Prohibitin is a pleiotropic protein that has roles in fundamental cellular processes, such as cellular proliferation and mitochondrial housekeeping, and in cell- or tissue-specific functions, such as adipogenesis and immune cell functions. The different functions of prohibitin are mediated by its cell compartment-specific attributes, which include acting as an adaptor molecule in membrane signaling, a scaffolding protein in mitochondria, and a transcriptional co-regulator in the nucleus. However, the precise relationship between its distinct cellular localization and diverse functions remain largely unknown. Accumulating evidence suggests that the phosphorylation of prohibitin plays a role in a number of cell signaling pathways and in intracellular trafficking. Herein, we discuss the known and potential importance of the site-specific phosphorylation of prohibitin in regulating these features. We will discuss this in the context of new evidence from tissue-specific transgenic mouse models of prohibitin, including a mutant prohibitin lacking a crucial tyrosine phosphorylation site. We conclude with the opinion that prohibitin can be used as a potential target for tyrosine kinase signal transduction-targeting therapy, including in insulin, growth factors, and immune signaling pathways. Unfortunately, global knockdown of PHB1 leads to embryonic lethality in multicellular organisms, such as Caenorhabditis elegans and Mus musculus, which has prevented researchers from identifying and illustrating the molecular functions of PHB1 at an organismal level.16 PHB1 is known to undergo various posttranslational modifications such as O-GlcNAc modification,17 palmitoylation,18 ubiquitination,20 phosphorylation21–23 and cysteine oxidation.24Prohibitin (PHB or PHB1) was first identified as an anti-proliferative gene and its cDNA was isolated by differential hybridization to RNA from normal versus regenerating liver tissue in rats.O-GlcNAc modification and serine/threonine phosphorylation that occur at the same residues in certain proteins have been shown to be regulated by tyrosine phosphorylation in nearby residues, and they can influence each other.17 Similarly, acetylation, ubiquitination and methylation may occur at the same lysine residue in a mutually exclusive manner and add diversity to protein functions and their regulation.25 Phosphorylation is the most extensively studied posttranslational modification and orchestrates a variety of cellular and molecular events, such as cell proliferation, differentiation and cell death. It has been estimated that more than 30% of cellular proteins contain covalently attached phosphate groups at a ratio of 1800:200:1 for serine, threonine and tyrosine residues, respectively.26 Phosphorylation is very important for normal cellular functions, while dysregulated phosphorylation has been implicated in various disease conditions such as cancer, diabetes and neurological disorders. Phosphorylation is a fundamental posttranslational modification of PHB1 that regulates its function, intracellular trafficking and its binding specificity for certain proteins. There are two major forms of phosphorylation known to occur in PHB1, serine/threonine phosphorylation and tyrosine phosphorylation. In this review, we highlight the roles of tyrosine phosphorylation of PHB1. In the entire human PHB1 protein there are only four tyrosine residues, at positions 28, 114, 249 and 259, and they are all highly conserved among different species.27 PHB1 is known to undergo tyrosine phosphorylation at multiple residues. There are several reports in the literature that show that PHB1 undergoes tyrosine phosphorylation upon activation by various stimuli.22 These stimuli include insulin, epidermal growth factor (EGF), platelet-derived growth factor (PDGF) and insulin-like growth factor (IGF).28 Recent discoveries have highlighted a crucial role for PHB1 in the MAPK/ERK pathway, PI3 kinase/Akt pathway, TGF-β signaling pathway and STAT3 signaling pathway.29–31 Herein, we describe the active role played by tyrosine phosphorylation of PHB1 in various signaling pathways and its mechanism of action in regulating these signaling molecules.Post-translational modification plays an important role in the regulation, stability, trafficking and cell signaling functions of proteins. Several of the commonly occurring posttranslational modifications in proteins are known to occur at the same residue in a mutually exclusive manner or regulate nearby modification sites. For example, 32 These events lead to increased tyrosine kinase activity of the insulin receptor towards its substrates. Upon activation, insulin receptor phosphorylates insulin receptor substrates (IRS) and Src homologous/collagen proteins (Shc). These dynamic events lead to the activation of subsequent signaling pathways; the PI3/Akt kinase pathway and MAPK/ERK pathway.32 PI3 kinase consists of two subunits: a catalytic subunit, p110, and a regulatory subunit, p85.33 PI3 kinase promotes the conversion of phosphatidylinositol 4,5-bis phosphate (PIP2) to phosphatidylinositol 3,4,5-triphosphate (PIP3). PIP3 acts as a second messenger and activates downstream target alpha serine/threonine protein kinase (Akt). Activation of Akt leads to various biological effects including the suppression of apoptosis by inactivating pro-apoptotic molecules such as BAD, BAX and caspase-9.34 The other arm of the tyrosine kinase signaling pathway is activated by binding of tyrosine-phosphorylated IRS or Shc to growth factor receptor bound protein −2 (Grb-2) and Sons of sevenless (Sos).32 These events lead to the activation of Ras and Raf-1 signaling which eventually activates MAPK and ERK signaling and the translocation of ERK to the nucleus.35 Upon translocation to the nucleus, ERK activate several mitogenic factors  that lead to the survival and proliferation of cells.Activation of the insulin signaling pathway is initiated by with insulin binding to its receptor resulting in its activation. Upon activation the insulin receptor undergoes dimerization and auto-phosphorylation of its cytoplasmic domains.21 Initially, PHB1 is phosphorylated at Tyr-114 upon stimulation with insulin. Once phosphorylated, it creates a binding site for SH2 domain-containing protein tyrosine phosphatase-1 (Shp1), which consequently alters the phosphorylation of Akt and glycogen synthase kinase-3β.21 This finding suggests that the insulin-induced tyrosine phosphorylation of PHB1 and subsequent recruitment of Shp1 may modulate insulin receptor substrates, PI3 kinase activity and subsequent downstream insulin signaling. In addition to the insulin receptor, other tyrosine kinases that can potentially phosphorylate PHB1 at tyrosine residues include the membrane tyrosine kinases EGFR, PDGFR and IGFR as well as Src domain-containing cytoplasmic tyrosine kinases such as Lyn and Syk. In this context, it is important to note that the Tyr-114 phosphorylation site is conserved in the homologous protein PHB2 and that the corresponding residue in PHB2 is Tyr-128 (O-GlcNAc transferase (OGT) and undergoes O-GlcNAc modification at Ser-121 and Thr-258,17 which are also phosphorylation sites for AMPK and Akt, respectively  indicates an important role in the regulation of PHB1 functions.36 This binary switch between tyrosine phosphorylation and O-GlcNAc modifications provides new mechanistic insight into various cell-signaling pathways, which warrants further investigation.We have reported that the phosphorylation of PHB1 at Tyr-114 plays a role in attenuating insulin signaling. Tyr-128 . Thus, iectively . These t in PHB1 . Althoug22 Analysis of the PHB1 protein sequence with BLAST for comparison with proteins with known lipid-binding domain revealed that PHB1 contains conserved lipid-binding PX domains, which may assist PHB1 in interacting with PIP3 and other lipids.22 The Tyr-114 phosphorylation site is in close proximity to the putative PIP3 binding domain of PHB1.22 Thus, it is possible that the phosphorylation status of Tyr-114 in PHB1 may influence other interacting partners of PHB1, either directly or indirectly, that have a role in PIP3 signaling. There are several reports in the literature showing PHB1 is translocated from mitochondria to the plasma membrane,37 and from the nucleus to mitochondria upon activation by various stimuli or agents.39 However, the mechanisms involved in the intracellular trafficking of PHB1 are not well understood. We have shown that PHB1 undergoes palmitoylation at Cys-69 and interacts with Eps 15 homology domain protein 2 (EHD2).18 This modification facilitates translocation of PHB1 to the membrane and its tyrosine phosphorylation.18 This was the first report that elucidated the mechanism behind translocation of PHB1 to the plasma membrane. Taken together, these studies suggest that the tyrosine phosphorylation of PHB1 plays an important role in modulating PI3K/Akt signaling pathways and has a relationship with neighboring post-translational modifications on the protein  to modulate insulin signaling. protein . Perturb40 For the activation of Akt, phosphorylation at the Thr-308 residue is required; to attain its maximal activity, phosphorylation at Ser-473 is also required.41 Dysregulation of Akt activity is known to be involved in various tumor malignancies and other diseases.42 Interestingly, PHB1 has been identified as a target protein for Akt.40 In human pancreatic cancer cells, PHB1 has been shown to be phosphorylated by Akt.40 In these cells, Akt-induced phosphorylation of PHB1 occurs at Thr-258, which is located within the Akt consensus motif R-x-R-x-x-S⁄T in PHB1 protein. Although Thr-258 is not conserved in PHB2, it has been reported that Akt can phosphorylate Ser-91 and Ser-176, which are present in Akt consensus motifs.43 Phosphorylation of PHB1 by Akt can potentially impact various cellular functions of PHB1 and its stability. It is also possible that the Akt phosphorylation of PHB1 can impact its tyrosine phosphorylation at Tyr-114 and vice versa. We have shown that the phosphorylation of PHB1 at Thr-258 and Tyr-114 has opposing effects on insulin signaling.22 The phosphorylation of PHB1 at Thr-258 upregulates insulin signaling, and substitution of Thr-258 by isoleucine results in both the downregulation of Thr-308 and Ser-473 phosphorylation in Akt.22 Furthermore, the phosphorylation of PHB1 at Tyr-114 downregulates insulin signaling, and substitution of Tyr-114 by phenylalanine results in upregulation of Akt phosphorylation at Thr-308 and Ser-473.22 These results provided evidence that Akt phosphorylation of PHB1 impacts its phosphorylation at Tyr-114 and vice versa. Consistent with previous findings, a recent report has demonstrated that Akt phosphorylates PHB1 at Thr-258 in human bladder cancer cells.44 Phosphorylation of PHB1 by Akt promotes its mitochondrial localization and leads to the proliferation of bladder cancer cells.44 Furthermore, substitution of Thr-258 by alanine induced cell death in bladder cancer cells.44 These results demonstrate that PHB1 is an important regulator during bladder cell tumorigenesis and that the phosphorylation of PHB1 at Thre-258 plays a key role in this process. In another report, the levels of plasma membrane-associated PHB1 and migration-inducting gene-7 (MIG-7) were positively correlated with advanced stages of cancer in human lung tissues.45 These results suggest that Akt phosphorylates PHB1 at Thr-258, and phosphorylation of PHB1 plays an important role in the development and progression of different types of human lung cancers. Thus, PHB1 serves as a target protein for Akt and plays a role in regulating Akt activity. Consistent with this notion, downregulation of PHB1 phosphorylation and MIG-7 expression results in reduced cancer invasion and metastasis in human lung cancer xenograft mice.45 These results suggest that targeting the phosphorylation of PHB1 and MIG-7 can be effective in treating patients with advanced lung cancers.Alpha serine/threonine protein kinase  is responsible for mediating various biological responses such as cell growth, cell differentiation, and apoptosis. It is a major signaling molecule present downstream of PI3 kinase.29 Raf-1 is normally phosphorylated at Ser-259 under inactive conditions, but upon EGF stimulation, Raf-1 is phosphorylated at Ser-338 and becomes activated. In this context, it is important to note that Thr-258 in PHB1 and Ser-259 in Raf-1 are located in the Akt consensus motif R-x-R-x-x-S⁄T.46 It is therefore conceivable that Thr-258 phosphorylation in PHB1 may compete with the Ser-259 phosphorylation of Raf-1 thereby reducing the phosphorylation of Raf-1 and facilitating its activation to promote cancer cell proliferation. In one recent report, it has been demonstrated that phosphorylation of PHB1 at Thr-258 and Tyr-259 correlated with the invasiveness and metastasis of human cervical cancer cells.37 A reduction in the invasiveness of cancer cells is observed when phosphorylation is absent on both of these residues. Interestingly, substitution of Tyr-259 for phenylalanine in PHB1 decreases the phosphorylation of Thr-258, but substitution of Thr-258 for isoleucine has no effect on the phosphorylation of Tyr-259.37 Thus, the phosphorylation at Tyr-259 in PHB1 appears to have a regulatory role in the phosphorylation at the Thr-258 site. It has been shown that a Thr-258 phospho-mutant form of PHB1 prevents Raf-1 activation,37 indicating that the phosphorylation at Thr-258 plays a crucial role in the induction of Raf-1 activation. Hence, activation of Raf-1/ERK signaling leads to an enhancement of the invasiveness and metastatic capabilities of cancer cells. It is anticipated that molecules targeting PHB1 and its phosphorylation on the plasma membrane may be effective in decreasing cancer cell metastasis and invasiveness. All of the above experimental findings highlight the crucial role played by PHB1 phosphorylation in cancer cells. However, more diligent studies are required to determine the role of PHB1 and its phosphorylation at serine/threonine residues by Akt as well as their relationship with tyrosine phosphorylation of PHB1.Furthermore, PHB1 has been shown to physically interact with Raf and activate Ras-induced Raf/MAP/ERK signaling upon EGF stimulation.47 Stimulation of murine T cells with anti-CD3 and anti-CD28 antibodies remarkably induces the cell surface expression of PHB1,12 which presumably participates in the T-cell receptor-mediated signaling cascade. PHB1 has been shown to be an endogenous ligand for sialic acid-binding lectin-9 (siglec-9) and negatively regulates T-cell signaling.47 Furthermore, Salmonella typhi, which causes systemic infection and typhoid in humans, targets PHB1 on T cells and downregulates T-cell-mediated immune signaling.49 In a separate study, to identify differentially expressed proteins upon activation of human T cells, Ross et al.23 found that the expression levels of PHB1 and PHB2 mRNA and protein were highly upregulated upon T-cell activation. Using proteomic analysis, the authors provided convincing evidence that PHB1 and PHB2 form a phosphocomplex in the inner mitochondrial membrane of primary human T cells.23 During this process, PHB1 is phosphorylated at serine residues, and PHB2 is phosphorylated at both serine and tyrosine residues.23 Furthermore, functional studies revealed that the PHB1/PHB2 phosphocomplex is required for the survival of differentiated T cells.23 It has been suggested that the phosphorylation of PHB2 at Tyr-248 may be important for protein-protein interactions because PHB2 Tyr-248 lies within a conserved NPXY motif, which could potentially interact with the phospho-tyrosine binding domain.23 Further analysis revealed that the PHB1/PHB2 phosphocomplex is required for mitochondrial homeostasis in T cells.23 It is possible that phosphorylation of PHB1/PHB2 at other tyrosine residues may also have a role in T-cell maturation, which warrants further investigations.There are a number of reports that have identified PHB1 as a membrane-associated protein in T cells and have demonstrated its role in T-cell receptor signaling.50 They are the major effector cells that drive allergic responses. Upon stimulation with antigens, mast cells secrete a large number of granules that contain histamine and other inflammatory mediators such as cytokines and eicosanoids.52 In antigen-stimulated mast cell signaling, antigen binds to the IgE bound to FcεRI at the plasma membrane.52 The presentation of antigen to IgE results in the aggregation of FcεRI complexes, and the rapid tyrosine phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAM) of the beta and the gamma chains of FcεRI by the Src family of tyrosine kinases, such as Lyn.52 Tyrosine phosphorylation of the gamma chain of FcεRI by Lyn leads to the recruitment of another cytoplasmic tyrosine kinase, Syk, to the gamma chain of FcεRI.52 The activation of Syk is essential for the activation of important signaling molecules such as transmembrane adaptor protein linker for the activation of T cells (LAT), phospholipase C (PLC), Src homology 2 (SH2) domain-containing leukocyte protein 76 (SLP-76) and Gab 2.52 These cascades of signaling events lead to the release of inflammatory markers that can induce allergic responses.Mast cells play a key role in the process of inflammation.52 Exposure of antigen to IgE results in translocation of PHB1 from the granules to the plasma membrane and subsequent tyrosine phosphorylation of PHB1 by Lyn.52 These experimental findings also suggest that PHB1 acts as a scaffold or adaptor protein to facilitate the phosphorylation of the FcεRI gamma complex by Lyn.52 Point mutation studies have revealed that phosphorylation at Tyr-114 and Tyr-259 is required for the recruitment of Syk to the FcεRI gamma complex and for the activation of mast cells.52 These experimental findings highlight that the phosphorylation of PHB1 at Tyr-114 and Tyr-259 plays a crucial role in the association of PHB1 with the membrane, where it acts as a scaffold protein for the FcεR gamma and Syk complex during antigen-stimulated signaling events in mast cells. Similar to our previous findings in 3T3-L1 and C2C12 cells,18 a Cys-69 mutant of PHB1 was unable to translocate to the plasma membrane and failed to participate in the antigen-stimulated mast cell signaling.52 Collectively, these studies suggest that the palmitoylation of PHB1 at Cys-69 is required for its translocation to the membrane and/or membrane association. More studies are required to understand the compartment-specific functions of PHB1 in different immune cell types.It has been shown that PHB1 is abundant in the intracellular granules of mast cells and has a critical role in antigen-stimulated allergic responses.53 PHB1 was shown to interact with the IgM receptor present on B-lymphocytes.53 However, the functional significance of PHB1-IgM association was not explored further until recently. It has now been shown that PHB1/PHB2 and the cytoplasmic domain of CD86 cooperate in mediating CD86 signaling in B-lymphocytes.54 CD86 is a transmembrane glycoprotein associated with B cells, dendritic cells and macrophages.55 In one study, Lucas et al.54 sought to identify potential signaling intermediates that associate with CD86, as well as their role in CD86 signaling pathway in B-lymphocytes. Under normal conditions, the expression level of CD86 was found to be low in resting B-cells. However, upon engagement with B-cell receptor CD-40 or IL-4 receptor, the levels of CD86 expression increase in B-cells.56–59 Unlike other transmembrane signaling molecules, CD86 contains a short cytoplasmic domain that is devoid of tyrosine phosphorylation motifs. By using a proteomic approach, the authors identified that in the CD40/IL-4-primed B cells, there is an interaction between PHB1, PHB2 and CD86.54 CD86 engagement on the surface of the primed B cells activates two signaling pathways, which leads to the activation of NF-κB.59 Inhibition of these signaling cascades in B cells decreases the expression of Oct-2, which in turn affects the rate of IgG1 production.59 Taken together, these results demonstrate that both PHB1/PHB2 and an intact CD86 cytoplasmic domain are required to mediate CD86 signaling, which regulates the level of IgG1 production by B cells. In these reports, however, the authors did not identify the residues that are phosphorylated in PHB1. Based on the available information in the literature as discussed above, it is possible that the phosphorylation of PHB1 at tyrosine residues (Tyr-114 and Tyr-259) may be critical in CD86 signaling in primed B cells. Future point mutation studies can provide clues regarding the role of tyrosine phosphorylation of PHB1 in primed B-cell signaling. In another report, Paris et al.60 have shown that Syk and PHB1 can interact with each other in a phosphorylation-dependent manner in antigen receptor-mediated B-cell signaling. It was found that PHB1 becomes phosphorylated at tyrosine residues and participate in immune signaling, but the specific phosphorylation site remains to be identified.60 As there are only four tyrosine residues in PHB1, it is likely that the phosphorylation occurs at previously known phosphorylation sites , which have been identified in a number of cell types and tissues including immune cells.52 In addition, whether the role of PHB1 in B-cell requires intracellular trafficking remains unclear. More studies are required to understand the compartment-specific functions of PHB1 in immune cell signaling in different immune cell types. Moreover, a role for PHB1 in TGF-β and STAT3 signaling pathways has also been reported.31 However, the potential involvement of tyrosine phosphorylation of PHB1 in these pathways has not yet been explored.The association of PHB1 with the cell membrane of B-lymphocytes was first discovered over 20 years ago.13 PHB1 is widely expressed in the ovary and its expression is dependent on the age of the organism and the stage in follicular development.13 In a recent study, it was shown that follicle-stimulating hormone (FSH) upregulates the expression of PHB1 in rat primary GCs.13 In addition, PHB1 was phosphorylated at Tyr-249, Thr-258 and Tyr-259 sites during GC differentiation.13 The observed level of phosphorylation at all three residues was found to be low in the absence of FSH stimulation, whereas enhanced phosphorylation was observed in the presence of FSH. Furthermore, phosphorylation of PHB1 was inhibited by the use of PD98059, which is an inhibitor of the MAPK-ERK pathway.13 These experimental results collectively suggest that PHB1 is a substrate of MEK1 and p38MAPK signaling during GC differentiation. It is interesting to note that Tyr-249 of PHB1 is located within the Raf-1 binding domain of PHB1, which spans through residues 243–275 in PHB1. Reports have demonstrated that PHB1 is indispensable for the activation of Raf-MAP-ERK pathway by Ras.29 A low level of Raf-1 Ser-338 phosphorylation and a high level of Ser-259 phosphorylation have been observed under low PHB1 conditions.29 Membrane targeting and activation of C-Raf by Ras require PHB1 in vivo.29 In the absence of PHB1, C-Raf kinase fails to interact with Ras upon activation by epidermal growth factor stimulation.29 These results indicate that there may be a hierarchical relationship between PHB1 and the MAPK-ERK pathway, and tyrosine phosphorylation of PHB1 might have an important role in regulating the Ras/Raf-mediated MAPK-ERK pathway. Cell fractionation studies have shown that only mitochondrial PHB1 is phosphorylated in rat granulosa cells,13 which would imply that the phosphorylation of PHB1 occurs in specific cellular compartments and/or has a role in the intracellular trafficking of PHB1. Taken together, these results provide new evidence that tyrosine phosphorylation of PHB1 plays an important role in regulating the FSH signaling pathway in ovarian granulosa cells.Ovarian granulosa cells (GCs) play a vital role in the growth and development of the ovarian follicles.61 The cellular proteins that are involved in iron homeostasis are often upregulated during oxidative stress. PHB1 has been reported to bind iron and be upregulated during oxidative stress. Specifically, Tyr-114 in PHB1 is an important residue for iron binding.62 There are numerous reports in the literature that provide support for PHB1 having a protective role in oxidative stress and mitochondrial dysfunction.64 It is possible that the iron-binding function of PHB1 plays a role in its protective effects against oxidative stress. We propose that the Tyr-114 residue in PHB1 has functions that are unique to each cellular compartment. For example, plasma membrane-associated PHB1 may be involved in cell signaling through tyrosine phosphorylation of PHB1, whereas the same tyrosine residue in mitochondrial PHB1 may aid in the iron binding function of PHB1. However, in light of new evidence showing the tyrosine phosphorylation of PHB1 in the mitochondrial fraction of granulosa cells,13 a possibility for the potential role of the phosphorylation of PHB1 at tyrosine residues in iron binding in mitochondria cannot be ruled out. This is because a number of tyrosine kinases, including EGFR, FGFR, ErB2, can translocate to the mitochondria, and the mitochondrial localization of Src family kinases has been reported.66 It is plausible that the phosphorylation status of Tyr-114 modulates the iron-binding property of PHB1 and thereby affects iron homeostasis in mitochondria or potentially other mitochondrial attributes of PHB such as chaperone/scaffolding functions  overexpressing PHB1 in adipocytes. As expected, Mito-Ob mice developed obesity independent of diet.70 Interestingly, female Mito-Ob mice had normal glucose homeostasis and insulin sensitivity, whereas male Mito-Ob mice developed impaired glucose homeostasis, hyperinsulinemia and insulin resistance.70 These results provide evidence that PHB1 has roles in regulating adipose tissue homeostasis and in sex-dimorphic functions.PHB1 has an important role in adipogenesis.21 Reports in the literature have also suggested that tyrosine phosphorylation of PHB1 has an important role in immune signaling, maturation and survival of T cells and in thymic growth.71 More recently, PHB1 has been identified as an adapter protein in antigen receptor signaling in mast cells.52 There, it facilitates protein-protein interactions in a Tyr-114 phosphorylation-dependent manner and modulates cytokine production. These results emphasize that phosphorylation of PHB1 at Tyr-114 is crucial for controlling cell signaling-mediated events. To explore the physiological relevance of phosphorylation of PHB1 in immune functions at the systemic level, we developed a mutant mouse (m-Mito-Ob) overexpressing Y114F-PHB1 from the aP2 gene promotor.28 The aP2 gene is primarily expressed in adipocytes, but is also expressed specifically in macrophages and dendritic cells.67 Similar to PHB1 mice, m-Mito-Ob mice developed obesity independent of diet in a sex-neutral manner, but only male mice developed glucose intolerance, hyperinsulinemia and insulin resistance, such as the Mito-Ob mice.28 These experimental results suggest that Tyr-114-mutant mice retain adipogenic functions and tyrosine phosphorylation may not have a role in mitochondria-associated adipogenic functions of PHB1. The Mito-Ob and m-Mito-Ob male mice exhibited similar metabolic features such as obesity, insulin resistance, and adipose tissue inflammation; however, male Mito-Ob mice developed non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) by 12 months of age,72 whereas male m-Mito-Ob mice developed lymph node tumors by 6 months of age.28 Development of histiocytosis with massive lymphadenopathy in m-Mito-Ob mice demonstrated that Tyr-114 phosphorylation of PHB1 had anti-proliferative effects on immune cells such as macrophages and dendritic cells,28 and consequently on B and T cells. This is consistent with previous reports that showed Tyr-114 phosphorylation has an inhibitory effect on P13K/Akt signaling.22 Interestingly, ovariectomy in female m-Mito-Ob mice resulted in glucose intolerance, hyperinsulinemia and insulin resistance and they developed lymph node tumors similar to male m-Mito-Ob mice.28 These events were not observed in ovariectomized Mito-Ob mice.28 These results demonstrate that PHB1 and mutant-PHB1 lacking a Tyr-114 phosphorylation site act differently in the presence and absence of estrogen.73 It would be interesting to determine if the tyrosine phosphorylation of PHB1 has a role in estrogen-related cell signaling events and functions.As mentioned before, PHB1 is phosphorylated at Tyr-114 in response to insulin, and this modification aids in the recruitment of Shp1, which is a negative regulator of insulin signaling.O-GlcNAc modification of PHB1 itself as well as insulin and immune signaling intermediates. This is because O-GlcNAc modifications of a number of insulin signaling intermediates have been shown to cause insulin resistance,74–76 and emerging evidence suggests that O-GlcNAc modification of immune signaling intermediates modify protein activity during cellular activation and differentiation.77 For instance, O-GlcNAc modification of the NF-κB subunit c-Rel is required for its DNA binding activity and subsequent cytokine expression in activated T cells.78 The inhibition of O-GlcNAc modification in this context is sufficient to repress IL-2 and IFNG expression following TCR stimulation. O-GlcNAc modification of STAT3 exerts an inhibitory effect on STAT3 phosphorylation, which results in a loss of IL-10 production in macrophages,79 thus demonstrating how O-GlcNAc modifications can control important transcriptional programs in immune cells. Of note, PHB1 has been shown to modulate NF-κB and STAT3 signaling.80 The focus has been on phosphorylation-mediated events. However, a potential role of O-GlcNAc modification and its crosstalk with phosphorylation has not yet been investigated.Although the mechanisms involved in PHB1- and mutant PHB1-induced metabolic and immune phenotypes in Mito-Ob and m-Mito-Ob mice remain to be elucidated, potential roles exist for the interaction of PHB1 with OGT and subsequent 81 Histological analysis of pancreatic islets from m-Mito-Ob mice revealed that there was a progressive increase in the infiltration of immune cells to the pancreatic islets. At early stage, immune cells were present around the islets in male m-Mito-Ob mice, indicating peri-insulitis.81 At advanced stages, immune cells migrated into the islets indicating features of intra-insulitis.81 Immunohistochemical studies revealed that immune cells present around the islets were positive for CD8 and F4/80,81 which indicated the presence of lymphocytes and macrophages around the pancreatic islets. However, pancreatic islets from the female m-Mito-Ob mice fed a high-fat diet and male m-Mito-Ob mice fed a low-fat diet were negative for CD8 and F4/80.81 These experimental findings indicate that PHB1 may play a role in immune checkpoints and that Tyr-114-mutant PHB1 may function as a genetic susceptibility factor in adult-onset T1D. More importantly, these findings provide evidence of both the crucial role played by phosphorylation of PHB1 at Tyr114 in the regulation of immune cell function and the role that a loss of tyrosine phosphorylation plays in the activation of diabetogenic effector T cells in response to environmental triggers or stress, such as a high-fat diet. Thus, Mito-Ob and m-Mito-Ob mice have created unique opportunities to gain new insights into the role of obesity-related abnormalities in the adult-onset T1D and for defining the role of PHB1 in antigen presenting cells such as dendritic cells and macrophages. Dendritic cells and macrophages are important cells in the immune system involved in antigen presentation and processing.56 These cells act as mediators between the adaptive and innate immune systems. In our transgenic models, both male and female Mito-Ob and m-Mito-Ob mice overexpress PHB1 and mutant PHB1, respectively, in dendritic cells and macrophages, but only male mice develop immune dysregulation suggesting a sexually dimorphic role of PHB1 in these cell types.73 These results also suggest an important role of PHB1 phosphorylation at Tyr-114 in regulating the function of dendritic cells and macrophages. It is possible that mutant PHB1 can affect macrophage and dendritic cell function in a sexually dimorphic manner or that mutant PHB1-overexpressing macrophages and dendritic cells respond differently in male and female organisms. In summary, these findings provide a proof-of-concept that that obesity-related abnormalities promote the development of adult-onset T1D by coupling environmental factors with genetic susceptibility. It is possible that similar factors are involved in the development of adult-onset T1D in humans. It is anticipated that the Mito-Ob and m-Mito-Ob mice will prove to be valuable tools for mechanistic studies uncovering the role of PHB1 in cell signaling pathways.Obesity and its associated abnormalities appear to be an important factor in the development of adult-onset type 1 diabetes (T1D). However, the mechanisms behind the association of obesity and adult-onset T1D are not clearly understood. One surprising finding from Mito-Ob and m-Mito-Ob mouse models is that male m-Mito-Ob mice developed autoimmune insulitis on a high-fat diet but male Mito-Ob mice did not.81 In this review, we have highlighted the regulatory role of tyrosine phosphorylation of PHB1 in the insulin signaling pathway, the MAPK/ERK pathway, the FSH signaling pathway, in immune cell signaling, and potentially in iron binding in mitochondria. Emerging evidence suggests that role of tyrosine phosphorylation of PHB1 in signaling mechanisms is complex. Moreover, several studies have shown that PHB1 upon stimulation with various stimuli, translocates to the plasma membrane and becomes phosphorylated22 and further traffics between the plasma membrane, mitochondria and the nucleus.39 It is possible that phosphorylation of PHB1 play a role in its intracellular trafficking. It is crucial to determine more regarding the cellular compartment-specific functions of PHB1, and the relationship between the PHB1 that is present on the plasma membrane, in the mitochondria and the nucleus. The two novel transgenic mouse models that we developed have revealed hidden roles of PHB1 and its tyrosine phosphorylation in adipocyte and immune crosstalk in immunometabolism. We anticipate that a fuller understanding of the roles and regulation of PHB1, especially the phosphorylation of its different tyrosine residues may lead to the discovery of new therapeutic targets including signal transduction-targeting therapy for metabolic- and immune-related diseases.PHB1 has pleiotropic functions, such as cell proliferation, cell growth, cell signaling and cell death. Most of these cellular events require proper signaling mechanisms to function effectively. This review emphasizes several of the recent developments in the study of PHB1 associated with cell signaling events and highlights the role of PHB1 tyrosine phosphorylation in mediating these cell-signaling processes . Defects


IgE-mediated wheat allergy is a gluten-related disorder. Wheat is one of the five most common food allergens in children. However, the natural history of IgE-mediated wheat allergy has seldom been described in the research literature. This study presents the current state of knowledge about the IgE-mediated wheat allergy in children. Triticum aestivum) were consumed by people in America as early as 11,000 years BC (before Christ). Wild wheat grains . Half of patients suffer from asthma (48%–67%) and/or allergic rhinitis (34%–62%) ,10. The The WA clinical picture depends on age . SymptomWA is usually diagnosed in young children, but it is rarely seen in infants, despite the fact that wheat proteins pass into breast milk, which was proven by Linn et al. in 1996 . In our WA can be accompanied by allergies to other cereals, most often to rye and/or corn .The prognosis of WA tolerance is not as poor as it is in the case of allergies to peanuts, shellfish, or fish that usually continue into adulthood. It is similar to the tolerance prognosis in children allergic to milk or egg. WA will prevail to maturity in about 10% of patients, with the most severe clinical forms of the disorder ,10,11. ISimilarly to other food allergies, the progress of tolerance can be assessed by means of the wheat IgE titers determined repeatedly during the elimination diet. When wheat IgE concentration increases, tolerance is unlikely to develop quickly. Conversely, it is likely to occur when the wheat IgE titers decline steadily. Moreover, such a procedure allows the determination of the maximum wheat IgE concentration. The higher the titers, the older the patient at the time when the tolerance develops. In our studies the tolerance age median was 3.5 years when the maximum wheat IgE concentrations were below 19.9 kU/L. When it was within the range of 20–49.9 kU/L, the median rose to seven years. It reached 16 years of age when the concentrations exceeded 50 kU/L .WDEIA is a rare syndrome . It is tAll of the wheat-induced diseases are caused by wheat proteins which constitute 10%–18% of the grain mass, depending on the strain. The main component (70%) of the wheat grain is starch.Depending on their dissolving agent, the wheat grain proteins are categorized into four main fractions: albumins (15%), globulins (7%), gliadins (33%), and glutenins (45%). Albumins are soluble in water; globulins, in salt solutions; gliadins, in alcohol; and while glutenins, in dilute acid and alkali. Albumins and globulins are structural proteins that contain many enzymes. Gliadins and glutenins are prolamins and are referred to as gluten. They are storage proteins.The wheat proteins that are regarded today as the WA major allergens will be discussed in detail in the next chapter. Wheat proteins triggering CD symptoms belong to gliadins. Wheat proteins responsible for non-celiac-gluten sensitivity (NCGS) have not been identified yet. One of the proteins under the researchers’ examination is a group of amylase-trypsin inhibitors (ATIs) that do not belong to glutens  .In the serum of patients with IgE-mediated wheat allergy numerous IgE antibodies are found that bind with proteins of all of the wheat grain fractions, most commonly with gliadins. However, in different examinations, they are not the same proteins and, therefore, they cannot be regarded as the major allergens in children with WA ,20. A prThe list of the World Health Organization includes 27 wheat allergens . ClinicaThe best-understood allergenic molecule of WA is rω-5 gliadin (Tri a 19) ,22,23. TThe second allergenic molecule of WA, for which commercial tests are available, is a non-specific lipid transfer protein Tri a 14) (nsLTP). Antibodies signaling the presence of the IgE specific to Tri a 14 are found in WA children and in patients with WDEIA. They are not very sensitive. It is now thought that they do not exhibit cross-reactivity with grass pollen, although there is not enough data to exclude this. Their assessment may help in differentiating wheat sensitization from pollen allergy, which is vital in patients with high levels of grass pollen-specific IgE [4 (nsLTP)In everyday allergological practice the possibility of swift and simple exclusion of cross-reactions is very important. Wheat is highly cross-reactive with other cereals; mainly rye and barley . It has Today, intense studies are conducted on some wheat protein components: glutenins with low and high molecular weight (LMW-glutenins and HMW-glutenins), α-, β-, and γ-gliadins, as well as non-specific lipid transport protein Tri a 14 ,33. HoweThe WA diagnosis is difficult because not all of the major wheat grain allergens are recognized. Similarly, to any other allergy, the gold standard of WA diagnosis remains the oral food challenge. It is usually performed in its open form, as the majority of the observed adverse reactions is of the objective nature. The patient is given whole wheat starting from small doses of wheat-specific protein 1–50 mg) followed by increasingly larger hourly doses (digestion of wheat can be slower than egg or milk), ending with a cumulative dose of up to 0.5–1 g of wheat protein –50 mg fo,34. WA iIn the next stage, allergological tests should be performed to confirm the elevated levels of wheat allergen-specific IgE. The first are skin tests (SPTs) to wheat flour. Generally, commercial wheat extract is used, the specificity of which is very low . Some alAnother step is the determination of serum concentrations of allergen-specific IgE to whole wheat extract. They are commercially available but their specificity is low despite high sensitivity ,33.Solutions used for skin tests to wheat flour and for the assessment of allergen-specific IgE to whole wheat extract consist of the mixture of grain albumins and globulins and, thus, do not contain the insoluble major wheat allergens, i.e., prolamins. This is why the utility of these tests in WA diagnosis is lower than in allergies to other foods, such as milk, egg, or peanuts . MoreoveGluten-specific IgE can also be assessed. Since the commercial test contains wheat gluten proteins, it is positive only in the case of a wheat allergy and negative in the case of allergies to other gluten-containing cereals. It is not known if it includes major wheat allergens. Gluten-specific IgE assays are positive in two thirds of children with WA .Currently, there are commercial tests for the IgE specific to two known allergenic molecules of wheat: Tri a 14 non-specific lipid transfer protein and Tri a 19 rω-5-gliadin [It is characteristic of children who had WA, and have developed a tolerance to wheat, that in most of them (approximately 80%) SPTs to wheat continue to be positive and IgE specific to whole wheat extract and gluten-specific IgE remain elevated, which is rare in other food allergies ,10. For It is worth emphasizing that even though IgE-mediated wheat allergy belongs to gluten-dependent disorders, it is induced solely by wheat proteins, thus being treated by a wheat-free diet. The remaining gluten cereals, such as rye, barley, and oats are well tolerated by most patients and should not be eliminated from their diet. In WA children, oat allergy is very rare. Rye and barley allergies are slightly more common.It is believed that different species of wheat have the same allergenicity, therefore, it is not recommended for the patients with severe WA to try different forms of wheat. There are no studies describing changes in allergenicity of wheat during processing .Moreover, it is not recommended to routinely administer a gluten-free diet to WA patients. Gluten-free products made from rice or corn flour, tapioca, millet, or sorghum, are usually well tolerated by WA patients. However, such products often contain wheat starch as the main ingredient which can be insufficiently purified of wheat proteins to be safe for WA patients. The gluten-free products dedicated to CD patients can contain no more than 20 mg of gluten proteins per 1 kg.Since 2009 the packaging of all products sold in the European Union must inform consumers about the wheat content.In 2013 the case was reported of two children with anaphylaxis after wheat ingestion who had developed prior tolerance to pressure-cooked whole wheat. The process of pressure-cooking changes the structure of the wheat husk. It is not known, however, if it damages the structure of the allergens . No otheThe research literature has provided the first report on the oral immunotherapy administered to older children with anaphylaxis triggered by wheat ingestion. After two years, the therapy resulted in the desensitization in 61% of the patients . FurtherIn the USA the rω-5 gliadin-free variety of wheat has been produced .It is presently believed that the time when foods are introduced into the infants’ diet is relevant to their tolerance. For half of the century, from 1955 to 2005, Europe, America, and Australia used nutrition charts for infants where the delayed introduction of strong allergens was strictly recommended. It is a general opinion that those schemes were responsible for the increased incidence of allergies in that period of time. Today, it is recommended to introduce foods containing strong allergens into the infants’ diets as early as between the 17th and 26th weeks of life.The initial exposure to wheat grains delayed until after six months of age may increase the risk of wheat allergy . So far In recent years the results of two extensive studies  have been published that indicate that the time of introducing wheat into the diet of infants at high risk of celiac disease has no effect on the CD prevalence ,42. In s


Balaenoptera musculus intermedia), fin whales , Antarctic minke whales  and leopard seals (Hydrurga leptonyx). The combined sound energy of a group or population vocalizing during extended periods contributed species-specific peaks to the ambient sound spectra. The temporal and spatial variation in the contribution of marine mammals to ambient sound suggests annual patterns in migration and behaviour. The Antarctic blue and fin whale contributions were loudest in austral autumn, whereas the Antarctic minke whale contribution was loudest during austral winter and repeatedly showed a diel pattern that coincided with the diel vertical migration of zooplankton.This paper describes the natural variability of ambient sound in the Southern Ocean, an acoustically pristine marine mammal habitat. Over a 3-year period, two autonomous recorders were moored along the Greenwich meridian to collect underwater passive acoustic data. Ambient sound levels were strongly affected by the annual variation of the sea-ice cover, which decouples local wind speed and sound levels during austral winter. With increasing sea-ice concentration, area and thickness, sound levels decreased while the contribution of distant sources increased. Marine mammal sounds formed a substantial part of the overall acoustic environment, comprising calls produced by Antarctic blue whales ( Wind In regions closer to anthropogenic sound sources, evidence is accumulating that marine mammal communication, among other behaviours, is likely to be affected by increased ambient sound levels, particularly for baleen whale populations that are thought to rely on long-distance (low-frequency) communication –12. Know2.2.1.Two autonomous underwater acoustic recorders  were moored in the Atlantic sector of the Southern Ocean from March 2008 to December 2010 at 66°01′ S and 00°05′ E (Mooring ID: AWI-230-6) and at 69°00′ S and 00°00′ E (Mooring ID: AWI-232-9) . HereinaShear currents can induce strumming or flow noise into underwater acoustic recordings. In our recordings flow and strumming noises could not be discerned from manual screening of the 5-min spectrograms and power spectra, and were consequently assumed to have negligible impact on the results of this study. However, the shear current occasionally induced impulsive shackle noise at 69° S that contaminated some recordings between May and August in 2008 and June and October in 2009. The shackle noise could easily be identified by characteristic peaks between 40 and 60 Hz and occurred in so few recordings that we choose to not remove them from the dataset. Other than being occasionally visible as peaks in the long-term spectrogram and 50th and 95th percentile spectra of Aural 69° S, the shackle noise did not impact the results of this study.−1. The self-noise of the hydrophones was reported by the manufacturer as 54 dB re 1 µPa2 Hz−1 at 10 Hz and 42 dB re 1 µPa2 Hz−1 at 100 and 1000 Hz. According to the Aural's manufacturer, the recorder's electronic self-noise is flat within ±1 dB over the usable frequency range from 10 Hz to 15 kHz [2 Hz−1 at 10 Hz and 45.4 dB re 1 µPa2 Hz−1 at 100 and 1000 Hz. In addition, the spectra measured by both recorders contained narrow peaks (approx. 3 Hz wide) between 80 and 2000 Hz caused by additional electronic noise. They remained constant over time and can be easily identified in the spectra.The recorders were equipped with HTI-68-MIN (High Tech Inc.) hydrophones with a factory calibrated sensitivity of −164.6 dB re 1 V µPao 15 kHz . Assumin2 Hz−1 for a 50 dB re 1 µPa2 Hz−1 signal and ±0.05 dB re 1 µPa2 Hz−1 for a 80 dB re 1 µPa2 Hz−1 signal. The recorders used UTC time and were set to record with a sample rate of 32 768 Hz for 5 min every 4 h starting 00.00 h daily, resulting in 486 h of acoustic recordings. As a gross of the recorded spectra hit the systems noise floor above 10 kHz, we limited our analysis to ambient sound between 10 Hz and 10 kHz. Data were stored losslessly in 16 bit wav files. Owing to internal data handling problems with the recorder, every 48th file was lost [The Aural's system gain was set to 22 dB, resulting in a saturation sound pressure level of 149 dB re 1 µPa. The recorded ambient sound spectra never reached saturation values. The sound level calibrations are solely based on factory calibration, no further pre- or post-calibration was performed, nor did we apply any frequency-specific correction of hydrophone sensitivity. According to the manufacturer the recorders' frequency response is flat within ±1 dB over the usable frequency range . Adding was lost . Additio2.2.marine mammal contributions (MMCs). Peaks in the spectra (MMCs) could be assigned to different species by manual perusal of the 5-min recordings and comparison with published records of marine mammal vocal repertoires. The following species' contribution could be detected in the PSD dataset  [Balaenoptera physalus) [Balaenoptera bonaerensis) [Hydrurga leptonyx) [fmin and fmax) that best characterized each MMC were chosen manually to avoid interference between the different MMCs (MMC) was only calculated over each MMC's core frequency band  was calculated using a pre-set averaging length, which was set to the full 5 min of each recording. The PSD was calculated and averaged after Welch's method  using a  dataset : Antarctermedia) ,24, fin hysalus) ,24, Antaerensis)  and leopeptonyx) . The nument MMCs . Both AnMMC in the four MMC core frequency bands was calculated by subtracting the estimated PSD without MMCs from the measured PSD. The hypothetical spectrum without the MMC (PSDinterpolated) was calculated by fitting an interpolation function to the measured PSD around each MMC frequency band . For the fin whale frequency band, the correlation coefficient r2 between PSDinterpolated and PSDmeasured was 0.99, for the Antarctic minke whale band 0.72 and for the leopard seal band 0.95. As the Antarctic blue whale MMC was present year-round, we could not calculate the correlation coefficient between PSDinterpolated and PSDmeasured, but manual inspection of the fitted spectra confirmed that the interpolation function provided robust estimates of the non-MMC spectrum. The average correlation coefficient r2 between PSDinterpolated and PSDmeasured in the frequency bands used to fit the interpolation function (coloured circles in MMC), we averaged PSDmeasured and PSDinterpolated over each MMC's respective core frequency band . This can be quantified as signal-to-noise ratio (SNR) usingFor each recording, the PSDncy band  and subtncy band  and nfmiMMC was higher than the pre-defined threshold of 1 dB.To ensure a rigid analysis, an MMC was defined as present when its SNR2.3.The zonal and meridional wind speed fields were extracted from the European Centre for Medium-Range Weather Forecasts interim climate reanalysis dataset (ERA-interim) . The temGridded sea-ice concentration data were obtained from the University of Bremen, based on their analysis of data from the Advanced Microwave Scanning Radiometer Earth Observing System (AMSR-E) . The datr in this study were calculated using Pearson's method [The Antarctic sea-ice extent time series was obtained from the National Snow and Ice Data Centre . Sea-ices method .3.3.1.The recorded spectral probability density over frequency is shown in b), the sound generated by moving shackles can be seen as faint dotted line at 40 and 57 Hz in 2008 and 2009.The 3-year time series of PSDs, referred to as long-term spectrograms hereinafter, are displayed in figure 43.2.MMC contributed by Antarctic blue, fin and Antarctic minke whales and leopard seals in their respective frequency bands (Lobodon carcinophaga) was occasionally present in the long-term spectrograms between 500 and 1000 Hz . Antarctic blue whale PSDs were similar at 66° S and 69° S from January to March and louder at 66° S compared with 69° S for the rest of the year.Between 27 and 28 Hz, the most persistent peak in the long-term spectrograms  is assocDecember a. Antarc3.2.2.b).Fin whales in the Southern Ocean emit pulsed calls with main energy around 20 and 89 or 99 Hz, depending on region ,23. In tfigure 53.2.3.c). At 66° S, Antarctic minke whale PSDs were highest between May and September, followed by a decrease in October and November. At 69° S, Antarctic minke whale PSD increased until October, followed by a sharp decrease in November. An exception to this pattern occurred in 2008, where Antarctic minke whale PSDs were stronger at 69° S compared with 66° S in the beginning of May, and increased at 66° S and decreased at 69° S until June. In the long-term spectrograms, the frequency characteristics of the Antarctic minke whale contribution varied from year to year  at 66° S, normalized for each day between 0 and 1. The diel cycle's phase remained stable throughout the recording period, with Antarctic minke whale PSDs being louder at midnight than midday. The diel cycle was strongest in austral winter 2009 .On a much shorter time scale, the Antarctic minke whale acoustic contribution followed a diel cycle from the end of April to the beginning of August each year. Figure 63.2.4.fmin to fmax) with the Antarctic minke whale contribution (d).Antarctic seals contribute distinctly to the underwater acoustic environment of the Southern Ocean . Leopardribution . The leoribution d.3.3.The impact of the physical environment on underwater sound levels was explored by comparing the average PSD in the frequency bands 30–80 and 500–1000 Hz with spatially averaged wind speed and sea-ice concentration as well as sea-ice draught and extent. These frequency bands were chosen to exclude interferences with the MMCs and to avoid the recorder noise floor. Both frequency bands show similar patterns over their respective bandwidth in the spectral probability density spectra : a bimoda,b, where each marker represents a 5-min recording and marker colour encodes the recording month. The time series and scatter plots indicate an inverse relationship between sea-ice concentration and PSD, which is clearest for the 30–80 Hz frequency band (dots in r ≈ −0.8) compared with 2000 km at 69° S (r ≈ −0.9).figure 8 dots in . Sea-icer > 0.5) with 500–1000 Hz PSD within a 200 km radius around both recorders' locations , whereas average PSD between 30 and 80 Hz correlates poorly with local wind speed . However, at 66° S wind speed and 30–80 Hz PSD correlate weakly (r ≈ 0.3) over an area between 50° S and 70° S (c) during ice-free conditions. During ice-covered conditions (average ice concentration in 200 km radius > 50%), wind speed correlates weakly (r ≈ 0.3) with 500–1000 Hz PSD within a 600 km radius for Aural 66° S (b) and within an area roughly indicating the coastal polynya for Aural 69° S. For both recorders, the 30–80 Hz PSD and wind speed correlate weakly in an area roughly indicating the coastal polynya, during ice-covered conditions. The relationship between local wind speed (averaged within 200 km radius) was also analysed as scatterplot . The response to increasing wind speed is similar at both locations: in the 30–80 Hz band, the PSD shows no substantial trend with increasing wind speed, both during ice-free and ice-covered conditions. In the 500–1000 Hz band, on the other hand, the PSD increases with increasing wind speed during ice-free conditions , and shows no substantial trend during ice-covered conditions.ocations a,e, whernd speed c,d. Howend 70° S c during al 66° S b and wittterplot c,d. The 4.Polarstern approached the moorings containing the recorders (i.e. for deployment and recovery of the moorings). We thus assume that local and distant sea surface processes are the major abiotic sources of ambient sound in the Southern Ocean.The recorded long-term spectrograms represent the prevailing natural sound conditions in the Atlantic sector of the Southern Ocean, consisting of the cumulative emissions from air–sea-ice interaction, marine mammals and icebergs or shelf ice . Ambient sound generated by shipping typically covers a frequency range from 10 to 1000 Hz and would be difficult to distinguish from sea-surface-generated sound in the ambient sound spectra . Ship-geMMC measurements were not affected. Owing to the self-noise's low amplitude and scarcity of the shackle noise, we assume that both had negligible impact on our analysis of the relation between environmental parameters and PSD. It did, however, limit our analysis to frequencies below 1000 Hz, as most spectra above that hit the systems noise floor.In the PSD dataset, the recorders' self-noise occurred as constant narrow peaks between 80 and 2000 Hz and broadband noise at frequencies above 1000 Hz. As the self-noise remained constant over time and the shackle noise occurred at frequencies away from the MMC bands, the PSD4.1.4.1.1.Wind stress at the sea surface generates sound between approximately 0.1 and 20 kHz . The inc4.1.2.South of the Antarctic Convergence Zone (i.e. south of 60° S), the sound speed minimum is located close to the sea surface, thus creating a surface sound duct . Both reGiven that wind-induced surface motion is one of the major sound sources over the observed spectrum , the cor4.1.3.The comparison between PSD and sea-ice concentration, draught and extent showed that a growing ice cover decreases ambient sound levels across the observed spectrum, with the strongest correlation in the 30–80 Hz band figures  and 8. LIn our observations, the net effect of the sea-ice cover is a reduction of ambient sound levels. However, sea ice is also a source of underwater sound, especially in the marginal sea-ice zone, where surface waves penetrate the ice floes ,44. Sea-4.2.MMC and PSDMMC. The two time series exhibit different temporal patterns, peaking at different times. If the abiotic sound were roughly constant over time, the two time series would show similar patterns. But as the abiotic ambient sound shows substantial temporal variation, the SNRMMC reflects both variation in the MMC  and abiotic ambient sound (noise). To avoid this interference with abiotic sound fluctuation, we choose to analyse the MMCs using PSDMMC as metric. Avoiding this interference is particularly relevant when sound energy contribution metrics are used to infer information on the animal's distribution and behaviour.Previous studies on the contribution of marine mammals to underwater ambient sound used relative metrics to describe the contribution . Furthermore, marine mammal PSDs only contain information about the marine mammal presence, not absence  and the vocalizing population could be sex or age segregated. For a given location, increased PSDsMMC could be caused by a combination of processes: an increase in number of vocalizing animals, increase in source level, increase in call rate, decreasing distance to the vocalizing animals or decreasing transmission loss between the vocalizing animals and the recorder.There are several other aspects that have to be borne in mind when deriving information on marine mammal occurrence and distribution from ambient sound spectra. The observed marine mammal PSDs only indicate periods of intense vocal activity, as sporadic calls will not be visible as peaks in the acoustic power spectra  and not each MMC's entire frequency range . Our observations match the Antarctic blue whale vocal activity pattern in the Drake Passage [Blue whale calls are recorded in all the world's oceans and distinct call types have been associated with specific subpopulations . A persiet al.  observed Passage  and the  Passage , where h Passage . Conside4.2.2.Owing to their low frequency (approx. 20 Hz) and high call rate, the pulsed vocalizations of fin whales are detectable as a peak in many recorded ambient sound spectra –56. West4.2.3.c). The difference in Antarctic minke whale PSD between 66° S and 69° S suggests latitudinal migration of vocalizing minke whales or local changes in vocal behaviour. A part of the annual and spatial variation of PSD could also be caused by changes in transmission loss due to sea-ice growth and melting. Annually, Antarctic minke whale PSD peaked during May and October at 66° S, and during October and December at 69° S (except a unique peak in PSD at 69° S in May and June 2008). This suggests a southward migration of vocalizing Antarctic minke whales, or southward shift in vocal behaviour in austral spring. Antarctic minke whales frequently feed on dense patches of krill under sea ice [Antarctic minke whale vocalizations have only recently been identified and were previously known as ‘Bioduck’ signals . Studiesfigure 5 sea ice ; the var sea ice .Euphausia superba), has been shown to occur in the polar night in the Atlantic sector of the Southern Ocean [b). Antarctic minke whales have been observed feeding directly under the sea surface, skimming the underside of sea ice for krill [A connection between feeding and vocal behaviour is supported by the finding that the Antarctic minke whale contribution followed a diel cycle from the end of April to August (rn Ocean . The occrn Ocean ,64. Thisrn Ocean ,63, and or krill . Given t4.2.4.Leopard seal vocalizations have been associated with mating: both sexes are known to produce a variety of calls . Owing t5.In contrast with the high levels of anthropogenic sound present in the Arctic , the SouThe temporal and spatial variability of the MMCs contains information about behavioural and distribution patterns. Recording MMCs with a higher spatial resolution and combining these measurements with statistical and acoustic propagation models can render it possible to estimate the spatial distribution of vocalizing animals, which will improve our understanding of their behaviour, migration and habitat use.


A model of ice floe break-up under ocean wave forcing in the marginal ice zone (MIZ) is proposed to investigate how floe size distribution (FSD) evolves under repeated wave break-up events. A three-dimensional linear model of ocean wave scattering by a finite array of compliant circular ice floes is coupled to a flexural failure model, which breaks a floe into two floes provided the two-dimensional stress field satisfies a break-up criterion. A closed-feedback loop algorithm is devised, which (i) solves the wave-scattering problem for a given FSD under time-harmonic plane wave forcing, (ii) computes the stress field in all the floes, (iii) fractures the floes satisfying the break-up criterion, and (iv) generates an updated FSD, initializing the geometry for the next iteration of the loop. The FSD after 50 break-up events is unimodal and near normal, or bimodal, suggesting waves alone do not govern the power law observed in some field studies. Multiple scattering is found to enhance break-up for long waves and thin ice, but to reduce break-up for short waves and thick ice. A break-up front marches forward in the latter regime, as wave-induced fracture weakens the ice cover, allowing waves to travel deeper into the MIZ. We observe a clear convergence trend towards a steady state for all rows of the array and for each thickness. The front rows consistently converge faster than the back ones and to a lower mean and a higher number of floes, so that the degree of break-up decreases with the level of penetration in the array. Interestingly, the evolution of the mean and number of floes of the FSD for the entire array almost coincides with those associated with the middle row of the array. This suggests a simple linear dependence of the FSD statistics with respect to the row number, which will be demonstrated later. The standard deviation of the FSD for the entire array is consistently at least as large as that of the last row, which in turn is the largest of all the rows. This reflects the larger spread of floe sizes on a large scale (entire array) compared with the local scale (each row). figure 10d,h,l. Observe first that the distributions look nearly normal. This is probably the result of effectively averaging over the wave periods. The bimodality seen in previous distributions does not completely disappear, however, as a shoulder-type feature can be seen on the right of the peak (i.e. for large radii), particularly for D=1 and 2 m. This suggests the existence of a second mode for large radii but with a small peak.We further display the PDF of the FSD for all cases discussed above in figure 10D=1, 2 and 4 m, respectively. This notwithstanding, it is unclear how the limited size of the array affects these estimates.The row dependence of the mean, standard deviation and number of floes after 50 break-up events is analysed further in 7.(i) a minimum floe diameter exists for each thickness below which break-up cannot occur, and(ii) this critical diameter depends on the wave period in a way that it reaches a minimum at a resonant wave period, for which the floe diameter is approximately equal to the open water wavelength and half the ice-covered wavelength.A new model of ice floe break-up under ocean wave forcing in the MIZ has been developed. It combines the time-harmonic multiple scattering theory for a finite array of floating elastic discs proposed by  with a p(i) Break-up of a single large floe causes the emergence of a bimodal FSD for most wave and ice parameters considered. Larger values of wave period and ice thickness correspond to FSDs with larger floe sizes and more separated peaks of the associated bimodal distribution. The convergence of the FSD towards its steady state under repeated break-up events is very quick for long waves and slower for short waves. Increasing values of ice thickness also tends to decrease the rate of convergence of the FSD, suggesting that multiple wave scattering within the array of broken floes enhances break-up and therefore influences the steady-state FSD.(ii) Break-up of an array of large floes under monochromatic forcing provides additional insight into the effect of multiple scattering on the FSD. First, the bimodality of the FSD observed for the single floe break-up simulations is either damped or removed, as the population of large floes associated with the second mode is consistently redistributed to smaller floes. Second, results of our simulations indicate that two scattering regimes exist, i.e. long waves/thin ice and short waves/thick ice. In the former regime, wave-induced ice break-up is enhanced compared with the single floe break-up, which is likely to be a consequence of constructive interference of wave fields radiated by the individual floes. In the second regime, multiple scattering causes sufficient wave energy attenuation through the array to prevent some ice floes from fracturing, resulting in broader FSDs compared with those obtained from the repeated break-up of a single floe.(iii) Investigation of the evolution of the FSD at different levels of penetration in the array indicates that enhanced floe break-up in the long-wave regime occurs throughout the array with a small upward trend with distance from the ice edge. In the short-wave regime, we observe the positive feedback between wave-induced ice break-up and ice-induced wave attenuation. Break-up originally only takes place in the front rows as waves are attenuated by scattering deeper in the array. After sufficient break-up, waves are less attenuated, carry energy at a higher level of penetration and cause break-up there. The overall effect is the observation of a break-up front marching forward in the MIZ as the structural integrity of the ice cover reduces under wave-induced break-up.(iv) Break-up of an array under a Bretschneider spectrum forcing generates near-normal FSDs for all ice thicknesses, which is likely to be a consequence of averaging with respect to the wave period. As opposed to the simulations with monochromatic forcing, break-up decreases with the level of penetration in the array, such that the mean floe size increases linearly with distance from the ice edge. The rate of floe size increase is larger for thicker ice.A closed-loop feedback algorithm has been proposed to model the evolution of the FSD in the MIZ under a sustained wave event. Each loop consists of (i) computing the MC stress in all the floes, (ii) breaking up each floe satisfying the MC criterion, and (iii) generating a new array of floes from the updated FSD, which is then used as the geometry of the wave interaction problem in the next loop. We conducted a number of numerical experiments to determine the evolution of the FSD towards a steady state through 50 break-up events (i.e. loops), for different wave and ice configurations. We simulated the break-up of (i) a single large floe for a monochromatic unit-amplitude plane wave forcing, (ii) an array of large floes for the same monochromatic forcing, and (iii) an array of large floes for a Bretschneider spectrum forcing. Key findings are summarized below.O(1 m) cannot be resolved accurately by current observation techniques, so that it is unclear whether the power law extends to very small scales. In any case, our analysis suggests that wave-induced floe break-up alone does not create or preserve the observed power-law FSD or the apparent increase in small floe numbers, but may partially contribute to it. Other processes acting on longer time and/or length scales  must be considered to explain this feature of the FSD. Although recent modelling work has shown the emergence of a power-law FSD [An important outcome of our investigation is that no power-law FSD was generated from the simulations. Our model consistently predicts that the number of floes decreases to zero for smaller floe sizes in an MIZ with uniform ice thickness. This results from the fact that small floes are less prone to elastic deformations than large floes, i.e. they behave similarly to a rigid body. Analysis of the data generated in §-law FSD ,28,50, iAlthough our findings provide much theoretical understanding of the wave-induced ice break-up process in the MIZ, the underlying model was constructed based on a number of simplifying assumptions, which may influence certain results. Specifically, the FSD resulting from the break-up of a more heterogeneous ice cover, i.e. governed by a floe shape and ice thickness distribution and possibly different ice types, under a realistic multidirectional random sea state, may spread the FSD over a wider range of floe sizes to the point where a power law could be fitted to a portion of the curve. In addition, the validity of our break-up model, which involves breaking a circular floe into two circular floes and time-harmonic wave forcing, is unclear and requires further investigation. It would be difficult to relax these assumptions in the context of the three-dimensional wave-scattering model considered here. We may envisage a simpler two-dimensional model, however, in which the one-dimensional ice cover is initialized as a semi-infinite beam and forced by a transient incident wave generated from a frequency spectrum. More theoretical investigation into this physical process is needed to be able to provide large-scale ice–ocean wave forecasting models with a quantitative parametrization of the two-way coupling between ocean waves and sea ice. Monitoring the evolution of the FSD in an area of ice-covered ocean during a wave break-up event either in the field or in a controlled laboratory setting, with the ability to resolve small floes accurately, would be needed to provide a clearer picture of the complicated processes investigated here.


Although one of these mutants was potentiated by a positive allosteric modulator, we showed that receptor activity was further potentiated in a trans-activation heterodimer containing a single allosteric site, however only when the allosteric site was located in the subunit responsible for G protein coupling. On the contrary, preventing activation in both subunits was necessary for obtaining full inhibition by a negative allosteric modulator. These findings correlate with the proposed activation mechanism of the metabotropic glutamate receptors (mGluRs), in which only a single transmembrane domain is activated at a time. CaSR and mGluRs belong to the class C G protein-coupled receptors, and our findings thus suggest that the activation mechanism is common to this subfamily.Allosteric modulators that are targeting the calcium-sensing receptor (CaSR) hold great therapeutic potential, and elucidating the molecular basis for modulation would thus benefit the development of novel therapeutics. In the present study, we aimed at investigating the mechanism of allosteric modulation in CaSR by testing dimers carrying mutations in the allosteric site of one or both of the subunits. To ensure measurements on a well-defined dimer composition, we applied a  The calcium-sensing receptor (CaSR) is essential for the maintenance of calcium homeostasis as it continually monitors the extracellular level of calcium3In GPCR drug discovery it can be advantageous to focus on the allosteric binding site, as the orthosteric site is highly conserved thereby making it difficult to achieve receptor selectivity for orthosteric ligands. Furthermore, allosteric drug compounds might be less likely to show adverse effects, since the modulatory effect is dependent on the presence of agonistB) receptors, the GABAB1 subunit is responsible for agonist binding while G protein coupling occurs only in the GABAB2 subunit26272913CaSR belongs to the class C GPCRs and contains the structural features that are characteristic for this receptor subfamily including a large amino-terminal domain (ATD) containing the orthosteric binding sites1112131516171920222324In this study, we aimed to investigate the role of allosteric modulation in the CaSR dimer. Specifically, we examined whether it is a necessity to have modulators bound in both subunits or if one modulator per dimer is adequate. Our data suggest that a single allosteric site per CaSR dimer is sufficient for obtaining positive modulation of activity, while full inhibition is only achieved if activation of both 7TM domains is prevented.trans-activation assay, which has previously been demonstrated for the mGluRs3133trans-activation mechanism thus allows for an assay in which a measured functional response can only arise from one specific dimer. Accordingly, two types of inactivating CaSR mutants were required in the present study in order to utilize the trans-activation system.When studying receptor dimers, it is of crucial importance to ensure that functional readouts arise only from a single, well-defined subunit composition. Here, we applied a 2+ in the IP-One assay, which measures activation of the Gq signaling pathway. None of the six constructs with mutations in the conserved orthosteric binding site displayed complete loss of function, however lower potency of Ca2+ was observed for all of them (2+ concentration. The two constructs with mutated ICL3 regions likewise demonstrated highly reduced potency of Ca2+ and only 25% (for L797A) and 5.9% (for F801A) of the WT maximum response  or decrease (F821A and I841A) in the EC50 of Ca2+ compared to the WT receptor  and negative allosteric modulators (NAMs), respectively, have been identified in the 7TM region of CaSR. As these sites are overlapping although not identical, it should be possible to identify a mutation impairing effects of PAMs and NAMs. Based on mutagenesis studies reported in the literature404142receptor , Table 3pt E837A , Table 3response , Table 3trans-activation control system was used to ensure measurements on the specific subunit composition in each; a dimer without any mutations in the allosteric sites, a dimer with an allosteric site mutation in one subunit, a dimer with a allosteric site mutation in the other subunit and a dimer with mutated allosteric sites in both subunits. The four dimers of interest are shown schematically in Because the E837A mutation eliminates the allosteric modulation in the subunit that contains the mutation, it was possible to identify whether the functional allosteric site in the other subunit was sufficient for obtaining the modulatory effect in the dimer. Four specific dimers were tested and the At first, receptor expression was validated for each mutant upon co-expression in the four dimer combinations of interest. In accordance with results described above, the S170A mutant displayed only 42% surface expression and 59% total expression compared to WT HA-CaSR upon co-expression with pEGFPN1. When introducing the allosteric site mutation E837A in the HA-S170A construct, expression levels increased to the level of WT HA-CaSR expression . Both myDimerization between a myc-tagged receptor construct and a HA-tagged receptor construct can be validated by use of a time-resolved fluorescence resonance energy transfer (TR-FRET) dimerization assayUpon co-expressing a myc-tagged and a HA-tagged version of each of the four CaSR mutants, increases in FRET signal were detected for each and homodimerization was thus verified . Althoug2+-mediated responses from the four heterodimers of interest was investigated by testing increasing concentrations of Ca2+ in the presence of 0.05 μM, 0.1 μM and 1 μM NPS R-568, respectively. The S170A:F801A heterodimer contains functional allosteric sites in both subunits and accordingly, leftward shifts of the Ca2+ curves were observed thus demonstrating a concentration-dependent potentiation by NPS R-568  was likewise tested at each mutant in the presence of 0.05 μM, 0.1 μM and 1 μM NPS R-568, respectively , at which the contribution from the S170A homodimer was highly limited, was chosen from the curves shown in 1 was observed only at the highest tested PAM concentration, whereas no significant agonist effect or potentiation was observed for any of the other mutants. On the contrary, robust and highly significant potentiation was detected for the responses of the S170A:F801A and S170A:F801A/E837A heterodimers  and a heterodimer with only one allosteric site (S170A:F801A/E837A) were both subjected to potentiation upon addition of NPS R-568, and our findings thus proved that one allosteric site was sufficient for obtaining potentiation of the agonist response in CaSR. Importantly, this potentiation was only achieved when the PAM was bound to the subunit in which G protein coupling occurred. If the allosteric site was located in a subunit with impaired G protein coupling, a small NPS R-568-mediated inhibition was instead observed. These findings are in perfect correlation with results obtained for an mGluR5/mGluR1 dimer, in which an mGluR5 PAM fully enhanced the agonist response in spite of the dimer having only one mGluR5 PAM site, while an mGluR1 PAM acted as a non-competitive antagonist, when G protein coupling was prevented in the mGluR1 subunitBy applying the 2+ was greatly reduced. It is well established that agonist binding in CaSR is highly cooperative with a Hill coefficient of 3–42+ sites were identified in CaSR by use of homology modeling47132+-mediated response of CaSRAlthough the applied S170A mutant was virtually inactive, substantial NPS R-568-mediated potentiation was observed, hence suggesting that the mutation did not completely eliminate agonist activation in CaSR, although the potency of CaNegative modulation of receptor activity was likewise studied by using CaSR dimers carrying mutations in one or both of the allosteric sites. Results showed that the activity of the S170A:F801A/E837A dimer with a single allosteric site was subjected to full inhibition by 5 μM NPS 2143, and the NAM thus seemed able to completely inhibit the activity of CaSR when binding in only one subunit. Noticeably, neither of the subunits in the S170A:F801A/E837A dimer was however able to trigger intracellular signaling, as the F801A mutation was impairing G protein coupling in the adjacent subunit. The S170A/E837A:F801A dimer also contained only one allosteric site, however NPS 2143 triggered only 30% inhibition of the max response of this receptor dimer, as the S170A/E837A subunit was still available for activation and G protein coupling. In conclusion, only a dimer in which activation and/or G protein coupling were prevented in both 7TM domains demonstrated full inhibition of CaSR activity. These results also correlate well with studies conducted using the mGluRs, for which it was shown that full inhibition required binding of antagonist in both subunitsAccording to the model of receptor activation that has been proposed for the mGluRs, only one 7TM domain in the dimer is activated at a time29In conclusion, we have shown that one allosteric site per dimer was sufficient for obtaining potentiation of the agonist-mediated activity of CaSR, while prevention of activation in both 7TM domains was required for full inhibition of the functional response. These findings correlate with the current model for mGluR activation, in which only one 7TM domain is activated at a time, and our study thus suggests that the activation mechanism is shared among the class C receptors. This asymmetry in the activation of the 7TM domains is however puzzling, as dimerization has proven crucial for the functional coupling between the ATDs and the 7TM domains, and more studies are therefore required in order to fully understand the molecular basis for activation and modulation in the 7TM domains of class C receptors. Such knowledge would be highly beneficial for the development of much needed novel therapeutics targeting CaSR.Dulbecco’s modified eagle medium (DMEM), 10.000 units/mL penicillin and 10.000 μg/mL streptomycin mixture (P/S), dialyzed fetal bovine serum (dFBS), Opti-MEM, Dulbecco’s Phosphate Buffered Saline (DPBS), Hank’s Balanced Salt Solution (HBSS) and Lipofectamine 2000 were all purchased from Thermo Fisher Scientific . NPS R-568 hydrochloride was purchased from Tocris Bioscience , while NPS 2143 hydrochloride was synthesized as previously publishedThe previously described myc-tagged rat GPRC6A-pEGFPN1 construct, which contains the mGluR5 signal peptide upstream of the myc tag2. 48 h prior to assay, transient transfection was performed using Lipofectamine 2000 as the transfection reagent. White opaque 96-well culture plates  were used for the ELISA and the TR-FRET dimerization assay, while clear tissue culture treated 96-well plates  were used for the IP-One assay. Both plate types were coated with poly-d-lysine before addition of cells and transfection mix. 0.25 μl/well of Lipofectamine 2000 were diluted in 25 μl/well of Opti-MEM and incubated at room temperature (RT) for 5 min. 0.03 μg/well of DNA was diluted in 25 μl/well of Opti-MEM, and subsequently mixed with the Lipofectamine 2000 dilution and incubated 20 min at RT before addition to the plates. Cell suspensions of 170.000 HEK293T cells/mL were prepared in complete growth medium and 100 μl/well (17.000 cells/well) was added to the plates, which were incubated for 48 h at 37 °C and 5% CO2 before performing assays.Human embryonic kidney (HEK) 293T cells were cultured in DMEM supplemented with 10% dFBS and 1% P/S at 37 °C and 5% CO2O with 3% skim milk, 1 mM Ca2+, 50 mM Trizma hydrochloride solution, pH 7.4) for 30 min after which primary antibody incubation for 45 min at RT was performed by addition of either anti-HA antibody  or anti-myc antibody  diluted 1:1000 in blocking solution. Subsequently, horseradish peroxidase conjugated anti-mouse antibody  diluted 1:1500 in blocking solution was added and plates were incubated for 45 min at RT. Receptor levels were detected by addition of 80 μl/well of DPBS supplemented with 1 mM CaCl2 and 10 μl/well of SuperSignal ELISA Femto Substrate . Chemiluminescence was measured on an EnSpire plate reader  and normalized to the level of WT CaSR.For assessment of surface and total expression of WT and mutant CaSR constructs, ELISA was performed as previously described1 accumulation assay1 antibody cryptate Terbium conjugate + 2.5% IP1-d2 conjugate ) was also added. The 384-well plate was incubated for 1 h at RT in dark before being measured on an EnVision plate reader . Upon excitation at 340 nm, emission at 615 nm and 665 nm was measured, and the FRET ratio (665 nm/615 nm) was converted to IP1 concentrations by interpolating values from an IP1 standard curve generated from a provided IP1 calibrator . When testing allosteric modulators, cells were pre-incubated for 30 min at 37 °C with 50 μl/well of ligand buffer supplemented with allosteric modulator and a final concentration of 1% DMSO. Afterwards, the buffer solution was removed and 50 μl/well of agonist solution in ligand buffer supplemented with allosteric modulator and a final concentration of 1% DMSO was added. Plates were incubated for 30 min at 37 °C followed by one wash in 100 μl/well of assay buffer. Cell lysis and detection was performed as described above.Functional activity was measured using the IP-One assay, which is an IP2, 0.5 mM MgCl2, 5 mg/mL bovine serum albumin, pH 7.4) before addition of the fluorophore-conjugated antibodies. Tb3+ conjugated anti-HA antibody  was used as the donor fluorophore and was diluted in 4 °C assay buffer to a final concentration of 1 nM, while d2 conjugated anti-myc antibody  was used as the acceptor fluorophore and was diluted in 4 °C assay buffer to a final concentration of 50 nM. 25 μl/well of each fluorophore was added to the plate, which was incubated for 24 h at 4 °C. Cells were washed four times with 100 μl/well of 4 °C assay buffer before being measured on an EnVision plate reader . Upon excitation at 340 nm, emission at 615 nm and 665 nm were measured, and the FRET ratio (665 nm/615 nm) was calculated for each condition and normalized to the FRET ratio of pEGFPN1.Dimerization of a HA-tagged receptor construct and a myc-tagged receptor construct was assessed by a TR-FRET-based assay as previously describedData were analyzed using GraphPad Prism version 6  and normalized as indicated in figure legends.How to cite this article: Jacobsen, S. E. et al. Investigating the molecular mechanism of positive and negative allosteric modulators in the calcium-sensing receptor dimer. Sci. Rep.7, 46355; doi: 10.1038/srep46355 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Ultrasonic acoustic emission analysis enables nondestructive monitoring of damage in dehydrating or freezing plant xylem.AE) in freezing stems during ice nucleation and propagation, by combining acoustic and infrared thermography techniques and controlling the ice nucleation point.We studied acoustic emissions , and the second (up to 2.5 AE s−1) after dissipation of the exothermal heat. Identical patterns were observed in other conifer and angiosperm species.Ultrasonic activity in freezing samples of AE patterns are explained by the low water potential of ice at the ice–liquid interface, which induced numerous and strong signals. Ice propagation velocities were estimated via AE (during the first phase) and infrared thermography. Acoustic activity ceased before the second phase probably because the exothermal heating and the volume expansion of ice caused decreasing tensions. Results indicate cavitation events at the ice front leading to AE. Ultrasonic emission analysis enabled new insights into the complex process of xylem freezing and might be used to monitor ice propagation in natura.The complex  Such nucleii might be bacteria, impurities, cell walls (heterogeneous nucleation) or even water molecules themselves when temperatures fall below −38°C (homogeneous nucleation). After nucleation, water molecules crystallize around the nucleus and ice propagates longitudinally and radially within the xylem  influences AE patterns.The process is further complicated as ice formation is an exothermal process diminishing the temperature decrease and solutes are concentrated in the liquid phase, increasing tensions in the remaining sap. Freezing normally starts and occurs in the apoplast, whereas intracellular sap usually freezes at lower temperature. Recently, freezing was demonstrated to induce a relevant flow of COT.We studied freezing in branches via thermocouples and infrared thermography, and recorded AEs by several sensors mounted at different positions along samples. Experiments were performed under controlled conditions in freezing chambers, which enabled comparison of spontaneous and induced ice nucleation, species‐specific freezing and AE patterns, as well as patterns at different ∆Picea abies (L.) H. Karst growing near the Department of Botany in Innsbruck, Austria, in April 2013, before budburst and resumption of cambial growth. Tension in samples was released overnight, the basal end in clear water and wrapped in a plastic bag at 5°C. Branches were bench‐dehydrated until they reached a Ψ of −2.8 MPa corresponding to 12% loss of conductivity: , Carpinus betulus L., 1753 (−3.2 MPa), Corylus avellana L., 1753 (−2.0 MPa) and Pinus mugo Turra, 1764 (−3.5 MPa).Branches, 1–1.5‐m long, were sampled from mature c. 360 mm in length and 10–20 mm in diameter, were cut from the main stem of branches and covered with parafilm  to avoid further dehydration. A small area (c. 25 mm²) was debarked for ice nucleation of the sample at the sap wood and covered by a removable piece of parafilm.Samples, P. abies and three branches in A. alba, C. betulus, C. avellana and P. mugo. The effect of initial conditions was tested independently on three branches of P. abies .The main experimental set‐up Fig.  was condet al., et al., Ultrasonic measurements were performed with a PCI‐8‐based system  and 150 kHz resonance sensors (R15) connected to a preamplifier set to 40 dB . The threshold was set to 45 dB, the gain to 40 dB . The camera was placed in an isolated box at the top of the freezing chamber and infrared images of the experimental set‐up were recorded at 100‐ms time resolution. Infrared images obtained during freezing of the samples were analysed by infrared differential thermal analysis  was almost negligible (< 1–2 AE s−1) before ice nucleation occurred. On ice nucleation, we observed a two‐phased spatial and dynamic pattern of AEs (Table c. 20 s), sensors close to the nucleation point revealed highest AE activities immediately after the exotherm indicated freezing. On one end of the sample (30 mm from the nucleation point), c. 540 AEs were recorded with a peak activity of 49.8 AE s−1 , but increased again after c. 20 min (phase II): maximum rates then were 0.9–2.5 AE s−1. Acoustic activity during phase II was similar on all sensors. However, we observed two‐fold more AEs in the centre of the sample (mean ± SE = 885 ± 222) than on the ends (mean ± SE = 358 ± 143) for both positions of ice nucleation  close to the ice nucleation point . A strong exponential relationship was found between the proportion of AEs observed during phase I and distance to ice nucleation (d) . Similar patterns were also observed in debarked samples or samples not covered with parafilm , phase I showed only few AEs (c. 10 AEs), whereas phase II was similar to nucleation at −6°C. Accordingly, we observed a strong power law relation between the energy of AEs recorded during phase I and ∆T (∑ energyphaseI = 1.4·(ΔT)5.5; R² = 0.732; P < 0.001; Fig. T (V = 0.78 · (ΔT)2.28; R² = 0.871; P < 0.001; Fig. P = 0.234) to velocities observed in debarked samples by infrared thermography (V = 0.09·(ΔT)3.34; R² = 0.649; P = 0.034). By contrast, infrared analyses in intact samples showed slightly lower velocities (V = 1.75·(ΔT)1.41; R² = 0.660; P < 0.001; Fig. P < 0.0001) or from debarked samples (P < 0.0001). For ∆T = 6 K, we observed an acoustic propagation rate of 36 mm s−1, whereas propagation monitored with infrared thermography was measured at 21 mm s−1.Artificially induced ice nucleation caused AE patterns similar to spontaneous nucleation . Two phases with similar intensity also were observed in the angiosperms Carpinus betulus and Coryllus avellana . This energy is recorded as an increase in temperature , closer to values calculated from acoustic signals.When water molecules turn from liquid into ice, the structure becomes more stable, which releases energy  after exotherm formation on the sensors close to the nucleation point  and started to decrease again, phase II of acoustic activity was observed in all sensors and lasted until temperatures stabilized (−10°C). These patterns were also observed in ugo Fig. a and A. lba Fig. b. Interelus Fig. c and C. ana Fig. d. AEs deet al., et al., mw and mi, mass of liquid and ice, respectively; Cpw, specific heat capacity of liquid water; ΔT, degree of supercooling; L, latent heat of water). According to Eqn L = 334 kJ kg−1 and Cpw = 4.18 kJ kg−1 K−1 at 25°C  to 0.4 kPa (6.5 K). This effect is minor compared with the Ψ induced by ice. According to Saclier et al.  induces c. 9% increase in local pressure at the ice–liquid interface ; the ice nucleation temperature influences phase I, whereas Ψ influences both phases. These results highlight the complexity of freezing in heterogenous tissues . Finally, we demonstrated that acoustic detection might be useful for localizing ice nucleation and the propagation front within wood and that propagation along the water column is not delayed by anatomy. Acoustic emission techniques would therefore warrant use on more complex tree architectures (e.g. ramified branches and/or large trunks) and potentially