S176 is a critical post-translational modification that suppresses YAP’s ability to promote cell proliferation and tumorigenesis by altering the subcellular localization of an essential YAP co-factor.The Hippo-YAP pathway is a central regulator of cell contact inhibition, proliferation and death. There are conflicting reports regarding the role of Angiomotin (Amot) in regulating this pathway. While some studies suggest a YAP-inhibitory function other studies indicate Amot is required for YAP activity. Here, we describe an Amot-dependent complex comprised of Amot, YAP and Merlin. The phosphorylation of Amot at Serine 176 shifts localization of this complex to the plasma membrane, where it associates with the tight-junction proteins Pals1/PATJ and E-cadherin. Conversely, hypophosphorylated Amot shifts localization of the complex to the nucleus, where it facilitates the association of YAP and TEAD, induces transcriptional activation of YAP target genes and promotes YAP-dependent cell proliferation. We propose that phosphorylation of AmotDOI:http://dx.doi.org/10.7554/eLife.23966.001 Cells in animals and other multi-cellular organisms need to know when and where they should grow and divide. Individual cells communicate with their surrounding environment and each other via signaling pathways such as the Hippo-YAP pathway, which stimulates cells to grow and therefore influences the size of organs. When the Hippo part of the pathway is active it causes a protein known as YAP to move out of a compartment in the cell called the nucleus. Inside the nucleus, YAP helps to activate genes that promote cell growth. If the Hippo pathway can no longer respond to cues from the environment, YAP becomes over-active and can contribute to the development of various cancers. Therefore researchers are trying to better understand how it is regulated.Many signals both from inside and outside the cell influence YAP activity. For example, some signals block YAP from entering the nucleus, whereas others cause YAP to be broken down entirely. Several studies have recently identified a signal protein called angiomotin as a regulator of YAP. However, the studies provide conflicting reports as to whether angiomotin promotes or inhibits cell growth.Like many other proteins, angiomotin can be tagged with a small molecule called a phosphate group that can alter its activity. Moleirinho, Hoxha et al. studied human cells containing versions of angiomotin that mimic different forms of the protein with or without the phosphate. The experiments indicate that when a phosphate is attached at a particular position (known as serine 176), angiomotin predominantly interacts with YAP and another protein called Merlin at the cell surface. On the other hand, when angiomotin does not have a phosphate attached to it, all three proteins can move into the nucleus, where YAP is able to activate genes and promote cell growth.Overall, these findings indicate that adding a phosphate group to angiomotin can act as a switch to regulate where in the cell it and YAP are found and thus, whether YAP is active. Future experiments will investigate which enzymes add the phosphate group to serine 176, and when they are able to do so.DOI:http://dx.doi.org/10.7554/eLife.23966.002 Angiomotin (Amot) was originally identified as an angiostatin-binding protein involved in the regulation of endothelial cell polarization, migration, proliferation, and angiogenesis . Amot is106LPTY109/239PPxY242 motifs present in the N-terminal domain of Amot-p130 and WW domain present in YAP  mice showed Amot is required for hepatic ductal cell proliferation and tumor formation in the context of either Nf2 loss or DDC -induced injury. Additionally, substantially increased expression of Amot was observed in NF2-null human schwannomas samples, which primarily displayed localization of Amot in the nucleus  as a binding partner of YAP and Merlin . We firsAs previous studies reported multiple cellular localizations for Merlin and YAP, we sought to determine the cellular compartments where YAP and Merlin co-localize. Flag-tagged Merlin and V5-tagged YAP were co-transfected in both HEK293 and hSC2λ cells and the localization and association of these proteins were examined by subcellular fractionation coupled to IP with an anti-Flag antibody. These subcellular fractionation studies showed an association between Merlin and YAP primarily in the nucleus and cytoplasm, and to a much lesser extent at the plasma membrane . FurtherSince Amot directly binds YAP and Merlin through two distinct domains , 2011, w239PPEY242 and 284PPEY287), and an unconventional LPTY motif (106LPTY109) that mediate the interaction with YAP  or HA-Amot-p130S176E (phosphomimetic mutant) and similar levels of the different Amot alleles were confirmed  . We theronfirmed . IPs weronfirmed . Furtheronfirmed . In addionfirmed . CollectS127A mutant retain their ability to associate with Amot/Merlin  in these cells compared to control 293-shAmot cells. As expected, transfection with the different Amot alleles resulted in increased Rac1-GTP levels. However, we found no significant differences in Rac1-GTP levels between the cells, suggesting the status of Amot serine 176 phosphorylation does not affect Rac1 activation  and ApoE (Apolipoprotein E) as previous findings suggest Amot plays a role in their regulation  is recruited to the plasma membrane where it is found co-localized and associated with Patj, Pals1 and E-cadherin at junctional structures. Our previous studies indicate that Amot is required for YAP function in the nucleus (S176A) is preferentially localized to the nucleus, where it facilitates YAP interactions with TEADs and concomitant activation of target gene transcription.The role of Angiomotin regulating the Hippo/YAP pathway has so far been elusive, mainly due to conflicting reports suggesting that YAP regulation by Angiomotin is either positive or negative. To gain a deeper understanding of Amot function, we employed multiple cell types originating from tissues in which dysregulation of the Hippo-YAP pathway is observed under pathological conditions. Our study reveals that Angiomotin phosphorylation at serine 176 mediates YAP localization. This post-translational modification is the key event determining a promoting or repressing regulation of YAP by Angiomotin. We found that phosphorylation at serine 176 does not mediate Angiomotin’s scaffolding functions but does impact the localization of a tertiary complex composed by Amot, YAP and Merlin, which is present in both the cytoplasm and nucleus for the wild type protein. The serine 176 phosphomimetic mutant . The following plasmids were previously described: psCMV-Pals1-Flag; PATJ-Flag . All experiments were carried out on cells grown to 70–80% confluency. Transfections were performed using Lipofectamine 2000  unless stated otherwise. Lentiviral infection of HEK293 cells was performed according to standard protocols. Briefly, HEK293T cells were co-transfected with packaging plasmids VSVG, Δ8.2, and either with pLKO.1-GFP or pLKO.1-shAmot constructs. Supernatants were collected 48 hr and 72 hr after transfection, and cells were infected with 4 mL of viral supernatant containing 4 mL of polybrene (8 μg/mL). After 48 hr, transduced cells were selected with puromycin (2 μg/mL) and this selection maintained for 72 hr.HEK293 and HepG2 cells were purchased from the ATCC. hSC2λ cells were obtained from the laboratory of Dr. Margret Wallace . All cel+/K+ATPase  from Developmental Studies Hybridoma Bank (University of Iowa). For immunofluorescence the following antibodies were used at the stated concentrations: rabbit monoclonal anti-YAP D8H1X , Cell Signaling Technologies; mouse monoclonal anti-Merlin , Santa Cruz Biotechnologies; Rabbit polyclonal anti-Angiomotin (1: 200).Rabbit polyclonal anti-Angiomotin was previously described  . Eluted proteins were separated on SDS/10% polyacrylamide gel and transferred onto Immobilon-P membranes (Millipore). To prevent nonspecific binding, membranes were incubated in blocking buffer  with agitation for 1 hr at room temperature, followed by immediate incubation with specific antibodies diluted in either 5% BSA or blocking buffer, overnight. Membranes were then washed three times in washing buffer , incubated for 1 hr at room temperature with goat anti-mouse HRP-conjugated antibody  or Protein A-HRP linked  and protein expression was detected by chemiluminescence using ECL .150 cm diameter dishes were transiently transfected with 10 μg of plasmid DNAs using Lipofectamine 2000 (Invitrogen). 48 hr later cell lysates were collected, washed twice in ice-cold PBS, and lysed with radioimmuno precipitation assay buffer ). 1 mg of protein was immunoprecipitated with Protein A/G resins  and indicated antibody with gentle rotation at 4Cells were collected and lysed with actin stabilization buffer . Biotinylated-phalloidin  was added to samples and incubated for 60’ at 4°C with constant rotation. Subsequently, 20 µL of streptavidin-coupled magnetic Dynabeads were added, incubated for 60’ at 4°C with constant rotation. The magnetic beads were isolated with a magnet, washed 3X with PBS and analyzed as described.Immunofluorescence staining was carried out as previously described . Briefly2, 1 mM PMSF, 1 mM DTT, 0.5%Nonidet P-40, protease inhibitor mixture), centrifuged at 4000 g for 5’ at 4°C. Supernatant was kept for the cytosolic and plasma membrane fractions. The pellet was washed five times with nuclear washing buffer , lysed on ice for 20 min in 400 μl of RIPA buffer and centrifuged at 13,000 g for 10 min at 4°C. Supernatant was kept as the nuclear fraction. For the cytosolic and plasma membrane fractions the supernatant was spun at 200,000 g for 30 min at 4°C and the resultant supernatant centrifuged again at 13,000g for 5 min at 4°C and kept as the cytosolic fraction. The pellet was washed in 500 μl lysis buffer  and re-sedimented at 200,000g for 30 min at 4°C. The pellet was then resuspended in ice-cold IP buffer  Triton X-100, and complete protease inhibitor mixture), incubated on a rocker for 30 min at 4°C, and cleared by centrifugation at 13,000g for 15 min at 4°C. The supernatant was kept as the plasma membrane fraction.Cells were incubated on ice for 20 min in ice-cold cytoplasmic buffer  and 48 hr later lysed in RIPA buffer and precipitated with indicated antibody overnight. Rac1-GTP was pulled down according to the manufacturer’s instructions .HEK293-shAmot cells were transfected with 8 μg of plasmid DNA (empty vector control/ wild type Amot-p130/Amot5 cells per well in 6-well plates. On the following day cells were co-transfected with 40 nM of siRNAs (targeting either YAP or a non-targeting siRNA duplex (control)), and 2 μg of plasmid DNA (empty vector control/ wild type Amot-p130/AmotS176A/AmotS176E) using TransIT-LT1 and TransIT-TKO  according to manufacturer’s instructions. After 24 hr, cells were trypsinized and 2 × 104 cells/well were reseeded in sterile 96-well tissue culture plates. 48 hr, 72 hr, and 96 hr after co-transfection, plates were processed and analysed using BrdU cell proliferation assay kit following manufacturer’s instructions . Plates were read using a spectrophotometer microplate reader set to 450 nm .HEK293-shAmot cells were seeded at a cell density of 2 × 103 cells/50 μl in 96-well plates. On the following day the indicated luciferase reporters and plasmids were transfected to a total of 160 ng and incubated overnight at 37°C. Luciferase activity was then measured with Dual Luciferase Reporter Assay System  according to manufacturer’s instructions. The reporter’s firefly luciferase activity was normalized to the levels of Renilla luciferase used as an internal control reporter. The relative luciferase activity displayed on the Y-axis indicates the ratio between Firefly/Renilla luciferase activities.The HIP/HOP-flash luciferase reporter system was a kind gift from Dr. Barry Gumbiner  and the −ΔΔCT algorithm (Extraction of RNA from cell lysates was performed using Qiagen RNeasy kit (Qiagen) followed by cDNA synthesis of 1 μg DNase-digested RNA, using SuperScript III First-Strand Synthesis System for quantitative RT–PCR (Invitrogen) according to manufacturer’s instructions. Quantitative PCR of the synthesized cDNA was conducted using SYBR Green 2x Master Mix (Applied Biosystems) according to the manufacturer’s protocol. 10 ng of each sample were used in each analysis. Real-time quantitative RT-PCR reactions were performed on StepOnePlus Real-Time PCR System (Applied Biosystems) and analysed using StepOne Software v2.2.2. All measurements were conducted three times in triplicate and standardized to the levels of GADPH. Relative changes in gene expression were calculated according to the 2lgorithm . Sequenc20 million HEK293T-shAmot cells were fixed with 1% formaldehyde for 10 min at RT. Fixation was halted with 125 mM glycine for 5 min at RT. Fixed cells were washed twice with cold PBS. Cell pellets were then resuspended in ChIP lysis buffer and chromatin was sheared with sonicator to obtain 0.3–0.5 kb DNA fragments. Angiomotin antibody (5 μg) and Dynabeads Protein A were added to the cell lysate and incubated overnight at 4°C. Beads were washed with buffer 1  and then washed with TE buffer. DNA was eluted by resuspending the beads in TE/1%SDS. ChIP DNA and Input were treated with RNase A (5 μg) for 1 hr at 37°C. Proteinase K (0.5 mg/mL) was added and incubated overnight at 65°C to reverse crosslinking. DNA was then purified phenol:chloroform and resuspended in a 30 μL of elution buffer. DNA was used for real time-PCR using SYBR Green PCR kit. A standard dilution curve was obtained for each Input and 1 μL of ChIP DNA was used in each PCR reaction. Melt curves were analyzed to confirm specificity of the amplified target. In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Anna Akhmanova (Senior Editor) and three reviewers, one of whom is a member of our Board of Reviewing Editors. The reviewers have opted to remain anonymous.Thank you for submitting your article "Regulation of localization and function of the transcriptional co-activator YAP by angiomotin" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.In this manuscript, the authors demonstrate that Amot acts as a scaffold to recruit YAP to Merlin, and that the formation of this complex is not affected by the phosphorylation of Amot at S176. Interestingly, however, the mutation of S176 to E induces a clear relocalization of Amot from the nucleus to the plasma membrane and the cytosolic fraction , while mutation to A result in strong nuclear signal. This is accompanied by a redistribution of YAP and Merlin (and in consequence of the complex) to the plasma membrane in the S176E cells, perhaps through augmented association with Pals1/Patj. The consequence of the phosphorylation status of Amot on proliferation / YAP activity was investigated, revealing strong activity of the S to A mutant in all assays, and inactivity of the S176E mutant. It provides evidence that AMOT facilitates YAP nuclear and transcriptional function, unless phosphorylated at ser 176, which facilitates AMOT interaction with plasma membrane proteins. The authors’ findings disagree with some previous conclusions about the role of AMOT, which they address well, and which makes the finding of importance.All reviewers thought that this work was novel and interesting. However additional controls are considered necessary to demonstrate some of the reagents are specific, quantification is needed, and some of the conclusions should be moderated or caveats considered in the Discussion.1) What is the expression level of the ectopically expressed Amot, compared to that endogenously expressed in HEK cells? Almost the entire analysis depends on exogenous expression of AMOT and AMOT mutants. It is well known that overexpression of proteins often leads to their accumulation in inappropriate compartments. Since so much of the conclusions and model depend on the idea of compartments and pools, it is essential that the authors ensure that their proteins are expressed at normal cellular levels, and that all mutants are expressed at similar levels2) Ectopically expressed PATJ and PALS1 co-Ips with Amot and AmotS>E, but not AmotS>A. This may be due to Amot localization at the membrane versus nucleus. Is association of Amot with Patj/PALS1 really needed for the membrane localization of the complex? The authors should knockdown Patj/PALS1  to support this conclusion.3) Could the interaction (or non-interaction) of the Amot constructs with Patj and Pals1 be only a reflection of the differential proportion of the Amot constructs in the plasma membrane fraction where Patj and Pals1 reside? . As presented, this set of experiments is supportive of the differential localization, but does not necessarily demonstrate a direct role for association with Patj/Pals1 . Is the association with Patj/Pals1 bringing Amot to the cortex? If more direct evidence for a preferential association of Amot S176E to Patj/Pals1 cannot be experimentally determined, the authors may want to revise the presentation of the associated results and conclusions.Specific comments/suggestions to address in the Discussion:4) Is it possible that overexpressed protein does not reveal a change in affinity upon altered phosphorylation?5) The authors should discuss the 5A YAP results. Do they believe this can be explained by increased nuclear localization of 5AYAP?6) The authors ignore the role of Lats, which is also thought to bind to AMOT, and the role of its phosphorylation of YAP in YAP regulation. Most studies on the hippo pathway agree that this phosphorylation event controls both the levels and nuclear-cytoplasmic distribution of YAP, but these two properties are not addressed. The authors do show that phosphorylation of YAP on ser 127 does not affect binding to AMOT, but do not show how AMOT regulates YAP phosphorylation or stability. Also, they do in fact see a change in binding of 5 ser > ala YAP mutations, which have been shown to regulate YAP function in addition to s127. This difference must be considered.7) There is no consideration of any upstream regulation of the pathway in either the experimental analysis or in the evaluation of the models and literature. What controls AMOT phosphorylation? Are Mst/hippo kinases involved in AMOT regulation? Are the authors imagining that this regulation has something to do with cell-cell contact control of the pathway? Or control by soluble growth factors and signaling receptors? In the absence of this broader context, it is very difficult to think about mechanisms and models. At the least this should be touched on in the Discussion.8) Their model implies a constitutive binding of YAP to AMOT in the control YAP localization by AMOT, without considering catalytic effects, especially those that might be conferred by Lats phosphorylation. Similar to other models, it invokes simple sequestration at the membrane via AMOT-PATJ binding to restrain YAP. Yet only a small fraction of S176 AMOT seems to associate with membranes or PATJ. Similarly, the model implies that the cytoplasmic and nuclear pools of YAP are all bound to AMOT. If a large fraction of YAP in the cytosol or nucleus is free, different mechanisms from the one proposed must be involved in controlling its localization.9) The authors discuss previous studies showing that AMOT interacts with the actin cytoskeleton and that this interaction is regulated by AMOT phosphorylation. However, they do not address this potentially important pool of AMOT in their study. How does the actin associated pool fractionate in their protocol – with cytoplasmic, nuclear, or membrane fractions?10) 11) Others have reported that cadherins can also interact with merlin-AMOT complexes at the membrane in addition to PATJ. Do the authors think that this may also be a significant interaction worth examining?12) The interpretation that the interaction between YAP and Merlin is predominantly occurring in the nucleus and the cytoplasm is supported by the data, but quantification of these experiments would help providing an assessment as to whether this simply reflect the relative levels of YAP across the different fractions or whether there is evidence for a truly differential interaction across fractions. . Perhaps an alternative way to provide support for a compartment-specific interaction would be to employ a fluorescence-based approach .13) The presentation of the western blots (IP/western) should be accompanied by quantification. I was surprised that experiments that I would assume should be loaded on the same SDS page gel (e.g. different constructs for the rescue experiments in 14) What is the intracellular localization of endogenous Merlin (and YAP)? The localization experiments currently presented all depend upon transient transfection.15) For 16) If the phosphospecific antibody works in IF, it would be nice to provide additional evidence that the phosphorylated form is enriched in the plasma membrane fraction . This would support the results obtained with the S to E mutation.17) What is the relative level of expression (in relation to the endogenous) of the different Amot constructs? Similarly, the authors may want to comment on the expression levels of the other constructs they used in relation to the respective endogenous proteins. Could some of the results observed due to overexpression / tagging artifacts?18) Concerning the 19) Other comments: – this likely happened through PDF generation during submission, but the quality of the images is problematic. Labels do not display clearly, and microscopy images are extremely difficult to evaluate. The authors need to provide high quality images with the revision. […] All reviewers thought that this work was novel and interesting. However additional controls are considered necessary to demonstrate some of the reagents are specific, quantification is needed, and some of the conclusions should be moderated or caveats considered in the Discussion.1) What is the expression level of the ectopically expressed Amot, compared to that endogenously expressed in HEK cells? Almost the entire analysis depends on exogenous expression of AMOT and AMOT mutants. It is well known that overexpression of proteins often leads to their accumulation in inappropriate compartments. Since so much of the conclusions and model depend on the idea of compartments and pools, it is essential that the authors ensure that their proteins are expressed at normal cellular levels, and that all mutants are expressed at similar levelsWe agree with the reviewers’ comments. Assessment of expression of the different Amot alleles and comparison to endogenous Amot levels are now included in 2) Ectopically expressed PATJ and PALS1 co-Ips with Amot and AmotS>E, but not AmotS>A. This may be due to Amot localization at the membrane versus nucleus. Is association of Amot with Patj/PALS1 really needed for the membrane localization of the complex? The authors should knockdown Patj/PALS1  to support this conclusion.In spite our extensive attempts, using several different shRNA/siRNAs, we were unable to achieve efficient knockdown of PATJ/PALS1 and thus unable to assess whether the membrane localization of AmotS>E is changed upon loss of these proteins. In addition, while responding to other review comments, we are able to detect enhanced association of the AmotS>E mutant with E-cadherin . Therefo3) Could the interaction (or non-interaction) of the Amot constructs with Patj and Pals1 be only a reflection of the differential proportion of the Amot constructs in the plasma membrane fraction where Patj and Pals1 reside? . As presented, this set of experiments is supportive of the differential localization, but does not necessarily demonstrate a direct role for association with Patj/Pals1 . Is the association with Patj/Pals1 bringing Amot to the cortex? If more direct evidence for a preferential association of Amot S176E to Patj/Pals1 cannot be experimentally determined, the authors may want to revise the presentation of the associated results and conclusions.Please see our response to question 2.Specific comments/suggestions to address in the Discussion:4) Is it possible that overexpressed protein does not reveal a change in affinity upon altered phosphorylation?While this is a possibility, we believe the likelihood for this is low. If one hypothesizes that phosphorylation at S176 has an impact on the affinity of Amot towards merlin or YAP, it would be expected that the S176A or S176E mutants would have different effects on affinity and association with their binding partners, which is not what we observed in our studies.5) The authors should discuss the 5A YAP results. Do they believe this can be explained by increased nuclear localization of 5AYAP?The literature indicates the S5A mutant is preferentially localized to the nucleus. The experiment shown in 6) The authors ignore the role of Lats, which is also thought to bind to AMOT, and the role of its phosphorylation of YAP in YAP regulation. Most studies on the hippo pathway agree that this phosphorylation event controls both the levels and nuclear-cytoplasmic distribution of YAP, but these two properties are not addressed. The authors do show that phosphorylation of YAP on ser 127 does not affect binding to AMOT, but do not show how AMOT regulates YAP phosphorylation or stability. Also, they do in fact see a change in binding of 5 ser > ala YAP mutations, which have been shown to regulate YAP function in addition to s127. This difference must be considered.Although we did briefly discuss the role of Lats in the paper, the role of Lats is complicated and not straightforward. Indeed Lats1/2 phosphorylate and regulate YAP localization and levels and a number of studies have shown Lats regulates phosphorylation of Amot on S176. However, the relationship between Lats, YAP and Angiomotin is complex. For example, a number of papers suggest that Lats phosphorylates Amot, leading to reduced binding to F-actin and increased YAP binding and inhibition . Moreover, Amot has been proposed to activate Lats2 and increase phosphorylation of YAP . In contrast, we have previously shown that in multiple systems, Amot antagonizes the association of Lats and YAP and inhibits phosphorylation of YAP by Lats . Given the complexity of the interactions between YAP, Amot and YAP, we believe this question requires a more extensive and in depth analysis which is beyond the scope of the current manuscript. Some of the issues raised in our response are also addressed in the Discussion of the manuscript.7) There is no consideration of any upstream regulation of the pathway in either the experimental analysis or in the evaluation of the models and literature. What controls AMOT phosphorylation? Are Mst/hippo kinases involved in AMOT regulation? Are the authors imagining that this regulation has something to do with cell-cell contact control of the pathway? Or control by soluble growth factors and signaling receptors? In the absence of this broader context, it is very difficult to think about mechanisms and models. At the least this should be touched on in the Discussion.We agree with this point. We initially left this out due to space considerations. In particular, while are many possible scenarios involving regulation of Amot activity by upstream events, since S176 has been shown to be phosphorylated by Lats, we can speculate that many of the upstream events regulating the Hippo cascade will also regulate Amot. We now include this in the Discussion.8) Their model implies a constitutive binding of YAP to AMOT in the control YAP localization by AMOT, without considering catalytic effects, especially those that might be conferred by Lats phosphorylation. Similar to other models, it invokes simple sequestration at the membrane via AMOT-PATJ binding to restrain YAP. Yet only a small fraction of S176 AMOT seems to associate with membranes or PATJ. Similarly, the model implies that the cytoplasmic and nuclear pools of YAP are all bound to AMOT. If a large fraction of YAP in the cytosol or nucleus is free, different mechanisms from the one proposed must be involved in controlling its localization.We agree that examining the dynamics of the association of the complex would be useful, however, given space and time limitations we did not yet get to do these types of analyses.We agree that a fraction of Amot-S176E appears to be localized to the PM, while additional protein is found in the cytoplasm and none in the nucleus (see example in 9) The authors discuss previous studies showing that AMOT interacts with the actin cytoskeleton and that this interaction is regulated by AMOT phosphorylation. However, they do not address this potentially important pool of AMOT in their study. How does the actin associated pool fractionate in their protocol – with cytoplasmic, nuclear, or membrane fractions?We thank the reviewer for this comment. In the revised version of the manuscript we include data assessing the association of the different Amot alleles with F-actin. While most of the other reports relied on co-localization by IF and in vitro studies using purified protein, we wished to directly assess the interaction of Amot with F-actin in cells. To achieve this, we used biotin-XX-phalloidin which preferentially binds F-actin. We then precipitated the F-actin from cells transfected with the different Amot mutants and assessed the levels of Amot that were co-precipitated. This analysis shows that there are no differences in the association of the different mutants with F-actin. While our findings contrast with some of the previous reports regarding the association of Amot with F-actin, we believe that the approach we employed directly assess the interaction between F-actin and Amot, while other reported efforts employed methods that are either indirect (IF) or in vitro. We include this new data in 10) We thank the reviewer for catching this. We now provide a revised figure in which the levels of IPed PATJ are similar between the different conditions.11) Others have reported that cadherins can also interact with merlin-AMOT complexes at the membrane in addition to PATJ. Do the authors think that this may also be a significant interaction worth examining?We thank the reviewer for this comment. We indeed examined this and find that Amot can co-IP with E-cadherin. Moreover, as in the case of PATJ/Pals1 the association is enhanced with the Amot-S176E mutant. We include this new information in 12) The interpretation that the interaction between YAP and Merlin is predominantly occurring in the nucleus and the cytoplasm is supported by the data, but quantification of these experiments would help providing an assessment as to whether this simply reflect the relative levels of YAP across the different fractions or whether there is evidence for a truly differential interaction across fractions. . Perhaps an alternative way to provide support for a compartment-specific interaction would be to employ a fluorescence-based approach .As noted by the reviewer, the data support the formation of the Merlin/YAP/Amot complex predominantly in the cytoplasm and nucleus  and this13) The presentation of the western blots (IP/western) should be accompanied by quantification. I was surprised that experiments that I would assume should be loaded on the same SDS page gel  What is the intracellular localization of endogenous Merlin (and YAP)? The localization experiments currently presented all depend upon transient transfection.The localization of endogenous Merlin and Amot can be seen in the IF experiments shown in 15) For We provide additional support in 16) If the phosphospecific antibody works in IF, it would be nice to provide additional evidence that the phosphorylated form is enriched in the plasma membrane fraction . This would support the results obtained with the S to E mutation.Unfortunately, we were unable to optimize the antibody for IF and therefore unable to carry out this experiment.17) What is the relative level of expression (in relation to the endogenous) of the different Amot constructs? Similarly, the authors may want to comment on the expression levels of the other constructs they used in relation to the respective endogenous proteins. Could some of the results observed due to overexpression / tagging artifacts?Please see our response to question 1.18) Concerning the Please see our response to question 5.19) Other comments: – this likely happened through PDF generation during submission, but the quality of the images is problematic. Labels do not display clearly, and microscopy images are extremely difficult to evaluate. The authors need to provide high quality images with the revision.Thank you for point this out. We redid the images at a higher resolution.


Allergic asthma is an inflammatory disorder of the airways that results from inappropriate production of IgE against harmless, environmental antigens. Sequestration of free IgE using humanized IgG anti-IgE is an effective therapy for asthma and other atopic disorders. However, the status of free IgE in subjects who have naturally developed immune tolerance to inhaled antigens has not been well studied.C57BL/6 mice were sensitized and challenged with ovalbumin (OVA) for 7 days to induce allergic airway disease (AAD) or 6 weeks to induce a state of local inhalational tolerance (LIT). Serum from AAD or LIT mice, diluted to achieve equivalent levels of total OVA-specific IgE, was used to sensitize rat basophil leukemia cells for allergen-mediated degranulation. Levels of degranulation were measured in relation to serum concentrations of free IgE and IgG anti-IgE/IgE immune complexes.Serum from AAD animals induced a greater degree of basophil degranulation than serum from LIT animals. These results correlated with higher levels of free IgE in AAD animals, whereas LIT mice demonstrated a significant increase in IgG anti-IgE/IgE immune complexes relative to their diseased counterparts.Sequestration of free IgE by naturally occurring IgG anti-IgE may aid in the development of immune tolerance against inhaled allergens. The decrease in bioavailability of free IgE may, in turn, contribute to the overall reduction of asthma symptoms via a mechanism that mimics the therapeutic effects of humanized IgG anti-IgE. Allergic asthma is an IgE-mediated hypersensitivity response that results from inappropriate immune activation (i.e. lack of immune tolerance) against innocuous environmental allergens . Current+ cells. However, total IgE levels remain elevated or may even increase following initiation of therapy due to the slow clearance of biologically inactive omalizumab-IgE complexes [Previous studies from our laboratory have shown that mice develop allergic airway disease (AAD) after short-term challenge with the model antigen ovalbumin (OVA), whereas long-term challenge results in the development of local inhalational tolerance (LIT) against OVA with subsequent resolution of the disease . Interesomplexes . This leFemale C57BL/6 mice (6–8 per group), 6–8 weeks of age, were purchased from Jackson Laboratory  and were housed and cared for as previously described . All aniThe biologic activity of IgE in serum samples was determined using a rat basophil mediator release assay . RBL-2H31 anti-IgE/IgE immune complexes were determined as previously described [Levels of IgGescribed . For detescribed . Serum sP ≤ 0.05 were used as the threshold for significance. All statistical analysis was performed using the GraphPad Prism  statistical software package.All statistical analyses between groups were made via one-way analysis of variance (ANOVA) followed by Newman–Keuls post hoc test. β-hexosaminidase release data were compared via area under the curve analysis. Data were expressed as mean ± SEM values for all comparisons; values of P < 0.001. OVA-specific IgE levels were virtually undetectable in naïve controls (0.1 ± 0.1 ng/ml). When evaluating the biological activity of the OVA-specific IgE using RBL cells, both AAD and LIT serum led to a greater degree of β-hexosaminidase release than naïve serum; however, the mediator release generated using AAD serum was significantly greater than that observed using LIT serum . This suggested that there was more non-complexed IgE (free IgE) in the serum of AAD mice. To confirm this possibility, free IgE levels were measured in the serum samples using recombinant FcεRIα protein in a method similar to that used to measure free IgE in humans [P < 0.001). However, concentrations of free IgE did not statistically differ between LIT mice and controls , which may account for the decrease in RBL mediator release observed in the latter group.To evaluate a potential cause for the reduced bioactivity of LIT serum, levels of IgGals Fig. a. In conn humans . Naïve aols Fig. b. Moreov1 anti-IgE/IgE immune complexes and a reduction in free IgE. Furthermore, these changes are linked to a diminished capacity of IgE to sensitize FcεRI-expressing cells such as basophils and mast cells. Thus, the importance of IgE may lie less in its absolute concentration and more in its capacity to bind FcεRI (free IgE).Naturally occurring autoantibodies directed against IgE are known to exist in a large proportion of subjects with atopic disease ; however1 anti-IgE/IgE immune complexes in LIT is particularly noteworthy as the efficacy of allergen-specific immunotherapy has been shown to rely partially upon the induction of IgG antibody subclasses that are capable of modulating IgE responses through direct blocking effects as well as through binding to inhibitory FcγRIIb receptors [The formation of IgGeceptors . In parteceptors , and at eceptors . That Igeceptors  contribueceptors  and co-eeceptors .1 anti-IgE/IgE immune complexes. Such changes may lead to a decreased level of free IgE, which reduces the potential for IgE to sensitize FcεRI-expressing cells such as basophils and mast cells. Presumably, such changes in the bioactivity of IgE would reduce overall symptomatology of allergic asthma in a manner that is analogous to the therapeutic administration of humanized IgG anti-IgE.Results from this study suggest that immune tolerance to inhaled allergens may be partially mediated by formation of IgG


Amyloids are polymeric structural states formed from locally or totally unfolded protein chains that permit surface reorganizations, stability enhancements and interaction properties that are absent in the precursor monomers. β-Parvalbumin, the major allergen in fish allergy, forms amyloids that are recognized by IgE in the patient sera, suggesting a yet unknown pathological role for these assemblies. We used Gad m 1 as the fish β-parvalbumin model and a combination of approaches, including peptide arrays, recombinant wt and mutant chains, biophysical characterizations, protease digestions, mass spectrometry, dot-blot and ELISA assays to gain insights into the role of amyloids in the IgE interaction. We found that Gad m 1 immunoreactive regions behave as sequence-dependent conformational epitopes that provide a 1000-fold increase in affinity and the structural repetitiveness required for optimal IgE binding and cross-linking upon folding into amyloids. These findings support the amyloid state as a key entity in type I food allergy. Amyloids are polymeric states of proteins defined by a cross-β sheet backbone, in which β-sheets that are mostly formed from β-strands of different molecules pack through their side chains12323562+-binding motifs12131415912171819Baltic cod (Gad c 1)21222324Atlantic cod (Gad m 1)Common carp (Cyp c 1)10272829Chub mackerel (Sco j 1)32262+ binding sites by mutagenesis in both Cyp c 1 and Sco j 1 resulted in soluble chains with reduced IgE-binding signals3034Fish β-parvalbumins represent the major allergens of IgE-mediated fish hypersensitive patients8910Here, we have addressed the role of Gad m 1 amyloid with respect to both its IgE-reactive properties and epitope architecture. Using surface-bound peptide arrays of Gad m 1, sera from fish allergic patients, recombinant wt and mutant Gad m 1 (rGad m 1) chains and biophysical approaches, we found that the relevant antigenic regions of the Gad m 1 chain fold into amyloids and, by doing so, optimize the IgE interaction.We first characterized the sequence elements to address how amyloid formation contributes to the IgE reactivity of Gad m 1. A set of 12-mer overlapping synthetic peptides with an offset of 2 that correspond to the sequence of Gad m 1 was used in an array-based immunoassay . This pe2+-binding loop of the EF domain (residues 88–96) and of the C-terminal (residues 95–109) regions was not detected by these sera, as probed by both IgE and IgG4 binding 373812+-bound folds with predominant α-helical secondary structures and a highly stable cooperative fold with a denaturation temperature of approximately 85 °C , which are flanked and overlapped by sequences with amyloid-forming capacity . We modi4ly 85 °C . These dact fold .2+, the fluorescence of ThT remains unaltered2+ remained unaltered for at least 70 h  resulted in I50 values of approximately 10−3.6 M. Using I50 as the apparent dissociation constant (KDapp), these values then yield KDapp of 4 × 10−7 M and 2 × 10−4 M for the amyloid and monomer forms, respectively. These data reveal that amyloid assembly provokes a 1000-fold enhancement of IgE affinity. On the other hand, using the dimensions of the rGad m 1 fibrils from the AFM images   , the RH urements  and by aDapp of the sera IgE binding to monomers and amyloids cannot simply be explained in terms of the polymer structural repetitiveness. We assayed the effect of sera on amyloid formation to analyze whether amyloid formation sculpts the architecture of nonnative epitopes, accounting for the high IgE affinity. The differences observed in the KWe took advantage of the protease resistance of amyloids to isolate and identify the assembly core and strengthen the observed functional overlapDespite the large number of sequences and 3D structures of allergens elucidated in the last few decades, the knowledge of how allergens recognize and cross-link cell-bound IgEs is still limited41263243364546474849364546474849The search for regions coding Gad m 1 amyloid assembly showed a complex chain organization. On the one hand, the ZipperDB algorithm putatively identified three regions  with favorable energetic fits for steric zipper formation. However, of these regions, only A and E exhibit OC-antibody binding as peptides. According to the ThT binding kinetics, region A plays a role in the initial steps of the polymerization process  with an outcome that is dependent on the sequences of the C and E regions. On the other hand, the regions identified as the two major IgE linear epitopes that formed part of pepsin-resistant fragments and displayed OC binding properties escaped the ZipperDB prediction analysis. The failure of the ZipperDB prediction suggests that these segments form cross-β spines that are different than the model steric zipper used as a reference in the algorithm. In fact, spines consisting of parallel β-turns resulting from β-hairpins that are distinct from the steric zipper model have been described for other amyloids122+-binding dependence of β-parvalbumins-IgE interaction30362+ removal is highly dependent on the chain sequence, as amyloid formation was triggered in wt and the ACE mutant, but not in the A mutant. Therefore, these data indicate that not all apo forms will form amyloids. Second, the hypoallergenic mrCyp c 1 with the two mutated Ca2+ binding sites exhibits a highly stable native fold that argues against its capacity to polymerize2+-binding properties, suggests that amyloids could also form in the presence of Ca2+ as a function of the chain sequence53Gad m 1 amyloids are formed from the apo form and their IgE reactivity argues against the well-established CaThe finding of amyloid folds for Gad m 1 IgE epitopes agrees with the unusual structural features of the Bos d 5/Fab immunocomplex455Dapp (mM range). The weakness of this interaction agrees with the loose contacts observed in the Bos d 5/Fab immunocomplex structureDapp in the μM range, revealing a 1000-fold enhancement of the interaction strength compared to the monomer. This tightening of the interaction can be explained by several factors. From a structural point of view, the folding of the epitope into a cross-β sheet may optimize the number and nature of contacts for optimal antibody recognition. From the stoichiometry point of view, the polymer nature of amyloids permits the saturation of the two IgE binding arms, whereas a monomer does not permit saturation. Moreover, binding of the first IgE arm consists of an intermolecular reaction, whereas the occupancy of the second site involves an intramolecular reaction.Both Gad m 1 monomers and amyloids bind IgE, but with significantly different affinities. The binding of sera IgE to monomers features a high KOn the other hand, allergens require the existence of at least two IgE epitopes for their deleterious IgE cross-linking on the surface of effector cells. This requirement presumes the development of either two different IgE molecules for monomeric allergens or single IgE molecules for an oligomeric allergen8The phase transition accompanying amyloid formation has also major implications in allergy diagnosis. Clearing amyloids  from solutions used for skin prick tests may result in false negatives compared with non-risk-free oral food challenges36Approval from the Ethics Committee (PI1950) of Hospital Universitario La Paz  was obtained. Parents signed written informed consent, and in the case of children aged 12 or older, assent from the children was also obtained. All methods were performed in accordance with the guidelines of the Hospital Universitario La Paz.TM (ThermoFisher)  from the Hospital Universitario La Paz with specific IgE antibodies to cod parvalbumin were selected. All patients had a history of symptoms suggestive of immediate hypersensitivity elicited by eating fish, with positive skin prick tests to fish extracts: cod and tuna ; swordfish and salmon ; hake  and megrim , as well as specific IgE antibodies to fish, as determined by the CAP-System FEIAoFisher) .Atlantic cod parvalbumin A518742+-affinity chromatography , followed by Q-Sepharose chromatography 2g for 20 min at 4 °C to remove any insoluble material. Protein concentrations were determined using the Bradford protein assay (Bio-Rad) calibrated with BSA . Aβ42 was obtained from GenScript and used as previously describedrGad m 1 (recombinant Gad m 1) was produced from a pET15b construct containing the synthetic ORF of Dodecapeptides spanning the whole sequence of Gad m 1, with an overlap of ten residues, were solid-phase synthesized and immobilized (≈10 nmol per spot) on an Amino-PEG500-UC540 sheet at the National Centre for Biotechnology . Before use, the membranes were rinsed with ethanol, washed three times with TBS , and incubated in TBS containing 1% BSA (w/v) and 2 mM EDTA for 1 h. The membranes were then probed for 2 h with sera from patients who are allergic to fish (1/10 dilution) and the anti-amyloid fibril OC antibody , prepared in TBST (TBS containing 0.05% Tween-20) supplemented with 0.5% BSA (w/v) or with rGad m 1 chains (2.5 μM) in TBS. After extensive washes with TBST, a 30 min incubation with either horseradish peroxidase-labeled anti-human IgE , anti-human IgG4 , goat anti-rabbit IgG  or anti-6X His tag® antibody  was performed. The signal was developed with the ECL-Western-blotting reagent (Bio-Rad) and detected with a ChemiDoc XRS instrument2. Thermal denaturation experiments were performed to follow the ellipticity changes at 222 nm upon heating from 15 °C to 90 °C at a 1 degree/min heating rate. Both the spectra and thermal unfolding curves were analyzed as previously described36Circular dichroism (CD) experiments were performed using a Jasco J-820 spectropolarimeter equipped with a Peltier-controlled thermostatted cell holder. Far UV CD spectra were recorded for a 25 μM protein concentration in 50 mM Tris-HCl, pH 7.5, supplemented with either 1 mM EDTA or 1 mM CaClH) and mass proportions (%) of the species were derived as previously described36HT for spherical rGad m 1 was calculated as 1.81 nm using 0.73 cm−1 and 0.35 g of H2O (g protein)−1 for the particle-specific volume and hydration36Dynamic light scattering (DLS) measurements were performed using a DynaPro spectroscatter (Wyatt Technology) with a 1.5-mm path length and a 12 μl quartz cuvette. The average of 20–25 acquisitions of buffers and protein solutions (160 μM protein concentrations) were filtered using a 0.2 μm Whatman Anodisc-3 filter. The hydrodynamic radii (R2. The kinetics of thioflavin T (ThT) binding was monitored by bottom reading the fluorescence intensity in a POLARstar microplate reader (BMG Labtech), as previously described3657Max Beckman ultracentrifuge. The resulting pellet and supernatant fractions were used for the protein and ThT determinations.The amyloid propensity and location of the segments in β-parvalbumin chains with adhesive properties was theoretically evaluated using the ZipperDB algorithm, as previously described2O, and dried with a stream of N2. AFM images were recorded in a MultiMode Veeco microscope and analyzed using WSxM (Nanotec), as previously described36rGad m 1 wt fibrils were deposited onto freshly cleaved mica surfaces for 10 min, washed with HThe immunoreactivity of the rGad m 1 wt and mutant species was assessed by dot-blot analysis using the anti-amyloid fibril OC antibody  and sera from patients who are allergic to fish (1/10 dilution). Briefly, aliquots containing 50–100 ng of protein in the different states were spotted in duplicate on a nitrocellulose membrane. Immunodetection was performed by incubating the membranes with the primary antibodies for 1 h, followed by extensive washes and 30 min incubation with horseradish peroxidase-labeled either mouse monoclonal B3102E8 anti-human IgE  or goat anti-rabbit IgG . The signal was developed using the ECL-Western-blotting reagent (Bio-Rad) and detected with a ChemiDoc XRS instrument2SO4 and the optical density (OD) was measured at 450 nm using a microplate reader (Bio-Rad 3550). The assays were performed in duplicate using blocking buffer as negative control and were statistically analyzed and fitted using Origin software. A parallel analysis using the Bradford protein assay (Bio-Rad) indicated similar extent of adsorption for all proteins used.Polystyrene 96-well plates (Costar 3590SA) were coated with 100 μl of rGad m1 chains in 0.2 M carbonate buffer, pH 9.4, by varying the concentrations from 0.1–500 μg/ml for 2 h at 37 °C. The coated wells were blocked with 1% BSA in TBS for 30 min and then incubated with 100 μl of a 1:10 dilution of the patients’ serum in TBST containing 1% BSA for 2 h at room temperature. After washing with TBST, the wells were incubated with peroxidase-labeled anti human IgE  for 1.5 h at room temperature. The plates were washed again and then developed with 100 μl of TMB-turbo ELISA substrate (Thermo Scientific). The reaction was stopped after 30 min with 10 μl of 2N HTypically, 50 μl of rGad m 1 fibrils (160 μM) were digested in 10 mM Gly-HCl, pH 1.3, with pepsin (1:50 w:w protease/substrate) at 37 °C for 1 h. The reactions were stopped by the addition of 1 M Tris-HCl, pH 8. After disaggregation with hexafluorisopropanol (HIFP), the hydrolyzed products were analyzed by mass spectrometry using a CHCA matrix and a MALDI-TOF/TOF 4800  for identification and HPLC  coupled to a Triple TOF 5600 (SCIEX) for fragmentation at the CNB Proteomics Facility .How to cite this article: Sánchez, R. et al. The amyloid fold of Gad m 1 epitopes governs IgE binding. Sci. Rep.6, 32801; doi: 10.1038/srep32801 (2016).


This unsupervised learning method recognizes lattice sites, is able to give the site type, and can identify Li hopping events. Results show that, while the cubic LLZO has a much higher hopping rate compared to its tetragonal counterpart, most of the Li hops in the cubic LLZO do not contribute to the diffusivity due to the dominance of back-and-forth type jumps. The hopping analysis and local Li configuration statistics give evidence that Li diffusivity in cubic LLZO is limited by the low vacancy concentration. The hopping statistics also shows uncorrelated Poisson-like diffusion for Li in the cubic LLZO, and correlated diffusion for Li in the tetragonal LLZO in the temporal scale. Further analysis of the spatio-temporal correlation using site-to-site mutual information confirms the weak site dependence of Li diffusion in the cubic LLZO as the origin for the uncorrelated diffusion. This work puts forward a perspective on combining machine learning and information theory to interpret results of molecular dynamics simulations.Understanding Li diffusion in solid conductors is essential for the next generation Li batteries. Here we show that density-based clustering of the trajectories computed using molecular dynamics simulations helps elucidate the Li diffusion mechanism within the Li Promising candidates include NASICON-type phosphates2/3-xLi3xTiO33ClO7P3S1173N4SiO410GeP2S1212159.6P3S12 and Li9.54Si1.74P1.44S11.7Cl0.3Making new Li batteries with higher capacity, better durability, and safety has been the object of a worldwide research effort since 1990s. In particular, replacing the organic electrolyte with a stable solid alternative has attracted tremendous interests from both industry and academia. Shifting from liquid electrolytes to solid electrolytes in Li battery potentially enables the use of Li metal as the anode, allows the application of high voltage cathodes, extends the lifetime, and greatly improves the safety12xLa3M2O12 7La3Zr2O12 (LLZO)2122232425ab inito molecular dynamics (AIMD), Jalem et al. found synchronous Li movement in the cubic phase and an unstable 24d tetrahedral sites may trigger the hopping events, which eventually contributed to a high Li conductivity, i.e., 10−4 S/cm at 300 Ket al.et al.a priori29303233Within the Li-conducting garnet family, Lidr. We note that by taking dr = 1 Å the density pattern from the simulation matches well its experimental counterpart as obtained from diffractionρ to separate the various spatial regions. As shown in ρ results in connected density, which cannot be used to differentiate the Li sites. On the other hand, when it is too large, the corresponding clusters are too small causing potential loss of information. We suggest that one could choose an intermediate ρ such thatThe DCT method developed in this article is based on the observation that ionic diffusive events in the crystal lattice are relative infrequent relative to the usual simulation scales. In addition, the ions mostly oscillate around their equilibrium lattice site, leading to high density therein as depicted in V0 is the entire domain of the simulation and Vρ is the region with density greater than ρ, i.e., Vρ the core domain and the corresponding voxels the core voxels. The voxels, whose density are nonzero and less than ρ, are named noise voxels. Third, we use a breadth-first search algorithm to find core voxels adjacent to interested core voxels and therefore to identify the voxel clusters. Starting from one core voxel, the breadth-first search method finds all the neighboring core voxels and then for each of them, the neighboring search is repeated until no connected core voxels are further found, see Scheme S1 for illustration. Lastly, in the mapping step, a reverse density calculation that map the density to the particles is performed to assign cluster labels of the voxel to the particles therein.where N atoms and T snapshots then it requires (NT)M calculations for a density map with M voxels. In addition, the nuclear density computation is ubiquitous in molecular dynamics simulations as a way to assist the interpretation of particle transport mechanism37383940The DBSCAN algorithm developed by Kriegel and co-workersThe clustering results on a 2 × 2 × 2 supercell give a total number of 576 clusters  for the cubic LLZO (c-LLZO) in the whole temperature range, and 448 clusters for the tetragonal LLZO (t-LLZO) from 300 K to 900 K and 576 cluster for temperatures greater than 900 K. The change of cluster number of t-LLZO at 900 K is accompanied by Li diffusivity change as t-LLZO starts to be similar to c-LLZO above 900 K, see 04, T13 and O11 as shown in As shown in The DCT allows us to obtain the statistics of the Li jumps, which, as noted above, is naturally encoded in a time series obtained by associating every Li to an identified cluster at a given time step. The hopping rate in c-LLZO is greater than that in t-LLZO, see Djump, from the jump statistics by the following formula29We notice that in c-LLZO, despite the high jump rate, it is highly probable that Li returns to the original site after hopping to the neighboring site as shown in l is the distance between neighboring site centers, calculated from the average Li coordinates within the given center, and ω is the Li hopping frequency. We filter out the back-and-forth Li hops and compute the jump diffusivity Djump. The comparison between Djump and Dself, is shown in where 04 and T13 configurations are the most frequent, Li diffusion is relatively short-ranged because continuously open channels are missing. In other words, the analysis suggests that one bottleneck to higher diffusion rate is the relatively low vacancy concentration. If more vacancies are created, the total number of back-and-forth Li hops could be reduced, thereby resulting in more effective Li diffusional rates. Therefore, it is expected that doping the LLZO with higher valence cations increases the Li vacancy concentration and enhances Li self-diffusion. This has been observed if LLZO is doped with, for example, penta-valent elements Ta5+ and Nb5+ 2344453+ and Ga3+ 47485.3La3Zr2O12.This result also supports our previous conjecture that, since TP(k) of observing k simultaneous jumps within 0.1 ps against the Poisson distribution et al.et al.The intriguing Li jump characteristics calls for a more detailed study, especially on the correlation between Li jumps and Li sites both in time and space. The nature of the temporal stochastic process can be studied by benchmarking the probability In c-LLZO, while the aggregate behavior of Li hopping appears to be qualitatively Poissonian in the temporal scale, studying the site-to-site interaction may provide further spatial scale information. In order to clarify the spatial correlation among Li sites, we calculated the site mutual information for all site combinations as defined by ref. X and Y are the random variables denoting corresponding sites and pij is the probability of the  to be in occupancy state , where i and j could be 0 or 1, e.g., p01 is the probability that site X is unoccupied and site Y is occupied. Detailed explanation is provided in Section S5. We note that such mutual information is the Kullback-Leibler distance of the individual site occupancy distributions, implying that it measures the amount of information into X is contained in Y, i.e., the site dependence. It is important to note that I > 0 indicates dependence between X and Y, and I = 0 if and only if X and Y are independent. Furthermore, the mutual information is positively related to the correlation. In the special case where X and Y are Gaussian random variables, I is related to the bivariate correlation ρb following the formula I, we also computed the delayed mutual information defined as where Iu,d when u = d = 0 corresponds to the entropy. Due to an occupancy close to 0.5 for the Td sites and greater randomness than the Oh sites, see The value of Iu,d for u > 0 and d = 0, measures the autocorrelation of a given X site. As a consequence of occupancy and in spite of considerable site randomness, the Td sites are slightly autocorrelated as shown in Iu,d versus d for fixed u are shown in The autoinformation, that is 11, occurred at a higher probability than others. Surprisingly, this does not mean that the neighboring Oh and Td site are highly correlated. On the contrary, the neighboring sites show decreased correlation in comparison to the Td-Td and Oh-Oh interactions at second nearest neighbors, as evidenced by the low mutual information between Oh and Td in Previous analysis on the site occupancy in Section S3 demonstrated that certain neighboring occupancy patterns, such as OLi diffusion in the cubic and tetragonal phase LLZO is studied by MD simulations. We develop a density-based clustering method to obtain the Li site shape, type and occupation information, and Li jump statistics. The results show that in c-LLZO, the Li occupancy is much higher at the Oh sites than the Td sites, and t-LLZO has an occupancy of 1 for three types of sites. Our study also shows that at RT the Li jumps in c-LLZO consist mostly of a back-and-forth type, and this type of jumps does not contribute to the effective diffusion. In line with the Li occupancy analysis, the reason for the back-and-forth jump is that when Li jump from an Oh to the neighboring empty Td site, the other diffusion paths are mostly blocked by the Li. Therefore, the main obstacle to Li diffusion in LLZO is the low vacancy concentration. Substituting cations with higher valence is an effective strategy to increase the vacancy concentration, as found in experimental observations. In addition, Li hops in c-LLZO are uncorrelated and fit well with the Poisson process while it deviates from the Poisson process in t-LLZO. Lastly, the information theoretic analysis of c-LLZO confirms that the low correlation between sites likely induces a Poissonian type of Li hopping. However, the Li sites still have some short-ranged time and space correlation and this correlation is stronger for the Td sites in comparison to the Oh sites.MD simulations were performed in LAMMPSHow to cite this article: Chen, C. et al. Data mining of molecular dynamics data reveals Li diffusion characteristics in garnet Li7La3Zr2O12. Sci. Rep.7, 40769; doi: 10.1038/srep40769 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In April 2016, southeast Asia experienced surface air temperatures (SATs) that surpassed national records, exacerbated energy consumption, disrupted agriculture and caused severe human discomfort. Here we show using observations and an ensemble of global warming simulations the combined impact of the El Niño/Southern Oscillation (ENSO) phenomenon and long-term warming on regional SAT extremes. We find a robust relationship between ENSO and southeast Asian SATs wherein virtually all April extremes occur during El Niño years. We then quantify the relative contributions of long-term warming and the 2015–16 El Niño to the extreme April 2016 SATs. The results indicate that global warming increases the likelihood of record-breaking April extremes where we estimate that 29% of the 2016 anomaly was caused by warming and 49% by El Niño. These post-Niño Aprils can potentially be anticipated a few months in advance, and thus, help societies prepare for the projected continued increases in extremes. et al. show that all April extremes occur after El Niño years, and that global warming has increased the likelihood of such extremes.Record temperatures in mainland Southeast Asia in April 2016 had severe impacts on the population. Thirumalai  Mainland southeast Asia (MSA) encountered its warmest monthly mean surface air temperatures (SATs) in April 2016 since record-keeping began in the mid-twentieth century1245While it is unmistakable that the Earth is warming globally due to the effect of increasing greenhouse gasesStudies using climate models suggest that tropical land areas, particularly Central Africa, the Maritime Continent and the Indian Ocean rim, will show the most rapid and robust intensification of peak seasonal temperatures, with at least 60% of regional land areas exceeding the late twentieth century maximum around the mid-twenty-first century1011121112131216In this work, we use observations as well as an ensemble of global warming simulations to understand the relationship between long-term warming and natural climate variability in MSA, and their combined impact on extreme SATs. We demonstrate that extreme April SATs in the region occur after the peak of El Niño years and characterize the impact of long-term warming on these events. Finally, we quantify the relative contributions of the El Niño/Southern Oscillation (ENSO) and long-term warming in producing the April 2016 event and demonstrate that with the continued march of ongoing global warming, record-breaking SATs in MSA will occur more frequently in the future.r=0.73) with the Niño-3.4 sea-surface temperature (SST) index, a common metric used to monitor ENSO variability1418Climate in the MSA region exhibits pronounced year-to-year variability that is strongly linked to the ENSO phenomenon7We explored the MSA April-El-Niño link further through a composite analysis of observed SATs during post-Niño Aprils. SAT anomalies from the GISTEMP22These composite analyses reveal that peak seasonal SATs in the MSA region are highly sensitive to El Niño. Previous studies proposed that changes in atmospheric circulation associated with El Niño reduce cloud cover over the land areas of the Maritime Continent and Indian Ocean rim causing the associated warming2024253et al. in prep) including the tropical SST patterns as well as circulation anomalies and teleconnections (see Methods for details on these simulations). The realism of CESM1 allows us to use data from the Large Ensemble, consisting of output from 39 simulations of twentieth and twenty-first century climateThe CESM1-LE and CMIP5 ensemble means simulate remarkably realistic patterns of surface temperature  and clouDo all El Niño events lead to hotter Aprils over MSA? We address this question by analysing the effect of El Niño and La Niña (El Niño 's cooler counterpart) on the distribution of April SATs. In both observations and simulations, after linearly detrending the data sets over their entire length to remove the long-term warming trend, we divided all April monthly mean SATs into distributions of post-El Niño and post-La Niña Aprils. The resulting distributions show that indeed the tails of the April MSA SATs are linked to ENSO variability. Both observations and model data show that post-El Niño Aprils are on average hotter (t-test (P<0.01). The distribution of SATs shifts due to this change in the mean and results in an increase of extreme SATs in the latter subperiod.Next, we looked into the effect of long-term regional warming on the observed and simulated distributions of all April SATs in MSA. Here, we divided the data sets into two equal subperiods and detrended the data over each subperiod . This ensures that the mean warming between the individual time periods is retained for each data set despite differing time periods see also . The lont-test; P<0.01) but statistically indistinguishable variance . Changes in the tails of the distribution caused by shifts in the mean, similar to the ones identified here, are the most straightforward way in which global warming can lead to increased frequency of extremes16f-test; P>0.05). Thus, the mean shift in post-Niño April SATs underpins long-term warming as the most likely cause of the increasing extremes in MSA.Lastly, we performed a similar analysis of only post-Niño Aprils . We findApril=αt+βt2+γT+δ, where April SATs in MSA (SATApril) are regressed upon linear and quadratic components of the regional long-term warming trend18t and t2, respectively), a term proportional to DJF Niño-3.4 SST Index (T) capturing variability correlated with ENSO and a residual term (δ) capturing the effect of other climate variability and weather. This analysis yielded a fit that has a high correlation coefficient (r=0.83) with April temperature observations from 1940 to 2016  from the entire GISTEMP data set (1940–2016) and based on our regression model, investigated the influence of long-term warming versus El Niño in each April extreme . All 15 The regression model indicates that the long-term warming trend caused ∼29% of the extreme 2016 April SAT in MSA . DespiteWith continued warming projected for the rest of this centuryThe median lead times across all ensemble members indicate that indeed, with future warming, record-breaking April SATs occur more frequently in MSA , a resulUnderstanding and projecting the impacts of global warming on regional extremes is of critical importance for adaptation purposes32We used four different observational data sets for analysing surface temperature in MSA: GISTEMP11We used model output from the Community Earth System Model Version 1 (CESM1) which consists of a large ensemble (hereinafter CESM1-LE) of 39 simulation of climate from 1920 to 2100 ref. 22). The large number of realizations allows us to perform a more robust statistical analysis of the link between El Niño and extreme April SATs for this study, as well attributes the changes to increasing anthropogenic forcings. All the simulations were run with historical forcings until the year 2005, after which the forcings follow the RCP8.5 scenario. The larENSO variability arises spontaneously in the CESM1-LE set of simulations where many aspects of El Niño and La Nina events are realistically simulated, although El Niño events show amplitudes that exceed those of observed eventsWe also performed similar analyses on a 10-member ensemble of simulations from the Community Atmosphere Model Version 5 run in Tropical Ocean-Global Atmosphere configuration  events as those years where peak SSTs were higher (lower) than 0.5 standard deviations of the overall timeseries in each member after subtracting the 10-year smoothed ensemble mean of Niño-3.4. Our results were not affected if we chose 0.5 standard deviations or 1 standard deviation to pick ENSO events. The trend line in 38SATApril=αt+βt2+γT+δ where t is time in years, T is the DJF Niño-3.4 SST anomaly and α,β, and γ are the regression coefficients. The fit yielded a high correlation coefficient , no quadratic long-term warming term and only a linear component (r=0.74), or global mean surface temperatures instead of April SATs in MSA (r=0.80). However, the regression configuration used in the main text provided the highest correlation coefficient. We used this regression to attribute April extremes in MSA to El Niño and long-term warming .The MATLAB codes that have contributed to the results and analysis reported in this study are readily available upon request from the lead author  simulations at https://esgf-node.llnl.gov/.The observational/reanalysis data sets used in this study are available here: the GISTEMP data setHow to cite this article: Thirumalai, K. et al. Extreme temperatures in Southeast Asia caused by El Niño and worsened by global warming. Nat. Commun.8, 15531. doi: 10.1038/ncomms15531 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures, Supplementary Discussion and Supplementary References


Treatment for medulloblastoma, the most common malignant brain tumor in children, remains limited to surgical resection, radiation, and traditional chemotherapy; with long-term survival as low as 50–60% for Sonic Hedgehog (Shh)-type medulloblastoma. We have shown that the transcription factor Atonal homologue 1 (Atoh1) is required for Shh-type medulloblastoma development in mice. To determine whether reducing either Atoh1 levels or activity in tumors after their development is beneficial, we studied Atoh1 dosage and modifications in Shh-type medulloblastoma. Heterozygosity of Atoh1 reduced tumor occurrence and prolonged survival. We discovered tyrosine 78 of Atoh1 is phosphorylated by a Jak2-mediated pathway only in tumor-initiating cells and in human SHH-type medulloblastoma. Phosphorylation of tyrosine 78 stabilizes Atoh1, increases Atoh1’s transcriptional activity, and is independent of canonical Jak2 signaling. Importantly, inhibition of Jak2 impairs tyrosine 78 phosphorylation and tumor growth in vivo. Taken together, inhibiting Jak2-mediated tyrosine 78 phosphorylation could provide a viable therapy for medulloblastoma. Medulloblastoma is the most common solid brain tumor that develops in children, with more than five hundred new cases diagnosed in the United States every year. There are four broad types of medulloblastoma. One of these is called the “Sonic Hedgehog” subtype, named after the biological pathway that becomes re-activated in these tumors. Only about half of patients with this subtype survive for more than 10 years. Moreover, medulloblastoma treatment combines surgery, chemotherapy and radiation, which can cause severe side effects including psychiatric disorders and cognitive impairment.Several drugs that treat medulloblastoma by targeting the Sonic Hedgehog pathway are currently being tested in clinical trials. However, these drugs are usually only effective for a limited time before the tumor evades the treatment. Therefore, there is a need to develop new treatment options for medulloblastoma, perhaps by targeting different signaling pathways in the cells.A protein called Atoh1 is needed for proper brain development in humans, but is not normally present after the first year of life. This protein is, however, re-expressed at high levels in medulloblastoma in mice and humans and is essential for Sonic Hedgehog-type medulloblastoma to form in mice.Klisch et al. used genetic techniques to reduce the amount of Atoh1 in mice that develop medulloblastoma. This intervention reduced the number of mice that developled tumors and increased their lifespan. Biochemical experiments showed that the tumor stem cells of the mice contain a modified version of Atoh1 where a phosphate molecule is bound to a particular region of the protein. This phosphorylation increased the amount and activity of Atoh1 in the cell, and so caused tumors to grow more quickly in mice. Phosphorylated Atoh1 was also detected in samples taken from human medulloblastoma tumors.Klisch et al. also found that an enzyme called Jak2 phosphorylates Atoh1. Inhibiting Jak2 reduced the levels of Atoh1 in medulloblastoma cells and slowed tumor growth in mice. Future work could investigate different ways of preventing Atoh1 phosphorylation, with the hope of finding new treatments for Sonic-Hedgehog-type medulloblastomas. Medulloblastoma (MB) is the most common malignant primary brain tumor in children  and is tTg line  Nd2::SmoA1Tg mice – heterozygous for Atoh1  – developed signs of disease  at similar levels , phospho-dead (Y78F) or phospho-mimetic (Y78D) Atoh1, using three different human-derived MB parental cell lines: DAOY (marked in blue), UW228 (orange) and ONS-76 (purple) . While tTo test the transcriptional activity of the Atoh1 variants we used an Atoh1-specific luciferase reporter system . OverexpTo test whether this phosphorylation drives tumor growth in vivo, we injected DAOY cells [known to form secondary tumors ] stably V617F) is associated with increased proliferation in human myeloproliferative diseases , as well as Protein Tyrosine Kinase 2 (Ptk2) in a bimolecular fluorescence complementation assay (BiFC) for an Atoh1 interaction . Only JaWe examined whether Jak2 influences Atoh1 activity through its canonical downstream cascades by using the Atoh1 transcriptional reporter assay. Co-transfection of Atoh1 and Jak2 dramatically increased Atoh1 activity in all three MB lines . InhibitHaving identified Jak2 as a critical kinase phosphorylating tyrosine 78, we asked if inhibition of Jak2 might be a viable strategy to decrease Atoh1 levels in vivo. We isolated Shh-type medulloblastoma cells, infected them with lentiviral particles harboring Jak2 shRNAs tagged with RFP, and grafted these into mice. Control shRNA-infected tumors grew rapidly, but both Jak2 shRNA-infected tumors showed dramatically reduced tumor growth , in accoTaken together, here we demonstrate that Atoh1, a protein not expressed in the post-natal normal brain, is expressed specifically in SHH-type medulloblastoma malignancies , and thaAll described assays as well as standard molecular biology techniques , nuclear-cytoplasmic fractionation and immunoblotting) can be requested in step-by-step, ‘protocol-style’ format. Plasmids used in this study are available upon request (see Key Resource table). The following assays were described elsewhere: virus production and stable cell line engineering , Atoh1-sAtoh1 lacZ , Atoh1-GFP , Nd2::SmoA1 (C57BL/6-Tg(Neurod2-Smo*A1)199Jols/J, RRID:IMSR_JAX:008831) and NOD/SCID  recipient. All mice in this study were Nd2::SmoA1 C57/Bl6 on either Atoh1-GFP homozygous  or Atoh1 heterozygous  background. All procedures were approved in advance under the guidelines of the Center for Comparative Medicine, Baylor College of Medicine and were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Both male and female mice were used. Mice were group-housed in temperature-controlled rooms on a 14–10 hr light–dark cycle, at constant temperature (22–24°C) and received food and water ad libitum. Mice of both sexes were randomly assigned to different treatment groups.Generation of mouse strains and genotyping procedures were previously described: oh1 lacZ  (B6.129Stoh1-GFP  (B6.129S2::SmoA1  (C57BL/6NOD/SCID  (NOD.CB1All patients provided written informed consent and tissues were collected under an IRB approved protocol at Baylor College of Medicine (BCM).Western blot (overnight incubation with a 1:5000 dilution): anti-p-Atoh1-Y78 , anti-GFP , anti-Jak2 , anti-p-Jak2 , anti-Vinculin , anti-beta Actin-HRP , anti-Rabbit-HRP , anti-FLAG M2-HRP , anti-Mouse-HRP ; Immunofluorescence: anti-p-Atoh1-Y78 (1:50), anti-GFP , anti-RFP , anti-CD15-PE , anti-chicken IgY-Alexa647 , anti-Mouse IgG2b-DyLight405 , anti-Rabbit IgG-Alexa647 ; FACS labeling: endogenous GFP signal from Atoh1-GFP KI mice, anti-CD15-Dylight649 ; Co-IP: anti-GFP , anti-Jak2 .CVCL_1167), 293T . UW228 (RRID:CVCL_4460) (CVCL_1624) : DAOY   and ONS-CL_1624)  were kinCL_1624) . For proCL_1624)  for 3 daCL_1624) . InhibitCL_1624) ], LY2940CL_1624) ], PD1843CL_1624) ], or AG4CL_1624) ]. All ch8 cells from Atoh1-positive/CD15-positive and Atoh1-positive/CD15-negative cell populations of Nd2::SmoA1Tg, Atoh1Atoh1-GFP/Atoh1-GFP tumors were used for immunoprecipitation. Cells were collected and lysed in lysis buffer , homogenized using 1 ml insulin syringes. GFP-trap beads  were added to cleared supernatant and immunoprecipitation reactions were performed overnight at 4°C with gentle head-to-tail rotation. After three washes, immune-complexes were eluted with glycine, vacuum dried and dissolved in 50 mM ammonium bicarbonate, pH = 10 for MS. MS was performed by Proteomics and Metabolomics Core Labs at BCM using standard protocols  as per manufacturer’s protocols.Tg, Atoh1Atoh1-GFP/Atoh1-GFP tumors FACS sorted for Atoh1-positive/CD15-positive, Atoh1-positive/CD15 negative and Atoh1-negative cells  and injected subcutaneously into the flank region of nude mice (n = 6 per group). Xenografts were left to grow for up to 1 months then harvested and weighed. When the inducible cell lines were used mice were fed doxycycline chow  ad libitum throughout the experiment.Xenografts were generated in immunocompromised mice (NOD/SCID) previously described . Cells uve cells ; (ii) tharticles . Briefly−bx) as previously described . Half-life was calculated based on normalized densitometric values obtained by ImageJ and solved for x when y = 0.5 for the following formula: y = a.Thank you for submitting your article "Jak2-mediated phosphorylation of Atoh1 is critical for medulloblastoma growth" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:The report describes the role of the transcription factor Atoh1  in the sonic hedgehog (shh)-driven subtype of medulloblastoma (MB). Using a genetic cross in mice, they initially demonstrated that heterozygous disruption of Atoh1 reduces the tumor burden resulting from shh-driven MB. They then determined that tumor-initiating cells within MB tumors demonstrate tyrosine phosphorylation on Y78 of Atoh1. They also showed that this phosphorylation is mediated by the JAK2 kinase and leads to stabilization of Atoh1 protein and increased transcriptional activity in a reporter assay. As expected, based on these observations, JAK2-specific shRNAs and a pharmacologic JAK inhibitor were found to reduce the tumorigenicity of grafted MB cells in mice. Demonstration that Y78 Atoh1 could be found in human MB tumors further supports the potential clinical relevance of the findings. Overall, this is a well-executed and well-documented study that implicates phosphorylation of Atoh1 on Y78 in the shh-driven subtype of medulloblastoma. As described below, the conclusion that JAK2 is the specific mediator of this phosphorylation event is less definitive.Essential Revisions:1) A limitation of the analysis is the lack of Jak2 deletion studies to show whether Jak2 is truly required for medulloblastoma growth. AG490, the inhibitor used to test the role of Jak2 is not highly specific. Additional targets affected by AG490 include EGFR, guanylyl and adenyl cyclases, and Hif1alpha . This lack of specificity complicates the conclusion that Jak2 plays a critical role in medulloblastoma progression. Ideally, the authors could breed a medulloblastoma-causing mutation into a Jak2 conditional background. Conditionally deleting Jak2 in the medulloblastoma cells of origin would test whether other kinases can assume the function of Jak2 when Jak2 is not present. For example, even if Jak1 does not phosphorylate Atoh1 in the presence of Jak2, Jak1 may nevertheless be able the phosphorylate Atoh1 if Jak2 is deleted. The best way to detect this type of complementation is to knock out Jak2 in medulloblastoma-prone mice and then see tumors grow and if Atoh1 gets phosphorylated. Without such genetic data, a firm conclusion should not be drawn that Jak2 is a critical node in medulloblastoma growth. However, if analyzing Jak2 conditional knock out mice is not feasible, the authors should consider focusing instead on the finding that Atoh1 phosphorylation seems to be very important for ATOH1 protein stability, and for medulloblastoma growth. The focus could then be on targeting Atoh1 phosphorylation, which the data strongly suggest would be an effective approach to treatment. Since AG490 decreases phosphorylation of Atoh1, a study testing for decreased tumor growth and increased survival in ND2:SmoA1 mice treated with AG490 could potentially identify a new direction for therapy, even if Jak2 is not necessarily the only target disrupted by the inhibitor.2) The characterization of the consequences of phosphorylation on Atoh1 activity is a little confusing. The authors claim that the effect of phosphorylation is to stabilize the protein; however, they go on to show in transfected cells using the various phospho-inactive, phospho-mimicking mutants that even though they are expressed at similar levels, the phosphorylation drives increased transcriptional activity in the reporter assay. So, the reader is left to wonder whether phosphorylation is specifically affecting protein levels to drive increased expression or if phosphorylation is more directly affecting Atoh1's activity as a transcription factor. This should be clarified.3) Related to the previous point, the authors should determine whether phosphorylation affects nuclear localization of Atoh1, which is a level of regulation of transcription factors that is frequently associated with phosphorylation.4) It would be useful to see that the JAK inhibitor affects Atoh1 half-life and/or transcriptional activity. The authors have relied largely on ectopic expression of Atoh1 mutants to support this connection, but inhibition of endogenous JAK2 to affect endogenous Atoh1 would provide a more compelling link.5) Despite the complexities that come from understanding Atoh1 within the context of multiple developmental molecular mechanisms, the authors provide minimal data to show Atoh1 expression and activation within the SHH subtype, essentially limited to 2 SHH tumors and 2 Wnt tumors. The manuscript would be strengthened by showing expression in a larger number of tumors, including tumors from Groups 3 and 4 medulloblastoma. There are a number of transcriptome databases that could easily be queried for mRNA expression. A larger number of samples should be assayed for protein expression.6) The cell lines used in this project have been shown to not be representative of primary medulloblastomas, especially of the SHH subtype. There are a number of groups that have established early passage cultures of primary tumors of all subtypes that can be considered for these experiments. Moreover, for the cell lines used data should be shown of endogenous Atoh1 expression or Y78 phosphorylation. Summary:The report describes the role of the transcription factor Atoh1  in the sonic hedgehog (shh)-driven subtype of medulloblastoma (MB). Using a genetic cross in mice, they initially demonstrated that heterozygous disruption of Atoh1 reduces the tumor burden resulting from shh-driven MB. They then determined that tumor-initiating cells within MB tumors demonstrate tyrosine phosphorylation on Y78 of Atoh1. They also showed that this phosphorylation is mediated by the JAK2 kinase and leads to stabilization of Atoh1 protein and increased transcriptional activity in a reporter assay. As expected, based on these observations, JAK2-specific shRNAs and a pharmacologic JAK inhibitor were found to reduce the tumorigenicity of grafted MB cells in mice. Demonstration that Y78 Atoh1 could be found in human MB tumors further supports the potential clinical relevance of the findings. Overall, this is a well-executed and well-documented study that implicates phosphorylation of Atoh1 on Y78 in the shh-driven subtype of medulloblastoma. As described below, the conclusion that JAK2 is the specific mediator of this phosphorylation event is less definitive.Essential Revisions:1) A limitation of the analysis is the lack of Jak2 deletion studies to show whether Jak2 is truly required for medulloblastoma growth. AG490, the inhibitor used to test the role of Jak2 is not highly specific. Additional targets affected by AG490 include EGFR, guanylyl and adenyl cyclases, and Hif1alpha . This lack of specificity complicates the conclusion that Jak2 plays a critical role in medulloblastoma progression. Ideally, the authors could breed a medulloblastoma-causing mutation into a Jak2 conditional background. Conditionally deleting Jak2 in the medulloblastoma cells of origin would test whether other kinases can assume the function of Jak2 when Jak2 is not present. For example, even if Jak1 does not phosphorylate Atoh1 in the presence of Jak2, Jak1 may nevertheless be able the phosphorylate Atoh1 if Jak2 is deleted. The best way to detect this type of complementation is to knock out Jak2 in medulloblastoma-prone mice and then see tumors grow and if Atoh1 gets phosphorylated. Without such genetic data, a firm conclusion should not be drawn that Jak2 is a critical node in medulloblastoma growth. However, if analyzing Jak2 conditional knock out mice is not feasible, the authors should consider focusing instead on the finding that Atoh1 phosphorylation seems to be very important for ATOH1 protein stability, and for medulloblastoma growth. The focus could then be on targeting Atoh1 phosphorylation, which the data strongly suggest would be an effective approach to treatment. Since AG490 decreases phosphorylation of Atoh1, a study testing for decreased tumor growth and increased survival in ND2:SmoA1 mice treated with AG490 could potentially identify a new direction for therapy, even if Jak2 is not necessarily the only target disrupted by the inhibitor.While using a Jak2 conditional knock out mouse model to delete Jak2 after the Shh-derived tumor has formed is an ideal experiment, this will take several months. To insure the specificity of Jak2 effects, we opted to use viral knock down of Jak2 in primary Shh-derived medulloblastoma tumors. Using xenografts, we show that downregulation of Jak2 indeed impedes tumor growth . MoreoveA second point raised pertains to whether another Jak family member, namely Jak1, could take over the role of Jak2 in its absence. While the in vitro kinase assay  and the In addition, we analyzed the Jak1 levels in our in vivo knock down of Jak2 . While w2) The characterization of the consequences of phosphorylation on Atoh1 activity is a little confusing. The authors claim that the effect of phosphorylation is to stabilize the protein; however, they go on to show in transfected cells using the various phospho-inactive, phospho-mimicking mutants that even though they are expressed at similar levels, the phosphorylation drives increased transcriptional activity in the reporter assay. So, the reader is left to wonder whether phosphorylation is specifically affecting protein levels to drive increased expression or if phosphorylation is more directly affecting Atoh1's activity as a transcription factor. This should be clarified.We show that the phosphorylation of tyrosine 78 on Atoh1 has two effects: an aberrant increase in Atoh1 transcriptional activity as well as a stabilizing effect on the Atoh1 protein. We do not believe that these two effects are in a linear cascade where higher stability leads to higher transcriptional activity as the steady state levels in the cell lines are comparable. It will be interesting to investigate whether phosphorylation effects the On- or Off-rates of Atoh1 on DNA, thereby leading to a longer promoter occupancy, however these experiments are beyond the scope of this paper. We edited the text to make it clear that these two effects are not connected in a linear manner.3) Related to the previous point, the authors should determine whether phosphorylation affects nuclear localization of Atoh1, which is a level of regulation of transcription factors that is frequently associated with phosphorylation.This is an excellent suggestion and we agree that this information strengthens the paper. In response, we evaluated the nuclear localization of the wildtype, phospho-mutant and phospho-mimetic Atoh1 constructs and saw no differences. We included the results in 4) It would be useful to see that the JAK inhibitor affects Atoh1 half-life and/or transcriptional activity. The authors have relied largely on ectopic expression of Atoh1 mutants to support this connection, but inhibition of endogenous JAK2 to affect endogenous Atoh1 would provide a more compelling link.Because Atoh1 is not only a proliferation factor but also induces numerous differentiation cascades, there is no cell line (to our knowledge) that expresses endogenous Atoh1. To identify a neuronal cell line that expresses endogenous Atoh1 we interrogated the Swedish Human Protein Atlas with 56 cell lines catalogued  as well as the Broad Institute Cancer Cell Line Encyclopedia with 1457 cell lines catalogued , but sadly failed to identify any. This is the primary reason for using the inducible expression system, which gives us the advantage of studying the effects of different mutants without the interference of the endogenous protein.endogenous Atoh1 by using mouse primary Shh-derived medulloblastoma cells, which express endogenous Atoh1. We have already used a short culture experiment to show a dramatic reduction in Y78 levels within one hour of AG490 inhibition  Despite the complexities that come from understanding Atoh1 within the context of multiple developmental molecular mechanisms, the authors provide minimal data to show Atoh1 expression and activation within the SHH subtype, essentially limited to 2 SHH tumors and 2 Wnt tumors. The manuscript would be strengthened by showing expression in a larger number of tumors, including tumors from Groups 3 and 4 medulloblastoma. There are a number of transcriptome databases that could easily be queried for mRNA expression. A larger number of samples should be assayed for protein expression.Atoh1 expression is almost undetectable in other medulloblastoma subtypes other than the Shh subtype. Accordingly, we re-reviewed existing expression studies and found Atoh1 to be expressed in SHH-type tumors but no other subtypes. To illustrate the exclusivity of Atoh1 expression in SHH-type medulloblastoma, we included a box plot of Atoh1 expression using the publicly available data from Dr. Pfister and colleagues . While our aim is to test Atoh1 protein levels in human medulloblastoma, we have yet to develop or identify an antibody that reliably detects Atoh1. We and others have tried for years to develop a reliable and specific antibody to no avail, for this reason we created the Atoh1-GFP fusion knock-in allele in the mice. While we acknowledge the limitations of our human data due to the mentioned antibody and sample availability, we believe that our in vivo mouse work is solid in supporting a Jak2-Atoh1 signaling axis in Shh-type medulloblastoma and our human data sufficiently support the notion that Atoh1 is phosphorylated on Y78 in human SHH-type medulloblastoma.6) The cell lines used in this project have been shown to not be representative of primary medulloblastomas, especially of the SHH subtype. There are a number of groups that have established early passage cultures of primary tumors of all subtypes that can be considered for these experiments. Moreover, for the cell lines used data should be shown of endogenous Atoh1 expression or Y78 phosphorylation.We take an active part in the brain tumor program at Texas Children’s in Houston. While Dr. Xiao-Nan Li and colleagues established patient tumor-derived orthotopic xenograft models for childhood brain tumors, there is a consensus that newly isolated medulloblastoma cell lines do not grow in culture .Moreover, while there is considerable discussion in the field about the origins and subtype of the different medulloblastoma cell lines used in this study, we would like to highlight for the reviewer a study by Dr. Fanayan and colleagues from earlier this year. This study provides a convincing argument that DAOY cells do have a SHH-type proteomic signature, whereas the UW228 cells display a signature more similar to the WNT subtype . This classification differs somewhat from earlier attempts to classify existing cell lines . It is noteworthy that DAOY cells originated from a desmoplastic cerebellar medulloblastoma, which is a hallmark of SHH-type medulloblastoma.As mentioned earlier, Atoh1 is not expressed in any of these cell lines probably due to its role in cellular differentiation . Therefore, our doxycycline-inducible system provides an opportunity to better study Atoh1 function. While we agree that these medulloblastoma cell lines do not fully recapitulate the tumor biology of medulloblastoma, it is noteworthy that this is an inherent flaw of all in vitro systems. Acknowledging this limitation, we opted to use three rather than just one cell line as well as in vivo studies to provide further support for our conclusions.


Stress-related Psychosocial Factors at Work, Fatigue, and Risky Driving Behavior in Bus Rapid Transport (BRT) Drivers”, published in Accident Analysis & Prevention.This Data in Brief (DiB) article presents a hierarchical multiple linear regression model that examine the associations between psychosocial work factors and risk behaviors at the wheel in Bus Rapid Transit (BRT) drivers (n=524). The data were collected using a structured self-administrable questionnaire made of measurements of wok stress , fatigue (need for recovery and chronic fatigue), psychological distress and demographics . The data contains 4 parts: descriptive statistics, bivariate correlations between the study variables and a regression model predicting risk behaviors at the wheel and the entire study dataset. For further information, it is convenient to read the full article entitled “ Specifications TableValue of the data•This data provides information on the psychosocial work factors associated with risk behaviors at the wheel in BRT drivers.•The data on the psychosocial work factors of BRT drivers can be compared with those of other groups of professional drivers.•The data could be generalized to other BRT-based transport systems (BRT systems exist in more than 160 cities in 33 countries).•The data can be used by other researchers to analyze the working conditions of BRT drivers.11.1In this cross-sectional study, the sample was made up of 524 male Bus Rapid Transit (BRT) operators from companies affiliated to the Transmilenio S.A. mass transport system in Bogota, Colombia. The mean age of professional drivers was of 40.6 years (SD=7.6) [20–65 range] and average driving experience was of 17.6 years (SD=7.3).1.2The Job Content Questionnaire (JCQ) *For further information, please refer to Useche, Cendales and Gómez 1.3Hierarchical linear regressions were used to examine the effect of the psychosocial work factors on the risk behaviors at the wheel. The “job strain score” was calculated through the ratio between psychological demands and decision latitude scales of the JCQ. Likewise, the effort-rewards imbalance score was calculated through the algorithm E/R*C, where “E” and “R” are the scores on the effort and reward scales of the ERI Questionnaire respectively, and “C” corresponds to the correction factor for the different number of items in the numerator and denominator. Driving experience, hours driven per day and days working per week were introduced in the first step of the regression model. Job strain and social support were included in the second step, effort-reward Imbalance in the third step, need for recovery (job-related fatigue) in the fourth step, general fatigue in the fifth step, and psychological distress in the sixth step.2The dataset of this article provides information on the psychosocial work factors associated with risk behaviors at the wheel on BRT drivers.