threshold  were specified in Additional file Functional enrichment tests were performed on the 37 potential therapeutic targets against Gene Ontology (GO) terms and KEGG pathways , 28, andp-value of 0.034  from the Comparative Toxicogenomics Database (CTD) . As the Here, the inferred genes in CTD are genes associated with muscle atrophy, which include not only genes curing the disease (therapeutic targets) but also genes causing the disease (causing genes) as well as genes effected by the disease (effected genes). It can be a reason for not very high predictive power. In addition, an imperfect network model also can be an explanation of this low performance because there are still unknown phosphorylation reactions in a cell.p-value 0.136. This indicates that the Petri net-based potential targets are not connected to the marker proteins within a short length. We noticed that their shortest path lengths yield a range from 2 to 5 and their average is 3.07, which is larger than the average shortest path length of all 331 candidate proteins, 3.06. Among the 37 Petri net-based targets, 15 proteins are longer in length than the overall average, 3.06. It could be frustrating because our results are not reproduced in a shortest path analysis, which is one of the most popular static network analysis. However, on the other hand, we think that Petri net simulation can characterizes atrophy-related gene sets, which cannot be predicted by the shortest measure.Various types of proximity measures in biological networks have been developed to elucidate drug efficacies or mechanisms . Among tWe validated several predicted therapeutic target genes by literature searches. BMPR1B is a member of the bone morphogenetic protein (BMP) receptor family, which is reported as a positive regulator of muscle mass in Winbanks’ work . Rho-assAmong several well-known simulation tools including Petri net, Random walk and ordinary differential equation (ODE), we consider that Petri net is the most appropriate tool for this work. The size of the constructed phosphorylation network is so large (7283 interactions) that we cannot employ ODE, whose kinetic parameters are incomplete. Additionally, it is not possible to employ Random walk for this study that particularly focuses on phosphorylation status, which involves two types of interactions and two types of nodes.We expected that myostatin direct targets, such as ACVR1 and ACVR1B, would be included in the predicted therapeutic targets because their inhibition produces almost the same effect as the inhibition of myostatin, which should recover the phosphorylation status from an atrophic state for the all five marker proteins; however, ACVR1 and ACVR1B are excluded. When they were inhibited, the numbers of markers recovered are 5, 5, 3 for ACVR1 and 4, 5, 3 for ACVR1B for the three simulations in the gene-inhibited state, which does not satisfy the selection criteria for potential therapeutic targets. We recognize that the criteria should be modified so as not to miss such obvious targets, and this will be carried out in further work.In this study, we suggested a new approach to predict potential therapeutic targets for muscle atrophy based on the phosphorylation status of the reference, atrophic and gene-inhibited states, which were simulated by Petri net. The simulation results between the reference and atrophic states were validated with Western blotting experiments detecting five marker proteins. We generated a list of the potential therapeutic targets whose inhibition recovers the five abnormally phosphorylated proteins in the atrophic state, and they were evaluated by various approaches, such as GO terms, the literature, and genes associated with muscle atrophy. We expect the new proposed strategy can provide an understanding of phosphorylation status in muscle atrophy and provide assistance towards identifying its therapeutics.Additional file 1:Figure S1. An average of rank correlations among ten simulations in pair-wise manner for each of eleven thresholds. We selected the enabling threshold 35 that show the highest rank correlation in the reference and atrophic states. (DOCX 51 kb)Additional file 2:Table S1. The 37 characterized therapeutic targets. Table S2. The enriched GO terms and KEGG pathways. Table S3. Hypergeometric test with gold standard from CTD. (XLSX 19 kb)


The antibody IgE plays a central role in allergic disease mechanisms. Its effector functions are controlled through interactions between the Fc region and two principal cell surface receptors FcεRI and CD23. The interaction with FcεRI is primarily responsible for allergic sensitization and the inflammatory response, while IgE binding to CD23 is involved in the regulation of IgE synthesis and allergen transcytosis. Here we present the crystal structure of a CD23/IgE-Fc complex and conduct isothermal titration calorimetric binding studies. Two lectin-like “head” domains of CD23 bind to IgE-Fc with affinities that differ by more than an order of magnitude, but the crystal structure reveals only one head bound to one of the two identical heavy-chains in the asymmetrically bent IgE-Fc. These results highlight the subtle interplay between receptor binding sites in IgE-Fc and their affinities, the understanding of which may be exploited for therapeutic intervention in allergic disease. For reasons poorly understood, IgE recognizes seemingly innocuous substances known as allergens KD of ~1 μM1617182+15Der p 1 generates a soluble monomeric form of CD23 consisting of just the lectin head domain, termed derCD23The second receptor for IgE, CD23, is unique among Ig receptors in that it belongs to the C-type lectin-like superfamilyH2) cell recognition and activation. The activated TH2 cells secrete cytokines that ultimately lead to an increase in IgE synthesis and exacerbation of allergic inflammation2629The interaction of CD23 with IgE is involved in regulating many allergic processesvice versaWe previously reported the crystal structure of derCD23 in complex with Fcε3-419Here we present the crystal structure of derCD23 in complex with the complete IgE-Fc region, including the Cε2 domains, which in the free molecule fold back onto the Fcε3-4 region in an asymmetrical fashioni.e., one IgE-Fc dimer bound to one molecule of derCD23) was unexpected, as it is well documented that one CD23 head domain binds to each heavy chain of IgE, whether the Fcε3-4 sub-fragment or the complete IgE-Fc151931The crystal structure contains two essentially identical complexes of derCD23/IgE-Fc within the asymmetric unit. Each complex consists of one IgE-Fc molecule bound to one molecule of derCD23 . The der2+-bound derCD23/Fcε3-4 crystal structure  10%, and the Cε4 domain 10%. The Cε2-Cε3 linker (Lys326 to Ser337) does not contribute to the interface. Relatively few well-defined residues are involved in the network of van der Waals contacts at the interface . Of these hydrophobic interactions, only one involves the Cε2 domain: Phe321 interacts with Gly180 of derCD23. No interactions are observed between derCD23 and the other IgE-Fc heavy chain.The derCD23/IgE-Fc interface shows an even poorer surface complementarity2 domain pair from the free IgE-Fc structure onto the derCD23/Fcε3-4 complex had suggested that only minor rearrangements of a few N-terminal residues in the Cε2 domain were required to prevent a steric clash with derCD232 domain pair move as a rigid unit, swinging out 16° to accommodate the derCD23 molecule .The binding affinity of derCD23 to IgE-Fc was determined using ITC, by titrating derCD23 into a fixed concentration of IgE-Fc. The data fitted best to a two-site binding model . One binKD = 1.2 μM and 14.4 μM), with distinct thermodynamic profiles (KD ~ 10 μM)The interaction of CD23 and IgE is critical in many allergic disease processes, including the regulation of IgE synthesis, facilitated allergen presentation and allergen transportation across the gut and airways. Here we present the crystal structure of a lectin-like head domain of CD23 in complex with the complete Fc receptor-binding region of IgE. The results of ITC experiments reported here confirm the 2:1 stoichiometry reported earlier19profiles . This isThe crystal structure of derCD23 bound to IgE-Fc unexpectedly revealed only a 1:1 complex, with the single derCD23 molecule bound principally to the heavy chain onto which the Cε2 domains are folded back. The derCD23 molecule primarily contacts Cε3 and, as in the complexes with Fcε3-42031vice versa2+-binding to CD23 causes local allosteric conformational changes that increase its affinity for IgEDespite the undoubted structural asymmetry between the two derCD23 binding sites in IgE-Fc, we cannot definitively assign the stronger and weaker affinity values to these sites. However, it is reasonable to assume that the derCD23 molecule seen in the crystal structure corresponds to the higher affinity site; it also has contributions from Cε2 which could not occur at the site in the other heavy chain. In any event, the binding of the first derCD23 molecule at least preserves, and perhaps even exacerbates, the asymmetry in IgE-Fc and in particular the Cε3-Cε4 interdomain angle; if the latter, then binding to the first site may influence the affinity of the second site . If ther2+, has recently been proposed to be involved in the mechanism that releases IgE/allergen complexes and recycles receptor back to the plasma membrane44Thus the CD23/IgE/FcεRI network of interactions involves at least two and perhaps three allosteric mechanisms within IgE-Fc. Together these permit an extraordinarily intricate level of control of protein-protein interactions regulating many aspects of the allergic response. Mutual exclusion of FcεRI and CD23 binding to IgE is essential to prevent mast cell and basophil activation by trimeric sCD23 in the absence of allergenExtrapolation of the results presented here to the binding of either one or two complete CD23 trimers to IgE is also important. We have previously reported that the two head domains that engage Fcε3-4 cannot come from the same trimerRecombinant human derCD23 Ser156-Glu298) and IgE-Fc  proteins were expressed, refolded and purified according to protocols previously described6-Glu298 3132·HCl (pH 7.5), 20 mM NaCl, 0.05% (w/v) sodium azide, followed by mixing of 0.56 mM derCD23 (8.6 mg/mL) with 0.28 mM IgE-Fc (20 mg/mL), and diluting the complex with an equal volume (100 nL) of 15% (w/v) PEG 8,000, 0.1 M sodium citrate, 0.05 M ammonium sulfate. A single needle-like crystal suitable for data collection grew after one and a half months at 295 K. The crystal was flash-cooled (using 18% (w/v) PEG 8,000, 0.1 M sodium citrate, 0.05 M ammonium sulfate, 20% (v/v) glycerol as cryoprotectant) and stored in liquid nitrogen. Diffraction data were collected at 100 K at beamline I03, Diamond Light Source.The derCD23-IgE-Fc complex was crystallized by vapor diffusion. IgE-Fc was concentrated to 37 mg/mL, and derCD23 to 18 mg/mL, both in 25 mM TrisMOSFLM48AIMLESSPHASER2+-free derCD23 molecules (using PDB 4J6J)PHENIXREFMAC5BUSTER-TNTCOOTFo–Fc composite omit, 2Fo–Fc and Fo–Fc electron density maps to minimize bias. Disruption of some disulfide bonds due to radiation damage was observed in the derCD23/IgE-Fc complex structure. Carbohydrate atoms were subsequently incorporated into the structures. During refinement, tight NCS restraints were initially used; these were gradually relaxed and finally local structure similarity restraintsTLSMD Web serverPISACONTACTSDynDomPyMOLIndexing and integration of data were carried out with ·HCl (pH 7.4), 125 mM NaCl supplemented with 4 mM CaCl2The derCD23 and IgE-Fc proteins were dialyzed overnight into 25 mM TrisHow to cite this article: Dhaliwal, B. et al. IgE binds asymmetrically to its B cell receptor CD23. Sci. Rep.7, 45533; doi: 10.1038/srep45533 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


N-Hydroxycinnamoylphenalkylamides (36H) exhibited both antioxidation and antityrosinase abilities. The compound was studied for its antioxidative properties, using a 1,1-diphenyl-2-picrylhydrazul- (DPPH-) scavenging test, a ferric ion-reducing antioxidant power assay (FRAP) assessment, and a metal-chelating power assay. The results showed that 36H had antioxidative capabilities in the DPPH-scavenging and ferric-reducing power examinations but the chelating power assay did not demonstrate antioxidative capability. 36H was also measured for tyrosinase inhibitory activity applying various species platforms, including in vitro mushroom, B16F10 mouse melanoma, and human melanocyte cells. In terms of in vitro mushroom tyrosinase suppression, 36H restrained the melanogenesis processes. It is assumed that 36H blocked the tyrosinase active site as a competitive inhibitor for mushroom tyrosinase, hence not decreasing the human normal melanocyte cellular viability. A quantitative real-time polymerase chain reaction (qRT-PCR) and western blot discovered that 36H downregulated melanogenesis-related RNA and proteins, including pigment production , melanosome maturation (Rab27a), and melanosome transportation . Overall, 36H displayed the biofunctions of antioxidation and melanin suppression, so there was a possibility for its application as a food additive or a skin-whitening agent. The human skin is the largest organ in the human body; it maintains bodily functions and prevents the loss of water, electrolytes, and biomolecules. The skin is composed of the epidermis, the dermis, and the hypodermis. The epidermis is further divided into five separated layers: the stratum corneum, the stratum lucidum, the stratum granulosum, the stratum spinosum, and the stratum basale . The basFree radicals are composed of an atom or a group of atoms that have one or more unpaired electrons. They are involved in physiological, metabolic, and immune reactions and signal transfer functions. Although they are a part of the normal healthy biochemical processes in the body, they also are responsible for damage. Reactive oxygen species (ROS) and various free radicals are stimulated by the generation of superoxide anions or hydrogen peroxides. This can cause confused messaging, damage to cell membranes, and damage to ion cell communications due to lipid peroxidation. This affects intracellular molecules that feature SH groups and acting proteins and DNA . The amoα-melanocyte-stimulating hormone to the melanocortin-1, which increases cyclic adenosine monophosphate (cAMP) and activates microphthalmia-associated transcription factors (MITF) 3. In the experiment, the color of the solution changed from light yellow to different shades of blue and green, depending on the reducing power of the target compound. μM had a reducing power value of 0.95 and 36H at 100 μM had a reducing power value of 0.85 compared to BHA. Therefore, 36H was shown to have good ferric-reducing antioxidant power.The second oxidation inhibitory assay is the ferric-reducing power test, which is a common and reliable way to measure the synthesis of Fe(III)–ferricyanide complex. A functional agent reduces FeμM) was used as a positive control. Fe2+ and ferrozine form complexes quantitatively. The formation of reagent complexes is often disrupted, because of chelating agents, which results in a reduction in the red color of the complex. 36H at a dosage of 10 μM had no significant effect on Fe2+-scavenging capacity, and at the concentration of 100 μM, it presented 43.2% inhibition. The positive control, EDTA, had approximately 80% ion-chelating capacity at 100 μM.The chelating activity for ferrous ion of 36H is shown in v versus 1/[s] gave a family of straight lines through the origin, which presented that 36H was a competitive complex. The kinetics of the enzyme are presented in The inhibition of tyrosinase activity was described in numerous reports, most of which used mushroom tyrosinase as the model. The advantages are being well-developed by scientists, easy procedures, low cost, rapid pigmentation process, similar structural and functional characteristics as mammals, and convenience in observing melanin development. The inhibition of mushroom tyrosinase activity by 36H was studied, and the inhibitory ability had a positive correlation with the concentration of 36H . In the μM, and the cell viability was determined to show low cellular toxicities in μM in a dose-dependent manner , TRP-1, and tyrosinase to form eumelanin. In HMC, 36H downregulated the cellular melanogenesis-related RNAs and proteins as demonstrated in The melanosome is an organelle that relies on melanin synthesis within melanocytes. The more melanosomes mature, the more melanin is formed. Therefore, melanosome maturation is important in the mechanism of skin whitening. Premelanosome protein 17 (Pmel17) is targeted to precursors of the pigment organelle, the melanosome, where it is proteolytically processed to several small fragments. Some of these fragments form nonpathological amyloids that assemble into sheets and form the striated pattern that underlies the melanosomal ultrastructure. This study demonstrated that 36H downregulated Pmel17 RNA and protein expression in HMC .Rab27a, MLPH, and Myo5a form a tri-protein complex to bind melanosomes at the melanocyte peripheries. In the process of melanosome transport, the ternary complex is the connection between actin cytoskeleton and melanosome. A lack of these proteins affects the transport, and melanoregulin (Mreg) drives melanosome transfer from melanocytes to keratinocytes via a regulated shedding mechanism. This study illustrated that 36H decreased the melanosome transport by affecting these related RNA and proteins .This study shows that antioxidants inhibit melanogenesis in two ways. In the melanin biosynthesis process , tyrosinBefore injecting the protein samples into SDS-PAGE, we normalized all protein levels. The protein normalization is a significant process applied to remove both experimental biological errors and artificial unexpected variabilities . Genes eα-melanocyte-stimulating hormone (α-MSH), adrenocorticotropic hormone (ACTH), and prostaglandins E2 (PGE2) [MITF RNA expression, but there was insignificant change in the amount of protein production. Tyrosinase affects melanin biosynthesis and TRP-2 and TRP-1 [When keratinocytes are exposed to UV , they re2 (PGE2) . These s2 (PGE2)  and thrond TRP-1 . Dopachrnd TRP-1 , which ind TRP-1 . In mushnd TRP-1 . Using aHuman skin melanin is driven by the intercellular movement of melanin-containing melanosomes from the extremities of HMC dendrites to neighboring keratinocytes. When it is carried by the actin filament, melanosome moves to the dendritic tail section, through exocytosis, and is transported into keratinocytes . The gre


Pain is a harmful sensation that arises from noxious stimuli. Transient receptor potential ankyrin 1 (TRPA1) is one target for studying pain mechanisms. TRPA1 is activated by various stimuli such as noxious cold, pungent natural products and environmental irritants. Since TRPA1 is an attractive target for pain therapy, a few TRPA1 antagonists have been developed and some function as analgesic agents. The responses of TRPA1 to agonists and antagonists vary among species and these species differences have been utilized to identify the structural basis of activation and inhibition mechanisms. The TRPA1 antagonist HC-030031 (HC) failed to inhibit frog TRPA1 (fTRPA1) and zebrafish TRPA1 activity induced by cinnamaldehyde (CA), but did inhibit human TRPA1 (hTRPA1) in a heterologous expression system. Chimeric studies between fTRPA1 and hTRPA1, as well as analyses using point mutants, revealed that a single amino acid residue (N855 in hTRPA1) significantly contributes to the inhibitory action of HC. Moreover, the N855 residue and the C-terminus region exhibited synergistic effects on the inhibition by HC. Molecular dynamics simulation suggested that HC stably binds to hTRPA1-N855. These findings provide novel insights into the structure-function relationship of TRPA1 and could lead to the development of more effective analgesics targeted to TRPA1. Pain usually arises from noxious stimuli and alerts us to potential danger, as well aids in the avoidance of similar experiences in the future. Considerable advances have been made over the last two decades in our understanding of peripheral pain mechanisms and the development of new analgesics. Mounting evidence suggests an important role in acute, inflammatory and chronic pain states for a subset of transient receptor potential (TRP) ion channels. TRP channels are nonselective cation channels that form a superfamily based on their structural similarity; this includes a six transmembrane (TM) domain with a pore region between TM5 and TM65Given that TRPA1 is a crucial nociceptive receptor, it is widely conserved among species. Characterization of TRPA1 from various species revealed that the sensitivity to different antagonists is species-specific because selective TRPA1 antagonists have been developed using mammalian TRPA1 7. Due to81011912Apart from A96, previous research1510X. laevis oocytes. Since CA-evoked responses were relatively fast and reversible50 values  were chosen10X. laevis oocytes expressing either wt-hTRPA1 or wt-fTRPA1 as previously reported for wt-hTRPA1X. laevis oocytes in a dose dependent manner  in t manner ,e, whilet manner ,f. Althot manner a, we uselar Ca2+ b,c2223, r Ca2+ 22d,e, suggX. laevis oocytes. We generated several chimeric combinations between hTRPA1 and fTRPA1 by introducing the N-terminus, different TM domains, and the C-terminus of fTRPA1 into hTRPA1  the CA concentration evokes similar current amplitudes during the first and second application.In order to narrow down the region responsible for the antagonistic effects of HC, chimeric hTRPA1 and fTRPA1 channels were constructed and their channel properties were examined in o hTRPA1 . We usedo hTRPA1 : (1) theThe N-terminus of hTRPA1 was first replaced with that of fTRPA1 [F-H (T1-Ct)], and HC significantly suppressed the CA-evoked currents in this chimera  and S4a.To further narrow down the region within the TM domains, F-H (T3-Ct) and F-H (T5-Ct) chimeras were constructed , and theWe searched for candidate amino acid residue(s) involved in the antagonistic activity of HC by comparing the amino acid sequence of TRPA1 from 5 different species within the region identified by the chimeric analyses above . We exam10X. laevis oocytes expressing either wt-hTRPA1 or hTRPA1-N855S - evoked currents for the first and second stimulation had an approximately similar size in A1-N855S  and S5a. oocytes  while hT oocytes . We alwa oocytes e and 3b, mM- evok oocytes . In cont2+]i in a dose-dependent manner in HEK293T cells expressing either wt-hTRPA1 or hTRPA1-N855S  was replaced with asparagine (fTRPA1-S880N). Although CA (0.5 mM) evoked similar sized currents in the first and second stimulation in A1-S880N b and 5a,A1-S880N .X. laevis oocytes  in wild type zebrafish (wt-z) TRPA1a and TRPA1b . To date oocytes b–e.X. laevis oocytes expressing either wt-hTRPA1 or hTRPA1-N855R  found in wt-zTRPA1b at the corresponding position. While CA (0.3 mM) evoked similar sized currents in the first and second stimulation in A1-N855R  and S8a,A1-N855R  which coA1-N855R . We obseA1-N855R , a phenoA1-N855R  that mayA1-N855R . In contA1-N855R  and S8e,We next examined the effect of HC in a reverse mutant by replacing arginine at position 860 in wt-zTRPA1b with asparagine (zTRPA1-R860N). We examined the effects of HC along with CA in the second application after confirming that repeated applications exhibited similar current activity . ConcurrGiven that we failed to observe complete attenuation of the HC effects in any of the above hTRPA1 point mutants, we searched for another action site of HC activity. Since a high concentration of HC exhibited significant inhibition of the F-H (Ct) chimera , we perfThe high-resolution structure of hTRPA1 has now been made available and can provide a platform for understanding channel functionδ atom in N855 at 0 ns, but it was formed immediately after the MD simulation began. This hydrogen bond was maintained during most of the simulation time. The definition of the secondary structure of the proteinδ atom of N855 as shown in Lastly, we performed an MD simulation to investigate whether HC binds to N855 in human TRPA1 for 100 ns. γ atom of S855 fluctuated between 0.2 nm and 0.5 nm. Although a hydrogen bond was sometimes formed for a short period, it was broken soon. A hydrogen bond was not formed stably in the N855S mutant.We also performed an MD simulation for the N855S mutant of the hTRPA1. X. laevis oocytes, whereas HC showed dose-dependent inhibition of hTRPA1 activity. Similar species-specific pharmacological activity of TRPA1 has been previously reported. Some examples include: (1) cysteine-attacking compound CMP1, activates rat TRPA1 but suppresses hTRPA1Given the localization of TRPA1, a polymodal non-selective cation channel, to sensory nerve endings and its well established presence in pain and inflammation research, it has become an intriguing clinical drug target. Many research groups, including our own, have previously attempted to identify the binding mechanisms or the effective sites for potent mammalian TRPA1 antagonists. While the action sites of A96 and AP-18, which are structurally similar, were discovered9121314Through chimeric and mutational studies with different species, we found that a single amino acid, N855, within the linker region of TM4 and TM5 is an important residue for the inhibition of hTRPA1 by HC. Mutants in which N855 in TRPA1 was replaced with serine or arginine, corresponding TRPA1 residues from frog and zebrafish, respectively, showed reduced HC sensitivity and reverse mutants (S880N and R860N), in which corresponding amino acids were changed to asparagine, led to increased HC sensitivity , 5 and 750 value of CA between wt-hTRPA1 and hTRPA1-N855S mutants in heterologous expression systems using either X. laevis oocytes or HEK293T cells. As reported, the hTRPA1 mutant (N855S) associated with the familial episodic pain syndrome is a gain-of-function mutantet al.Remarkably, a missense mutation in the same residue N855 to serine was previously shown to be associated with an autosomal-dominant heritable familial episodic pain syndrome triggered by cold, fasting and fatigue, resulting in upper body pain in humansMany groups, as reported above, have predicted the action sites of A96. The high-resolution 3D structure of hTRPA1 was recently published and showed an actual A96 binding site based on its unique density within a pocket formed by TM5 and TM6 and the first pore helix which is surrounded by the previously predicted sites. A96 could thus inhibit channel activity via an “induced fit” mechanism involving movement in nearby residues that comprise the pocketWhile the single amino acid residue (N855 in hTRPA1) located in the linker region between TM4 and TM5 exhibited a partial but significant role in the effects of HC, the mutation of this amino acid together with the C-terminal replacement in fTRPA1 caused almost complete inhibition by HC , similarIn the last decade, a few TRPA1 antagonists have been developed and entered into pre-clinical trialsX. laevis oocytes. Mature X. laevis females were purchased from Hamamatsu Seibutsu Kyozai  and reared at ~18 °C. Oocytes were surgically excised from anesthetized X. laevis, and follicular membranes were enzymatically removed from oocytes and ionic currents were recorded using the two-electrode voltage clamp method described previously2, 1 mM MgCl2, 5 mM Hepes, pH 7.4 (with NaOH), and applied to the oocytes by perfusion. ND96 Ca2+ free solution was prepared by removing 1.8 mM CaCl2 from the solution. The zTRPA1a and zTRPA1b expression vectors were kindly provided by Dr. ProberChimeras and mutants for fTRPA1, hTRPA1, zebrafish (z) TRPA1a, and zTRPA1b, were heterologously expressed in Xenopus laevis oocytes as described previouslyWe used expression vectors for wild type fTRPA1, hTRPA1, zTRPA1a, and zTRPA1b, all of which were expressed in either human embryonic kidney (HEK) 293 cells and/or in Full length chimeric TRPA1 cDNA was amplified by polymerase chain reaction (PCR). In the first step, different DNA fragments of TRPA1, F-H (T1-Ct) [TRPA1 (715–1119)], F-H (T1-T6)-F [hTRPA1(715–975)], F-H (T3-Ct) [hTRPA1(789–1119)], F-H (T5-Ct) [hTRPA1(844–1119)], and F-H (Ct) [hTRPA1(975–1119)] were amplified by PCR with an expression vector containing human TRPA1 cDNA as a template using the combination of primers listed in 2+ imaging was described previously2+]i imaging experiments after incubation for approximately 24 h. Fura-2 was loaded into the cells by incubating at 37 °C for 0.5–1 h with fura-2 acetoxymethyl ester (5 μM) in D-MEM  containing heat-denatured 10% FBS (Gibco), 0.5% penicillin/streptomycin (Invitrogen) and 1% GlutaMax (Invitrogen). Cells were transferred to recording chambers mounted on the stage of an inverted microscope equipped with an image acquisition and analysis system. All chemicals were applied by perfusion of bath solution.The procedure for Ca2+]i, cells were illuminated every 3 s with light at 340 and 380 nm. The intensity of the fluorescent signals at 500 nm emitted by light, excited at 340 (F340) and 380 (F380) nm were recorded, and their ratios (F340/F380) were calculated. Cells were continuously superfused with bath solution, and drugs were applied through the same tube. All experiments were carried out at room temperature (~25 °C).To measure [CaCinnamaldehyde, HC-030031 and A-967079 were purchased from Wako, Sigma, and Santa Cruz Biotechnology respectively. All chemicals were dissolved in DMSO as stock solutions (0.1–2 M).δ and Hδ atoms of the asparagine and replaced the Cγ atom and the Oδ atom with the Oγ atom and the Hγ atom, respectively.We prepared the initial hTRPA1conformation for the molecular dynamics (MD) simulations employing the PDB structure (ID: 3J9P). Because several amino acid residues were missing in the PDB structure, we complemented the residues 664–679, 748–763, 786–802, and 1007–1030 using the modeling program MODELLER35HC is a non-standard organic molecule for MD simulation of biomolecules. We therefore determined the parameters for calculating the potential energy and force of HC. To derive the partial atomic charge of HC, we followed the RESP charge methodΔt = 0.5 fs for the bonding interactions and Δt = 2.0 fs for the non-bonding interactions. To maintain the atomic structure of the TRP channel in a vacuum, N, Cα, and C atoms of the residues that form α-helix structures were restrained with a harmonic potential.The Generalized-Ensemble Molecular Biophysics (GEMB) program developed by one of the authors (H.O.)404145462+ imaging experiments were obtained from at least three independent transfections. The data from current recordings were obtained from oocytes collected from at least three different frogs. The data are presented as the mean ± SEM (n = number of observations). Statistical analyses were tested using a one-way ANOVA followed by the Tukey-Kramer test with p values < 0.05. EC50 was determined using Origin software (OriginLab). The amino acid sequences of TRPA1 from the various vertebrate species were aligned using ClustalW implemented in MEGA5 and the amino acid similarities were calculated49Data for the CaHow to cite this article: Gupta, R. et al. Structural basis of TRPA1 inhibition by HC-030031 utilizing species-specific differences. Sci. Rep.6, 37460; doi: 10.1038/srep37460 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Due to their ease of fabrication, facile use and low cost, ice valves have great potential for use in microfluidic platforms. For this to be possible, a rapid response speed is key and hence there is still much scope for improvement in current ice valve technology. Therefore, in this study, an ice valve with enhanced thermal conductivity and a movable refrigeration source has been developed. An embedded aluminium cylinder is used to dramatically enhance the heat conduction performance of the microfluidic platform and a movable thermoelectric unit eliminates the thermal inertia, resulting in a faster cooling process. The proposed ice valve achieves very short closing times (0.37 s at 10 μL/min) and also operates at high flow rates (1150 μL/min). Furthermore, the response time of the valve decreased by a factor of 8 when compared to current state of the art technology. Microfluidic technologies are widely used in chemical and biological analysis because they offer high analytical throughput, low sample consumption and have high integration capabilities127Ice valves switch flow by controlling the state of the fluid phase. To stop flow, a section of the fluid in the channel is cooled to below its freezing point, and it hence solidifies, which causes the channel to be blocked. To reopen the valve, the ice is allowed to thaw by stopping the cooling process. Ice valves were first reported in 1995To solve these problems, in this study an aluminium cylinder was embedded under the channel in the microfluidic platform, and a movable refrigeration source was designed to increase the response speed. The experimental results showed that the response speed was increased by a factor of 8 when compared with current state of the art ice valves (the response time is compared at the same flow rates), as well as the maximum flow rate by a factor of 10.In brief, the platform consists of a PMMA (poly(methyl methacrylate)) microfluidic device embedded with an aluminium cylinder covered with a layer of anti-freeze solution, a movable TE unit and a water cooling system . When a A layer of anti-freeze solution, consisting of 45% ethylene glycol and 55% water (with a freezing point below −45 °C), is inserted between the TE unit and the device to reduce the contact thermal resistance. Without the anti-freeze solution, the TE unit will condense moisture from the air to form an ice layer on the top of the cooling head, which will reduce the heat transfer.The characterization of the valve was performed by measuring its closing time as a function of flow rate. To characterize the ice valve, a channel with a rectangular cross section of 300 μm × 200 μm (width × height) was fabricated in a PMMA substrate using a tri-axis CNC machine . The aluminium cylinders were made of alloy 7050-T7451 with both a height and a diameter of 1 mm. To measure the closing time of the valve, the Teflon tubing  at the outlet of the device was taped to a ruler. The closing time of the ice valve is defined from the point when the device contacts with the TE unit to the point that the valve closes (the flow stops). These experiments were conducted at room temperature (19.6 °C) and the cooling head was cooled to −45.2 °C (as measured using a k-type thermocouple) prior to contact with the device. The voltage applied to the TE unit remained at 12 V and the current was 5.2 A.To keep the anti-freeze solution in a liquid state, a custom made hollow aluminium cooling head  was set To measure the closing time of the valve, multiple air bubbles were manually generated in the tubing connected to the outlet of the device , and the111311We built a mathematical model to allow the prediction of the cooling time, at the same flow rate, in two different types of ice valves to allow us to compare their efficacy. Due to super cooling and the coupling of phase change and the temperature and fluid fields, it is difficult to build an accurate model to calculate the closing time of the valve mathematically. Herein we consider the cooling rate at the bottom of the channel at the same convection coefficient, which represents the ability of the system to take the heat away from the liquid in the channel. The cooling rate is determined by three main factors: the initial temperature, the heat conduction performance and the temperature difference between the cooled bottom of the channel and the flowing bulk liquid.Cwater = 4.2 × 103 J·kg−1·K−1 and Lwater = 3.35 × 105 J·kg−1. When water is cooled from room temperature (Troom) to pre-cooling temperature (Tpre), the energy released per unit mass, E1 in J·kg−1, is defined as:The initial temperature of the system determines the energy released from the liquid during the valve closing operation. In previously published work, pre-cooling the deviceE2 in J·kg−1, is:During the crystallisation stage, the energy released per unit mass, Tsuper) is −17 °C, the maximum energy released by pre-cooling is E1 = 1.55 × 105 J·kg−1, while the total energy released during the entire cooling process is E1 +  E2 = 4.9 × 105 J·kg−1. As mentioned above, E2 (which includes the latent heat) accounts for a large proportion of the released energy, and it cannot be released by pre-cooling. Therefore, transferring the energy released during crystallization in the shortest amount of time possible is key to increasing the response speed of the ice valve. Practically, this means that better heat conduction and a higher temperature difference are important design considerations.Assuming that the super-cooling temperature  for both traditional ice valves and the ice valve proposed in this study. The response speed of the ice valves is determined by the heat conductivity of the substrate in contact with the TE unit and the temperature of the cold side of the TE unit. To highlight the performance enhancements achieved by our ice valve design, the traditional ice valve is simulated in a glass microfluidic device, which should theoretically show a better performance than a PMMA microfluidic device with the same structure. The traditional ice valve has a non-movable TE unit integrated into the device and the thermal contact resistance can therefore be ignored. For both platfroms, the length and height of the channel are 20 mm and 0.2 mm, respectively, and the widths of the TE unit and the aluminium cylinger for our platform are 6 mm and 1 mm respectively. The governing equation for the simulation model is the heat equation for conductive and convective heat transfer:Cp is the specific heat capacity (J·kg−1·K−1), T is the temperature (K), k is the thermal conductivity (W·m−1·K−1), ρ is the density (kg·m−3), u is the velocity vector (m·s−1), and Q is a sink or source term (which is set to zero because heat is neither produced nor consumed in the device).where y direction are set to zero. The expression:In the channels, the velocity field of the fluid flow is defined by an analytical expression that approximates a fully developed laminar flow. The velocity components in the x component of the velocity, where vmax is the maximum velocity (ms−1) that arises in the middle of the channel, r is a variable defined as the distance from the center of the channel to the measurement point, and R is equal to half the channel height such that rmax = R. The simulation accounts for heat conduction in the device, as well as convection in the aluminium cylinder and in the anti-freeze solution. This COMSOL model allows us to examine the effects of various factors on valve performance. The COMSOL model was defined as a fully insulated system, except for the inlet and outlet boundaries, and the boundary that comes in contact with the TE unit in the channel. At the inlet, a constant temperature was specified as 20 °C (room temperature). At the boundary that comes in contact with the TE unit, a constant temperature was specified at −45 °C since that is the approximate temperature at the top of the cooling head. At the outlet, convection dominates the transport of heat, and thus, the convective flux boundary condition, gives the At any given flow rate, and given sufficient time, the COMSOL model shows that the flow within the channel reaches a constant temperature. The equilibrium temperature at each flow rate is shown in The design of this platform such that the TE unit is a separate module from the microfluidic device increases the integration density of the ice valves. In traditional ice valves, the TE units are fixed to the device and therefore the size of the TE unit limits the integration density. In our platform, the cold area is concentrated solely in the zone contacting the aluminium cylinder and is therefore not constrained by the TE unit itself. Thus, decreases in the size of the Al cylinder will further increase the integration density. Finally, the enhanced performance exhibited by our ice valve is not dependant on the material from which the microfluidic device is fabricated since the thermal conductivity of the Al alloy is over 100 times higher than that of most commonly used materials for device fabrication .Herein we show a simple and robust ice valve with enhanced thermal conductivity and a movable refrigeration source. This ice valve is capable of very short closing times (0.37 s) when compared to current literature and is able to operate at extremely high flow rates (1150 μL/min). We provide mathematical and COMSOL models to describe the design and behaviour of our ice valve compared to traditional ice valves. The response speed of our valve was over than 8 times faster than current ice valves operating at similar flow rates and it has a series of other advantages such as improved integration density of the valves on device and no limitation with regards to type of device material required.How to cite this article: Si, C. et al. High response speed microfluidic ice valves with enhanced thermal conductivity and a movable refrigeration source. Sci. Rep.7, 40570; doi: 10.1038/srep40570 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Warm subtropical-origin Atlantic water flows northward across the Greenland-Scotland Ridge into the Nordic Seas, where it relinquishes heat to the atmosphere and gradually transforms into dense Atlantic-origin water. Returning southward along east Greenland, this water mass is situated beneath a layer of cold, fresh surface water and sea ice. Here we show, using measurements from autonomous ocean gliders, that the Atlantic-origin water was re-ventilated while transiting the western Iceland Sea during winter. This re-ventilation is a recent phenomenon made possible by the retreat of the ice edge toward Greenland. The fresh surface layer that characterises this region in summer is diverted onto the Greenland shelf by enhanced onshore Ekman transport induced by stronger northerly winds in fall and winter. Severe heat loss from the ocean offshore of the ice edge subsequently triggers convection, which further transforms the Atlantic-origin water. This re-ventilation is a counterintuitive occurrence in a warming climate, and highlights the difficulties inherent in predicting the behaviour of the complex coupled climate system. Warm Atlantic water circulates cyclonically around the Nordic Seas while gradually cooling. Here, the authors show that the retreat of the ice edge toward Greenland has led to further transformation of this water mass, which is no longer situated underneath sea ice when transiting the western Iceland Sea in winter. Most of the North Atlantic deep waters originate from the Nordic Seas2, as result of a warm-to-cold transformation that takes place primarily in the eastern part of that region5. The resulting product, referred to as Atlantic-origin water6, is returned southward by the East Greenland Current7 , in which warm waters are transported northward near the surface and cold waters are returned to the south at depth10. Proximity to the ice edge, where the highest ocean to atmosphere fluxes of heat occur due to frequent intense cold air outbreaks in which frigid, dry air masses are advected over comparatively warm surface water, is an important factor14. Diminished heat loss from this region owing to a retreat of the ice edge and different rates of warming of the ocean and the atmosphere will likely reduce the formation of Arctic-origin water15. In the Iceland Sea this water mass is primarily produced on the north-western outskirts of the cyclonic gyre, where the atmospheric forcing is more intense16. Farther to the west . Instead, the western end of the February 2016 glider transect has an approximately 400 m deep mixed layer with a density of σθ = 28.01–28.02 kg/m3, more than sufficient to supply the dense Denmark Strait overflow plume  were deployed with the purpose of mapping the extent of convection in the Iceland Sea during the subsequent winter. Here we focus on measurements obtained near the ice edge in the western Iceland Sea. Vertical sections of potential temperature and salinity from a cruise in summer 2012 and the two glider transects that approached the ice edge in winter 2015–2016 are shown in Figs. me Figs.  and 3b. er Figs.  and 3c. 21 in combination with historical measurements13, the monthly mean hydrographic properties averaged over the upper 50 m of the water column demonstrated a pattern that is consistent with the shipboard and glider measurements  was employed. The August 2012 profile closest to the turnaround point was used as initial conditions and a constant heat loss of 120 W/m2, typical for winter 2015–2016  were discarded. Each dive/climb cycle was subsequently inspected for density inversions, and measurements causing inversions exceeding 0.05 kg/m3 were excluded33. The sensors were laboratory calibrated prior to deployment. In addition, the glider data were calibrated against shipboard measurements at the times of deployment and recovery. A rendezvous partway through the deployment was used for data intercomparison. Corrections were estimated in potential conductivity–potential temperature space34. Offsets corresponding to 0.006 and 0.016 in salinity were applied to two of the gliders , while a drift over the entire deployment period corresponding to 0.0008 in salinity was corrected for one glider (sg559). No corrections were applied to the conductivity sensor of the third glider (sg564) or any of the temperature sensors.The wintertime transects were obtained by autonomous, buoyancy-driven Seagliders that are capable of diving to 1000 m depth35.Vertical sections of potential temperature and salinity were constructed using Laplacian-spline interpolation7 and the calibrated seal data set in ref.21.Details about the August 2012 shipboard transect can be found in ref. Wind stress, surface sensible and latent heat fluxes, as well as sea ice concentration for the climatological analyses Fig.  were takτ(t) is the 6-hourly average mean wind stress in the ice-free portion of the western Iceland Sea region . Since the Ekman transport distance is inversely proportional to Ekman depth, a decrease of h by 50% would result in an increase of the estimated distance by a factor of 2, whereas an equivalent increase of h would result in a lower distance by one-third.The Ekman transport distance was estimated from22 mixed-layer model, turbulent heat fluxes, which provide the dominant contribution to mixed-layer deepening37, were imposed at the surface at each time step. Initial conditions were obtained from the August 2012 shipboard and February 2016 glider measurements. The depth and properties of the mixed layer were then adjusted until three stability criteria, chief among which is static stability, were satisfied.To implement the one-dimensional PWPhttp://kogur.whoi.edu. The seal data can be found in the MEOP database (www.meop.net). The ERA-Interim reanalysis data were obtained from the European Centre for Medium-Range Weather Forecasts and the passive microwave sea ice product38 was obtained from the National Snow and Ice Data Center.The glider data can be accessed at the Pangaea repository (10.1594/PANGAEA.884339). The shipboard data can be obtained from Peer Review File


Global Navigation Satellite System (GNSS) signals can be exploited to remotely sense atmosphere and land and ocean surface to retrieve a range of geophysical parameters. This paper proposes two new methods, termed as power-summation of differential Delay-Doppler Maps (PS-D) and pixel-number of differential Delay-Doppler Maps (PN-D), to distinguish between sea ice and sea water using differential Delay-Doppler Maps (dDDMs). PS-D and PN-D make use of power-summation and pixel-number of dDDMs, respectively, to measure the degree of difference between two DDMs so as to determine the transition state  and hence ice and water are detected. Moreover, an adaptive incoherent averaging of DDMs is employed to improve the computational efficiency. A large number of DDMs recorded by UK TechDemoSat-1 (TDS-1) over the Arctic region are used to test the proposed sea ice detection methods. Through evaluating against ground-truth measurements from the Ocean Sea Ice SAF, the proposed PS-D and PN-D methods achieve a probability of detection of 99.72% and 99.69% respectively, while the probability of false detection is 0.28% and 0.31% respectively. Knowledge of sea ice coverage and thickness of the polar region is very important for ship routing , oil andGlobal Navigation Satellite System (GNSS) Reflectometry (GNSS-R) has been investigated for many years as an innovative remote sensing technique using GNSS signals reflected off the Earth’s surface . GNSS-R www.merrbys.co.uk [GNSS-R-based sea ice detection was first investigated by Komjathy et al.  by compays.co.uk . TDS-1 Dys.co.uk , ocean wys.co.uk , Tsunamiys.co.uk , soil moys.co.uk , sea surys.co.uk ,39 and sys.co.uk ,41.The first result of sea ice detection using TDS-1 GNSS-R DDMs was presented in , which pThe next section of this paper presents the details of the two proposed ice detection methods, including the definition of the new observation variable, the derivation of the formulas, the data processing techniques, and the detection procedures. The TDS-1, launched in July 2014, was placed into a quasi-Sun synchronous orbit with an altitude of ~635 km and an inclination of 98.4°. The Space GNSS Receiver Remote Sensing Instrument (SGR-ReSI) built by Surrey Satellite Technology Ltd.  is one of the key payloads carried on TDS-1. The ReSI supports the GPS L1 frequency band using a downward pointing antenna with a gain of ~13 dBi. More details about TDS-1 and the SGR-ReSI are introduced in .The TDS-1 receiver produces DDMs through cross correlating received scattered signals with locally generated and time-synchronized code replicas for different path delays and Doppler shifts . SpeckleGNSS-R DDMs are usually processed to produce observables that could describe the scattering strength of reflected surface. Different surface roughness related observables have been proposed in ,45. ThosThe standard DDM observable is usually denoted as One existing observable derived from fined in  and latefined in , which cA similar observable modified from an observable defined in  is definIf In this paper, an observable called dDDM observable is proposed for distinguishing sea ice from water. Details of the proposed observable are provided as follows.Two adjacent normalized DDM observables can be expressed as PS represents the power summation and PN depicts abstract pixel number of the grids with absolute values .N represents the number of pixels of the noise box. The noise box chosen in this paper spans twenty Doppler bins and five delay chips starting from 1st to 20th in the standard DDM . The examples of the resultant DDMs of sea ice and water with 5-s incoherent averaging are shown in Let Considering that the values of As illustrated in surfaces a,c basicsurfaces b,d have The power values of (a) Power Summation of dDDM (PS-D)PS) of the grids with absolute values (PS’) of the grids with absolute values (If If ➢If ➢If If The power summation ((b) Pixel Number of dDDM (PN-D)PN and PN’) of the grids with absolute values  and same-surface . For PS-D based method, if the maximum absolute power summation value of same-surface  are displayed in The three rows of dDDMs presented in https://www.ecmwf.int) for validating our results. The SP ground-track of 57 DDMs (DDM 1 to DDM 57) is marked in the WH map with a red line, while there is no valid WH data from DDM 58 to DDM 131. The WH of these 57 SP was retrieved through a 2-D linear interpolation according to the SP locations. The values of WH were scaled down as we only concentrate on the variability, which may explain the variability of dDDMs. The relationship between relative wave height and PS of dDDM is presented in There is a large variability in PS and PN from dDDM 1 to dDDM 37, then a relatively stable variability from dDDM 38 to dDDM 58, and again large change from dDDM 59 to dDDM 61. This phenomenon may be associated with the sea wave height (WH) over the studied area. The significant wave height data shown in  of 26 OcAs introduced in In order to assess the performance of the proposed methods, another two observables  , pixel nIt is useful and interesting to illustrate sea ice detection results directly on a geographical map. There was a significant decline in the coverage of sea ice over Arctic region from March to August 2015 due to the increase of temperature. On the other hand, as the temperature decreased from August 2015 to March 2016, the coverage of sea ice increased up to almost the same coverage of March 2015.As shown in Although the proposed methods hold great potential for monitoring sea ice, a small quantity of false detection still occurs around the open drift ice and multi-year ice areas. The received signals reflected from both sea ice and water may lead to false detection. Thus, more in-depth research into the relationship between reflected signals and sea ice concentration in needed in the future. Having a good knowledge about sea ice concentration will benefit sea ice detection a lot.In this paper, GNSS-R DDM measurements collected by the SGR-ReSI on board the TDS-1 satellite are used for distinguishing between sea ice and water. A dDDM observable is proposed for the first time and two methods (PS-D and PN-D) based on power-summation and pixel-number of dDDMs are proposed to detect sea ice. Meanwhile, several data processing schemes including noise subtraction, incoherent averaging and overall normalization are employed to improve significantly the performance of proposed sea ice detection approaches. The feasibility of PS-D and PN-D methods is validated with ground-truth sea ice edge data provided by OSI SAF and the results indicate the good performance of PS-D and PN-D with a detection probability of 99.72% and 99.69% respectively and a probability of false detection of 0.28% and 0.31% respectively. The proposed methods considerably outperform the existing methods with an improved detection probability of more than 1.5% and significantly reduced probability of false detection.The dDDMs of water-water transition show larger spreading characteristics than those of ice-ice transition due to the larger variability of reflected surface. This characteristic allows us to distinguish water-water from ice-ice transition, while it does not allow us to model the relationship between received signals and the roughness as the TDS-1 data was not calibrated and the instrument onboard the TDS-1 satellite was not optimized for altimetry purposes.In the future, it is desirable to further reduce the sea ice false detection probability to obtain more accurate sea ice coverage. Open ice affects sea ice detection and new techniques are needed to handle this issue. Moreover, data coverage rate of TDS-1 is rather limited but CYGNSS launched in 2016 and the potential GEROS-ISS  mission


Finite-element method was used to evaluate biomechanics stability of extraforaminal lumbar interbody fusion (ELIF) under different internal fixation.  The L3–L5 level finite-element model was established to simulate decompression and internal fixation at L4-L5 segment. The intact finite model was treated in accordance with the different internal fixation. The treatment groups were exerted 400 N load and 6 N·m additional force from motion to calculate the angular displacement of L4-L5.  The ROMs were smaller in all internal fixation groups than those in the intact model. Furthermore, the ROMs were smaller in ELIF + UPS group than in TLIF + UPS group under all operating conditions, especially left lateral flexion and right rotation. The ROMs were higher in ELIF + UPS group than in TLIF + BPS group. The ROMs of ELIF + UPS + TLFS group were much smaller than those in ELIF + UPS group, and as compared with TLIF + BPS group, there was no significant difference in the range of experimental loading.  The biomechanical stability of ELIF with unilateral pedicle screw fixation is superior to that of TLIF with unilateral pedicle screw fixation but lower than that of TLIF with bilateral pedicle screws fixation. The stability of ELIF with unilateral fixation can be further improved by supplementing a translaminar facet screw. Lumbar degenerative disease has been the main cause of lower back pain and leg pain in adults –3. ThereIn the extraforaminal lumbar interbody fusion (ELIF) technique, only the superior facet joint is resected and the inferior facet joint and soft tissues attached behind it are retained. Compared with conventional transforaminal lumbar interbody fusion (TLIF), ELIF technique retains the posterior structures more completely and could potentially improve the immediate stability. Therefore, we analyzed the lumbar biomechanical stability of ELIF surgery by using 12 cadaveric spine specimens [This study has been reviewed and approved by the ethics committee of Shanghai East Hospital, Tongji University School of Medicine.In September 15, 2015, a 26-year-old man  diagnosed with L4-L5 lumbar disc herniation was recruited for a preoperative lumbar computed tomography scan from T12 level to pelvic inlet using a dual source scanner . This participant provided the written informed consent to participate in this study. The scanning parameters were as follows: tube current = 250 mA, tube voltage = 120 kV, scanning slice thickness = 1.0 mm, and reconstruction slice thickness = 1.0 mm. The data in the commonly used DICOM 3.0 format were read using the medical finite-element modeling software Simpleware 2.0 , and an L3–L5 3D geometric model was established. We utilized the finite-element preprocessing software HyperMesh  and select the appropriate element types and materials for mesh generation. The mesh model includes 124,528 elements and the mesh size is 2 mm. The materials for the various parts of the input model and their characteristics including elastic modulus and Poisson ratio are listed in Images of the pedicle screw system and intervertebral fusion cage  in the IGES format were imported into HyperMesh, and the finite-element model was constructed for ELIF and TLIF based on the requirements listed below. The elastic modulus for screws in ELIF and TLIF groups was 110,000 MPa and the Poisson ratio was 0.3. The intervertebral fusion cage was a bullet-type cage with dimensions of 9 mm × 11 mm × 27 mm, and the elastic modulus and Poisson ratio were 3700 MPa and 0.25, respectively. The screw diameter was 6.0 mm, and length was 45 mm. The implanting angle of fusion cage was 80° at the spine sagittal plane in the ELIF group and 45° in the TLIF group. In both groups, the fusion cage was placed into the intervertebral space obliquely from the right side. The experimental groups were divided into as follows: ELIF with unilateral pedicle screw (ELIF + UPS), TLIF with unilateral pedicle screw (TLIF + UPS), TLIF with bilateral pedicle screws (TLIF + BPS), and ELIF with unilateral pedicle screw + translaminar facet screw (ELIF + UPS + TLFS). The designs of the experimental models were exactly based on the clinical surgical approaches. In the ELIF + UPS group, the superior facet joint of the L5 vertebra was removed along with the entire nucleus pulposus in the L4-L5 disc and the right posterior two-thirds of the fibrous ring. The posterior supraspinous ligament, interspinous ligament, spinous process, and the left side structures were retained. The pedicle screws were placed in the L4 and L5 pedicles on the right side , and 7 types of ligaments, yielding a total of 13 types of materials . The modThe ROMs were smaller in all internal fixation groups than those in the intact group . FurtherThe maximum stress concentration point on the connecting rod was the junction between the screw and its head in the ELIF and TLIF models. The stress on the proximal end was greater than that on the distal end. The stresses on the connecting rod under all operating conditions were greater in the case of unilateral pedicle screw fixation than those in the case of bilateral pedicle screw fixation. Furthermore, the stresses on the connecting rods were smaller in the ELIF + UPS group than those in the TLIF + UPS group, especially under right lateral flexion . With adTLIF has gained increasing popularity in the treatment of degenerative lumbar disease due to its advantages including direct decompression on the nerve root and less risk of complications and good fusion result –22. HoweFinite-element models are widely applied in understanding of biomechanical function of the spine due to the effective stimulation, high repeatability, and less cost . Before Although this study has achieved primary success, more work needs to be done. Firstly, material properties in these 3D models were simplified and idealized. Although these simplifications were reasonable, it influenced the exact ROM value. Thus, the results obtained in these analyses reflect the difference of ROMs among groups instead of the exact ROM value. Secondly, the soft tissue environment was different between these models and human lumbar spines because the whole spinal model including lumbar spine muscle failed to be established.


NOS) and cyclooxygenase (COX) in the regulation of sweating during a passive heat stress in young adults. Ten trained athletes and nine untrained counterparts were passively heated until oral temperature  increased by 1.5°C above baseline resting. Forearm sweat rate (ventilated capsule) was measured at three skin sites continuously perfused with either lactated Ringer's solution (Control), 10 mmol/L NG‐nitro‐L‐arginine methyl ester , or 10 mmol/L ketorolac  via intradermal microdialysis. Sweat rate was averaged for each 0.3°C increase in Tor. Sweat rate at the L‐NAME site was lower than Control following a 0.9 and 1.2°C increase in Tor in both groups . Relative to the Control site, NOS‐inhibition reduced sweating similarly between the groups (P = 0.51). Sweat rate at the Ketorolac site was not different from the Control at any levels of Tor in both groups (P > 0.05). Nevertheless, a greater sweat rate was measured at the end of heating in the trained as compared to the untrained individuals (P ≤ 0.05). We show that NOS contributes similarly to sweating in both trained and untrained individuals during a passive heat stress. Further, no effect of COX on sweating was measured for either group. The greater sweat production observed in endurance‐trained athletes is likely mediated by factors other than NOS‐ and COX‐dependent mechanisms.The aim of our study was to determine if habitual endurance training can influence the relative contribution of nitric oxide synthase ( It is well‐established that endurance trained individuals show an enhanced sweat production relative to their untrained counterparts during a passive  inhibitor during exercise  and untrained adults  participated in the study. The endurance‐trained individuals were competitive distance runners who were members of the athletic club at Niigata University and were engaged in endurance running training at least five times per week for at least 3 years. Their best competitive 5000 m times were 15.44 ± 0.22 min and 21.07 ± 1.31 min for the males (n = 8) and females (n = 2), respectively. It has previously been reported that long‐ and middle‐distance runners with similar training backgrounds show a greater sweating response relative to the untrained counterparts during a passively induced heat stress , and (3) 10 mmol/L ketorolac, a non‐selective COX inhibitor  in a counter‐balanced manner. The infusion rate was maintained at 4 μl/min via a micro‐infusion pump . The concentrations of L‐NAME and ketorolac employed were based on previous studies using a comparable technique . Upon arrival to the laboratory, the participant's body weight was measured. They subsequently donned a water perfusion suit  under which they wore a cotton T‐shirt. The participants then rested in a semirecumbent position during which time skin temperature was clamped at 33–34°C by circulating water regulated at 34°C. During this time, using aseptic techniques, three microdialysis fibers were inserted into the dermal layer of skin on the dorsal side of the left forearm. A 25‐gauge needle was first introduced into the unanesthetized skin and exited ~2.0 cm distally from the insertion point. The microdialysis fiber   was then threaded through the lumen of the needle, which was subsequently withdrawn leaving the fibre in place. Each of the fibers were separated by at least 2.0 cm. Ten minutes after the last fibre insertion, the microdialysis probes were perfused with (1) lactated Ringer's solution , (2) 10 mmol/L or) increased by 1.5°C above the pre‐heating baseline resting levels.After a 5‐min baseline data collection period, whole‐body heating was initiated by circulating water regulated at 44°C through the perfusion suit and simultaneously immersing the lower legs in warm water maintained at 41°C. Thereafter, the water temperature perfusing the suit was increased by 2°C increments every 10 min to a maximum of 52°C which was maintained for the duration of the heating protocol. Ten minutes after the initiation of heating, the temperature of the lower leg water bath was increased to 43°C and was maintained at this temperature for the duration of the trial. The heating protocol was continued until oral temperature  was used as an estimate of T2 plastic capsule was affixed at each site using adhesive tapes and topical glue . The participant was required to rest their forearm on a table maintained at heart level throughout the experiment. Dry nitrogen gas was passed through each capsule and over the skin surface at a rate of 0.8 L/min. Water content from the effluent air was measured using a capacitance hygrometer . All temperature and sweat rate data were recorded at 1‐sec intervals using a data logger  and simultaneously displayed on a computer screen (MX100 standard software). Heart rate (HR) was monitored continuously using a telemetry transmitter strapped around the chest .Local sweat rate was measured continuously using the ventilated capsule method. A 1.0 cmor. Mean body temperature was calculated as 0.8 × Tor  + 0.2 × mean skin temperature , level of increase in Tor above baseline resting levels , and group (two levels: endurance‐trained and untrained). HR, Tor, mean skin temperature, and mean body temperature were analysed using a two way mixed‐design ANOVA with factors of Tor  and group. When a significant main effect or an interaction was observed, a post hoc for multiple comparison was conducted with a Student's pairwise or nonpairwise t‐test. Sweat rates at L‐NAME and Ketorolac sites were compared to the Control sites for the within treatment sites analysis. P values for the multiple comparisons were adjusted using the Bonferroni's procedure. A Student's nonpairwise t‐test was employed to compare NOS‐ or COX‐dependent sweating between groups. Linear regression analysis was performed to determine the relationship between individual changes in sweat rate at the L‐NAME or Ketorolac sites from the Control site and the individual sweat rate achieved at the Control site. The participant's characteristics  were compared between the groups by a Student's non‐pairwise t‐test. Data were presented as means ± SD, and statistical significance was set at P ≤ 0.05. All statistical analyses were performed using a statistical package (SPSS) version 24.0.Sweat rate was analyzed using a three‐way mixed‐design ANOVA with factors of treatment site  were similar between untrained and trained groups, respectively . No differences in heating time were measured between groups . A main effect of group was observed on HR during passive heating such that a lower HR was measured in the endurance‐trained relative to the untrained group .Age (20 ± 1 and 21 ± 3 years), height (1.68 ± 0.08 and 1.71 ± 0.07 m), weight (59 ± 5 and 59 ± 6 kg), and body surface area  from the Control site were observed  in both groups. However, contrary to our hypothesis, the magnitude of this NOS‐mediated reduction in sweating was not different between groups. Furthermore, we showed that relative to the Control site, COX inhibition did not influence sweat rate in either group or for any level of heat stress. These results suggest that endurance training does not affect the relative contribution of NOS to the sweating response which occurred at moderate‐to‐high levels of hyperthermia only in young adults. Moreover, we show that COX is not involved in the observed endurance‐training‐induced increases in sweat production.To our knowledge, this is the first study to evaluate the relative contribution of NOS and COX in the regulation of the sweating in endurance‐trained athletes during a passive heat stress. We showed that NOS inhibition similarly attenuated sweat rate relative to the Control site in both untrained and endurance‐trained individuals following Tor only in both groups   of 0.28 in the relationship between NOS‐dependent sweating and sweat rate at the Control site   during passive heating  is not necessarily associated with improvements in sweat production (Amano et al. We did not measure In conclusion, we show that NOS‐dependent sweat production during passive heating does not differ between young endurance‐trained and untrained individuals. In addition, we show that irrespective of the individuals training status, COX does not mediate changes in sweating during progressive increase in core temperature associated with passive heating. Finally, our findings demonstrate that the greater sweat production observed in endurance‐trained athletes is likely mediated by factors other than NOS‐ and COX‐dependent mechanisms.None declared.