The control of cellular growth is central to multicellular patterning. In plants, the encapsulating cell wall literally binds neighbouring cells to each other and limits cellular sliding/migration. In contrast to its developmental importance, growth regulation is poorly understood in plants. Here, we reveal that the phytohormone auxin impacts on the shape of the biggest plant organelle, the vacuole. TIR1/AFBs-dependent auxin signalling posttranslationally controls the protein abundance of vacuolar SNARE components. Genetic and pharmacological interference with the auxin effect on vacuolar SNAREs interrelates with auxin-resistant vacuolar morphogenesis and cell size regulation. Vacuolar SNARE VTI11 is strictly required for auxin-reliant vacuolar morphogenesis and loss of function renders cells largely insensitive to auxin-dependent growth inhibition. Our data suggests that the adaptation of SNARE-dependent vacuolar morphogenesis allows auxin to limit cellular expansion, contributing to root organ growth rates.DOI:http://dx.doi.org/10.7554/eLife.05868.001 In plants and animals, the way that cells grow is carefully controlled to enable tissues and organs to form and be maintained. This is especially important in plants because the cells are attached to each other by their cell walls. This means that, unlike some animal cells, plant cells are not able to move around as a plant's organs develop.Plant cells contain a large storage compartment called the vacuole, which occupies 30–80% of a cell's volume. The volume of the vacuole increases as the cell increases in size, and some researchers have suggested that the vacuole might help to control cell growth. A plant hormone called auxin can alter the growth of plant cells. However, this hormone's effect depends on the position of the cell in the plant; for example, it inhibits the growth of root cells, but promotes the growth of cells in the shoots and leaves. Nevertheless, it is not clear precisely how auxin controls plant cell growth.Arabidopsis thaliana. The experiments show that auxin alters the appearance of the vacuoles in these cells so they become smaller in size. At the same time, auxin also inhibits the growth of these cells.Here, Löfke et al. studied the effect of auxin on the appearance of vacuoles in a type of plant cell—called the root epidermal cell—on the surface of the roots of a plant called Löfke et al. found that auxin increases the amount of certain proteins in the membrane that surrounds the vacuole. These proteins belong to the SNARE family and one SNARE protein in particular, called VTI11, is required for auxin to be able to both alter the appearance of the vacuoles and restrict the growth of root epidermal cells. Enzymes called PI4 kinases were also shown to be involved in the control of the SNARE proteins in response to the auxin hormone.Löfke et al.'s findings suggest that auxin restricts the growth of root epidermal cells by controlling the amount of SNARE proteins in the vacuole membrane. The next challenge will be to understand precisely how the shape of the vacuole is controlled and how it contributes to cell growth.DOI:http://dx.doi.org/10.7554/eLife.05868.002 Symplastic growth, characterised by cells that do not alter their relative position to each other, is typical in plant tissue expansion . Such deThe phytohormone auxin is a crucial growth regulator and central in differential growth processes. TRANSPORT INHIBITOR RESISTANT1 (TIR1) and its homologs AUXIN F-BOX PROTEINS (AFBs) have been unequivocally demonstrated to be auxin receptors . GenomicIntriguingly, auxin-signalling events promote and inhibit cellular growth in a cell-type- and auxin concentration-dependent manner. Physiological auxin levels induce growth in light grown aerial tissues, while most root tissues show growth repression in response to the very same auxin concentrations . The ‘acThe higher plant vacuole is, due to its size, the most prominent plant organelle, and shares its lytic function with its counterparts in yeast and animal lysosomes . It may Here we use this cell biological model system to reveal and subsequently characterise auxin-dependent vacuolar morphogenesis and its requirement for limiting cellular growth.Epidermal cells show smaller and larger vacuolar structures in shorter tricho- and longer atrichoblast cells, respectively  Figure . We hypoTo specifically investigate the role of auxin in limiting cellular size in roots, we initially assessed how auxin impacts on late meristematic epidermal cells. To allow cellular development under high auxin conditions, we exogenously applied synthetic auxin, 1-naphtylacetic acid (NAA), in nanomolar ranges for 20 hr and screened for subcellular effects that showed differential regulation in neighbouring cells. Remarkably, exogenous application of auxin (NAA [250 nM]) or endogenous elevation of YUCCA-dependent auxin biosynthesis led to a dramatic change in vacuolar appearance in root epidermal cells, which consequently displayed smaller luminal vacuolar structures .We established a vacuolar morphology index in epidermal cells based on the biggest luminal structure to further evaluate the apparent auxin effect on vacuolar shape . This anThis data shows that auxin differentially affects vacuolar shape in neighbouring epidermal cells.Notably, the differential effect of auxin on vacuoles correlated exactly with a differential effect on cellular size. High auxin levels reduced the cell length of atrichoblast cells , whereasOur data shows that the auxin effect on vacuolar morphology correlates with its negative effect on late meristematic epidermal cell size. Auxin treatments manifestly did not reverse vacuolar morphology of fully elongated epidermal root cells in the differentiation zone . This fiNext we investigated the auxin effect on vacuoles in the course of time. As the auxin effect on vacuoles was most pronounced in atrichoblasts, we focused our analysis (from here onwards) mainly on this cell-type. Notably, auxin imposed in time steadily increasing effects on vacuolar appearance . Auxin iBased on our time course experiments we conclude that the auxin effect on vacuolar morphology precedes the auxin impact on late meristematic cell size.tir1 afb1 afb3 triple mutants prompted partial resistance to the auxin-induced changes in vacuolar appearance /AUXIN F-BOX PROTEINS (AFB) auxin receptors  are requpearance .10.7554/This set of data indicates that TIR1/AFBs-dependent auxin signalling is required for the auxin effect on vacuolar morphogenesis.SNAP (Soluble NSF Attachment Protein) Receptor (SNARE) complexes at the vacuole. Proximity of adjacent membrane allows the interaction of v (vesicle)- and t (target)-SNAREs to form a complex, allowing the fusion of vesicles to specific target membranes. SNAREs are essential for eukaryotic vesicle trafficking and according to structural features SNAREs are divided in R (arginine)- and Q (glutamine)-SNAREs -SNAREs . In yeas)-SNAREs . Ergo, wYP22-GFP . Auxin sYP22-GFP .10.7554/Exogenous application of auxin does not detectably impact on cytosolic pH . HoweverTo assess whether auxin treatments affect the overall cellular abundance of vacuolar SNAREs, we performed defined z-stack imaging. Subsequent maximum projections and intensity measurements confirmed that auxin increases cellular SNARE abundance at the tonoplast . To furtNotably, tonoplast marker NET4A-GFP  did not Induction of a single SNARE component, such as VAMP711, did not affect vacuolar morphogenesis , possibltir1 afb1 afb3 triple mutant  -dependent processes have been previously shown to play a role in vacuolar biogenesis in yeast  and alsopi4kß1/2 double mutants. Compared to wild-type seedlings, pi4kß1/2 double mutants showed partially auxin resistant vacuolar appearance  . This danditions . We accopi4kß1/2 double mutants  in  mutants . Notablyn levels . Based opi4kß1/2 double mutants were similarly resistant to the auxin-dependent inhibition of cellular expansion  with PI kinases  and genetic interference . vamp711 (N673991), obtained from the Nottingham Arabidopsis stock centre and the decrease of VAMP711 transcript was shown by RT-PCR. Other plant lines were described previously, pUBQ10::VAMP711-YFP/RFP (Wave 9Y/R) (pER8 and pER8::YUC6 (tir triple: tir1-1/afb1-3/afb3-4 (pi4kß1/2 (35S::SYP21-YFP (SYP22::SYP22-GFP in syp22 background (syp21 (pep12) (syp22 (vam3-1) (vti11 (zigzag)  supplemented MS plates. Seeds were stratified at 4°C for 2 days in the dark and grown on vertically orientated ½ Murashige and Skoog (MS) medium plates under a long-day regime (16 hr light/8 hr dark) at 20–22°C. Gateway cloning was used to construct pMDC7_B(pUBQ)::VAMP711  from the Wave line vector harbouring VAMP711 (Wave 9Y). The primers were designed using life technologies OligoPerfect (www.lifetechnologies.com/oligoperfect) and are given below. The fragment obtained was introduced into the pDONR221 . Coding VAMP711 sequence from the entry vector was subsequently introduced in the gateway compatible destination vector pMDC7_B(pUBQ) using the Invitrogen LR clonase(+) and the resulting construct was transformed into Col-0 plants by floral dipping in Agrobacterium tumefaciens liquid culture. The T1 generation obtained was selected on hygromycin B supplemented MS plates.We used ve 9Y/R) , pER8 anR8::YUC6 , tir tri3/afb3-4 , pi4kß1/pi4kß1/2 , 35S::SYYP21-YFP , SYP22::ckground , syp21 ( (pep12) , syp22 ((vam3-1) , vti11 ((zigzag) , VTI11::in vti11 , NET4A::ET4A-GFP . The pi4All chemicals were dissolved in DMSO and were applied in solid ½ MS-medium. Only dyes were applied in liquid ½ MS-medium before imaging. 1-naphthaleneacetic acid (NAA) was obtained from Duchefa (Netherlands), 5-F-IAA, estradiol, FM4-64, L-kynurenine (Kyn) and propidium iodide (PI) from Sigma , wortmannin (WM) from Cayman Chemical , MDY-64 from life technologies  and auxinole was kindly provided by Ken-ichiro Hayashi .t-test using graphpad . Figures display a representative experiment (out of three independent repetitions) utilising eight individual roots. The cell length change in the late meristematic zone . Figures show a representative experiment (out of three independent repetitions) utilising 10 individual roots. To estimate the position for the cell length measurements in the elongation zone, seedlings were stained in propidium iodide for 5 min and subsequently imaged choosing the position were no propidium iodide (0.02 mg/ml) entered the vasculature, showing fully developed endodermal diffusion barriers. Length measurements of epidermal root hair cells were performed in imageJ by quantifying the length of individual cells which were averaged. Means and standard error were calculated and statistical significance was evaluated by the Student's t-test using graphpad . Figures show a representative experiment (out of three independent repetitions) utilising 10 individual roots. In each root approximately three cells were quantified. For analysis of the root length, seedlings grown in vertically orientated plates were scanned on a flat-bed scanner and measurements were performed in imageJ. Per condition, 20–25 seedlings were analysed (for each experiment) 8 days after germination. Means and standard error were calculated and statistical significance was evaluated by the Student's t-test using graphpad . Figures show a representative experiment (out of three independent repetitions).For analysing the vacuolar morphology and cell length change, all the experiments were carried out on 7 days old seedlings. For analysing the vacuolar morphology index, optical confocal sections (above the cell nucleus) of the root epidermis were acquired and processed in imageJ. Images were taken in the late meristematic zone  were added and immediately incubated at 95°C for 5 min. After centrifugation, 15 µl of the extracts were separated by SDS-PAGE (10% gel) and blotted onto a 0.2 µm polyvinylidene difluoride membrane (BioRad). After blocking with a solution of 5% skim milk powder in TBS-T  the membrane was probed with an anti-GFP antibody  diluted 1:2000 in skim milk TBS-T solution or anti-alpha-tubulin  diluted 1:50000 in skim milk TBS-T solution. Horseradish peroxidase-conjugated goat anti-mouse antibody  was employed as secondary antibody (1:20000). For detection, the SuperSignal West Pico chemiluminescent detection reagent  was used. Three independent root samples were quantified with imageJ and normalized to alpha-tubulin, means and standard error were calculated and statistical significance was evaluated by the Student's t-test using graphpad . Figure shows a representative experiment (out of three independent repetitions).Root samples (∼50 mg each) of 7 days old http://rsb.info.nih.gov/ij/) and data was statistically evaluated by Student's t-test using graphpad . Representative images are shown.For live cell imaging, wherever possible, roots were mounted in a propidium iodide (PI) solution (0.02 mg/ml) for counterstaining the cell walls. MDY-64 and FM4-64 staining was performed as described . For 3D VAMP711_ATTB1_fwd, GGGGACAAGTTTGTACAAAAAAGCAGGCTCGATGGCGATTCTGTACGCC, rev, GGGGACCACTTTGTACAAGAAAGCTGGGTCTTAAATGCAAGATGGTAGAGTAGGTC; UBQ5 fwd, GACGCTTCATCTCGTCC, rev, GTAAACGTAGGTGAGTCCA.Destination vector: pMDC7_B(pUBQ) . review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent  and 3 reviewers, as well as a member of our Board of Reviewing Editors.Thank you for sending your work entitled “Auxin regulates SNARE-dependent vacuolar morphology restricting cell size” for consideration at The Reviewing editor and the reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.As you can see from their comments, the reviewers have substantive concerns regarding your work, and we would like to ask you to address them comprehensively. Especially:1) Please pay particular attention to the issues raised by reviewer 1, regarding the quantification of your markers. This issue is at the very heart of your manuscript, and we consider that it must be resolved to render your work publishable. Western blots for the other fusion proteins might be one way to provide more evidence, along with the reviewer's suggestions.2) Please also clarify the involvement of TIR1-type auxin signaling, which suggests that there must be a transcriptional effect in this regulation somewhere. If it is not directly targeting the SNARE expression, but something regulating it, then do the time scales allow the conclusion that the auxin regulation is nevertheless direct and not a secondary effect?3) The mutants that you present appear to have largely been published, nevertheless we would like to see evidence that the effects you report are due to those mutations, i.e. that you do not observe them in transgenically complemented plants.Reviewer #1:vti11 does not support the formation of the compact multi-lobed structures. Overall, this paper could make an interesting contribution to eLife.The authors describe an interesting series of experiments suggesting that auxin increases the abundance of the vacuolar SNARE protein VTI11 and leads to a contraction of the vacuole resulting in a complex multi-lobed structure. These two observations are linked by the fact that a non-functional mutant allele Critical points to be fixed:syp21, which is actually on a pre-vacuolar compartment. My concern is that the contraction of the vacuoles into compact structures might lead to erroneous measures of the fusion proteins, due to the fact that the authors evaluated only a single confocal section, probably through the center plane of these cells, rather than a stack of images covering the entire depth. A consequence could be that in cells with voluminous vacuoles much of the vacuolar membranes lies outside the confocal section and will not be measured. In the contracted state, a greater share of this membrane should be closer to the center of the cell, leading to an increase in the fluorescence in the single confocal section.The increase in VTI11 abundance in the analyzed cell types is not convincing. The authors show a strong increase in the signal of a YFP fusion protein upon NAA treatment and conclude that this means increased protein levels. This conclusion is challenged by the fact that artificial overexpression of VAMP711 did not influence vacuolar morphology and, more importantly, that the lipid marker MDY64 shows exactly the same NAA-induced increase in fluorescence as the VAMP11-YFP . This drThe authors could rule out that their results are artefacts by addressing several points:1) Integrate the fluorescence signals of the various SNARE proteins through a stack of sections covering the entire cell.2) Do the same analysis for the lipid marker MDY64 and discuss the strong effect observed.3) Present negative controls that do not show an NAA-induced increase of fluorescence. YFP-fusions of other vacuolar membrane proteins should be analyzed.4) Re-decorate the blot in Minor comments:Many experiments use a YFP-fusion of VAMP11 and the fluorescence emission of YFP is very sensitive to pH changes. The authors should at least provide good arguments indicating that NAA does not change the intracellular pH to which the YFP tag is exposed.Are the fluorescent protein tags on the cytosolic or on the lumenal side of the membrane?It is not self-evident how an increase in vacuolar SNAREs could lead to a collapse to the vacuoles into a very compact morphology. The Discussion would be more stimulating if it presented at least some speculations on this issue.The vacuolar morphology index appears too simplified in order to represent the complex morphological changes in a meaningful way. It arbitrarily selects only the largest vacuole in the picture as a measure, leaving aside the many small and medium-sized structures. Given this, the statistical analysis of such results appears as formally correct but of questionable relevance. I would have preferred to see an analysis of the total vacuolar volume in these cells, which may be the parameter that is most relevant for cell expansion.Reviewer #2:Arabidopsis root epidermis to show how auxin controls growth of growth-competent cells via stabilising protein components of the SNARE complex, which in turn regulate vacuole morphology. A range of genetic and chemical approaches has been taken and the results generally support the conclusions made by the authors. The manuscript is well written and the unravelling of this auxin-dependent control of vacuole morphology and cell growth will be interesting to a wide audience of cell and developmental biologists not restricted to the plant field. Below are some points for the authors to address, not in any particular order.Löfke et al. use vti11 mutant is mentioned as displaying roundish vacuoles. Does this mean that vti11 is the only mutant in a SNARE-encoding gene that has a phenotype? Also it would be useful to explain earlier what it means that VTI11 is a Qb-SNARE (see point on SNARE background below). Finally, since the vacuole morphology is clearly different in the vti11 mutant from that in wild type, it is puzzling why the authors indicate that the defects in SNARE-dependent vacuolar morphology regulation in vti11 are relatively mild. Perhaps they mean that the altered vacuolar morphology in vti11 has little effect on cell growth in A and T cells?In the subsection headed “Vacuolar SNARE function is required for the auxin-dependent modulation of vacuolar morphology”, paragraphs 1 and 2 could be made clearer. In the reference to the redundant action of SNARE components, the vti11 mutant  could be due to cell wall limitations. I think they can expand their reasoning to include that the increased growth observed upon Kynurenin treatment in The authors explain in the discussion that lack of increased growth by WM treatment  and in tAlong the same line, it seems to me a discussion could be included on coordination between tricholast and atrichoblast cell files as well as cell divisions within a cell file. For example, do any of the genetic and pharmological treatments lead to a difference in the ratio of trichoblasts:atrichoblasts in the meristematic zone or total number of cells?In the subsection headed “Auxin effect on vacuolar morphogenesis interrelates with auxin-dependent cellular growth inhibition” a paragraph describes the effect of VTI11 and PI4K activity on root epidermal cell length (elongation zone) and overall root growth. Two points: 1) It is mentioned in the legend to Minor comments:Some of the headings could be made more specific. For example, “Interference with the auxin effect on vacuolar SNAREs impedes auxin-dependent cell size control”, this heading should refer to the connection made with PI-dependent processes.I appreciate that space is limiting, but the minimal background information on the SNARE complex makes it difficult to relate to their role in this process. A few additional sentences to introduce SNARE would be useful. For example: 1) one gets the impression that SNAREs only exist in yeast and plants, although they indeed are common to all multicellular eukaryotic organisms, 2) mention their function in controlling membrane fusion, and 3) it would be useful to let the reader know what they are composed of such that the reader is prepared for what the Qb-SNARE VTI11 is.Reviewer #3:This paper reports on the dramatic effect auxin can have on the shape of root tip cell vacuoles. The data indicate that via auxin receptors, auxin can regulate the abundance of vacuolar SNARES in particular the QbSNARE VTI11. The authors have quantified the effect by designing a vacuolar morphology index, so that they can compare vacuolar morphology in the various mutants and treated root tips. The method of quantification is relatively simple and crude but does effectively reflect the changes in vacuolar morphology which in some cases are visually very striking. I'm sure with time the group will develop better methods of assaying the changed in vacuolar morphology.In general the study is well presented and the paper well written and is a substantial contribution to the field. I feel that the authors have been a little cautious in their Discussion and have made little attempt to hypothesis as to why a vacuolar SNARE may have such a dramatic effect on vacuolar morphology. It is difficult to tell if in some of the micrographs we are looking at many individual vacuoles which have not fused together due to lack of SNARE complexes or whether they are all interconnected but just change in morphology. A bit of speculation would be good.Minor comments:In the beginning of the subsection headed “Vacuolar SNARE function is required for the auxin-dependent modulation of vacuolar morphology”, perhaps you need to introduce VTI11 here or at least put it in the section heading.In the Discussion section, I really wonder whether turgor exerted by the vacuole in the root tip is important. The vacuole appears very pleomorphic at this stage of development. 1) Please pay particular attention to the issues raised by reviewer 1, regarding the quantification of your markers. This issue is at the very heart of your manuscript, and we consider that it must be resolved to render your work publishable. Western blots for the other fusion proteins might be one way to provide more evidence, along with the reviewer's suggestions.We thank particularly reviewer 1 for raising these concerns. We accordingly paid particular attention to the quantification of vacuolar SNAREs and performed several experiments. 1) We experimentally excluded that membrane crowding accounts for the observed increase in fluorescent intensity. 2) Moreover, we performed detailed z-stack imaging, confirming our previous single section results on auxin induced increase in cellular abundance of vacuolar SNAREs. 3) In agreement, we also performed additional western blots, confirming that auxin increases the abundance of several vacuolar SNARE proteins. 4) Finally, we included an additional vacuolar marker NET4a-GFP in all our experiments, which fluorescent intensity or protein abundance is not affected by auxin. This set of experiments illustrates that auxin affects SNARE abundance at the tonoplast. The additional data is shown in 2) Please also clarify the involvement of TIR1-type auxin signaling, which suggests that there must be a transcriptional effect in this regulation somewhere. If it is not directly targeting the SNARE expression, but something regulating it, then do the time scales allow the conclusion that the auxin regulation is nevertheless direct and not a secondary effect?We once again thank you for the very valuable suggestion. We focused on the time scale of the auxin effect on vacuolar morphology as suggested. Accordingly, we emphasized on detailed time course experiments. First of all we reveal that the auxin effect on vacuolar morphology increases in time. Moreover, we could detect the first auxin induced morphological change of the vacuole within 15-30 minutes. This time frame is within in the time scale of fast genomic responses. Moreover, this finding also allowed us to illustrate that the auxin effect on vacuolar morphology preceded the auxin impact on cell size, which required ∼ 45 minutes. We included the data in the revised manuscript .3) The mutants that you present appear to have largely been published, nevertheless we would like to see evidence that the effects you report are due to those mutations, i.e. that you do not observe them in transgenically complemented plants.vti11 mutant phenotype is indeed central in our manuscript and, hence, we followed the suggestion, utilizing a functional pVTI11::VTI11-GFP construct to complement the vti11 mutant phenotypes. Notably, VTI11-GFP expression in vti11 mutant epidermal cells did not only rescue vacuolar morphology in untreated conditions, but also reversed auxin sensitivity in regards to vacuolar morphology and organ root growth. The new set of data and its quantification is now shown in The interpretation of the Reviewer #1:1) Integrate the fluorescence signals of the various SNARE proteins through a stack of sections covering the entire cell.We followed the reviewer’s suggestion and imaged again all used SNARE markers and integrated the signals through a stack of sections covering the entire cell. The new data confirms our previous single section data and is now shown in 2) Do the same analysis for the lipid marker MDY64 and discuss the strong effect observed.We followed the reviewer’s suggestion and performed simultaneously image acquisition of VAMP711-Y/RFP and lipid markers MDY-64 and FM4-64. We did not detect any signal increase of MDY-64 or FM4-64 as compared to VAMP711-Y/RFP. The previous pictures must have been misleading and we apologize for not choosing representative pictures in the previous version. The new data is now shown in 3) Present negative controls that do not show an NAA-induced increase of fluorescence. YFP-fusions of other vacuolar membrane proteins should be analyzed.We appreciate the suggestion and included the tonoplast marker NET4A-GFP, which was not affected by auxin. This suggests certain specificity for the auxin effect on vacuolar SNAREs. The new data is now shown in 4) Re-decorate the blot infor several other vacuolar marker proteins in order to demonstrate specificity of the effect of VAMP11-YFPWe followed the reviewer’s suggestion and have performed western blot analysis including additional SNARE proteins and the NET4A-GFP (as a negative control). The data is now included in Minor comments:Many experiments use a YFP-fusion of VAMP11 and the fluorescence emission of YFP is very sensitive to pH changes. The authors should at least provide good arguments indicating that NAA does not change the intracellular pH to which the YFP tag is exposed.We are grateful for this comment and assume many readers will have similar concerns. Therefore, we included a discussion on this aspect in the revised version of the manuscript. In our experiments we used VAMP711 fusion proteins to RFP and YFP. RFP and YFP proteins have very distinct sensitivities to pH changes, but both fusion proteins showed comparable auxin induced increase in fluorescent intensity. Moreover, previous work  suggesteAre the fluorescent protein tags on the cytosolic or on the lumenal side of the membrane?The fluorescent tags are facing the cytosol. This information is now included in the revised version of the manuscript.It is not self-evident how an increase in vacuolar SNAREs could lead to a collapse to the vacuoles into a very compact morphology. The Discussion would be more stimulating if it presented at least some speculations on this issue.This is indeed a very interesting question. We assume that auxin could trigger a conformational change of SNAREs, which may affect its activity. Alternatively, vacuolar SNAREs could also provide a structural component required for vacuolar organisation. We currently investigate these options. At the current stage we can only speculate and we followed the reviewer’s suggestion and elaborated on our Discussion.The vacuolar morphology index appears too simplified in order to represent the complex morphological changes in a meaningful way. It arbitrarily selects only the largest vacuole in the picture as a measure, leaving aside the many small and medium-sized structures. Given this, the statistical analysis of such results appears as formally correct but of questionable relevance. I would have preferred to see an analysis of the total vacuolar volume in these cells, which may be the parameter that is most relevant for cell expansion.The vacuole is a highly complex structure, which is visually distinct in every cell. Long term auxin treatments and its effect on vacuolar shapes are very easily detectable by eye. However, shorter time points require a solid quantification method to capture morphological changes. We implemented the vacuolar morphology index to detect such major changes in vacuolar morphology. Notably, the measurement of the biggest vacuolar structure turned out to be a very reproducible approximation for vacuolar morphology. Importantly the measured values are quite stable in between independent experiments (both untreated and treated). Moreover, the index is quite sensitive to auxin induced morphological changes, which guided us to depict earliest morphological changes following short-term auxin treatments. Notably, the vacuolar morphology index robustly correlates with the auxin effect on cell sizes. Therefore, we feel that this simple quantification method is straightforward and very suitable for the here conducted work. In this respect, we kindly agree also with reviewer 3 . Said that we also would like to mention that we would indeed further develop tools to depict and quantify the plant vacuoles. In our on-going research, we are also investigating the potential effect of auxin on vacuolar volume and whether it is a parameter that is most relevant. We will develop in the future more sophisticated 3D imaging approaches, which will allow us to address additional questions, such as raised by reviewer 1.Reviewer #2:In the subsection headed “Vacuolar SNARE function is required for the auxin-dependent modulation of vacuolar morphology”,paragraphs 1 and 2 could be made clearer. In the reference to the redundant action of SNARE components, the vti11 mutant is mentioned as displaying roundish vacuoles. Does this mean that vti11 is the only mutant in a SNARE-encoding gene that has a phenotype? Also it would be useful to explain earlier what it means that VTI11 is a Qb-SNARE (see point on SNARE background below).We thank the reviewer for pointing out the lack of discussion and improved the Introduction and Discussion on SNARE proteins. It has been previously shown that several SNARE complex components indeed function redundantly. Some mutants display defects in storage vacuoles and mild growth phenotypes . However, based on our analysis most mutants did not show any defects in vacuolar morphology.Finally, since the vacuole morphology is clearly different in the vti11 mutant from that in wild type, it is puzzling why the authors indicate that the defects in SNARE-dependent vacuolar morphology regulation in vti11 are relatively mild. Perhaps they mean that the altered vacuolar morphology in vti11 has little effect on cell growth in A and T cells?vti11 mutant vacuolar morphology is still differentially regulated in tricho- and atrichoblast. In the revised version of the manuscript, we reformulated the sentence: “Despite these apparent defects, vacuoles remained differentially controlled in vti11 mutant tricho- and atrichoblast cells, indicating that the cell type-dependent regulation of vacuolar morphology is at least partially operational in vti11 mutants.”This formulation was indeed confusing. We intended to point out that in the The authors explain in the discussion that lack of increased growth by WM treatment  and in the atrichoblasts of the vti11 mutant  could be due to cell wall limitations. I think they can expand their reasoning to include that the increased growth observed upon Kynurenin treatment insuggests that auxin acts not only on SNAREs/vacuolar morphology, but may also restrict growth by inhibiting availability of cell wall components.Yes, we agree with the reviewer, this data is in agreement with a dual role of auxin. Upcoming research in our group will further decipher this interplay.Along the same line, it seems to me a discussion could be included on coordination between tricholast and atrichoblast cell files as well as cell divisions within a cell file. For example, do any of the genetic and pharmological treatments lead to a difference in the ratio of trichoblasts:atrichoblasts in the meristematic zone or total number of cells?Indeed pharmacological treatments (high and low auxin conditions) lead to a change in the ratio of tricho- and atrichoblasts . We currcould be amalgamated into one figure.panels are identical topanels except for the higher amount of NAA. Same goes forand. I suggest addingtoand turnintoWe followed the advice and amalgamated In the subsection headed “Auxin effect on vacuolar morphogenesis interrelates with auxin-dependent cellular growth inhibition” a paragraph describes the effect of VTI11 and PI4K activity on root epidermal cell length (elongation zone) and overall root growth. Two points: 1) It is mentioned in the legend tothat trichoblasts were measured. Why not atrichoblasts?These were rather technical reasons. Visual limitations of single sections imposed the practical depiction of trichoblast cells. We used medium sections to depict the start of the differentiation zone . In these sections, a cell “without” hairs could be still a trichoblast cell whose root hair was simply not in focus. Therefore, we considered for our measurments only cells showing a root hair, which ensured that we measured only one cell type. We assume that relative tissue expansion is constant between tricho- and atrichoblasts. Therefore, the relative outcome would be the same if we had used atrichoblasts.And 2) It is surprising that WM treatment alone has a negative effect on growth . If anything, it would be expected to be positive. Can the authors explain this?Besides its effect on vacuolar morphology, WM interferes with several intracellular processes and it is likely to interfere with growth. It indeed has been previously shown that high levels of WM (33µM) have a negative impact on growth . We used very low WM concentrations and observed only a mild effect on growth. Besides its mild effect on growth, this concentration range did not only reveal auxin resistant vacuoles, but also reduced the auxin effect on cell size and root organ growth.Minor comments:Some of the headings could be made more specific. For example, “Interference with the auxin effect on vacuolar SNAREs impedes auxin-dependent cell size control”, this heading should refer to the connection made with PI-dependent processes.We thank the reviewer for this suggestion and revised the heading accordingly: “Interference with phosphatidylinositol homeostasis affects vacuolar SNAREs and impedes auxin-dependent cell size control.”I appreciate that space is limiting, but the minimal background information on the SNARE complex makes it difficult to relate to their role in this process. A few additional sentences to introduce SNARE would be useful. For example: 1) one gets the impression that SNAREs only exist in yeast and plants, although they indeed are common to all multicellular eukaryotic organisms, 2) mention their function in controlling membrane fusion, and 3) it would be useful to let the reader know what they are composed of such that the reader is prepared for what the Qb-SNARE VTI11 is.We appreciate the very valuable suggestion and included more detailed background information on the SNAREs in the revised version of the manuscript.Reviewer #3:I feel that the authors have been a little cautious in their Discussion and have made little attempt to hypothesis as to why a vacuolar SNARE may have such a dramatic effect on vacuolar morphology.We thank the reviewer for this suggestion. We indeed were very cautious in the previous version, because we did not directly address these questions. However, we agree with the reviewer and substantiated the discussion on vacuolar SNAREs in the revised version of the manuscript.It is difficult to tell if in some of the micrographs we are looking at many individual vacuoles which have not fused together due to lack of SNARE complexes or whether they are all interconnected but just change in morphology. A bit of speculation would be good.We again thank the reviewer to address this very interesting question. We included a paragraph discussing the possibility of interconnected vacuoles and cited also the latest results by Viotti et al., 2013. Ongoing research in our group is actually addressing this eminent question.Minor comments:In the beginning of the subsection headed “Vacuolar SNARE function is required for the auxin-dependent modulation of vacuolar morphology”, perhaps you need to introduce VTI11 here or at least put it in the section heading.We followed the reviewer’s suggestion and included VTI11 in the section heading.In the Discussion section, I really wonder whether turgor exerted by the vacuole in the root tip is important. The vacuole appears very pleomorphic at this stage of development.We agree with the reviewer and discussed this issue accordingly: “Hence, the potential role of auxin in turgor regulation should also be carefully assessed. Just such a role has been insinuated in recent papers related to lateral root emergence. Auxin responses have been reported to reduce cellular pressure in cell files facing lateral root primordia  and vacu


Despite the multiple available pharmacological and behavioral therapies for the management of chronic phantom limb pain (PLP) in lower limb amputees, treatment for this condition is still a major challenge and the results are mixed. Given that PLP is associated with maladaptive brain plasticity, interventions that promote cortical reorganization such as non-invasive brain stimulation and behavioral methods including transcranial direct current stimulation (tDCS) and mirror therapy (MT), respectively, may prove to be beneficial to control pain in PLP. Due to its complementary effects, a combination of tDCS and MT may result in synergistic effects in PLP.The objective of this study is to evaluate the efficacy of tDCS and MT as a rehabilitative tool for the management of PLP in unilateral lower limb amputees.A prospective, randomized, placebo-controlled, double-blind, factorial, superiority clinical trial will be carried out. Participants will be eligible if they meet the following inclusion criteria: lower limb unilateral traumatic amputees that present PLP for at least 3 months after the amputated limb has completely healed. Participants (N=132) will be randomly allocated to the following groups: (1) active tDCS and active MT, (2) sham tDCS and active MT, (3) active tDCS and sham MT, and (4) sham tDCS and sham MT. tDCS will be applied with the anodal electrode placed over the primary motor cortex (M1) contralateral to the amputation side and the cathode over the contralateral supraorbital area. Stimulation will be applied at the same time of the MT protocol with the parameters 2 mA for 20 minutes. Pain outcome assessments will be performed at baseline, before and after each intervention session, at the end of MT, and in 2 follow-up visits. In order to assess cortical reorganization and correlate with clinical outcomes, participants will undergo functional magnetic resonance imaging (fMRI) and transcranial magnetic stimulation (TMS) before and after the intervention.This clinical trial received institutional review board (IRB) approval in July of 2015 and enrollment started in December of 2015. To date 2 participants have been enrolled. The estimate enrollment rate is about 30 to 35 patients per year; thus we expect to complete enrollment in 4 years.This factorial design will provide relevant data to evaluate whether tDCS combined with MT is more effective than each therapy alone, as well as with no intervention (sham/sham) in patients with chronic PLP after unilateral lower limb amputation. In addition, this randomized clinical trial will help to investigate the neurophysiological mechanisms underlying the disease, which could potentially provide relevant findings for further management of this chronic condition and also help to optimize the use of this novel intervention.Clinicaltrials.gov NCT02487966; https://clinicaltrials.gov/ct2/show/NCT02487966 (Archived by WebCite at http://www.webcitation.org/6i3GrKMyf) Phantom limb pain (PLP) belongs to a group of neuropathic pain syndromes characterized by pain in the amputated limb -4. In WePLP is experienced by 50% to 80% of the amputees. Although PLP may decrease or disappear over time, prospective studies indicate this is often not the case. Even 2 years after amputation, 59% of the patients reported PLP with only 5% to 10 % decrease in the intensity, exemplifying how it still remains a significant clinical problem that impairs quality of life ,4.The precise mechanisms underlying development of pain in patients with limb amputation are not well elucidated. It has been demonstrated that long standing limb amputation can cause structural reorganization of the brainstem, thalamic nuclei, or the somatosensory cortex leading to maladaptive plastic changes -11. Give. Using functional magnetic resonance imaging (fMRI), Lotze et al showed that the shift in the lip representation into the primary motor and somatosensory cortex is correlated with the amount of PLP [. These findings led to the current view that this reorganizational change represents a main pathophysiological mechanism of PLP [. Current rehabilitative therapies to treat PLP do not take into account such maladaptative plastic changes. An ideal therapeutic approach to treat PLP should aim to modulate and reverse the maladaptive plastic changes involved in the development of chronic PLP [After an upper limb amputation, either shrinkage of the upper-limb region or expansion of the surrounding areas  is found in the primary somatosensory (S1) and motor (M1) cortex ,8,10,13 t of PLP ,15. Cortt of PLP -18 . Them of PLP ,20 . Curonic PLP .In this context, given that current options for pain treatment have insignificant or no effect on brain plasticity, the investigation of alternative approaches such as neuromodulation techniques can be used not only to alleviate pain but also to revert maladaptive plasticity. One candidate to promote plastic changes is transcranial direct current stimulation (tDCS). tDCS delivers a low intensity current that can modulate (facilitate or inhibit) spontaneous neuronal activity, its long term effects are likely to be mediated by mechanisms of synaptic long term potentiation and depression affecting neuroplasticity ,23.Recent studies have confirmed the therapeutic potential of tDCS in treating PLP. In 2013, Bolognini et al showed that a single session of anodal tDCS  targeting M1 induced a selective short-lasting decrease of PLP . In addiSoler et al  conducteTherefore, combining tDCS with a behavioral intervention may optimize PLP rehabilitation. Mirror therapy (MT) seems to be the optimal behavioral intervention to activate sensorimotor cortex as shown by several studies ,29-31. RIn summary, there is a great unmet need for non-invasive treatments for chronic PLP. In this protocol, we will test a novel rehabilitation approach combining a behavioral therapy (MT) with a method of brain modulation (tDCS) to treat and investigate the mechanisms of PLP.The primary aim of this clinical trial is to perform a comparative analysis of the efficacy of tDCS and MT as a rehabilitative tool for the management of chronic PLP in unilateral lower limb amputees.The secondary aim of the study is to examine the mechanisms underlying PLP using two neurophysiological techniques. Single-pulse and paired-pulse transcranial magnetic stimulation (TMS) will be utilized to assess cortical mapping and cortical excitability changes associated with cortical reorganization. In addition, fMRI will be employed to assess brain changes, including the quantification of maladaptive cortical reorganization.We hypothesize that the combination of tDCS and MT will achieve greater effects when compared with the isolated use of either tDCS or MT, as well as with the sham tDCS combined with sham MT with regard to improvement (greater pain reduction) of chronic PLP, as indexed by the VAS scale in participants with unilateral lower limb amputation.Our second hypothesis is that the combined group (tDCS and MT) will have a greater activation than any therapy alone and the no therapy group (sham tDCS and covered mirror) in the TMS and fMRI evaluations. In addition, neurophysiological and hemodynamic changes will be correlated with pain reduction.A prospective, randomized , placebo-controlled, double-blind, factorial superiority study will be carried out .Patients will be recruited from the Limb Loss Clinic of the Spaulding Rehabilitation Hospital/Network and additional recruitment around the Boston, MA area. All study procedures will be performed at the Spaulding Neuromodulation Center in the Spaulding Rehabilitation Hospital, Charlestown, MA, USA.The eligibility criteria (inclusion and exclusion) for the study are shown in Inclusion criteriaAble to provide informed consent to participate in the studySubject is older than 18 yearsUnilateral lower limb amputation3 months of PLP after the amputated limb has completely healedAverage pain of at least 4 on a numeric rating scale (NRS), ranging from 0 to 10 in the previous weekIf the subject is taking any medications, dosages must be stable for at least 2 weeks prior to the enrollment of the studyExclusion criteriaPregnancy or trying to become pregnant in the next 2 monthsHistory of alcohol or drug abuse within the past 6 months, as self-reportedPresence of the following contraindication to tDCS and TMSFerromagnetic metal in the head Implanted neck or head electronic medical devices History of chronic pain previous to the amputationHead injury with post-traumatic amnesia for greater than 24 hours, as self-reportedUnstable medical conditions Uncontrolled epilepsy or prior seizures within the last 1 yearSuffering from severe depression (as defined by a score of >30 in the Beck Depression Inventory)History of unexplained fainting spells or loss of consciousness as self-reported during the last 2 yearsHistory of neurosurgery, as self-reportedMT within 3 months prior to enrollment2) and delivered by a specially developed, battery-driven, constant current stimulator with a maximum output of 10 mA.tDCS will be performed during the MT session, as this technique may facilitate behavioral changes by enhancing neuroplasticity and increasing functional connectivity. The Soterix Medical 1×1 tDCS stimulators device  will be utilized. This device sends a low-level current from the positive electrode (anode) to the negative electrode (cathode). During tDCS, low amplitude direct currents will be applied via scalp electrodes and penetrate the skull to enter the brain. Direct current will be transferred by a saline soaked pair of surface sponge electrodes . Participants will be allowed to reschedule up to 3 stimulation visits (maximum of 2 consecutives). During each active anodal tDCS session, an anodal electrode will be placed over M1, contralateral to the amputation side and the cathode over the contralateral supraorbital area and tDCS will be applied for 20 minutes at 2 mA . For theFor the active MT sessions, participants will be asked to perform movements (15 minutes daily) using the unaffected limb while watching its mirrored reflection superimposed over the affected limb. During MT, participants will be asked to consciously relate the movement observed in the mirror to their phantom limb and to keep their attention focused on the task. Instructions will be explained verbally, demonstrated by a therapist, and performed by the subject in front of the therapist during the first 2 weeks (the MT sessions will be scheduled at the same time as the tDCS sessions). After the training, participants will continue MT everyday for 2 more weeks at home. Participants will be instructed to stop MT if it intensifies their pain, and to document if this happens. For the sham MT (covered MT), participants will be asked to perform movements in the same way as the active group but with a covered mirror.The participants in each group will be evaluated by an experienced researcher in the evaluation procedures and blinded to which group (active vs sham tDCS) each participant belongs. The following 14 evaluations will be carried out: (1) evaluation 1 will be carried out one week prior to the intervention, (2) evaluations 2-11 will occur before and after the intervention, (3) evaluation 12 will take place right after the home-based MT is finished, (4) evaluation 13 will take place 4 weeks after the home-based MT is finished, and (5) evaluation 14 will take place 8 weeks after the home-based MT is finished.Pain assessment will be indexed by the VAS for pain. This scale is commonly used to obtain self-reported ratings of pain level on a visual scale . Participants will rate the intensity of their PLP from 0  to 10 (indicating the worst pain felt). They will also report the frequency of PLP paroxysms, when PLP clearly increases above the background level from 0 (never during the day) to 10 (very frequently) ,25,34. TA pain and medication diary will be filled out daily by each participant during the total duration of the trial. This assessment tool will help to monitor daily changes in pain levels, medication dosage information, as well as safety. Participants will be asked to record the number of PLP paroxysms  on a daily basis using a pain diary. In addition, the participants will record the intensity of the strongest episode as well as non-painful phantom limb sensation, phantom movements and stump pain on different colored VAS included in the diary. Moreover, participants will record their current medications and dosages daily in a pain medication diary, until completion of the study.Participants will undergo assessments of neurocognitive and psychological aspects such as depression or anxiety. In the case of depression the subjects will be assessed with the Beck depression inventory . This seIn order to assess potential cognitive decline, participants will undergo evaluation with the Mini Mental State examination (MMSE). This is a sensitive, valid, and reliable 30-point questionnaire that is used extensively in clinical and research settings to measure cognitive abilities. It will be used as a baseline evaluation .SF-36) [Each participant will undergo assessments to evaluate changes in quality of life and safety before and after the intervention. Quality of life will be assessed using the Short Form Health Survey (SF-36) . SF-36 iSF-36) -46. In aSF-36) ,48. HereSF-36) .fMRI will be used to quantify patterns of activation associated with maladaptive cortical reorganization before and after treatment of each participant. Structural and functional imaging data will be acquired on a 3 Tesla Philips Achieva System  with a 32-channel phased array coil. Structural T1-weighted scans will be acquired using a turbo spin echo sequence . Functional scans will be acquired with a single-shot EPI sequence . Two functional runs will be collected each lasting 360 seconds in duration (see below for details regarding task design and contrasts of interest).A repeated measures design (baseline and post-treatment) will be conducted for each participant. Additional baseline data will be obtained for secondary correlations. In addition, a sensitivity analysis testing the comparison of post-treatment will be conducted.This design is based on previous work investigating the task-based activation of cortical motor networks associated with the observation and imagination of lower limb movements . The tasFor the analysis, the region of interest (ROI) will be selected. The ratio of activation between the MOV-LEG and MIR-LEG  will be determined previously. Using this coefficient, a comparison across the 4 different conditions will be performed. For this sub study, a standard safety screening questionnaire will be administered prior to participation by the attending technician or study investigator (NINDS CDE) ,52.motor-evoked potentia l (MEP) amplitudes over the rectus femoris [Transcranial magnetic stimulation (TMS) will be used to assess cortical excitability and cortical reorganization. For the TMS assessments, we will use Magstim Bistim2 stimulator and a figure 8 coil . The most distal muscle will be studied, in the case of the presence of the quadriceps we will place the electrodes over the rectus femoris, another pair of electrodes will be placed in the first dorsal interossei muscle (FDI), and a ground electrode will always be placed over the participant’s distal prominence of the ulna bone. Electromyogram (EMG) recordings will be processed using Powerlab 4/30  with a band pass filter of 20-2000 kHz. Offline analyses will be performed using LabChart . First, head measures will be taken to identify the approximate spot of the motor cortex (using the vertex as the reference) . Then, t femoris -56. The  femoris . Changes femoris .TMS measurements will include short-interval intracortical inhibition (SICI) and intracortical facilitation (ICF) using the paired-pulse technique . For paiCoGx = (Σxi *MEPi) / ΣMEPi (1)where MEPi represents the mean amplitude of the MEPs produced at one site. The sum of the average MEP amplitude will be calculated for each active site, where an active site was defined as a site at which the mean MEP amplitude was at least 0.05 mV.The study will take place for 13 weeks . All stuVisitsVisit 1: Consent and screeningVisit 2: BaselineVisits 3-12: Intervention visitAfter visit 12, the participants will continue MT  at home for 2 more weeksVisit 13: End of home intervention (MT)Visit 14: Follow-up 1, 4 weeks after visit 13Visit 15: Follow-up 2, 8 weeks after visit 14The participants randomized to receive sham tDCS will have the opportunity to enroll into an open label portion of the study at the conclusion of their participation in the randomized portion of the trial.We will recruit 132 subjects with PLP of traumatic etiology; congenital or diabetic amputees will be excluded since these patients may have different neuroplastic profiles. Participants will need to meet all of the inclusion criteria and none of the exclusion criteria.The sample size was calculated utilizing STATA 11 program. We based our calculation on a study carried out by Soler et al (2010) entitled “Effectiveness of transcranial direct current stimulation and visual illusion on neuropathic pain in spinal cord injury” . Using VIndividuals with chronic PLP after a unilateral lower limb amputation will be primarily recruited through the Limb Loss Clinic at Spaulding Rehabilitation Hospital/Network in Charlestown, MA. The Amputee Program at Spaulding Rehabilitation Hospital has more than 120 in-patients with amputations per year, and its Limb Loss Clinic has more than 200 amputees with PLP. Spaulding Rehabilitation Hospital was the primary rehabilitation center that received patients with traumatic amputations from the Boston Marathon Bombings. In addition, we will approach colleagues at the other Harvard teaching hospitals, including Brigham and Women’s Hospital (BWH) and Massachusetts General Hospital (MGH), and outpatient clinics in the greater Boston area.Once eligibility and consent have been approved and completed, randomization will occur using the randomized list generated by an automatic Web-based randomization program. Participants will be randomly assigned to 1 of the following 4 groups: (1) Group 1 will receive active tDCS and MT, (2) Group 2 will receive sham tDCS and MT, (3) Group 3 will receive active tDCS and covered MT, and (4) Group 4 will receive both sham tDCS and covered MT.The participants randomized to receive sham tDCS will have the opportunity to enroll into an open label portion of the study at the conclusion of their participation in the randomized portion of the trial.Participants will be randomly assigned to 1 of the 4 groups in a 1:1:1:1 allocation ratio. We will use stratified randomization methods with random block sizes of blocks of 4 and 8. Stratification will be based on the participant’s baseline pain levels using 2 strata: less than or equal to an average 6 in VAS or greater than 6 in VAS). The randomization order will be kept in sealed envelopes; therefore participants will get their assignment according to the order of entrance in the study .This process will be carried out by a member of the research team who is not involved in the recruitment process or development of the study.Eligible participants will be randomized based on an allocation sequence generated by an independent person not involved in the study through a true randomization process.A series of numbered, sealed, opaque envelopes will be used to ensure concealed allocation. The order of entrance to the study will determine the allocation of the participant.A researcher not involved in the study will be in charge of the allocation.Participants will be blinded by receiving sham tDCS stimulation with the same electrode montage as the active group. All trial researchers involved in the analysis and collection of data will be blinded to the treatment allocation group until after analyses are performed at the completion of the trial.The tDCS blinding questionnaire will be performed after the stimulation session. Each participant will complete a questionnaire to determine if the blinding methods were effective .Data forms and questionnaires will be coded in a standardized manner, and double-entered into our database. Digital measures and recordings will be similarly tracked in our database and regularly backed up. Analyses will be conducted using standard statistical software such as SAS and Matlab.P values for secondary and exploratory outcomes will be determined without corrections for multiple comparisons. Furthermore, Pearson’s correlation analyses will be performed to assess the association between PLP relief and changes in non-painful phantom sensations, phantom movements, and telescoping, as measured with VAS. Finally, we will apply a path analysis [The primary outcome is PLP indexed by VAS. PLP will be analyzed using intensity of pain over time. To analyze these data, we will initially adopt a mixed analysis of variance (ANOVA) model in which the dependent variable will be the outcome of PLP (such as VAS) and the independent variables will be group , time (baseline and after treatment and follow-up), and the interaction group time. In addition, we will add the random variable ID to account for within participant’s variability and the repeated measures on time. Whenever necessary, post-hoc comparisons with Bonferroni correction for multiple comparisons will be carried out initially to explore significant main effects or interactions. analysis  to the pWe propose that a direct effect of tDCS and MT on PLP can be assumed if the treatment effect cannot be explained by changes in psychological or functional outcomes.Statistical models for pain will be developed using covariates that include baseline pain, psychological changes, functional changes, and the covariate treatment (main effect of treatment). To complete the path analysis, separate regression models will be run to model the effects of treatment on each outcome alone including all the secondary outcomes. Analyses of the secondary outcomes will be conducted in an exploratory manner (no correction for multiple comparisons). Secondary outcomes are other pain measurements, psychological, neuropsychological and quality of life measurements, and neurophysiological markers (as indexed by TMS and fMRI).For the intention-to-treat analysis, we will use a conservative method and assume that participants will not improve from the last measured point. We will also perform a sensitive analysis for the missing data using other methods such as completers only.Image processing and analysis of functional data will be performed using standard analysis procedures in FSL version 5.0.5 . Preprocessing includes head motion correction, B0 unwrapping, brain extraction, intensity normalization, high pass temporal filtering with a frequency cutoff of 120 seconds, and Gaussian spatial smoothing . Registration will be performed with FMRIB's Linear Image Registration Tool (FLIRT). Each functional image will be registered to the representative T1-weighted anatomical image (using a 6 degree of freedom boundary based linear registration) and to the MNI 152 template (using a 12-parameter nonlinear affine transformation with a warp resolution of 10 mm). Individual time series analysis will be carried out using a general linear model (GLM). Both active (leg moving and viewing) and passive (rest) conditions will be convolved with a Gaussian hemodynamic response function and their temporal derivatives will be used to model the data. The primary contrasts of interest will be conducted for leg moving versus leg viewing, as well as leg moving versus rest and leg viewing versus rest. The leg moving versus rest condition will be used to identify and define a ROI associated with movement of the contralateral leg during the baseline condition. Activation within this ROI will be compared between pre and post conditions as well as within the same ROI transposed to the opposite hemisphere. As PLP is known to be associated with pathological ipsilateral activation ,15,19, cTMS data will be analyzed offline using LabChart . Offline analyses will include measures of peak-to-peak amplitude, the area-under-the-curve of all MEPs, ICF, and SICI, and the relative duration of CSPs . We will perform a mixed ANOVA model in which the dependent variable is the measurement of cortical excitability  and the independent variables are the groups  and time (pre and post intervention). Furthermore, Pearson’s correlation analyses will be performed to assess the association between PLP relief (VAS change) and cortical excitability changes, as derived from the TMS evaluations.At the end of each stimulation session, participants will complete a side effects questionnaire for tDCS in order to evaluate potential adverse effects of tDCS  and MT  on a 4-point scale . The participants will be asked whether they have experienced any side effects in an open-ended manner and they will then be specifically asked about headache, neck pain, scalp pain, scalp burns, tingling, skin redness, sleepiness, trouble concentrating, and acute mood change. If any side effects are reported, the degree of relatedness to the intervention will be assessed on a 5-point scale. This type of adverse events questionnaire has been used frequently in our previous tDCS studies , includiIn addition, the Side Effects Questionnaire for TMS will be applied at each TMS assessment session. Participants will complete a questionnaire to evaluate potential adverse effects of rTMS  on a 5-point scale .The present study complies with the principles of the Declaration of Helsinki and received approval from the ethics committee  of Spaulding Rehabilitation Network under the protocol number 2015P001065. Participants will agree to the participation by signing a statement of informed consent. The participants will be allowed to abandon the study at any time with no negative repercussions. All data will be collected in a de-identified manner. Each participant will be identified by an identification number referent to the enroll order. Data will be recorded in hardcopy and electronic form. Hardcopies will be stored in a secured filing cabinet at the administering institution. Electronic copies will be stored in encrypted files on a password-protected computer. All data will be kept for 7 years; following this time, hardcopies will be destroyed by shredding or burning and electronic copies will be deleted by formatting. Participant records will not contain any directly identifiable information.This clinical trial received IRB approval in July of 2015 and enrollment started in December of 2015. Currently, 6 participants have been screened and 2 of them successfully met the eligibility criteria. The first participant completed the entire protocol. The second participant is undergoing stimulation sessions. In addition to that, 2 participants are scheduled for screening in the next 2 months. The estimate enrollment rate is about 30 to 35 patients per year; thus we expect to complete enrollment in 4 years.To the best of our knowledge, this is the first study to combine tDCS and MT for the treatment of PLP. This paper offers a detailed description of a randomized, placebo-controlled, double blind, factorial trial aimed to evaluate the effects of tDCS combined with MT as a rehabilitative tool to decrease PLP, as well as to examine the neural mechanisms underlying PLP in unilateral lower amputees. The results will be published and will provide evidence regarding the use of tDCS combined with MT on this population.Some concerns regarding the study design should be discussed. One of them is the choice of using a factorial design; on one hand, this type of design was the best option given that there is no gold standard treatment for PLP. On the other hand, this design increases the number of groups and sample size. For the trial, a conservative approach was used to calculate the sample size in order to avoid a common limitation of clinical trials such as a difficult enrollment and recruitment. It is still possible that our sample size will not be adequate. We do consider that a sample of 132 participants will be appropriate to test our hypothesis, especially taking into account previous effects sizes and the addition of the mechanistic aims (TMS and fMRI).On the other hand, it should be noted that if we find an effect size that is smaller than the one proposed in the study it will not be considered clinically meaningful. This is a 4-year clinical trial; therefore, an expected recruitment rate of 33 subjects per year is appropriate, taking into account the amount of traumatic amputation in the New England area and that the Spaulding Rehabilitation Hospital is a major rehabilitation center for this population. In addition, the factorial design in this trial gives also the possibility of additional secondary analyses between groups. Finally, given the selection of a homogeneous sample (only lower limb and traumatic amputation), it is expected that the study will provide robust results regarding the effects of these treatments in isolation and in combination.This is a promising clinical trial, given that the previous results reported by our research group showed a decrease in PLP levels indexed by VAS after anodal tDCS when compared with sham . Additio


An aging population could substantially enhance the burden of heat-related health risks in a warming climate because of their higher susceptibility to extreme heat health effects. Here, we project heat-related mortality for adults 65 years and older in Beijing China across 31 downscaled climate models and 2 representative concentration pathways (RCPs) in the 2020s, 2050s, and 2080s. Under a scenario of medium population and RCP8.5, by the 2080s, Beijing is projected to experience 14,401 heat-related deaths per year for elderly individuals, which is a 264.9% increase compared with the 1980s. These impacts could be moderated through adaptation. In the 2080s, even with the 30% and 50% adaptation rate assumed in our study, the increase in heat-related death is approximately 7.4 times and 1.3 times larger than in the 1980s respectively under a scenario of high population and RCP8.5. These findings could assist countries in establishing public health intervention policies for the dual problems of climate change and aging population. Examples could include ensuring facilities with large elderly populations are protected from extreme heat (for example through back-up power supplies and/or passive cooling) and using databases and community networks to ensure the home-bound elderly are safe during extreme heat events. An increase in global mean surface temperature of 0.3 °C to 4.8 °C has been projected for 2081–2100 relative to 1986–2005345679Global warming and a rapid increase in age in the population are two major global challenges of the 212. The significant and rapid increase in life expectancy during 1990 to 2013 in different regions of China has occurred during the transition period of economic growth and demographic changesChina represents an iconic example of future heat exposure among the elderly due to demographic and climate change. China has the largest population in the world and will be confronted with major challenges related to its aging population in coming decades. According to a report from China’s 2010 census, the population of persons 65 years and above rose to 118.8 million from 100.5 million in 2000These demographic changes will interact with increasing heat hazards as well. According to the 3rd National Assessment Report on Climate Change, the annual average temperature of the Chinese mainland area has increased by 0.21–0.25 °C/decade in the past 50–60 years. This warming is projected to accelerate during the 21st century, leading to more frequent extreme heat eventsBeijing is a microcosm of rapid urbanization in China and is representative of the development mode followed by other growing Chinese cities in the future. This city is vulnerable to global warming due to the high concentration of older adults, as well as the urban heat island effectA number of prior studies have projected future heat-related mortality in a changing climate359101535718198421222324We first estimated the exposure-response relationship between observed daily mortality of persons 65 years of age and older and daily temperature in Beijing. We obtained downscaled temperature projections from 31 climate models and 2 RCPs, and population projections under three demographic scenarios. These inputs were then combined to estimate future heat-related mortality, which were compared with heat-related mortality in a baseline period.2.5 data covering the period between 2008 and 2011 and the O3 data covering the period between 2009 and 2011 were obtained from the Beijing Meteorological Bureau, which provides daily PM2.5 and O3 concentrations from the Beijing Baolian station. Historical data of deaths and mean temperature for the years 2008 through 2011 were collected in Beijing. Daily mortality data were obtained from the Chinese Center for Disease Control and Prevention. Daily death counts of persons 65 years of age and older from all internal causes (ICD-10 codes A00-R99) were pooled, excluding accidental causes. Daily Tmean data and other meteorological data were obtained from the China Meteorological Data Sharing Service System for Beijing (station number: 54511), which is the only official meteorological station to represent Beijing meteorological information in the world weather station network. To address potential impacts of air pollution, PMWe used a Poisson regression model combined with a distributed lag non-linear model (DLNM) to examine the impact of temperature on mortality26We used a DLNM to examine the non-linear and delayed effects of temperature on mortality because prior studies have shown that temperature can have a lagged effect on mortality2829We modeled the exposure-response curve with a natural cubic spline with 3 internal knots placed at equally spaced values, and the lag-response curve with a natural cubic spline with the 3 internal knots placed at equally spaced values in the log scale. We selected a lag of 14 days to model the effects of temperature on mortality because previous studies have suggested that the effects of temperature on heat usually have lag effects, which are potentially affected by mortality displacement2830312.5 and ozone).Sensitivity analyses for exposure-response relationship investigated potential modifications related to modeling choices, exposure metrics, and air pollution  and the DLNM package.Temperature projections were developed by first extracting downscaled outputs from 31 global scale general circulation models (GCMs) used in the Intergovernmental Panel on Climate Change Fifth Assessment report, in conjunction with 2 representative concentration pathwaysThe fifth IPCC report introduced a set of new scenarios named RCPs, which are the product of an innovative collaboration between integrated assessment modelers, climate modelers, terrestrial ecosystem modelers, and emission inventory experts1The climate model outputs from the WCRP CMIP5 multi-model datasetThe resulting 31 temperature projections for daily Tmean from 2010 to 2099 are based on three 30-year time slices, which are 2020s (2010–2039), 2050s (2040–2069) and 2080s (2070–2099), and for a baseline period 1980s (1970–1999). The 31 GCMs are described in The population of persons 65 and older in the baseline period (1980s) was obtained from the Beijing Municipal Bureau of StatisticsFuture heat adaptation was also applied in this study. Recent evidence suggests that the heat-mortality relationship may be diminishing over time due to adaptation phenomena3591540Projected mortality impacts were estimated using modeled daily Tmean. For any day with Tmean greater than MMT, the change in mortality was calculated relative to MMT for the heat effect. Daily additional heat-related deaths were computed as:Mortality is daily temperature-related additional deaths, Y0 is baseline daily mortality rate , and POP is population. ERC is attributable percentage change in mortality for a specified change in temperature, derived from the calculated relative risk at each temperature from the statistical analysis of observed data as described above.Where ΔWe computed heat-related daily deaths in this way for each time period , and then computed the average number of heat-related deaths per year. We also computed percent changes in annual average deaths from the 1980s to future time periods.We used the ANOVA-type estimation of variance components (VC) method in the uncertainty analysis. We performed the analysis in different period respectively for understanding the influence of different factor on heat-related mortality projection. These factors include the 2 RCPs, 31 GCMs, 4 population scenarios and 5 adaptation scenarios.The analyses were conducted using R software (version 3.2) and the VCA package.Projected annual heat-related deaths taking into account projected growth in the population of persons 65 years and older are shown in Projections with different adaptations to heat are shown in This study is the first to project the health effect of rising temperatures under a changing climate on the fast-aging population in the world’s most populous country over the 21st century. Previous works have projected increases in heat-related mortality under the changing climate in Europe and the U.S., but seldom with age-specific population projectionsth percentile across the range of temperatures, consistent with previous studies, which reported MMTs in the 60–80th percentile range4434445Our exposure response function was qualitatively similar to previous studies. The minimum mortality occurred at approximately the 70Nearly all previous studies assumed a constant population to experience the health risk3485th century, especially for individuals 65 years of age and older463187The temperature-mortality relationship may not remain stable over time because of adaptation to heat and other factors. For example, the heat effect in New York City declined dramatically over the 20Climate change is another key uncertainty in heat-related mortality projections421222324While we incorporated a rich range of uncertainties related to climate change, population demographics and adaptation, our work has some limitations. We assumed a constant population mortality rate in our projection. Changes in general health, access to health care, socio-economic status, and public health education and prevention are additional important uncertainties5An aging population will substantially enhance the burden of heat-related health risks in a warming climate in China. This study provides the first evidence of an important hybrid question of the aging population and global warming in this century. These findings strongly support the need for countries in making public health intervention policy for the dual problems of climate change and aging population. A range of models and multiple RCPs here enable a future health risk projection with articulation of key uncertainty, which could provide more credible information support in implementing policies that mitigate climate change and promote public health. In addition, future research may be needed to consider the effects of rapid urbanization of the city, which may be on the same order of magnitude as effects from global-scale greenhouse gas-forced climate changeHow to cite this article: Li, T. et al. Aging Will Amplify the Heat-related Mortality Risk under a Changing Climate: Projection for the Elderly in Beijing, China. Sci. Rep.6, 28161; doi: 10.1038/srep28161 (2016).


To examine the changes in the maternal mortality ratio (MMR) and causes of maternal death in Taiwan based on nationwide linked data sets.We linked four population-based data sets  to identify maternal deaths for 2004–2011. Subsequently, we calculated the MMR  and the proportion of direct and indirect causes of maternal death by maternal age and year.Based on the linked data sets, we identified 236 maternal death cases, of which only 102 were reported in officially published mortality data, with an underreporting rate of 57% [(236−102) × 100 / 236]. The age-adjusted MMR was 18.4 in 2004–2005 and decreased to 12.5 in 2008–2009; however, the MMR leveled off at 12.6 in 2010–2011. The MMR increased from 5.2 in 2008–2009 to 7.1 in 2010–2011 for patients aged 15–29 years. Women aged 15–29 years had relatively lower proportion in dying from direct causes (amniotic fluid embolism and obstetric hemorrhage) compared with their counterpart older women.Approximately two-thirds of maternal deaths were not reported in officially published mortality data. Routine surveillance of maternal mortality by using enhanced methods is necessary to monitor the health status of reproductive-age women. Furthermore, a comprehensive maternal death review is necessary to explore the preventability of these maternal deaths. The maternal mortality ratio (MMR) is a crucial indicator of the health status of reproductive-age women . The fifWhy were the maternal deaths of official published mortality data in most countries underreported? Because official published mortality data were based on information recorded on the death certificate only. Medical certifiers might intentionally  or unintentionally (to report only the mechanism of death) omit the pregnancy-related information on the death certificate. For example, women died from amniotic fluid embolism or postpartum hemorrhage might often been recorded as respiratory failure or disseminated intravascular coagulopathy on the death certificate. The underlying cause of death would be attributed to disease of respiratory system  or disease of blood or blood forming organ (Chapter III in ICD-10) instead of pregnancy, childbirth and the puerperium (Chapter XV in ICD-10).The most commonly used method to assess the extent of MMR underreporting is to link the death certificates of reproductive-age women to their birth or fetal death certificates –8,10–12.Relatively few studies from middle-income countries have linked nationwide data sets to estimate the MMR ,15. A stIn 2012, Taiwan had a population of 23 million and a gross national income per capita of US$21,967, which was lower than that of South Korea US$30,178) and Japan , two countries with a similar Confucian background 8 and Jap. The totA maternal death was defined as reproductive-age women (15–49 years) registered in COD mortality for years 2004 through 2011 and also registered in BR or BN for the same period. Because of some errors in registration of basic information, we further focused on deaths in which the birth and maternal death dates were within 42 days, according to the definition of maternal death established by the World Health Organization.To obtain more information to determine the cause of maternal death, we further linked to the NHI inpatient claims data set to get discharge diagnoses. We considered both discharge diagnoses (ICD-9-CM codes) registered in NHI inpatient claims and CODs (ICD-10 codes) recorded in the mortality to determine the causes of maternal death. Some cases had identical diagnoses in the two data sets, whereas the diagnoses for some cases differed between the two data sets because some medical certifiers might intentionally or unintentionally exclude pregnancy-related information while issuing a death certificate. For example, “amniotic fluid embolism” was recorded in the NHI inpatient claims data set, whereas “respiratory failure” was recorded as the underlying COD in mortality data set. In these discrepant cases, we determined the cause of maternal death according to the pregnancy-related discharge diagnoses recorded in the NHI claims data. If two or more pregnancy-related diagnoses were recorded in the NHI claims data, we selected the first-reported diagnosis.Causes of maternal death were classified into two types: direct and indirect causes. Deaths resulting from obstetric complications, such as hypertension , amniotic fluid embolism , or postpartum hemorrhage , were defined as direct causes. Deaths resulting from a preexisting disease or a disease that developed during pregnancy and was not due to any of the direct obstetric cause but was aggravated by the physiological effects of pregnancy, such as infectious diseases , neoplasm , or circulatory system-related diseases , were defined as indirect causes.First, we calculated the underreporting rate between the officially reported number of maternal deaths and those determined from the linked data. The underreporting rate (%) was calculated according to the following formula:  × 100 / the number of deaths estimated using the linked data. Second, we computed the MMR  according to the maternal age and year. Because the proportion of reproductive-age women has markedly increased in the past two decades, we calculated the age-adjusted MMR according to the age groups of 2004–2011 combined as a standard population. Finally, we compared the proportions of direct and indirect causes of maternal death by maternal age and year.The study design was approved by the Institutional Review Board of National Cheng Kung University Hospital (B-ER-102-120-t). The data in the administrative data sets used in our study were scrambled, and the researchers were provided identification numbers of those insured to protect patient privacy. Informed consent was not obtained because patient information was anonymized and de-identified prior to analysis.We initially identified 266 potential maternal death cases from the four linked data sets. Of these, the birth date was inaccurately recorded after the death date in seven cases, and incident deaths were reported in 23 cases . After excluding these cases, we included 236 maternal death cases in the study. The cases from the different data sets are illustrated in Of the 236 maternal deaths, only 102 were recorded in the officially published mortality data, with an average underreporting rate of 57% [(236−102) × 100 / 236]. The underreporting rates were similar for the different age groups: 58%, 58%, and 54% among women aged 15–29, 39–34, and 35−49 years, respectively. The underreporting rate decreased from 61% in 2004–2005 to 42% in 2008–2009 but increased to 65% in 2010–2011.The age-adjusted MMR was 18.4 in 2004–2005 and decreased to 12.5 in 2008–2009, but leveled off at 12.6 in 2010–2011. Conversely, according to the officially published mortality data, the age-adjusted MMR trends were stable from 2004–2005 to 2008–2009 and declined in 2010–2011 . Table 2Approximately two-fifths of maternal deaths were attributed to direct causes. Amniotic fluid embolism and postpartum hemorrhage were the two leading direct causes of maternal death. Cardiovascular diseases and stroke were the two main indirect causes of maternal death . The numIn this nationwide population-based data linkage study, we observed that approximately two-thirds of identified maternal deaths were not recorded in the original officially published mortality data. The MMR according to linked data declined between 2004 and 2011 for all groups of reproductive-age women. However, an increase in the MMR and number of deaths caused by amniotic fluid embolism and indirect causes were observed for women aged 15–29 years from 2008–2009 to 2010–2011. This change might be due to a lower number of events.The underreporting rate estimated in this study was 57%, which is similar to that estimated in previous studies –15; howeAnother reason for the discrepancy between the two estimated rates was that many women with fatal pregnancy-related complications were treated in intensive care units for a certain period before death. The physicians of the intensive care units who issued the death certificates might have unintentionally omitted mentioning pregnancy on the death certificate. The third reason for the discrepancy was that physicians in the 2000s in Taiwan were more prone to intentionally forgot reporting pregnancy status on the death certificates than their counterparts in the 1980s because the levels of litigation regarding maternal death were higher in the 2000s. Karimian-Teherani et al suggested that countries with lower levels of litigation and higher rates of autopsy for forensic reasons were associated with a lower underreporting rate of maternal death because the doctors readily mentioned the pregnancy status of the deceased women [To tackle the problem of underreporting of the MMR in the officially published mortality data, numerous countries have conducted routine surveillance linking different data sets to estimate the MMR accurately and to monitor the health status of reproductive-age women –15. In tAccording to the estimation of the Global Burden of Disease Study, the MMRs of Taiwan were 17.1 in 2000, 13.5 in 2003, and 12.2 in 2011 ,26. The Regarding the causes of maternal death, amniotic fluid embolism was the leading cause of maternal death followed by postpartum hemorrhage, according to the findings of the current study. The leading cause of maternal death was embolism in France , Canada This study had several limitations. First, despite having an ID in each data set, some maternal deaths could not be identified because of errors in transcription, a common problem encountered in using administrative data sets. Second, many women with severe obstetric complications were treated in intensive care units and died more than 42 days after delivery (late pregnancy death according to the ICD-10 codes); hence, these cases were not included in this study. Third, we could not further analyze the causes of maternal death by using the ICD codes because the results based on few cases (less than three cases) cannot be publicly revealed.Through the linkage of four nationwide administrative data sets, this study indicated that officially published mortality data had 57% fewer maternal deaths reported than the linked data. We suggest that routine surveillance of maternal mortality by using sophisticated methods is necessary to monitor the health status of reproductive-age women. Furthermore, a comprehensive review of maternal deaths is necessary to explore the possible preventive factors for designing relevant prevention programs ,29.


Two signal components with different lifetimes were obtained by processing the transverse relaxation curves with a numeric program based on the Inverse Laplace Transformation. The long lifetime components of the transverse relaxation time T2eff and longitudinal relaxation time T1 were chosen to monitor the hydraulic fluid aging. The results demonstrate that an increase of the service time of the turbine oils clearly results in a decrease of T2eff,long and T1,long. This indicates that the T2eff,long and T1,long relaxation times, obtained from the unilateral magnetic resonance measurements, can be applied as indices for degradation of the hydraulic fluid in power station turbines.A three-magnet array unilateral NMR sensor with a homogeneous sensitive spot was employed for assessing aging of the turbine oils used in two different power stations. The Carr-Purcell-Meiboom-Gill (CPMG) sequence and Inversion Recovery-prepared CPMG were employed for measuring the  In poweetc. may all change  mak mak2eff  the oil . The agi3.2.1 of the oils. The CPMG echo train was summed on the spectrometer before being returned to the software Prospa. The integrals of the CPMG echoes were fitted to Since in an inhomogeneous magnetic environment it is practically impossible to obtain FID signals, inversion recovery as described by Hurliman in  with CPM1,long was chosen to represent the longitudinal relaxation time of the turbine oils. From For clarity, only the first 30 points of the curves are shown in 4.1H relaxation time measurements with a three-magnet array unilateral magnet as a sensor. We demonstrate that the aging results in a decrease in T2eff,long and T1,long relaxation times. Therefore, T2eff,long and T1,long can be used as the indices of turbine oil aging. This method is simple and produces reliable results. The next step will focus on the measurements of more turbine oils to establish a statistical data base and measurements of other sample parameters, such as molecular diffusion, with unilateral magnetic resonance. We propose this method to predict when the turbine oils should be eliminated to prevent unexpected accidents in power stations.The aging status of phosphate ester hydraulic fluids with different service times, from two different power stations, has been studied by