In contrast, germinal center B cells did not increase during secondary infection. Unexpectedly, the memory IgE response was lost in mice where the extracellular part of IgG1 had been replaced with IgE sequences. Adoptive transfer studies revealed that IgG1+ B cells were required and sufficient to constitute the memory IgE response in recipient mice. T cell-derived IL-4/IL-13 was required for the memory IgE response but not for expansion of B cells from memory mice. Together, our results reveal a close relationship between the IgE and IgG1 repertoires in vivo and demonstrate that the memory IgE response is mainly conserved at the level of memory IgG1+ B cells. Therefore, targeting the generation and survival of allergen-specific IgG1+ B cells could lead to development of new therapeutic strategies to treat chronic allergic disorders.IgE-mediated activation of mast cells and basophils contributes to protective immunity against helminths but also causes allergic responses. The development and persistence of IgE responses are poorly understood, which is in part due to the low number of IgE-producing cells. Here, we used next generation sequencing to uncover a striking overlap between the IgE and IgG1 repertoires in helminth-infected or OVA/alum-immunized wild-type BALB/c mice. The memory IgE response after secondary infection induced a strong increase of IgE This study reveals that repertoires of IgE—the class of antibody that mediates allergic reactions—closely resemble those of IgG1, suggesting that the memory IgE response unfolds from IgG1-switched B cells (and not from IgM-expressing B cells) in response to T cell-derived cytokines. Allergic inflammation is initiated when IgE antibodies bind to high-affinity receptors on the cell surface of mast cells and basophils, thereby triggering the release of proinflammatory mediators. The development and persistence of IgE responses in vivo is poorly characterized because of the low number of IgE-producing B cells and plasma cells. Naïve mature B cells produce IgM antibodies. Upon activation, they “switch” class to produce IgG, IgA, or IgE antibodies. It is currently highly debated whether IgE-expressing B cells are generated by direct switching from IgM-expressing B cells or by sequential switching via IgG1-expressing B cells. Using next generation sequencing, we compared thousands of IgE, IgG1, and IgM sequences after immunization of mice with parasitic worms and found a striking overlap between the IgE and IgG1 repertoires. We further show that the memory IgE response to a secondary encounter with the same parasitic worms was dependent on T cell-derived cytokines. Genetically modified mice and adoptive transfers of B cells revealed that the memory IgE response is conserved at the level of IgG1-expressing B cells. These results favor the concept that bona fide IgE-expressing B cells do not exist, and memory IgE responses unfold from IgG1-expressing B cells, which undergo a secondary switch reaction and differentiation to plasma cells. Nippostrongylus brasiliensis is a well-established model to study general mechanisms of IgE production in vivo. Serum IgE levels of N. brasiliensis-infected BALB/c mice increase up to 1,000-fold by day 14 after N. brasiliensis infection [N. brasiliensis is abolished in IL-4-depleted [Schistosoma mansoni eggs [FH) cells, natural killer T (NKT) cells, basophils, eosinophils, mast cells, and type 2 innate lymphoid cells (ILC2). Although Th2 cells and TFH cells are generally considered to be the most relevant cell types for induction of IgE-CSR in B cells, it remains unclear to what extent innate IL-4/IL-13-expressing cell types contribute to this process, especially during secondary infection when basophils and mast cells can be rapidly activated to release large amounts of IL-4/IL-13.IgE probably emerged during mammalian evolution to defend hosts against parasites, since a correlation between high IgE levels and protection against helminths has been recognized ,2. Surprnfection . The IgEdepleted  or IL-4-depleted  mice, inoni eggs ,13. IL-4+ B cells inside GCs by using fluorescent IgE reporter mice [Immunohistological stainings indicated that IgE-CSR occurs outside GCs , while oter mice –17. Repoter mice –18. In tter mice , was conN. brasiliensis infection as compared to control mice, but the memory IgE response was not investigated [On the molecular level, IgE-CSR was found to either occur directly from IgM to IgE or sequentially from IgM to IgG1 followed by a second switch reaction to IgE . The relstigated . Furtherstigated ,21,22. Sstigated . However+ memory B cells do exist or not continues to be controversially discussed [+ memory B cells exist and directly respond to antigen challenge [+-sorted memory B cells from N. brasiliensis-infected IgE-GFP reporter mice into B cell-deficient hosts gave rise to serum IgE levels after N. brasiliensis infection [+ B cells, which may have contaminated the population of transferred B cells, and these mice express an engineered membrane IgE molecule that contains 52 amino acids of human IgE, which may alter the behavior of IgE+ B cells [+ memory B cells from T/B monoclonal mice indicated that the memory IgE response does not develop from bona fide IgE+ memory B cells but rather depends on an IgG1+ precursor population [The question whether bona fide IgEiscussed . Mice wihallenge . Howeverhallenge . Others nfection . However B cells . Studiespulation ,15,17.N. brasiliensis-infected or OVA/alum-immunized wild-type BALB/c mice. Competitive adoptive transfers further revealed that T cell-derived IL-4/IL-13 was required for the memory IgE response but not for expansion of memory B cells. IgG1+ B cells were required and sufficient to establish the memory IgE response after transfer in IgH allogeneic recipients. Interestingly, the memory IgE response was also impaired in mice where the extracellular parts of IgG1 had been replaced by IgE domains. Collectively, our results demonstrate that the memory IgE response is largely dependent on clonal expansion and affinity maturation of IgG1-expressing B cells that require a second IL-4/IL-13 signal from T cells to subsequently switch to IgE and differentiate into IgE-secreting plasma cells.Using NGS analyses, we observed a striking overlap between the IgG1 and IgE repertoires in + B cells and plasma cells (PCs) in vivo, we analyzed wild-type, IL-4/IL-13-deficient (4-13ko), and 4-13Tko mice after N. brasiliensis infection by flow cytometry.Mice with selective deletion of IL-4/IL-13 in T cells (4-13Tko mice) are unable to mount a serum IgE response and show impaired GC formation after helminth infection ,26. To f+ B cells and PCs were missing in 4-13ko and 4-13Tko mice compared to wild-type (WT) controls, and IgG1+ B cells were reduced 20-fold  and by a smaller part of PCs .We first treated cells isolated from the draining lymph nodes (LN) with an acidic wash buffer to remove cytophilic IgE bound to the low affinity IgE receptor FcεRII (CD23) and then stained for surface and intracellular IgE and IgG1. In our hands, this procedure is comparable to an alternative IgE staining protocol where extracellular IgE is first blocked by anti-IgE antibodies followed by intracellular staining for IgE  S1 Fig)S1 Fig).  20-fold . It is ient mice . The acient mice . We obse+ B cell or sequentially via an IgG1+ B cell intermediary [N. brasiliensis infection using a promiscuous 5’ primer that binds to VH1, 2, 3, 5, and 14 family sequences and thereby picks up most of the expressed VH genes [H, DH, and JH segments was very similar among the three isotypes and the majority of sequences used in the VH1/J558 family [H1 or DH2 families  between the IgE and IgG1 repertoire revealed a similar average number of SHMs in different parts of the Vequences . The avedentical . Alignmedentical . To figupertoire . IgE+ GCturation .+ memory B cells or rather depend on memory IgG1+ B cells that switch to IgE after secondary antigen encounter.Taken together, these findings clearly demonstrate that the sequential pathway for IgE-CSR dominates the in vivo IgE response and points to an important role of the GC as the site where affinity maturation probably occurs at the level of IgG1-expressing B cells that have the capacity to further switch and differentiate to IgE-producing PCs. This raises the question whether memory IgE responses are driven by IgEN. brasiliensis, and this effect was dependent on the presence of CD4+ T cells [−CD138+FSChiSSChi) when compared to the primary response (+CD38loGL-7hi) was observed after secondary N. brasiliensis infection, indicating that the new antibody-secreting cells originate mostly from pre-established memory B cells N. brasilresponse . Interes B cells . Eosinop B cells .N. brasiliensis infection. At day 13 after primary infection, the peak of the humoral response, PCs could be found mainly in the draining LN and spleen of infected mice  with N. brasiliensis. Five to six weeks later, we isolated total lymphocytes from LN, spleen, and bone marrow of these N. brasiliensis memory mice and the corresponding cell populations from naïve Ly5.1 mice that were of the Ig heavy chain “b” allotype (Ly5.1/IgHb mice). The samples from naïve and memory mice were adjusted to equal numbers of B cells, mixed, and transferred to Rag1–/–mice, which were infected with N. brasiliensis 1 d later  as compared to the CD38+IgD+ population (largely reflecting naïve B cells)  . Most IgB cells) . In contnsferred . This in5 purified CD4+ T cells from WT or 4-13Tko N. brasiliensis memory mice together with 2 x 105 purified and equally mixed B cells from memory IgHa and naïve IgHb mice into Rag1−/− recipients  in which the extracellular part of the IgG1 heavy chain had been replaced by the extracellular part of the IgE heavy chain [N. brasiliensis infection, wild-type mice had about 43% IgG1+ and 1.6% IgE+ GC B cells, while IgEki/ki mice had no IgG1+ and about 27% IgE+ GC B cells (+ GC B cells in IgEki/ki mice did not result in the same increase of IgE+ PCs (ki/ki mice as compared to control mice at the peak of the primary response to N. brasiliensis (ki/ki mice were comparable to the IgE levels after primary infection and much lower compared to IgE levels in control mice (ki/ki mice (IgHa) or normal IgHa mice to C57BL/6 mice (IgHb). Due to allelic exclusion, about 50% of B cells in these mice express the IgHa allele while the other 50% express the IgHb allele. We found that IgEb dominated the memory IgE response to N. brasiliensis in IgEki(a)/wt(b) mice illustrating the competitive advantage of the wild-type allele encoding the extracellular domains of IgG1  expression levels, a poor BCR signaling response, decreased expression of costimulatory molecules, and enhanced apoptosis suggesting that IgE+ GC B cells are prone to die rather than giving rise to memory B cells and PCs [Interestingly, the number of GC B cells did not increase during secondary infection, while PCs showed massive expansion. Neither eosinophils nor basophils were required for secondary PC expansion or for the memory IgE response, although both cell populations are sources of IL-4 and IL-13 and despite previous reports that described critical roles of eosinophils and basophils for PC survival in bone marrow and spleen, respectively ,33. The henotype . Principhenotype . Furtherhenotype  or in Omhenotype . These “henotype . However B cells ,21. The  B cells . IgE+ GC and PCs . However+ GC B cell population is present in IgEki/ki mice indicating that surface IgE+ B cells can participate in the GC reaction and are not immediately prone to die. However, we also observed that IgE+ GC B cells contain fewer SHMs as compared to IgG1+ GC B cells indicating that affinity maturation is impaired in IgE+ GC B cells. Interestingly, IgEki/ki mice failed to mount a normal IgE memory response suggesting that the extracellular part of the chimeric IgE-IgG1 receptor does not allow the formation of memory B cells. One possible explanation for this finding would be that IgE+ B cells are actively removed or silenced in vivo, although the mechanisms behind such a scenario remain unclear. An alternative explanation would be that antigen-independent binding of the extracellular part of IgG1 to a yet-to-be-identified target structure induces a prosurvival signal in memory IgG1+ B cells, which then give rise to the memory IgE response. Achatz et al. demonstrated that the memory IgE response is blunted when B cells express transmembrane IgE with a truncated cytoplasmic tail favoring the concept that the memory IgE response develops directly from IgE+ B cells [+ precursors. For instance, it could well be that IgE-secreting PCs develop first from IgG1+ memory B cells and the cytoplasmic tail of IgE is then required for survival of IgE+ PCs since transmembrane IgE is clearly expressed on these cells [We further demonstrate that a prominent IgE B cells . Howeverse cells .+ GC B cells as compared to IgE+ GC B cells. Early IgE+ PCs show very few SHMs and are often generated in extrafollicular foci, while PCs with SHMs are thought to be derived from GC B cells that arise later during an immune response [+ PCs of N. brasiliensis-infected mice indicates that the repertoire of these PCs was generated during the GC response. Taken together, this set of experiments provides strong evidence that the majority of the IgE response to primary N. brasiliensis infection in mice with a normal polyclonal B and T cell repertoire is constituted by PCs originating from sequentially switched B cells.We show here, to our knowledge, for the first time an extensive NGS analysis of the IgE and IgG1 repertoires in normal BALB/c mice after helminth infection or during allergic inflammation. We found largely overlapping IgE and IgG1 repertoires based on the comparison of CDR3 regions from several thousand IgE and IgG1 sequences. The analysis of SHMs in selected pools of IgE and IgG1 sequences with the same CDR3 region revealed that the majority of SHMs was identical in both isotypes. Furthermore, we found that the Ig repertoire of IgE-producing PCs was more closely related to IgG1response . The lar+ memory B cells) is probably established after the first infection and does not require a second GC phase, since we observed no GC response upon secondary infection.The IgE repertoires between lung, spleen, and LN were very similar after primary and secondary infection, while an IgE repertoire overlap between bone marrow and the peripheral organs could only be observed after secondary infection. We unexpectedly observed a high IgE repertoire diversity with 300–400 different sequences among 1,000 analyzed sequences in lung, spleen, and LN after primary and secondary infection. This shows that many different clones of IgE-expressing cells disseminate into different tissues and the diversity was maintained after secondary infection, excluding the possibility that only few memory B cells participate in the memory response. The average number of SHMs in IgE increased after secondary infection and was remarkably constant in all four tissues. The pool of IgE memory precursor cells .PtprcaPepcb/BoyJ), and Rosa26-YFP reporter mice (B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J) were originally obtained from The Jackson Laboratory. Cγ1Cre/Cre mice [Cγ1Cre/+Rosa26loxP-STOP-loxP-eYFP/+ mice. We further used IgE–/–_BALB/c mice [−/−_BALB/c (4-13ko) mice [ki/ki_C57BL/6 mice have been described [BALB/c, C57BL/6_Ly5.1  mice , conditiko) mice , Mcpt8Crko) mice , and Δdbko) mice . IgEki/kescribed . In thesN. brasiliensis as described [Mice were infected subcutaneously at the base of the tail with 500 L3 stage larvae of escribed .3) and incubated with anti-CD16/CD32 blocking antibody  for 5 min at room temperature followed by staining with the following antibodies: Fluorescein isothiocyanate (FITC)- or Alexa Fluor 647 (A647)-labeled anti-CD45R (B220) (clone RA3-6B2), FITC-labeled anti-CD4 (clone RM4-5), PerCP-Cy5.5-labeled anti-CD45.2 (Ly5.2) (clone 104), A488- or A647-labeled anti-GL-7 (clone GL-7), were purchased from eBioscience ; Biotin-, BV510- or FITC-labeled anti-IgE (clone R35-72), and allophycocyanin (APC)-labeled anti-CD138 (clone 281–2) were purchased from BD Biosciences ; Biotin- or phycoerythrin (PE)-labeled anti-IgG1 (clone RMG1-1), pacific blue (PB)-labeled anti-CD45.1 (Ly5.1) (clone A20), PB-labeled anti-CD4 (clone RM4-5), PE-Cy7-labeled anti-CD38 (clone 90), and A647-labeled anti-CD49b (clone HMα2) were purchased from Biolegend ; VioBlue-labeled B220 (clone RA3-6B2) was purchased from Miltenyi Biotec . Permeabilization of cells was achieved using intracellular staining buffers from Biolegend. To detect biotinylated antibodies PE-Cy7–labeled streptavidin (BD Biosciences) or APC-eFluor780-labeled streptavidin (eBioscience) were used. Dead cells were excluded during acquisition on FACSCanto II . Sorts were performed with FACSAria II (BD Biosciences) or S3 Cell Sorter . Data were analyzed with FlowJo software .Cytophilic IgE was efficiently removed by short treatment with acetate buffer as described . Single a (UH297) and biotin-labeled anti-IgEb (JKS-6) were used (Biolegend). IgG1 ELISA was performed with a commercial ELISA kit (SouthernBiotech).IgE levels in the serum of naive and infected mice were determined with purified anti-IgE  for coating and biotinylated anti-IgE  for detection. For detection of allotype-specific IgE, biotin-labeled anti-IgEN. brasiliensis, and bone marrow, lung, spleen, and LN were collected at day 15 after primary and day 9 after secondary infection. Organs or sort-purified PCs and GC B cells were homogenized in RLT buffer , and RNA was isolated from lysates by RNeasy Mini kit (Qiagen). cDNA synthesis was performed with SuperScript III Reverse Transcriptase with oligo(dT)20 primer . To amplify the coding regions for the variable parts of the heavy chains of IgG1 and IgE, PCRs were performed with Platinum Taq DNA Polymerase (Life Technologies) and forward primer VHall 5´- cgtatcgcctccctcgcgccatcag(MID)AGGTSMARCTGCAGSAGTCWGG-3´ [AGAGGTCAGACTGCAGGACAG-3´ or Cε region 5´-ctatgcgccttgccagcccgctcagTCTGAATACCAGGTCACAGTC-3´. The gene-specific primer sequences (underlined) were modified by addition of 454 adaptors, which are necessary for NGS analysis. MID (multiple identifier) specifies a 4-nt sequence used in six different variations to identify samples within single lanes. Amplicons were prepared with the GS FLX Titanium SV emPCR kit (Lib-A) for ultra-deep 454 pyrosequencing on the Genome Sequencer FLX system  as described by the manufacturer.Mice were infected subcutaneously with 500 L3 larvae TCWGG-3´  specificAGGTSMARCTGCAGSAGTCWGG-3´ in combination with reverse primers binding in the constant Cμ region 5´-ATGGTGCTGGGCAGGAAGTC-3´, Cγ1 or Cγ2a region 5´-AGAGGTCAGACTGCAGGACAG-3´, Cε region 5´-TCTGAATACCAGGTCACAGTC-3´, or Cα region 5´-ATCAGGCAGCCGATTATCAC-3´. PCR conditions were as follows: 94°C, 5 min; 35 × ; and 72°C, 5 min. Amplicons were purified by gel extraction with QIAquick Gel Extraction kit (Qiagen) and quantified by Quant-iT dsDNA HS Assay kit and the Qubit fluorometer (both from Life technologies). Amplicons were sequenced by Microsynth  with the Genome Sequencer FLX system after 454 adaptors and MID sequences had been added.For one set of samples from primary infection, PCRs were run with a forward primer VHall 5´-1 p2) = 2 p1 p2 / (p12 + p12) where p1 and p2 are the two normalized populations that should be compared to each other. IMGT sequence files are accessible at the Dryad repository: http://dx.doi.org/10.5061/dryad.8bj97 [Sequences were assigned to individual samples according to their MID, and sequences shorter than 320 bp and Cγ2a sequences were excluded. Sequences were further analyzed with ImMunoGeneTics (IMGT) HighV-QUEST ,50, a wead.8bj97 .p-values were calculated with unpaired two-sided Student's t test using SigmaPlot . p < 0.05 was considered statistically significant.S1 Data(XLSX)Click here for additional data file.S1 FigN. brasiliensis infection of wild-type BALB/c (WT) or IgE-deficient mice (IgE-ko). Cells were either incubated with excess amounts of unlabeled anti-IgE antibody to block surface IgE (surface block) or washed with acidic buffer to remove cytophilic IgE from the cell surface (acid treatment) followed by intracellular IgE staining. (A) Samples are gated on GC B cells Click here for additional data file.S2 FigBALB/c mice were immunized intraperitoneally (i.p.) with OVA/alum on day 0 and day 7, challenged intranasally on days 13 and 14 before Ig repertoires were analyzed on day 15 by NGS. (A) Number of different CDR3 sequences among 1,000 randomly selected sequences from IgE, IgG1, and IgM pools. (B) Heat maps demonstrate that the most abundant CDR3 sequences in the IgE repertoires of each mouse are often shared with the IgG1 but not the IgM repertoire. The brightest green means that this CDR3 sequence was found in at least 0.5% of all sequences. (C) Morisita-Horn indices as a measure for the relatedness between 1,000 randomly picked sequences of the IgG1 and IgE repertoires or the IgM and IgE repertoires. (D) Number of somatic mutations in the VH genes of IgG1 and IgE. (E) Distribution of somatic mutations over indicated regions of the VH genes. Bars show the mean + SEM from three mice.(TIF)Click here for additional data file.S3 FigN. brasiliensis, and Ig repertoires were analyzed on day 15 after infection. (A) Repertoire diversity displayed as mean + SD different sequences in 1,000 randomly chosen sequences. (B) Relative usage of different VH, DH, and JH segments among indicated isotypes. Bars show the mean + SD from five mice. (C) Heat maps show the overlap between the first 50 most frequent CDR3 sequences in the IgE repertoire with the same CDR3 sequences in the IgG1 and IgM repertoires from five individual mice. Each row indicates one unique CDR3 sequence ordered by decreasing frequency in the IgE pools. The brightest green indicates CDR3 sequences with an abundance of ≥ 1%. Bar graph shows the Morisita-Horn Index for the relatedness between the IgE and IgG1 repertoires and the relatedness between the IgE and IgM repertoires based on 1,000 randomly chosen sequences from each isotype. (D) Same analysis as in (C), but IgA was used instead of IgE. (E) Direct comparison of the IgE and IgA repertoires. ** p < 0.01 by Student’s t test.Five BALB/c mice were infected with (TIF)Click here for additional data file.S4 FigN. brasiliensis infection for usage of indicated VH, DH, and JH segments among 1,000 randomly chosen IgE and IgG1 sequences from bone marrow (BM), lung, spleen, and mesenteric LN.Two individual mice were analyzed at day 15 after primary (TIF)Click here for additional data file.S5 FigN. brasiliensis infection for usage of indicated VH, DH, and JH segments among 1,000 randomly chosen IgE and IgG1 sequences from bone marrow (BM), lung, spleen, and mesenteric LN.Two individual mice were analyzed at day 9 after secondary (TIF)Click here for additional data file.S6 Figb/Ly5.1 mice were infected with N. brasiliensis 4 wk before cell transfer to establish memory mice. Cell suspension from SP or LN from memory IgHb/Ly5.1 and naïve IgHa/Ly5.2 mice were mixed at a 1:1 ratio of B cells from each mouse and transferred into Rag1–/–mice. Mesenteric LN and serum were analyzed 12 d after N. brasiliensis infection of Rag1−/− recipient mice. (B) Representative plots showing transferred CD4+ T cells and B220+ B cells (left) and percentage of naïve (Ly5.2+) and memory (Ly5.1+) CD4+ T cells (middle plot) or B220+ B cells (right plot). (C) Bar graph shows the percentage of B cells from naïve or memory donor cells from LN and spleen (SP) in the mesenteric LN of infected Rag1−/− recipient mice. (D) Frequency of Ly5.1+ and Ly5.2+ B cells within the CD38+IgD+ gate (mainly naïve B cells) and CD38+IgD− gate (mainly memory B cells). Dot plots are gated from the parental gate shown in b) or B cells from naive mice (detected as IgEa) in the serum of infected Rag1–/–recipient mice. Bars in (C) and (E) show the mean + SD from four mice per group.(A) Outline of transfer experiment referring to data in B–D. IgH(TIF)Click here for additional data file.S7 FigThe indicated sorting gates were used to purify IgG1-expressing B cells and IgG1-negative PCs (upper part) or to remove IgM-, IgD-, and IgG1-expressing B cells or IgM-, IgD-, and IgE-expressing B cells (lower part) in order to transfer enriched and untouched IgE- or IgG1-expressing B cells for the experiment shown in (TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S9 Fig(TIF)Click here for additional data file.S10 Fig(TIF)Click here for additional data file.S11 Fig(TIF)Click here for additional data file.S12 Fig(A) Gating strategy to distinguish transferred Ly5.1 and Ly5.2 B cells. (B) Gating strategy to analyze CD38 expression on Ly5.2+ and Ly5.2- B cells.(TIF)Click here for additional data file.S13 Fig(TIF)Click here for additional data file.


Background: Mutations in the chloroquine resistance transporter (PfCRT) change the susceptibility of Plasmodium falciparum to diverse antimalarial drugs.Results: In addition to chloroquine, PfCRT transports quinine, quinidine, and verapamil, which bind to distinct but antagonistically interacting sites.Conclusion: PfCRT is a multidrug carrier with a polyspecific drug-binding cavity.Significance: These findings could be used to develop high affinity inhibitors of PfCRT. Plasmodium falciparum. We have previously shown that mutant PfCRT transports the antimalarial drug chloroquine away from its target, whereas the wild-type form of PfCRT does not. However, little is understood about the transport of other drugs via PfCRT or the mechanism by which PfCRT recognizes different substrates. Here we show that mutant PfCRT also transports quinine, quinidine, and verapamil, indicating that the protein behaves as a multidrug resistance carrier. Detailed kinetic analyses revealed that chloroquine and quinine compete for transport via PfCRT in a manner that is consistent with mixed-type inhibition. Moreover, our analyses suggest that PfCRT accepts chloroquine and quinine at distinct but antagonistically interacting sites. We also found verapamil to be a partial mixed-type inhibitor of chloroquine transport via PfCRT, further supporting the idea that PfCRT possesses multiple substrate-binding sites. Our findings provide new mechanistic insights into the workings of PfCRT, which could be exploited to design potent inhibitors of this key mediator of drug resistance.Mutations in the “chloroquine resistance transporter” (PfCRT) are a major determinant of drug resistance in the malaria parasite  Plasmodium falciparum to several important antimalarial drugs  or that possess structural moieties present in quinoline drugs  but also encompass a diverse range of compounds that have not been deployed as antimalarial treatments (–Changes in the “chloroquine resistance transporter” (PfCRT)al drugs , 2. Inital drugs , a synthal drugs . These ieatments –12. Givein vitro parasite assays as well as characterizations of PfCRT in heterologous expression systems. In the parasite studies, the efflux of radiolabeled drugs from parasite-infected red blood cells was linked to PfCRT (––Dd2) at endosomal membranes within Dictyostelium discoideum reduced the accumulation of chloroquine and quinine within these vesicles, consistent with the mutant protein mediating the transport of these two drugs  playing a pivotal role in the binding and translocation of drugs , 27. Indin vitro  was expressed in Xenopus oocytes and shown to mediate the transport of radiolabeled chloroquine, quinine, quinidine, and verapamil. The results of an in depth kinetic examination of the inhibition of chloroquine or quinine transport by another drug suggest that PfCRTDd2 possesses at least two distinct binding sites that antagonistically affect one another.Here we investigated the interaction of PfCRT with chloroquine, quinine, quinidine, and verapamil. The latter compound can partially reverse chloroquine resistance in vitro . The PfCX. laevis frogs was obtained from (i) the Australian National University Animal Experimentation Ethics Committee  in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and (ii) the Regierungspräsidium Karlsruhe (Aktenzeichen 35-9185 81/G-31/11) in accordance with the German “Tierschutzgesetz.”Ethical approval of the work performed with the 3H]Chloroquine , [3H]quinine , [3H]quinidine , and [3H]verapamil  were obtained from American Radiolabeled Chemicals or GE Healthcare.chloroquine (50 nm), [3H]quinine (62.5 nm), [3H]quinidine (62.5 nm), or [3H]verapamil (25 nm). Where specified, one or more unlabeled drugs were present at the indicated concentrations. The influx assays were terminated by removing the reaction medium and washing the oocytes three times with 3 ml of ice-cold ND96 buffer. Each oocyte was transferred to a separate scintillation vial and lysed by the addition of 5% sodium dodecyl sulfate (200 μl). The radioactivity in the sample solution was measured using a β-scintillation counter. The direction of radiolabeled drug transport in these assays is from the acidic extracellular medium  into the oocyte cytosol (pH ∼7.2), which corresponds to the efflux of drug from the acidic digestive vacuole (pH 5–5.5) into the parasite cytosol (pH 7.3). Water-injected oocytes and oocytes expressing PfCRTHB3 take up [3H]chloroquine to similar (low) levels via simple diffusion of the neutral species; this represents the “background” level of chloroquine accumulation in oocytes  were obtained by least-squares fit of the equation, Y = Ymin + ((Ymax − Ymin)/(1 + ([inhibitor]/IC50)C), where Y is PfCRTDd2-mediated drug transport, Ymin and Ymax are the minimum and maximum values of Y, IC50 is the half-maximum inhibitory concentration, and C is a constant.Measurements of radiolabeled drug uptake were made over 1 h at 25 °C and in a ND96 medium that contained chloroquine or [3H]quinine to each of the unlabeled chloroquine/quinine mixtures . Both drugs can permeate membranes as free bases and will partition between membrane-bound compartments according to the prevailing pH gradient. The uptake of radiolabeled quinine and quinidine was measured in an acidic medium (pH 5.0) into Xenopus oocytes expressing either PfCRTDd2 or the wild-type protein from the HB3 strain (PfCRTHB3) as well as into water-injected oocytes. Oocytes expressing PfCRTDd2 took up significantly more quinine and quinidine relative to water-injected oocytes or oocytes expressing PfCRTHB3 . Similar results were obtained when the time courses were performed at pH 6.0 (data not shown). The level of uptake in oocytes expressing PfCRTHB3 was not significantly different from that measured in water-injected oocytes, indicating that the accumulation of quinine and quinidine in these oocytes and in the water-injected oocytes was most likely due to the simple diffusion of the uncharged forms of these drugs. Consistent with this observation, quinine transport was shown to be dependent on the pH of the medium (C). That is, the accumulation of quinine in the control oocytes increased as the pH was raised from 4.0 to 6.0, which correlated well with the pH dependence of the concentration of the uncharged quinine present in the medium (D). Nevertheless, under each of the conditions tested, the accumulation of quinine in oocytes expressing PfCRTDd2 was significantly higher than that measured in water-injected oocytes and in oocytes expressing PfCRTHB3 (p < 0.05).Quinine and its stereoisomer quinidine are diprotic weak bases with pe medium C. That ie medium D. NevertDd2 was calculated by subtracting the uptake in oocytes expressing PfCRTHB3 from that measured in PfCRTDd2-expressing oocytes . The resulting data indicated that the PfCRTDd2-mediated uptake of quinine and quinidine was approximately linear with time for at least 60 min at both pH 5.0 and pH 6.0.The portion of quinine or quinidine accumulation attributable to PfCRTDd2 at pH 5.0 (E). Verapamil has previously been shown to inhibit chloroquine transport via PfCRTDd2 . A least squares fit of the Michaelis-Menten equation to the data yielded apparent Michaelis constants (mK) of 28 ± 4 and 23 ± 4 μm and maximal velocities (Vmax) of 14 ± 1 and 13 ± 1 pmol/h/oocyte for quinine and quinidine, respectively.Given that the time dependence of PfCRTt pH 6.0 , 25), thDd2 transports both quinine and chloroquine led us to undertake a series of kinetic analyses to assess the mechanism by which PfCRT accepts different substrates. Substrate competition experiments were conducted in which the uptake of radiolabeled chloroquine was determined at 60 min , and at each of these chloroquine concentrations, the effects of seven concentrations of unlabeled quinine (ranging from 0 to 500 μm) were tested. The rate of PfCRT-mediated chloroquine uptake was calculated, and each of the seven quinine data sets was plotted as a function of the chloroquine concentration (A). To determine the nature of the interaction between PfCRT, chloroquine, and quinine, we performed a least-squares global fit of 16 different models of inhibition (cAIC) and the Akaike weight . The resulting rankings were assessed using the heuristic criteria devised by Burnham and Anderson  suggested that both models were equally credible. We therefore applied an F-test to discriminate between full mixed-type inhibition and partial mixed-type inhibition, the results of which indicated that the former model provided the better fit to the data .The finding that PfCRTe uptake ) in watentration A. To dethibition  to the de weight   indicated that neither model could be immediately dismissed. However, cases of noncompetitive inhibition are rare in nature and appear to be primarily restricted to small inhibitors, such as protons or other inorganic ions  and also cause a decrease in the apparent maximum rate of transport (i.e. the Vmax). Hence, if quinine behaves as a mixed-type inhibitor of chloroquine transport, we would expect it to affect both the apparent mK and apparent Vmax of chloroquine transport via PfCRTDd2. To test this possibility, we reanalyzed the data presented in Fig. 2A by fitting the Michaelis-Menten equation to each of the seven data sets (which differed in the concentration of quinine that was present). The resulting apparent Vmax values as well as the ratios of Vmax to mK were plotted as a function of the quinine concentration . Both of these values decreased with increasing concentrations of unlabeled quinine, resulting in hyperbolic curves. The half-maximum inhibitory concentration derived from Fig. 3A, which equates to the dissociation constant for the binding of quinine to the chloroquine-PfCRTDd2 complex  gives the dissociation constant for the binding of quinine to the empty transporter (KS). Taken together, these data indicate that (i) the presence of chloroquine in the substrate-binding site reduces the transporter's affinity for quinine by a factor (α) of ∼3, and (ii) although quinine's inhibition of chloroquine transport is mostly a competitive process, there is also an uncompetitive component  regardless of whether the substrate-binding site is occupied or empty but exhibits greater potency against one binding state over the other. Mixed-type inhibitors affect the apparent affinity of the transporter for the substrate (lex (αKS ), was 12omponent .Dd2. In assays similar to those described above, the uptake of radiolabeled quinine was measured in the presence of different concentrations of unlabeled quinine (ranging from 0 to 100 μm), and at each of these quinine concentrations, the effects of seven concentrations of unlabeled chloroquine (ranging from 0 to 3 mm) were measured. The 16 models of inhibition were fitted to the resulting data  and ranked according to ΔcAIC and the Akaike weight . These analyses suggested that the most plausible models were again full mixed-type inhibition and partial mixed-type inhibition , with an F-test indicating that full mixed-type inhibition best described the data . Moreover, increasing the concentration of unlabeled chloroquine decreased the apparent Vmax of quinine transport (A) and increased the apparent mK of PfCRTDd2 for quinine (B). The corresponding αKS and KS values were 650 ± 50 and 300 ± 50 μm, respectively. These findings further support the idea that the affinity of PfCRTDd2 for one drug decreases when the carrier is occupied by a second drug.A complementary set of experiments was undertaken to investigate the ability of chloroquine to inhibit quinine transport via PfCRTransport A and inc quinine B. The coDd2, as well as of their inhibition of the transporter, we modeled the kinetics of the interaction between PfCRTDd2, chloroquine and quinine using the full mixed-type inhibition equation. Three-dimensional plots of the modeled system are presented in A and B, with the experimentally derived data  superimposed. To assess how well the model fitted the data, we calculated the difference between the observed and expected uptake values and plotted the resulting residual values as a function of quinine or chloroquine concentration   . Within m quinine treatment of Fig. 2A) and a quinine concentration  that gave the same rate of drug transport (P). If two substrates bind to the same site, the total rate of drug transport will be independent of P. However, if the substrates bind to different but antagonistically interacting sites, the total rate of drug transport will vary with P, resulting in a concave curve , and at each of these chloroquine concentrations, the effects of six concentrations of unlabeled verapamil  were tested (A). The data were analyzed as outlined for the quinine and chloroquine data sets, and the results are presented in Fig. 8B. A partial mixed-type inhibition model was found to describe the data with high statistical confidence (ΔcAIC of 0 and Akaike weight of 0.889), and an F-test confirmed that this model was preferred over full mixed-type inhibition . Increasing the concentration of unlabeled verapamil decreased the apparent Vmax of chloroquine transport (A) and increased the apparent mK of PfCRTDd2 for chloroquine (B), with the verapamil αKS for the chloroquine-PfCRT complex (110 ± 10 μm) ∼3-fold greater than the verapamil KS for the empty transporter (35 ± 4 μm). Finally, we evaluated the partial mixed-type inhibition model by superimposing the experimentally derived data over a three-dimensional plot of the modeled system (C). A plot of the residual values as a function of the chloroquine or verapamil concentration (D) produced, in both cases, a relatively random distribution of points across the x axis. These results suggest that verapamil acts as a partial mixed-type inhibitor of chloroquine transport via PfCRTDd2.Verapamil is an inhibitor of PfCRTin vitro , 24, 30.e tested A. The daransport A and incoroquine B, with td system C. A plotntration D produceDd2 to translocate verapamil was investigated by measuring the uptake of radiolabeled verapamil in water-injected oocytes and in oocytes expressing PfCRTDd2 or PfCRTHB3. As shown in Dd2 relative to water-injected oocytes or oocytes expressing PfCRTHB3. The low signal obtained for the transport of verapamil via PfCRTDd2, which is most likely due to the relatively high lipophilicity of this drug (at pH 5.2 verapamil has a distribution coefficient (log D) of 0.52, whereas chloroquine has a log D of −3.44; , precluDd2. By contrast, these drugs do not appear to be substrates of the wild-type form of the protein (PfCRTHB3). These observations are consistent with the findings of Lehane et al. , was not different from zero . By comparison, analyses of the kinetics of verapamil inhibition suggested that the verapamil-chloroquine-PfCRTDd2 complex remains active, albeit with a chloroquine transport rate that is 30-fold lower than that measured in the absence of verapamil . The very low level of activity detected for the verapamil-chloroquine-PfCRTDd2 complex suggests that the transporter is occasionally able to translocate chloroquine and verapamil in symport, which is a phenomenon that is unlikely to occur when chloroquine and quinine are the bound substrates. The reaction schemes describing the interactions of PfCRTDd2 with (i) chloroquine and quinine and (ii) chloroquine and verapamil are shown in A and B, respectively.Our detailed kinetic analyses of PfCRT-mediated drug transport in the presence of a second (inhibiting) drug indicated that these drugs interact with PfCRTeriments , and 8, rom zero . This fihibition B. By comet al.  could fulfill these roles, forming a site of interaction where compounds compete for binding and translocation   . These fctivity) . The worctivity) –43.Dd2 could influence the properties of other substrate-binding sites. First, the binding of one substrate could result in conformational changes that alter the protein's ability to bind a substrate at a second site. This allosteric mechanism is consistent with the induced fit model of protein function, which has been remarkably successful in explaining the functions of a range of proteins, including transporters and channels . The attachment of a protonated drug would add one or more positive charges to the protein's substrate-binding cavity, resulting in a reduction in the electrostatic attractions between this domain and the second protonated drug. In this regard, it is worth noting that of the eight amino acids that differ between PfCRTHB3 and PfCRTDd2, four result in the gain of a negative charge or the loss of a positive charge . Hence, it is highly likely that electrostatic interactions play an important role in the transport activity of PfCRTDd2. This electrostatically driven model of inhibition is similar to the one proposed by Warhurst  have largely resulted in significantly improved cure rates in regions where drug-resistant malaria is prevalent . MoreovDd2 possesses more than one drug-binding site and that these binding sites, although distinct, are interdependent and hence may be located within a large polyspecific substrate recognition cavity. Our findings suggest that the mutations that confer resistance to chloroquine allow the protein to interact with and transport a range of structurally diverse compounds. However, the additional substrate attachment points generated by these changes could be exploited for the design of potent resistance-reversing agents.The work described here provides direct evidence of the ability of a chloroquine resistance-conferring form of PfCRT to transport quinine, quinidine, and verapamil. In a series of detailed kinetic analyses, we showed that chloroquine and quinine and also chloroquine and verapamil are mixed-type inhibitors of one another. The patterns of inhibition that we observed indicate that PfCRT


Percutaneous sacroiliac screw fixation under fluoroscopic control is an effective method for posterior pelvic ring stabilization. However, sacral dysmorphism has a high risk of L5 nerve injury. This study describes a simple method for the preoperative assessment of the sacral morphology using CT scans with widely available tools.CT scans of 1000 patients were analyzed. True inlet, outlet, and lateral views of the sacrum were obtained using a two-dimensional reconstruction tool to align the sacrum in a reproducible manner. Corridor morphology in the inlet view was measured to calculate different morphological types: (1) Ascending type, (2) Horizontal type, and (3) Descending type. In a second step, the corridor was analyzed for the presence of an anterior indentation of the sacrum between the SI joint and the midsagittal plane with proximity to the nerve root L5, which, therefore, may be harmed during screw misplacement.A notch was found in the majority of cases with relative frequencies ranging from 69 % (upper quartile of S1) to 95 % (upper quartile of S2). Descending types were, by far, the most frequent corridor type with one exception: In the upper quartile of S1, the ascending type was the most frequent corridor (71 %). Horizontal types were less frequent with a relative incidence between 2 and 14 %.This study should increase the awareness for sacral dysmorphism, emphasize the importance of a preoperative assessment of the osseous corridor, and provide a simple method for the preoperative assessment with widely available tools. Percutaneous sacroiliac (SI) screw fixation is a minimally invasive and effective method for the stabilization of the posterior pelvic ring –6. ScrewMajor complications of fluoroscopically controlled percutaneous SI screw fixation include nerve and vessel lesions following screw misplacement. The relative risk of lesions of the fifth lumbar nerve (L5), for example, is up to 8 % , 16–20. The aim of this study, therefore, was to present a simple method for the preoperative assessment of the sacral morphology using CT scans with widely available tools. In addition, it was our goal to classify the morphology of the upper sacrum based on the risk of iatrogenic nerve lesions during percutaneous SI screw fixation.Age <18 years.Recent or consolidated pelvic fractures.Osteolytic pelvic lesions.CT scans of a consecutive series of 1000 patients were analyzed in this study. The patients were included irrespective of gender, health conditions, or premorbidities. Exclusion criteria were:All patients underwent abdominal CT scans, which include the osseous pelvis, in a supine position with hip and knee joints extended. CT scans were performed during clinical routine. Indications for the CT scans were different from the aim of this study. Accordingly, the patients were not exposed to additional radiation for the purpose of this study. A 64-section CT scanner  was used to perform the CT scans. The CT images were taken with 0.6 mm slice depth and saved in slices of 1.25 mm in thickness. 2D reconstructions and measurements were performed with both a standard picture archiving and communication system (PACS) and software .True inlet, outlet, and lateral views of the sacrum were obtained by using a two-dimensional reconstruction tool in a standardized and reproducible manner. In a first step, the midsagittal plane was selected in the lateral view Fig. a. The axThe assessment of the width of the corridor in the inlet view (inlet corridor) is shown in Fig. Subsequently, the diameters were used to calculate different types of inlet corridors. In a first step, the corridors were either defined as ascending, horizontal, or descending corridors. In an ascending corridor, the diameter 1 (SI joint) is smaller than the diameter 3  was used for the statistical analysis. Metric scaled data are reported as arithmetic mean ± standard deviation and categorical data as absolute frequency and percentage distribution. Correlations for metric scaled data were quantified using the Pearson coefficient and for non-parametric samples using the Spearman coefficient. The Kolmogorov–Smirnov test was used for the determination of the distribution form. The probability level was set at CT scans of 1000 patients were analyzed in this study. Measurements were performed both on the right side and left side resulting in 2000 measurements. The mean age of the patients was 68.8 ± 21.5 years. There were 623 female (62.2 %) and 387 male patients (37.8 %) included.The results of the assessment of the different corridor types are shown in Table Percutaneous SI screw fixation under fluoroscopic control is an effective and widely used method for the stabilization of the posterior pelvic ring. Screw misplacement with consecutive nerve lesions, mainly of the nerve root L5, is a major complication of this technique , 16–20. To avoid screw misplacement in percutaneous SI screw fixation, CT guided and navigated procedures have been developed , 15, 29.SI screw fixation under fluoroscopic control not only relies on sound intra-operative imaging, but also on the surgeon´s ability to interpret the two-dimensional images in a 3D context. Several authors have provided different approaches for the interpretation of these images in an inlet, outlet, and lateral views , 30–32. Miller  summarizThe fluoroscopic control during SI screw fixation is adequate when drilling in a strict transverse direction. Transverse corridors of adequate width, however, are present in a minority of patients only. Ascending corridors with a notch have a very narrow intraosseous corridor in a transverse direction. These types require a more posterior entry point  and a more ascending drilling direction, which increases the risk of nerve lesions particularly in the additional presence of a notch. The aim of our study, therefore, was to develop a simple method for the preoperative assessment of the corridor type and the presence of a notch with widely available tools as shown above. We consider horizontal and descending corridor types without a notch to be feasible for fluoroscopically controlled SI screw fixation, while ascending types with notch bear a higher risk of iatrogenic nerve lesions. In these cases, we recommend navigated SI screw fixation. One interesting finding of our study was the high frequency of ascending corridors in the upper quartile of S1 with a much lower frequency in the middle quartile and lower quartile of S1. An ascending corridor in the upper quartile is mainly determined by the inclusion of the sacral promontory in the corridor analysis. In the upper quartile, there was an additional notch in 69 % of the cases as well. Accordingly, we consider a screw trajectory to the promontory to be associated with a higher risk of L5 nerve root lesions than more flat screw trajectories.One limitation of our study has to be noted. It was our goal to classify the morphology of the upper sacrum based on the risk of iatrogenic nerve lesions during percutaneous SI screw fixation. Accordingly, we focused on the corridor morphology in the inlet view and defined different inlet corridor types as well as the presence of a notch. For a safe overall screw placement additional parameters, such as the absolute diameter of the corridor in mm and corridors in the outlet view are relevant as well. The assessment of these parameters, however, was not the goal of this study.In conclusion, our concept obviously is not a “one-size-fits-all’’ concept neither. It aims at three goals: (a) to increase the awareness of the surgeon for the problem of sacral dysmorphism, (b) to emphasize the importance of a preoperative assessment of the osseous corridor, and (c) to provide a simple method for the preoperative assessment with widely available tools.


ΔF508), the most common mutation causing cystic fibrosis, both S1P receptor antagonism and AMPK inhibition enhance CFTR activity, without instigating discernable correction. In summary, we demonstrate that S1P/AMPK signaling transiently attenuates CFTR activity. Since our previous work positions CFTR as a negative S1P signaling regulator, this signaling link may positively reinforce S1P signals. This discovery has clinical ramifications for the treatment of disease states associated with enhanced S1P signaling and/or deficient CFTR activity . S1P receptor/AMPK inhibition could synergistically enhance the efficacy of therapeutic strategies aiming to correct aberrant CFTR trafficking.The cystic fibrosis transmembrane conductance regulator (CFTR) attenuates sphingosine-1-phosphate (S1P) signaling in resistance arteries and has emerged as a prominent regulator of myogenic vasoconstriction. This investigation demonstrates that S1P inhibits CFTR activity via adenosine monophosphate-activated kinase (AMPK), establishing a potential feedback link. In Baby Hamster Kidney (BHK) cells expressing wild-type human CFTR, S1P (1μmol/L) attenuates forskolin-stimulated, CFTR-dependent iodide efflux. S1P’s inhibitory effect is rapid (within 30 seconds), transient and correlates with CFTR serine residue 737 (S737) phosphorylation. Both S1P receptor antagonism (4μmol/L VPC 23019) and AMPK inhibition (80μmol/L Compound C or AMPK siRNA) attenuate S1P-stimluated (i) AMPK phosphorylation, (ii) CFTR S737 phosphorylation and (iii) CFTR activity inhibition. In BHK cells expressing the ΔF508 CFTR mutant (CFTR Sphingosine-1-phosphate (S1P) is a key endogenous regulator of resistance artery myogenic vasoconstriction . PressurOur previous work in resistance arteries has characterized Sphk1 and CFTR/SPP1 as the principal counteracting signaling elements within a signaling framework that precisely controls S1P bioavailability and consequently, its pro-constrictive actions. In principle, inversely regulating this signaling tandem  would enable more efficient control of S1P signal onset, amplitude and duration. However, while we have defined a rapid mechanism that enhances S1P synthesis in response to transmural pressure elevation , a simil1/S1P3 receptor subtypes [737 and Ser768) within CFTR’s regulatory domain (R-domain) [Although AMPK is primarily considered an energy-sensing enzyme regulated by cellular AMP levels , non-metsubtypes ,8, a mecsubtypes ,14. Acti-domain) ,10. We hUnfortunately, assessing the effect of S1P signals on S1P uptake is impractical, due to significant confounds. Specifically, labeled S1P  is both intrinsically active  and competes with unlabeled S1P for CFTR-dependent transport: this makes it impossible to discern between signaling- and competition-based transport inhibition. We therefore utilized iodide efflux to measure total CFTR activity , a strategic maneuver that eliminates these confounds and permits testing the core hypothesis: that S1P signaling modulates CFTR activity. This investigation characterizes a rapid, non-transcriptional mechanism that putatively amplifies stimulated S1P signals by transiently suspending S1P degradation.Guide for the Care and Use of Laboratory Animals published by the NIH . All animal care and experimental protocols were approved by the Institutional Animal Care and Use Committee at the University of Toronto and were conducted in accordance with Canadian animal protection laws.This investigation conforms to the tm1EUR) [Hospital for Sick Children . Prior to tissue collection , animals were fully anesthetized with isoflurane and humanely euthanized by decapitation. Mesenteric arteries were micro-dissected and lysed for western blotting, as previously described [Wild-type mice  were purchased from Charles River Laboratories . Mice homozygous for the ΔF508 CFTR mutation (CFTRtm1EUR)  and the escribed . A subseescribed . Mice we2). BHK cells stably expressing wild-type CFTR (CFTRwt) [ΔF508) [Naïve Baby Hamster Kidney (BHK) cells  were maintained in DMEM/F12 media containing 5% fetal bovine serum under standard culture conditions   or the Δ [ΔF508)  were maiS737A) or a glycine to aspartic acid mutation at residue 551 (CFTRG551D); siRNA reagents  were purchased from Dharmacon, Inc. .Cells (at 40–50% confluency) were transfected with plasmid DNA (2μg DNA per 35mm dish) or siRNA (25nmol/L per 22mm dish) using FuGene 6 transfection reagent , according to the manufacturer’s instructions. Cells were transfected for 48 hours, at which point cell confluency was approximately 95%. This investigation utilized plasmids encoding mutated CFTR proteins containing either a serine-to-alanine mutation at residue 737 (CFTR2), followed by several washes with iodide-free buffer (NaNO3 substituted for NaI). Iodide efflux is minimal under basal/non-stimulated conditions  were scraped, pelleted, washed and re-suspended into 250μl of efflux buffer. Iodide efflux was stimulated with forskolin  and real-time iodide electrode traces were recorded with a Digidata 1320A data acquisition system and Clampex 8 software . In terms of kinetics, efflux was evident within 30 seconds (post-stimulation), was reliably maximal after 60 seconds and remained maximal for at least 2 minutes  were sequentially transferred through 8 separate volumes of 2mL efflux buffer for 1 minute each. The efflux buffer contained 10μmol/L FSK, 1mmol/L isobutylmethylxanthine and 100μmol/L cpt-cAMP (in 1% v/v DMSO), a cocktail that more potently activates PKA compared to FSK alone. Supernatant iodide levels (cell free) were determined (iodide electrode) for each 1-minute interval : these antibodies were used in the validation process. All other antibodies used in this study are commercially available.We utilized standard procedures for western blotting; specific methodological and reagent details are provided in the wt; Band C is widely accepted as the functional form at the cell surface.The 67D4 and 570 CFTR antibodies specifically recognize a non-phosphorylated CFTR epitope containing serine 737. Consequently, the loss of antibody binding indicates increased phosphorylation. Western blots display the mature, fully-glycosylated form of CFTR (Band C), which predominates in BHK cells expressing CFTRn is the number of independent experiments. A non-parametric Mann-Whitney test with computation of exact p-values was utilized for the comparison of two independent groups. For comparison of multiple independent groups, a non-parametric one-way ANOVA  followed by a Mann-Whitney test with exact p-value computation as a post-hoc test for pair-wise comparisons was employed . Differences were considered significant at error probabilities of P<0.05.All data are expressed as means ± SEM, where wt (n = 6): this result confirms that FITC-labeled S1P has intrinsic cell-signaling activity, a critical confound that precludes using FITC-S1P uptake to assess S1P-dependent effects on CFTR activity. We therefore measured iodide efflux during maximal CFTR channel activation [G551D) [As shown in tivation  as the p [G551D)  abolishe [G551D)  and S1P wt . CFTR must be expressed at the cell surface in order to release iodide to the extracellular compartment; accordingly, FSK does not stimulate iodide efflux in BHK cells stably expressing CFTRΔF508  [ΔF508 integration into the plasma membrane and confers FSK the ability to stimulate iodide efflux  , a CFTR , n = 5)  and exhi, n = 5) . As prev, n = 5) –24, low x n = 6; . Taken twt-expressing BHK cells. Representative tracings are displayed in ΔF508  significantly attenuates FSK-stimulated iodide efflux in CFTR8 n = 6; . Althoug8 n = 6; .CFTR serine 737 (S737) is phosphorylated by several serine kinases, including PKA , AMPK 1 and lemuS1P (1μmol/L) stimulates rapid (within 30 seconds) and transient  CFTR S737 phosphorylation n = 5–6; . We confS737A) abolishes S1P’s inhibitory effect on FSK-stimulated iodide efflux .Mutating the CFTR serine 737 phosphorylation site  attenuates S1P-stimulated AMPK phosphorylation -specific agonist SEW-2871 and the S1P3 receptor (S1P3R)-specific agonist CYM-5541 (both used at 1μmol/L). Selective S1P1R activation (SEW-2871) mimics the effects of S1P: we observe rapid and transient phosphorylation of both AMPK (3R activation (CYM-5541) stimulates only mild AMPK phosphorylation (n = 9–10), which does not induce CFTR S737 phosphorylation  prevents S1P-stimulated AMPK phosphorylation Fig 1E), rylation ; n = 6. rylation ; n = 6 arylation ; n = 5–7wt  isolatecontrols ; n = 4. e efflux ; n = 5–6x . Since reatment ; n = 3.The present investigation demonstrates that S1P regulates CFTR activity (defined here as the product of surface expression and channel gating). Given the ubiquitous and highly integrative nature of S1P signaling , this se1R-dependent AMPK activation we document in the present study matches observations from a distinctly separate model of cultured endothelial cells [in vivo settings.We strategically selected the BHK expression system to address our mechanistic hypothesis, so that human CFTR constructs  could be expressed in an environment with minimal endogenous CFTR expression. Although BHK cells do not natively express CFTR, the S1P and AMPK signaling pathways are ubiquitous and have highly conserved links in virtually all cell types ,14. Indeal cells . Furtheral cells . Taken tIodide efflux provides a rapid, convenient and sensitive assessment of cell surface CFTR channel activity ,30,31. A1R, AMPK and CFTR S737 phosphorylation. Although antagonizing S1P1R/S1P3R (VPC23019) prevents exogenously-applied S1P from attenuating CFTR activity, receptor antagonism alone  has no effect: BHK cells expressing CFTRwt, therefore, must possess sufficient S1P transport capacity to sequester all constitutively produced/released S1P . S1P exerts maximal effects on CFTR channel activity at the physiologically relevant concentration (200nmol/L) [S1P markedly attenuates PKA-stimulated CFTR channel activity, with requisite involvement of S1P0nmol/L) .both phosphorylate and persistently associate with CFTR: if the AMPK α1 subunit is mutated to reduce either kinase activity  or CFTR interaction , then AMPK is rendered incapable of attenuating CFTR channel activity [CFTR S737 phosphorylation is crucial for attenuating CFTR activity and yet, this does not entirely explain the inhibitory effect: as shown in activity . Thus, tΔF508, where only a small proportion of CFTRΔF508 localizes to the cell surface [ΔF508 residing at the cell surface . Several factors may contribute to this observation: as examples, it is possible that (i) the small fraction of CFTRΔF508 channels localized to the cell surface [wt when S1P/AMPK signaling is inhibited; and/or (ii) non-canonical transport mechanisms localize the Band B form  to the plasma membrane [The high level of CFTR surface  is more membrane . InteresΔF508 channels that reside at the cell surface has significant clinical ramifications, given that most the prevalent form of cystic fibrosis is caused by the ΔF508 mutation. Therapeutics currently in development primarily target the defect of insufficient trafficking to the membrane. However, the ultimate efficacy of these correction compounds is limited, with rescued CFTRΔF508 expression/activity generally representing only 10–30% of the wild-type level [1 receptor modulators are clinically available  and second-generation compounds are currently in clinical trials [ΔF508 channel activity , inflammatory-based CFTR down-regulation should reduce S1P degradation, thereby activating the S1P/AMPK signaling pathway and ultimately inhibit the residual CFTR channels. Indeed, elevated S1P levels ; we therefore proposed that CFTR therapeutics possess unexplored utility for treating heart failure-associated vascular dysfunction and multi-organ injury . AnalogoFig E in  and enha1R blockade exceeds simple antagonism at the receptor level: it also unleashes S1P degradation via CFTR disinhibition.In summary, we demonstrate the presence of a S1P/AMPK signaling mechanism that transiently attenuates CFTR activity and positively reinforces the S1P signal through the suspension of S1P degradation. This new signaling connection could enable a myriad of upstream cellular signals to converge on CFTR, through these ubiquitous and highly conserved signaling foci. Pathological reductions in CFTR expression/activity trigger a vicious cycle of the mechanism’s inhibitory actions: our data, therefore, support complementing CFTR correction/potentiation strategies with the inhibition of S1P/AMPK signaling for greater efficacy. Our mechanistic concept offers the intriguing premise that pharmacological S1PS1 File3 receptor activation does not induce CFTR S737 phosphorylation ; and Lung S1P levels in mice with heart failure .This complete supplemental information file contains: (i) information about reagents; (ii) methodological details, including custom CFTR antibody production and validation, western blot procedures, heart failure induction and S1P quantification; and (iii) supplemental data figures A-E. Supplemental data figures include: Measuring CFTR-dependent iodide efflux ; Validation of the 67D4 antibody for serine 737 phosphosensitivity ; Antibody 570 detects S1P-dependent CFTR phosphorylation ; S1P(PDF)Click here for additional data file.


C. elegans homolog of ROR, has an active tyrosine kinase domain, suggesting a divergence in the signaling processes of the ROR family during evolution. In addition, we show that substitution of the non-consensus residues from ROR1 or ROR2 into CAM-1 and MuSK markedly reduce kinase activity, while restoration of the consensus residues in ROR does not restore robust kinase function. We further demonstrate that the membrane-bound extracellular domain alone of either ROR1 or ROR2 is sufficient for suppression of canonical Wnt3a signaling, and that this domain can also enhance Wnt5a suppression of Wnt3a signaling. Based on these data, we conclude that human ROR1 and ROR2 are RTK-like pseudokinases.Receptor tyrosine kinase-like orphan receptors (ROR) 1 and 2 are atypical members of the receptor tyrosine kinase (RTK) family and have been associated with several human diseases. The vertebrate RORs contain an ATP binding domain that deviates from the consensus amino acid sequence, although the impact of this deviation on catalytic activity is not known and the kinase function of these receptors remains controversial. Recently, ROR2 was shown to signal through a Wnt responsive, β-catenin independent pathway and suppress a canonical Wnt/β-catenin signal. In this work we demonstrate that both ROR1 and ROR2 kinase domains are catalytically deficient while CAM-1, the  C. elegans to humans Receptor tyrosine kinase-like orphan receptors (ROR) 1 and 2 are among the most widely studied non-canonical Wnt receptors of the receptor tyrosine kinase (RTK) family and ROR genes are conserved in animals from The RORs share significant domain similarity to Muscle Specific Kinase (MuSK) receptor , which iWnt signaling is mediated through several pathways: a canonical β-catenin dependent pathway, and multiple non-canonical β-catenin independent pathways which remain poorly understood Non-canonical Wnt pathways can be stimulated by the ligand Wnt5a, however, signaling proceeds independently of β-catenin, through several alternate downstream effectors, many of which have been linked to the generation and maintenance of planar cell polarity (PCP) (7). ROR1 and ROR2, which share approximately 50% sequence identity Nematostella vectensis (sea anemone), is predicted to be inactive based on substitution of a valine residue for the catalytic lysine in the VAIK ATP binding motif. In contrast, the closely related human receptor, MuSK, or ROR proteins in lower organisms, like the C. elegans CAM-1 D. melanogaster ROR retain consensus kinase motifs. The kinase domains of these receptors share 40 to 45% sequence identity with ROR.The kinase domains of the vertebrate ROR proteins contain non-consensus cysteine, aspartic or glutamic acid residues in the glycine rich P-loop that may hinder ATP binding and disrupt enzymatic activity  [22]. LiThe catalytic effects of substitutions of larger amino acids in the second glycine of the kinase P-loop are incompletely explained by the available structural data. Structural alignments of the C-terminal lobes of the kinase domain of ROR2 and the ATP-free, apo form of MuSK An accurate understanding of the function and enzymatic activity of the ROR kinase domain is essential to defining its role in disease. We therefore evaluated the enzymatic activity of isolated ROR1 and ROR2 kinase domains which have been overexpressed in insect cells and purified to greater then 95% homogeneity. Previous studies with crudely purified, immunoprecipitated ROR1 that may contain co-factors or associated kinases have produced conflicting results, with one study demonstrating the presence of ROR1 kinase activity in vitro biochemical activity of the highly purified cytoplasmic domains of ROR1 and ROR2, or the positive control CAM-1 and MuSK indicate extremely weak activity for either human ROR kinase domain; comparable to that of classic kinase-inactivating mutants of MuSK and CAM-1. In fact, this lack of robust kinase activity of human ROR is characteristic of a pseudokinase, as suggested from the analysis of the protein sequence, unlike CAM-1 and MuSK. Further, we demonstrate using quantitative mass spectrometry that Wnt5a activation of ROR1 is not associated with kinase activation of the receptors in mammalian cells. Full-length and truncation constructs of ROR1 and ROR2 were evaluated in a cell-based assay for Wnt reporter activity under identical conditions to the quantitative mass spectrometry experiments, demonstrating that enhanced inhibition of canonical Wnt signaling by either ROR does not require the presence of the kinase domain. These biochemical data, combined with the phylogenetic data, suggest a divergence of selection pressure on mammalian ROR during evolution to retain the robust phosphotransferase activity of CAM-1 and MuSK, and point to the dominant function of the ROR kinase domain as a pseudokinase scaffold for recruiting other proteins active in cell polarity processes.In the current work, in vitro assays was purified from L cells transfected with mouse Wnt3a (American Type Culture Collection) as previously described Wnt3a and Wnt5a protein used for 2, 5 units benzonase/mL, Roche complete protease inhibitor cocktail without EDTA) and disrupted by nitrogen decompression. After ultracentrifugation of the lysate, the supernatant was filtered and then purified by nickel-affinity chromatography followed by size-exclusion chromatography. All lysis and purification steps were performed on ice or at 4°C. Protein identities were confirmed by mass spectrometry.A recombinant baculovirus/Sf9 cell system was used to express the wild type and mutant intracellular domains (ICD) of ROR1, ROR2, MuSK or CAM-1, encoding residues C428-L937, C428-A943, R519-V869 or R491-D928 respectively , with an2, and 0.2 mM sodium orthovanadate. The controls were incubated likewise, without ATP. The proteins were then analyzed by immunoblotting.For detection of autophosphorylation, the wild type and mutant ICDs of receptors ROR1, ROR2, CAM-1 and MuSK were incubated overnight, approximately 18 hours, at 4°C, with 2 mM ATP, 20 mM MgClm(peptide)K 0–5 mM activation loop peptide , 5 mM ATP and 20 mM MgCl2 were used. For determination of m(ATP)K, 0–10 mM ATP and 20 mM MgCl2 or 0–5 mM ATP and 5 mM MnCl2 were used, both in the presence of 5 mM peptide. All reactions were carried out at 20°C and included [γ−32P]ATP. Kinetic parameters were determined by fitting the initial rate data  to the Michaelis-Menten equation using GraphPad Prism software. Phosphorylated CAM-1 was generated by pre-incubating purified enzyme with 2 mM ATP, 20 mM MgCl2, and 0.2 mM sodium orthovanadate overnight at 4°C. Phosphorylation was confirmed by anti-phosphotyrosine immunoblot and intact mass spectrometry.Steady-state kinetic measurements were carried out by phosphocellulose paper binding assay For detection of tyrosine phosphorylation, the proteins were resolved on 4–20% SDS-polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were probed with anti-phosphotyrosine primary antibody , followed by IRDye 800CW Rabbit anti-HRP secondary antibody (LI-COR). The blots were imaged and quantitated using an Odyssey Infrared Imaging System (LI-COR) and Image Studio software.3VO4, 1 mM DTT with protease inhibitors) and the cytosolic fraction used for electrophoresis. The proteins were electrophoresed on 4–20% TGX gels (Bio-Rad), followed by transfer to PVDF membrane. Blots were incubated with the primary antibodies (overnight), and anti-mouse or rabbit IR conjugated secondary antibodies  and the proteins visualized and quantitated using the Odyssey Infrared Imaging System (LI-COR).Expression of receptor constructs in cells used for the luciferase assay was determined by immunoblot detection of receptor proteins using anti-Flag M2 antibody (Sigma) and 10 µl of the lysate from the luciferase assays. Cytosolic β-catenin, in cells transfected with empty vector, ROR1, ROR2 or MuSK constructs and treated with Wnt3a and/or Wnt5a was detected using the anti-β-catenin monoclonal antibody (BD Biosciences). For this assay, cells were lysed in a hypotonic buffer . Peptides before and after phospho-enrichment were analyzed by capillary reverse phase liquid chromatography tandem mass spectrometry on an orbitrap mass spectrometer . Mass spectra were recorded in data-dependent experiments whereby eight collision-induced dissociation product ion spectra were collected per full MS scan Coomassie Blue stained SDS-PAGE gel bands corresponding to ROR1, ROR2 and CAM-1 ICDs were excised, reduced, cysteine-alkylated, and digested with trypsin The ROR1 tryptic peptide (EIYSADYYR) was quantified in its unphosphorylated form and as potentially singly phosphorylated on each of its three tyrosines by stable isotopes dilution mass spectrometry. Synthetic peptides labeled with stable isotope that gave each form of the peptide a distinct mass were procured . These were added to ROR1 tryptic digests to yield 133 fmol per duplicate LC-MS injection, performed as above. Peak areas were extracted from the Orbitrap full MS scans and endogenous peptide amounts were calculated relative to the synthetic peptide standards. Where no peak corresponding to the endogenous phosphopeptide was observed, an upper bound for its concentration was calculated based on the values of background peaks of the same mass to charge ratio.The ROR1 FL (Q30-L937), ROR1 ECD (Q30-E403), ROR1 ECD-TM (Q30-C428), ROR1 TM-ICD (D392-L937), ROR2 FL (E34-A943), ROR2 ECD (E34-G403), ROR2 ECD-TM (E34-C428), ROR2 TM-ICD (S395-A943) and MuSK FL (E22-V783 of isoform 2)  construc4 HEK293 cells were plated per well of a 24-well dish (Nunc) 24 h before transfections. Cells were transfected with 0.125 mg of the indicated receptor construct and 0.125 mg of TB23 mix with Fugene 6 (Roche) as described 2, 1 mM DTT, 50 mM NaF, 1 mM Na3VO4 and protease inhibitor cocktail) and 10 µl samples were assayed in duplicate using the Dual-Glo Luciferase Assay kit (Promega) and detected in an Envision (Perkin-Elmer). Firefly luciferase activity was normalized against Renilla luciferase activity and the data normalized to this ratio obtained in untreated cells.Human kidney epithelial 293 (HEK293) cells were grown in 50∶50 high-glucose DMEM and Ham’s F12 with 10% fetal bovine serum (FBS). For luciferase assays, 0.6×10C. elegans CAM-1  from human ROR1, ROR2, MuSK and ns CAM-1 . Furtherns CAM-1 . All purns CAM-1 . MuSK haAfter overnight incubation with magnesium and ATP, the wild type kinase domains of ROR1 and ROR2 show 100 and 1000 fold less tyrosine phosphorylation than CAM-1 and MuSK, respectively  & S2. Rein vitro steady state kinetic parameters for peptide phosphorylation activity using [γ−32P]ATP in a phosphocellulose paper binding assay. Peptides from the activation loop of CAM-1, ROR1 or insulin receptor kinase (IRK) were evaluated as substrates. CAM-1 was able to phosphorylate all three peptides. In contrast, neither ROR1 nor ROR2 demonstrated any phosphorylation activity in identical conditions (m(ATP)K in the presence of magnesium is, within experimental error, identical to that previously reported for unphosphorylated MuSK and the m(ATP)K is further reduced in the presence of manganese, as has been observed for other tyrosine kinases m(peptide)K and the null phenotype observed for the kinase dead version of CAM-1 To further characterize the catalytic activity of ROR1 and ROR2, we determined nditions . We werein vitro.In order to determine the specific locations of the phosphorylation detected by immunoblot analysis, the phosphosites of wild type ROR1, ROR2 and CAM-1 were mapped using electrospray mass spectrometry. Tyrosine phosphorylation within the respective activation loops and additional phosphorylation sites within the kinase and serine/threonine-rich domains identified from the eukaryotic expression source are shown in To evaluate if Wnt5a could activate the kinase domain of ROR1 expressed in HEK293 cells, we transfected full-length FLAG tagged ROR1 and treated cells with 100 µg/ml of Wnt5a. Isotopically labeled synthetic peptides (described in Methods) identified from the phospho-mapping of the ROR1 catalytic domain, including singly phosphorylated fragments from the kinase domain activation loop, were then used to determine the extent of tyrosine phosphorylation in the activation loop of ROR1. Following anti-FLAG immunoprecipitation, phosphorylation of tyrosine residues 641, 645 and 646 of the activation loop of ROR1 was not detected above a limit of 1 part per 100 , Table 2ROR1 and ROR2 are known to act as Wnt5a receptors C. elegansIn vitro, the efficiency of ROR1 and ROR2 tyrosine phosphorylation catalysis, as determined by anti-phosphotyrosine immunoblot is 100 to 1000 fold less than that observed for the C. elegans ROR homolog CAM-1 or human MuSK. In addition, enzyme kinetics experiments for peptide tyrosine phosphorylation by the kinase domains ROR1 and ROR2 demonstrated no activity within the quantification limits of the assay (KM(ATP)>15 mM), in contrast to CAM-1 and MuSK (33). While these data alone are not conclusive evidence of a complete lack of catalytic potential, the biological significance of such a high KM(ATP) at typical cellular ATP concentrations is dubious, at least on common receptor tyrosine kinase substrates. Previous reports have associated kinase activity with ROR that has been immunoprecipitated from crude cell extracts, a condition where cofactors or associated kinases might also co-precipitate ++-binding nor ATP-binding activity in a thermal-shift assay trans-phosphorylation of ROR by kinases has been noted by researchers A number of molecular events have been proposed to explain ROR signaling mechanisms, including tyrosine phosphorylation by the ROR kinase domain in vitro data that ROR1 lacks robust kinase activity. These cellular phosphorylation experiments were performed under identical conditions to the TOPbrite reporter assays, providing a positive control of ROR1 functionality. While we did not observe autophosphorylation of ROR1 in response to Wnt5a stimulation, it is possible that another ligand for ROR1 exists which could activate the receptor in a different context.The kinetics and autophosphorylation experiments in the current report were performed with isolated protein of greater than 95% purity. It is possible that, in contrast to CAM-1, human ROR kinase activity may require a trans-activating cellular cofactor or that an autoregulatory domain inhibits activity of the constructs used in our biochemical analysis, as has been shown for other RTKs in vitro, it is possible that the protein preparations contained a small amount of active tyrosine kinase contaminant from the host cell lysate and we cannot rule out the possibility that wild type ROR1 and ROR2 completely lack catalytic activity. We were unable to isolate soluble cytoplasmic ROR protein containing classic catalytic inactivating mutations, as a negative control  As a pseudokinase, it is possible and perhaps likely that ROR may allosterically activate a functional kinase, as has been demonstrated with the pseudokinase STRADα and LKB1 C. elegans CAM-1 is an active kinase in vitro while human ROR1 and ROR2 lack comparable catalytic activity in similar assays, suggesting an evolutionary divergence in at least some gene functions. Our data support the hypothesis that human ROR functions principally as an RTK-like pseudokinase. A more detailed study of these events will be essential to understand the physiological functions of ROR and may lead to the identification of the interaction targets of the kinase domain.In conclusion, we have shown that Figure S1ROR intracellular domains are pure and monomeric. a) Coomassie stained SDS-PAGE gels of the ROR1, ROR2, ROR2 D482G and CAM-1 ICDs, 5 µg per lane. b) Comparison of the theoretical and experimental molecular weight of the wild type ROR1 and ROR2 ICDs as determined by MALLS.(TIFF)Click here for additional data file.Figure S2Experimental replicate data of tyrosine autophosphorylation quantitation are consistent with .(TIFF)Click here for additional data file.Figure S3Validation of expression of ROR constructs in HEK293 cells. a) HEK293 cells were transfected with various ROR constructs and FACS analysis performed using the anti-FLAG monoclonal antibody and PE-conjugated anti-mouse secondary. b) Lysates of HEK293 cells transfected with the various ROR constructs in (TIFF)Click here for additional data file.Table S1Observed phosphorylation sites in ROR1, ROR2 and CAM-1. Phosphosite mapping was performed on the isolated ROR1, ROR2 and CAM-1 ICDs following incubation with magnesium and ATP. Tyrosine phosphorylation was observed in ROR1 and ROR2 only after phosphopeptide enrichment using TiO2 affinity chromatography, which was not needed for CAM-1. Phosphorylated residues are indicated in bold. aBracketed sequences contain a phosphosite that could not be localized to a specific residue. bPeptides containing activation loop tyrosine residues.(DOC)Click here for additional data file.


These results suggest a potential rolefor meteoritic phosphorus in the origin and development of early life.Phosphorylation of the nucleosides adenosine and uridine by the simple mixing andmild heating of aqueous solutions of the organic compounds with synthetic analogs ofthe meteoritic mineral schreibersite,  Phosphorylation of glycerol by Fe3P to give glycerolphosphate (a component of cell membranes) has been reported previously3P orFe2NiP, and stirred and heated to 80 °C for 2days to 2 weeks  used inthis study was determined to be structurally identical to its meteoritic counterpart byX-ray diffractometry, X-ray photoelectron spectroscopy, and Raman spectroscopy20The resulting solutions were analyzed by UPLC-MS, MS/MS, and 3P, and Fe2NiP. Since 2′-, 3′-, and5′-adenosine monophosphate are isomers, the elution times and massfragmentation patterns 2+ added exceeded the solubility ofboth magnesium phosphate and magnesium phosphite. This result could imply that thephosphorylating agent is possibly a phosphorus species in an oxidation state other than+5 and thus would have sufficient aqueous solubility in the presence of a divalentcation. It was also conjectured that the pH drift from “neutral”to basic was due to the hydrolysis of urea and the formation of ammonium carbonate.Indeed, in the presence of potassium carbonate or ammonium hydroxide  in place of urea, phosphorylation of adenosine was also observed and up to 9.5% (with K2CO3), in agreement with measuredcorrosion rates of 0.1% per week without added solutes23The mechanism of phosphorylation is still unknown and is being actively investigated. Itis possible that the process occurs in solution or on the surface of the schreibersite.As was mentioned previously the yield of organophosphates from these experiments is nothigh, and the concentration of AMP ranges from being undetectable to about600 μM. The yield seems to be controlled by the release of Pfrom phosphide corrosion, which is slow−7 s−1,To determine the relevance of our model environment (schreibersite corroding in anorganic-rich aqueous solution) to prebiotic chemistry, the steady state concentration ofnucleotides is equal to the production rate divided by the hydrolysis rate constant. Inthe absence of biological enzymes hydrolyzing these organophosphates, the rate ofhydrolysis is typically slow(~102 gas3O4) and phosphorus oxyanions is a strongly exergonicprocess releasing 100 kJ·mol−1 at80 °C and it is possible that some portion of this energy isdirected to phosphorylation.Prior work on nucleoside phosphorylation has shown that inorganic phosphate can serve asboth a catalyst and reactant in nucleotide synthesis242 was not excluded from the experiments, prior work24262 in the headspace of theseexperiments or dissolved within the solutions was insufficient to promote oxidation ofthe schreibersite to release the concentrations of P observed.Although OA source of reactive phosphorus may have been an important part of the prebiotic earth,and possibly of Mars3P (0.7 g), and for the other reagents (if used); urea(0.50 g), MgSO4 (0.75 g), 25% soln.NH4OH (0.29 g), K2CO3(1.15 g). For sample U4, 1.5 g of Fe2NiP was usedinstead of 0.7 g Fe3P. The sample of Fe2NiP wassubjected to a series of wet/dry cycles (5×), leading to substantialsurface corrosion. The reagents were added in a glass vial containing7 mL of deionized water, to which the nucleoside was added.


Steinernema feltiae is a moderately freezing tolerant nematode, that can withstand intracellular ice formation. We investigated recrystallization inhibition, thermal hysteresis and ice nucleation activities in the infective juveniles of S. feltiae. Both the splat cooling assay and optical recrystallometry indicate the presence of ice active substances that inhibit recrystallization in the nematode extract. The substance is relatively heat stable and largely retains the recrystallization inhibition activity after heating. No thermal hysteresis activity was detected but the extract had a typical hexagonal crystal shape when grown from a single seed crystal and weak ice nucleation activity. An ice active substance is present in a low concentration, which may be involved in the freezing survival of this species by inhibiting ice recrystallization. Cold tolerant ectotherms have evolved a number of strategies to survive low temperatures , 2. ShorSome authors consider that there is a third category of ice active protein called a ‘recrystallization-inhibiting protein’, found in some freeze-tolerant animals . RecrystSteinernema feltiae is a freeze-tolerant nematode and either thermal hysteresis or recrystallization inhibition activity or both could be associated with its freezing survival. When frozen relatively rapidly S. feltiae shows modest abilities to survive freezing with a lower lethal temperature of -5°C [Steinernema carpocapsae is the only entomopathogenic nematode so far demonstrated to have recrystallization inhibition activity [S. feltiae. of -5°C . Steineractivity . No otheSteinernema feltiae was reared in bee wax moth larvae, Galleria mellonella at 22°C. Freshly harvested third-stage infective juveniles of S. feltiae were passed through two layers of tissue paper to obtain active nematodes. Nematodes were washed in artificial tap water [ap water  and centRecrystallization inhibition was assessed using the splat freezing technique  and as dwww.otago-osmometers.com) measures changes in optical transmittance of a frozen sample at a preset annealing temperature. Samples having RI activity do not change their level of optical transmittance with time whereas in samples with no RI transmittance increases with time as the ice crystals grow and scatter less light [The optical recrystallometer . Drops of each sample  were transferred to mineral oil  in watch glasses. The sample wells of the osmometer were filled with oil (Cargille’s B) and droplets of the samples transferred to it using a micropipette system. They were rapidly frozen by cooling the osmometer to ~ −40°C. The temperature was then raised rapidly until close to the expected melting temperature, and then slowly (0.01°C min-1) until the last ice crystal melted, which was taken as the melting point. Then the temperature was decreased rapidly to refreeze the sample and then increased to melt the sample to a single ice crystal. Just before the ice crystal melted, the temperature was decreased until a discernible growth of the ice crystal was observed. This was taken as the hysteresis freezing point. Thermal hysteresis was measured as the difference between melting and hysteresis freezing points. The shape of ice crystal was noted and photographed. The melting points of 1000 mmolKg-1 standard and Milli-Q water were used to correct observed nematode extract melting points and their osmolality calculated.et al [-1 and the temperature of crystallization (Tc: where spontaneous freezing occurs) was read as the start of each sample exotherm.Ice nucleation activity was determined using an ice nucleation spectrometer similar to that described by Wharton et al . A 10 μlSteinernema feltiae extract (25.3 mg dry weight/ml) showed moderate recrystallization inhibition (RI) activity. The mean crystal diameter after 30 min annealing at −8°C of nematode extract  was significantly smaller  than that of a buffer control (117.9±7.84 μm) in the splat freezing technique , and the activity is lost (reduced to that of the buffer control) after a 1:3 dilution. However, heating did not reduce the RI activity of the nematode extract significantly  in terms of increase in crystal size  but the level of transmittance in the buffer control were not significantly different  from the second serial dilution (1:3) of the nematode extract.In general, the pattern observed was that nematode extracts showed little or no change in optical transmittance with time, indicating RI activity; whereas in buffer, or diluted samples optical transmittance increased, indicating no RI activity. Since recrystallization is temperature sensitive, different annealing temperatures were tested. The change in the optical transmittance of the extract was less at −8°C than at −7°C and −6°C . PartialA similar pattern of RI activity is shown by both the splat freezing assay and optical recrystallometry . This coS. feltiae showed no thermal hysteresis but had a typical hexagonal crystal shape when grown from a single seed crystal  of the extract occurred at –14.7 ± 1.37°C which was significantly different (P < 0.05) to that of the buffer control (–20.8 ± 1.83°C). Serial dilution had a significant effect on the activity  and after a 1:3 dilution it did not differ from that of the buffer control (P > 0.05) ( > 0.05) .S. feltiae showed a moderate level of RI activity in the splat freezing assay. This was indicated by smaller ice crystals after the annealing period in comparison to the buffer control. Heating the extract did not produce significant loss of RI suggesting the activity was relatively heat stable. The RI activity of P. davidi IAP is also relatively heat stable [et al [S. carpocapsae. The annealing period and the concentration used  were different than that in the present study , so the results cannot be directly compared. However, despite the lower concentration and shorter annealing time, the crystal size was smaller in two of the six species (S. carpocapsae and P. davidi) than that of the S. feltiae extract shown here. This places S. feltiae in a moderate RI category. However, a moderate level of RI may be of importance for this moderately freeze tolerant species which survives intracellular freezing to -3°C [et al [S. feltiae may play a role in the size and shape of ice crystals, as indicated by their appearance in freeze-substituted specimens [P. davidi, which has strong RI activity and survives to much lower temperatures [Infective juveniles of t stable  and so it stable . The leve [et al  compared to -3°C . Ramløv C [et al  suggest C [et al , 24. Thepecimens . This preratures . Howevereratures , suggestet al [The optical transmittance of nematode extract did not change during annealing at -8°C, whereas that of buffer controls and diluted extract increased significantly. There was good correspondence between crystal size in the splat freezing assay and optical transmittance in the recrystallometer in similar samples. This indicates that both assays are detecting recrystallization, although the physical processes involved in the two assays are likely to be different. Recrystallization is both temperature and time sensitive, so both assays are influenced by these factors . Whartonet al  measuredNo thermal hysteresis was detected in the nematode extract. An hexagonal crystal shape, however, suggests the binding of a substance, most probably a protein, to the ice. In most cases this is due to an antifreeze protein , 25. TheSteinernema feltiae had weak ice nucleating activity, allowing the extracts of the nematode to freeze at −14.6°C. However, this activity is unlikely to be involved in the survival of the nematode, as this species can survive freezing down to −3°C at the most. Thus, nucleation of the whole nematode seems to be exogenous as nematodes are essentially aquatic and rely on inoculative freezing [Celatoblatta quinquemaculata, for example, freezes at −5°C having strong nucleators [P. davidi, has no ice nucleating activity, rather produces ice nucleation inhibitors [S. feltiae is thus probably an incidental property of some of its component molecules or of its intestinal contents.freezing . Once thfreezing . Most frfreezing . An extrcleators . Convershibitors . The weaS. feltiae has no thermal hysteresis or strong ice nucleating activity. The recrystallization inhibition activity found may assist the freezing tolerance of the nematode but there could be other factors involved, such as production of low molecular weight cryoprotectants [In conclusion, tectants , that ma