Here, we describe three high-resolution cryo-electron microscopy structures of a fungal scramblase from Aspergillus fumigatus, afTMEM16, reconstituted in lipid nanodiscs. These structures reveal that Ca2+-dependent activation of the scramblase entails global rearrangement of the transmembrane and cytosolic domains. These structures, together with functional experiments, suggest that activation of the protein thins the membrane near the transport pathway to facilitate rapid transbilayer lipid movement.The lipid distribution of plasma membranes of eukaryotic cells is asymmetric and phospholipid scramblases disrupt this asymmetry by mediating the rapid, nonselective transport of lipids down their concentration gradients. As a result, phosphatidylserine is exposed to the outer leaflet of membrane, an important step in extracellular signaling networks controlling processes such as apoptosis, blood coagulation, membrane fusion and repair. Several TMEM16 family members have been identified as Ca We determine the structures of a functionally characterized TMEM16 scramblase, afTMEM16 from Aspergillus fumigatus, in lipid nanodiscs in an inactive (Ca2+-free) and an active (Ca2+-bound) conformation. These structures allow us to define key conformational rearrangements that underlie Ca2+-dependent scramblase activation. Additionally, we show that scrambling is inhibited by the lipid C24:0 ceramide (Cer24:0) and determine the 3.6 Å resolution structure of the Ca2+-bound afTMEM16/Cer24:0-nanodisc complex. These structures, together with functional experiments, suggest that the Ca2+-dependent conformational rearrangements described here allow the scramblase to locally thin the membrane at the opened lipid pathway to facilitate lipid transport.Phospholipid scramblases are unusual membrane proteins in the sense that their environment serves as their substrate. Activation of scramblases results in the fast and passive transbilayer movement of lipids. Thus, scramblases should affect the surrounding membrane to create a conduit between leaflets though which lipids can diffuse. Current models of lipid translocation, based on the structure of the Caane core . However2+ to resolutions of 4.0 and 3.9 Å, respectively  and TM3 slides by ~6 Å to reach the Ca2+-free conformation , three polar residues , and the unpaired main chain carbonyl of G441   , Video 3ing site , Video 3o-EM map , confirm2+-dependent interactions between the scramblase and the nanodisc membrane . Protein was extracted using 1% (w/v) digitonin (EMD biosciences) at 4°C for 2 hr and the lysate was cleared by centrifugation at 40,000 g for 45 min. The supernatant was supplemented with 1 mM MgCl2 and 10 mM Imidazole, loaded onto a column of Ni-NTA agarose resin (Qiagen), washed with buffer A + 30 mM Imidazole and 0.12% digitonin, and eluted with buffer A + 300 mM Imidazole and 0.12% digitonin. The elution was treated with Tobacco Etch Virus protease overnight to remove the His tag and then further purified on a Superdex 200 10/300 GL column equilibrated with buffer A supplemented with 0.12% digitonin (GE Lifesciences). The afTMEM16 protein peak was collected and concentrated using a 50 Kdmolecular weight cut off concentrator .afTMEM16 was expressed and purified as described . Briefly pDDGFP2  with afT2, washed with pentane and resuspended at 20 mg ml−1 in buffer B  with 35 mM 3-[(3-cholamidopropyl)dimethylammonio]−1- propanesulfonate (CHAPS). afTMEM16 was added at 5 µg protein/mg lipids and detergent was removed using four changes of 150 mg ml−1 Bio-Beads SM-2 (Bio-Rad) with rotation at 4°C. Calcium or EGTA were introduced using sonicate, freeze, and thaw cycles. Liposomes were extruded through a 400 nm membrane and 20 µl were added to a final volume of 2 mL of buffer B + 0.5 mM Ca(NO3)2 or 2 mM EGTA. The fluorescence intensity of the NBD (excitation-470 nm emission-530 nm) was monitored over time with mixing in a PTI spectrophotometer and after 100 s sodium dithionite was added at a final concentration of 40 mM. Data was collected using the FelixGX 4.1.0 software at a sampling rate of 3 Hz. All experiments with added ceramide lipids were carried out in the background of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG) at a ratio of 3:1. Ceramides tested include: N-stearoyl-D-erythro-sphingosine (Cer18:0), N-behenoyl-D-erythro-sphingosine (Cer22:0), N-lignoceroyl-D-erythro-sphinganine (Cer24:0), and N-nervonoyl-D-erythro-sphingosine (Cer24:1) all of which were tested at 1 mole% and 5 mole%. Chain length experiments were done in the background of 7PC:3 PG due to the availability of the long chain lipids. Lipids used include 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine , POPG (16:0-18:1C), 1,2-dioleoyl-sn-glycero-3-phosphocholine , 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) , 1,2-dierucoyl-sn-glycero-3-phosphocholine  and 1,2- dierucoyl-phosphatidylglycerol .Liposomes were prepared as described , brieflytot(t) is the total fluorescence at time t, LiPF is the fraction of NBD-labeled lipids in the inner leaflet of protein free liposomes, γ=γ’[D] where γ’ is the second order rate constant of dithionite reduction, [D] is the dithionite concentration, f0 is the fraction of protein-free liposomes in the sample, α and β are the forward and backward scrambling rate constants, respectively, andQuantification of the scrambling rate constants by afTMEM16 was determined as recently described . Briefly0, α and β while LiPF and γ are experimentally determined from experiments on protein-free liposomes. In protein-free vesicles a very slow fluorescence decay is visible likely reflecting a slow leakage of dithionite into the vesicles or the spontaneous flipping of the NBD-labeled lipids. A linear fit was used to estimate the rate of this process was estimated to be L=(5.4 ± 1.6).10−5 s−1 (n > 160). For WT afTMEM16 and most mutants the leak is >2 orders of magnitude smaller than the rate constant of protein-mediated scrambling and therefore is negligible. All conditions were tested side by side with a control preparation in standard conditions. In some rare cases, this control sample behaved anomalously, judged by scrambling fit parameters outside three times the standard deviation of the mean for the WT. In these cases, the whole batch of experiments was disregarded.The free parameters of the fit are f−1) to an OD600 of 0.8 and expression was induced with 1 mM IPTG for 3 hr. Cells were harvested and resuspended in buffer C  supplemented with 1% Triton X-100, 5 μg ml−1 leupeptin, 2 μg ml−1 pepstatin, 100 μM phenylmethane sulphonylfluoride and protease inhibitor cocktail tablets (Roche). Cells were lysed by sonication and the lysate was cleared by centrifugation at 30,000 g for 45 min at 4° C. The lysate was incubated with Ni-NTA agarose resin for 1 hr at 4°C followed by sequential washes with: buffer C + 1% triton-100, buffer C + 50 mM sodium cholate +20 mM imidazole and buffer C + 50 mM imidazole. The protein was eluted with buffer C + 400 mM imidazole, desalted using a PD-10 desalting column (GE life science) equilibrated with buffer D  supplemented with 0.5 mM EDTA. The final protein was concentrated to ~8 mg ml−1 (~250 μM) using a 30 kDa molecular weight cut off concentrator , flash frozen and stored at −80°C.MSP1E3 was expressed and purified as described . Briefly2, washed with pentane and resuspended in buffer D and 40 mM sodium cholate (Anatrace) at a final concentration of 20 mM. Molar ratios of 1:0.8:60 MSP1E3:afTMEM16:lipids were mixed at a final lipid concentration of 7 mM and incubated at room temperature for 20 min. Detergent was removed via incubation with Bio-Beads SM-2 (Bio-Rad) at room temperature with agitation for 2 hr and then overnight with fresh Bio-Beads SM2 at a concentration of 200 mg ml−1. The reconstitution mixture was purified using a Superose6 Increase 10/300 GL column (GE Lifesciences) pre-equilibrated with buffer D plus 5 mM EDTA or 0.5 mM CaCl2 and the peak corresponding to afTMEM16-containing nanodiscs was collected for cryo electron microscopy analysis.Reconstitution of afTMEM16 in nanodiscs was carried out as follows, 3POPE:1POPG lipids in chloroform (Avanti) were dried under N−1) supplemented with 3 mM Fos-Choline-8-Fluorinated (Anatrace) was applied to a glow-discharged UltrAuFoil R1.2/1.3 300-mesh gold grid (Quantifoil) and incubated for one minute under 100% humidity at 15°C. Following incubation, grids were blotted for 2 s and plunge frozen in liquid ethane using a Vitrobot Mark IV (FEI). For the +C24:0 Ceramide/+Ca2+and EDTA samples as well as +Ca2+Dataset D (–/Å2 distributed over 45 frames (1.39 e–/ Å2/frame) was used with an exposure time of 9 s (200 ms/frame) and defocus range of −1.5 μm to −2.5 μm. For the +Ca2+datasets B and C (–/Å2 distributed over 50 frames (1.39 e–/ Å2/frame) was used with an exposure time of 10 s (200 ms/frame) and a defocus range of −1.5 μm to −2.5 μm. For all samples, automated data collection was carried out using Leginon  were subjected to 3D classification using the nhTMEM16 crystal structure low-pass filtered to 40 Å as an initial model. For the 0.5 mM CaCl2 sample, datasets B and C were used combined to make dataset A; particles selected from the first round of 2D classification from each dataset were combined and subjected to a second round of 2D classification and the resulting 302,092 particles were subjected to the same 3D classification procedure. Particles from 3D classes with defined structural features  were combined and re-extracted without binning and refined without enforcing symmetry using an initial model generated in CryoSPARC , the first refinement resulted in a map with a resolution of ~6 Å. As with the +Ca2+ sample, the protein was two-fold symmetric with the exception of the resolved portion of TM6 and the overall afTMEM16/nanodisc complex was not symmetric  = 0.143 criterion using Relion Post Processing . BlocRes2+datasets  in cyroSPARC, the particles originally picked in Relion were used. Extracted particles were classified in 2D and classes with structural features consistent with afTMEM16-nanodisc complexes were selected for ab initio model generation and classification. For each dataset, the best model and the associated particles were selected for homogeneous refinement followed by b-factor sharpening. Only the C24:0 dataset had a resolution better than 6 Å from cryoSPARC so these models were not used for analysis other than to confirm the observed membrane bending and thinning , and mutated to match the afTMEM16 sequence. The final model contains residues 13–54, 60–119, 126–259, 270–312, 316–399, 418–461, 490–597, 602–657 and 705–724 and the following residues were truncated due to missing side chain density: L43, Q49, R53, K69, K70, E94, K100, Q102, K129, H130, D137, K257, E258, L316, E461, H555, F705, K713, E714, and E717. The model was improved iteratively by real space refinement in PHENIX imposing crystallographic symmetry and secondary structure restraints followed by manual inspection and removal of outliers. The model for the +Ca2+structure was built using the refined +C24:0 Ceramide structure (PDBID 6E1O) as an initial model, that was docked into the +Ca2+density using the jigglefit script in COOT (https://www2.mrc-lmb.cam.ac.uk/groups/murshudov/index.html), manually inspected, and refined using real space refinement in PHENIX as above  or rigid body refined where smaller rearrangements were observed. The final model contains residues 13–54, 60–119, 128–258, 270–312, 316–400, 420–460, 490–594, 602–659 and 705–724, and the following residues were truncated due to missing side chain density: D17, F36, R53, K70, E94, K100, Q102, E132, K241, H247, R250, K257, E310, K317, K634, K642, Y654, R704, R708, E714, E717. For all three structures, the density in the regions of TM3 and TM4 and the connecting loop (residues 274–352) was less well-defined than remainder of the structure and the model may be less accurate in this area.The maps were of high quality and we built atomic models for each structure for the transmembrane domain, most of the cytosolic region and connecting loops , as well in COOT   . To eval2+ and +Ca2+ structures, we calculated a difference map between the two volumes using the volume operation subtract function in chimera , vortexed for 1 min, and incubated at room temperature for at least 3 hr while shielded from light. The solutions were sonicated for 1 min at low power and subjected to six freeze-thaw cycles. The samples then were extruded 21 times with an Avanti Polar Lipids mini-extruder (Avanti) and 0.1 mm polycarbonate filter. The samples were extruded at 35–40°C. Extravesicular ANTS was removed with PD-10 desalting column . The average LUV diameter was ~130 nm, with an average polydispersity index (PDI) of 10%, as determined using dynamic light scattering. LUV stock solutions were stored at room temperature with an average shelf life of 7 days.The gramicidin channel-based fluorescence assay for monitoring changes in lipid bilayer properties was implemented using large unilamellar vesicles (LUVs) incorporating gramicidin (gA) and loaded with 8-aminonaphthalene-1,3,6-trisulfonic acid, disodium salt (ANTS), following published protocols . VesicleoC with an SX-20 stopped-flow spectrofluorometer (Applied Photophysics), with an LED light source. Excitation was at 352 nm and emission was recorded above 450 nm with a high-pass filter; the deadtime of the instrument is ≈ 2 ms with a sampling rate of 5000 points/s. Samples were prepared by diluting the stock lipid concentration with buffer solution (140 mM NaOH and 10 mM HEPES) to 125 μM LUV; each sample was incubated for at least 10 min before several 1 s mixing reactions. Each sample was first mixed with the control buffer (no Tl+), followed by mixing with the quench solution (50 mM TINO394 mM NaNO3 and 10 mM HEPES). Experiments are conducted with two independently prepared populations of vesicles per lipid/ceramide combination and traces are analyzed in MATLAB (MathWorks).The time course of the ANTS fluorescence was measured at 25The fluorescence quench rate therefore was determined as described  by fittiF(t) denotes the fluorescence intensities at time t; β (0 < β ≤1) is a parameter that accounts for the dispersity of the vesicle population; and τo is a parameter with dimension of time. F(0), F(∞), β and τo were determined from a nonlinear least squares fit of t = 2 ms. The ceramide-induced changes in the quench rate then was evaluated as2+-bound in the presence of C24:0 Ceramide afTMEM16/nanodisc complexes have been deposited in the Electron Microscopy Data Bank under accession numbers EMD-8948, EMD-8931, and EMDB-8959, respectively. The deposition includes corresponding masked and unmasked maps, the mask used for the final FSC calculation, and the FSC curves. Coordinates for the models of the calcium-bound, calcium-free, and Ca2+-bound ceramide inhibited states have been deposited in the Protein Data Bank under accession numbers 6E0H, 6DZ7, and 6E1O respectively. All other data are available from the corresponding author upon reasonable request.The three-dimensional cryo-EM density maps of the calcium-bound, calcium-free, and Ca In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.2+ activation of a TMEM16 scramblase" for consideration by eLife. Your article has been reviewed by three peer reviewers, including X as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by a Reviewing Editor and Richard Aldrich as the Senior Editor. The following individuals involved in review of your submission have agreed to reveal their identity: Stephen Barstow Long (Reviewer #3).Thank you for submitting your article "Cryo-EM structures reveal bilayer remodeling during CaThe reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:2+. The structures thereby reveal Ca2+-dependent conformational changes that are mechanistically satisfying, consistent with existing functional and mutational data, and allow the authors to propose a plausible model for lipid scrambling by this class of enzymes. There are two novel conclusions: first, gating of the afTMEM16 scramblase by Ca2+ involves global conformational changes that affect the organization of the lipid bilayer and, second, that scrambling is dependent on the species of lipids present in the reconstitution. In terms of presentation, these are two independent stories that could be better integrated and focused. The first story about TMEM16 gating is solid, but the second story is not as well developed.This interesting manuscript presents three nice cryo-EM structures of afTMEM16 in nanodiscs. The cryo-EM is well-done. The densities support the atomic models and are consistent with FSC-indicated resolution limits. The three structures are a significant advance in our understanding of TMEM16 scramblases. First, they represent the first structures of TMEM16 scramblases determined in lipid nanodiscs (a previous structure of nhTMEM16 scramblase had been determined in detergent). Second, they are determined in the presence and absence of CaEssential revisions:2+ and the lipid-scrambling pathway that becomes present only when Ca2+ is bound. The authors certainly make these points, but they could be strengthened and clarified. They certainly warrant a movie showing the transition and the formation of the lipid pathway when Ca2+ is bound. Furthermore, we found the manuscript somewhat confusing and a bit disorganized at times. A series of suggestions below (see point 4) should help clarify these issues.1) The major focus of the manuscript should be on the remarkable conformational changes in the protein that are induced by the binding of Ca2+ was included. This off-centered nature is an interesting point, and we agree that it potentially provides a clue to the mechanism, but we would not be comfortable saying that this is direct evidence that the protein causes bilayer remodeling in the presence of Ca2+. For example, is it possible that the classification of the particles identified those that were off-center? Perhaps the large size of the nanodisc allows the TMEM16 protein to "rattle around" within it such that its distribution within the nanodisc is perturbed by minor effects. The authors state (subsection “Ca2+-dependent remodeling of the nanodisc upon scramblase activation”): "We… observed clear, Ca2+-dependent remodeling of the membrane around the protein in both the 2D class averages and 3D reconstructions ." We could not identify in the 2D class averages what the authors were referring to: both 0 Ca2+ and 0.5 mM Ca2+ seemed to show asymmetry within the nanodisc. Thus, it is unclear what is meant by "nanodiscs containing afTMEM16 are more distorted in the presence than in the absence of Ca2+". To our mind, there are many possible explanations for the asymmetry, and "bilayer remodeling" should not be a major focus of the manuscript or its Title. Perhaps the emphasis could be shifted to the Ca2+-dependent conformational changes that are observed in a lipid environment.2) We feel uneasy about the emphasis placed upon what the authors refer to as "bilayer remodeling". The structures are determined in lipid nanodiscs. Therefore, the density surrounding the transmembrane region of afTMEM16 would be due to combination of the nanodisc scaffold protein and lipid molecules. The authors observe that the TMEM16 protein is off-center in the nanodisc in the datasets in which Ca2+-dependent scrambling, but also inhibits Ca2+-independent scrambling . The scrambling with 5% C24:0 ceramide in the absence of protein seems to be significantly augmented as if the liposome integrity has been compromised.3) The data on the effects of lipid chain length and ceramide require additional work. The lipid effects are more complicated than appears at first glance. The experiments on the effects on chain length are weakened by the limited variety of lipids tested and the fact that the C22 lipids not only differ in chain length but also in saturation. Figure 6B was hard to decipher because the denotation 3PE:1PG and 7PG:3PG made it seem that both mixtures contained saturated acyl chains, but Methods indicates that the lipids in C22 PC:PG (DEPC and DEPG) contain two unsaturated fatty acids, while the lipids in C16-18 PC:PG (POPC and POPG) contain only one. Thus, we do not know whether the inhibition of scrambling in Figure 6B is due to chain length or saturation. Is this inhibition actually related to the membrane thickness as the authors suppose or does DEPC or DEPG inhibit afTMEM16 directly? What is the effect of addition of 5% DEPC or DEPG to POPE:POPG liposomes? The observation that C24:1 ceramide has no effect is fascinating, but does C22:1 also have no effect? Also, ceramide not only affects CaWe see two ways of addressing these concerns. The authors might solidify the suggested mechanism by showing (1) scrambling rates in PC:PG with C22 saturated acyl chains, (2) the effect of 5% DEPC/DEPG in POPE:POPG liposomes, and (3) the effect of C22:1 ceramide. We realize that some of these experiments may be constrained by the commercial availability of lipids. Thus, if saturated C22 PC:PG is not available commercially, the authors could drop (1), but show the concentration dependence of DEPC/DEPG in a POPE:POPG background and try to relate this to membrane thickness . Alternatively, the authors could remove most of the functional data, and just show inhibition by ceramide and acknowledge that more work will need to be done to clarify its mechanism. The cryo-EM structure of nhTMEM16 with ceramide is intriguing, but unfortunately does not provide any mechanistic insight into ceramide inhibition of the scrambling.4) Suggestions for improving presentation clarity:2+ 4.1) To benefit the reader who is unfamiliar with the other structures of TMEM16 proteins, it would be beneficial to provide additional context to the current structures. E.g.: They represent the first structures of TMEM16 scramblases determined in lipid nanodiscs – a previous structure of nhTMEM16 scramblase had been determined in detergent. They also represent first structural information for afTMEM16. It could be emphasized that a lipid environment is particularly important to understand the conformational changes +/- Ca2+-bound afTMEM16 scramblase in nanodisc", but the section goes on to discuss both the Ca2+-bound structure and conformational changes in the absence of Ca2+. Moreover, the first paragraph of this section is a bit confusing because it was difficult to discern the important information. Terms were introduced without context (e.g. the "hydrophobic dimer cavity"). The discussion of C1 vs. C2 might be better placed elsewhere.4.2) The first subsection is titled "Structure of the Ca2+ binding induces global rearrangements in afTMEM16" Ca2+-induced conformational changes are described partly as a movement going from the Ca2+-free to the Ca2+-bound state (sentences starting with "The cytosolic domains of afTMEM16 are translated…" and "In the absence of Ca2+…"), but partly as a movement going from the Ca2+-bound to the Ca2+-free state (sentence starting with "From the Ca2+-bound conformation…"). This is confusing to the reader. It might be better to describe conformational differences between the two structures consistently as going from one particular conformation to the other.4.3) In subsection "Ca2+ structure). If mentioned (for the 0 Ca2+ or ceramide structures), it would be more accurate to refer to the "lipids" as "density consistent with the acyl chains of lipids" or something similar since densities for intact lipid molecules are not observed. The atomic coordinates of the 0 Ca2+ structure do not include lipids – they should either be included in the coordinates or removed altogether.4.4) It is questionable whether lipids  can be resolved at 3.9Å resolution  should help clarify these issues.1) The major focus of the manuscript should be on the remarkable conformational changes in the protein that are induced by the binding of CaAs suggested by the reviewers, we extensively re-organized the manuscript, harmonized the presentation of the results and added emphasis to the protein conformational changes.2+ was included. This off-centered nature is an interesting point, and we agree that it potentially provides a clue to the mechanism, but we would not be comfortable saying that this is direct evidence that the protein causes bilayer remodeling in the presence of Ca2+. For example, is it possible that the classification of the particles identified those that were off-center? Perhaps the large size of the nanodisc allows the TMEM16 protein to "rattle around" within it such that its distribution within the nanodisc is perturbed by minor effects. The authors state (subsection “Ca2+-dependent remodeling of the nanodisc upon scramblase activation”): "We… observed clear, Ca2+-dependent remodeling of the membrane around the protein in both the 2D class averages and 3D reconstructions ." We could not identify in the 2D class averages what the authors were referring to: both 0 Ca2+ and 0.5 mM Ca2+ seemed to show asymmetry within the nanodisc. Thus, it is unclear what is meant by "nanodiscs containing afTMEM16 are more distorted in the presence than in the absence of Ca2+". To our mind, there are many possible explanations for the asymmetry, and "bilayer remodeling" should not be a major focus of the manuscript or its Title. Perhaps the emphasis could be shifted to the Ca2+-dependent conformational changes that are observed in a lipid environment.2) We feel uneasy about the emphasis placed upon what the authors refer to as "bilayer remodeling". The structures are determined in lipid nanodiscs. Therefore, the density surrounding the transmembrane region of afTMEM16 would be due to combination of the nanodisc scaffold protein and lipid molecules. The authors observe that the TMEM16 protein is off-center in the nanodisc in the datasets in which CaAs suggested, we removed bilayer remodeling from the Title, overall de-emphasized remodeling and the asymmetry of the complexes, and more clearly separate the description of the data from our interpretation.We renamed the subsection on membrane remodeling to “Structural analysis of the afTMEM16/nanodisc complex”, we significantly shortened it and moved some of the more speculative portions to the Discussion section. We apologize for not clearly explaining what we mean by membrane remodeling. We now state that “Inspection of these maps suggests the membrane in nanodiscs containing afTMEM16 is bent along the two dimer cavities of the scramblase  and that there is a region of low density at the open lipid pathway .” We added several figures to better illustrate how our data supports these statements. Briefly:2+, one in Ca2+ free and one with 0.5 mM Ca2+ and 5 mole% C24:0 ceramide). This is shown in Figure 5A, C and Figure 5—figure supplements 1, 2 and 3.i) We see the membrane bending along both dimer cavities in 5 datasets (3 in the presence of 0.5 mM Ca2+ (when the pathway is open). This is shown in Figure 5B, and Figure 5—figure supplements 1 and 2.ii) We see weakening of the density at both lipid pathways in the 3 datasets in the presence of Ca2+ with Relion, cryoSPARC or cisTEM yields similar looking maps of the afTMEM16/nanodisc complex. This is shown in Figure 5—figure supplement 3. The different processing strategies for all datasets are reported in Supplementary file 2.iii) These effects are not dependent on the data processing algorithm. We show that re-processing the high-resolution dataset in 0.5 mM Ca+ channel in nanodiscs  in Figure 8—figure supplement 3 as an example and note that the channel-only homologue TMEM16A does not distort nanodiscs . This is now discussed in the Discussion section.iv) These effects are specific to the afTMEM16 scramblase. Reconstitution of other membrane proteins in nanodiscs is not generally associated to membrane distortions. We show the low pass filtered unmasked map of the SthK KWe note that a recent report from the Dutzler and Paulino labs describes similar findings for the nhTMEM16. They report bending of the nanodisc membrane and of the detergent micelle at the dimer cavities similar to what we see. This is reported in a manuscript currently available online in bioRxiv .The reviewers raise 3 main technical concerns:i) “The structures are determined in lipid nanodiscs. Therefore, the density surrounding the transmembrane region of afTMEM16 would be due to combination of the nanodisc scaffold protein and lipid molecules.”We agree with the reviewers that the nanodisc density is the combination of the nanodisc and MSP1E3 protein and that it is likely that the afTMEM16 scramblase distorts both to a similar extent. This is now noted in subsection “Structural analysis of the afTMEM16/nanodisc complex”.2+ . Thus, the same effects are seen regardless of the distance from the nanodisc edge suggesting that the density of the MSP does not contribute significantly to them.However, we do not think that the effects we describe are due to the scaffold protein, which is localized at and defines the outer edge of the nanodisc. If the MSP protein contributed to the membrane density, then we would expect that its effect should depend on the relative position of the protein within the nanodisc. In contrast, we observe bending at both dimer cavities in 5 datasets, regardless of the positioning of the protein within the nanodisc . Similarly, the weakening of the density is seen at both open pathways of the 3 datasets obtained in the presence of 0.5 mM Ca2+ was included. This off-centered nature is an interesting point, and we agree that it potentially provides a clue to the mechanism, but we would not be comfortable saying that this is direct evidence that the protein causes bilayer remodeling in the presence of Ca2+.”ii) “The authors observe that the TMEM16 protein is off-center in the nanodisc in the datasets in which Ca2+ is interesting, we cannot draw mechanistic insights from it. To minimize confusion, we describe this observation only at the beginning of the manuscript, where we discuss the processing of the data in C1 vs. C2 symmetry .We agree that while the acentric position of the protein within the nanodisc in the presence of Ca2+ and 0.5 mM Ca2+ seemed to show asymmetry within the nanodisc.”iii) “We could not identify in the 2D class averages what the authors were referring to: both 0 Ca2+. This is consistent with the observation that the membrane bending along the dimer cavities is seen independently of Ca2+.We thank the reviewers for pointing this out. We now clarify that the 2D classes show that the nanodisc is bent both in the presence and absence of Ca3) The data on the effects of lipid chain length and ceramide require additional work. The lipid effects are more complicated than appears at first glance. The experiments on the effects on chain length are weakened by the limited variety of lipids tested and the fact that the C22 lipids not only differ in chain length but also in saturation. Figure 6B was hard to decipher because the denotation 3PE:1PG and 7PG:3PG made it seem that both mixtures contained saturated acyl chains, but Methods indicates that the lipids in C22 PC:PG (DEPC and DEPG) contain two unsaturated fatty acids, while the lipids in C16-18 PC:PG (POPC and POPG) contain only one. Thus, we do not know whether the inhibition of scrambling in Figure 6B is due to chain length or saturation. Is this inhibition actually related to the membrane thickness as the authors suppose or does DEPC or DEPG inhibit afTMEM16 directly?We thank the reviewers for raising this excellent point. We carried out additional experiments where we form liposomes using a 7:3 DOPC:DOPG mixture. These lipids have two chains with 18 carbons and with one unsaturated bond, therefore directly allow us to test the effect of saturation on scrambling. Our results show that afTMEM16 is fully functional in these lipids, indicating that presence of two saturated tails does not inhibit scrambling. These data have been added to Figure 6 and Figure 6—figure supplement 1 and are discussed in subsection “Lipid dependence of scrambling by afTMEM16”.What is the effect of addition of 5% DEPC or DEPG to POPE:POPG liposomes? The observation that C24:1 ceramide has no effect is fascinating, but does C22:1 also have no effect?Unfortunately, the C22:1 ceramide lipid is not commercially available preventing us from experimentally testing its effect.2+-dependent scrambling, but also inhibits Ca2+-independent scrambling .Also, ceramide not only affects Ca2+ is now discussedThe effect of the long chain ceramides on scrambling in 0 Casubsection “Lipid dependence of scrambling by afTMEM16”.The scrambling with 5% C24:0 ceramide in the absence of protein seems to be significantly augmented as if the liposome integrity has been compromised.Addition of 5% C24:0 leads to an asymmetric initial distribution of the NBD-PE probe, as shown by the fact that the steady state value of the fluorescence decay of the protein-free liposomes dips below the 50% mark. This asymmetry does not reflect compromised liposome integrity, as evidenced by the fact that the fluorescence reaches a plateau and remains stable for the duration of the experiment. In the case of leaky liposomes we observe a slow decay of fluorescence towards 0 as all fluorophores become bleached. This is now noted in the figure legend to Figure 6—figure supplement 2. We observe this asymmetric distribution in a variety of different lipid compositions  without adverse effects on liposome integrity. Indeed, a similar asymmetric initial distribution is visible in the long chain DEPC:DEPG liposomes . While do not know the origins of this preferential direction of incorporation of the lipids, we speculate it is likely due to headgroup packing and membrane curvature. Importantly, this asymmetry does not affect our estimate of the effects of the C24:0 on the scrambling rate constants. In fitting the time course of fluorescence decay in proteoliposomes to Equation 1, the fraction of labeled lipids in the inner leaflet is fixed to the value measured in the protein free liposomes from a same-day reconstitution. Please see Malvezzi et al. (2018) for a complete description of this analysis.We see two ways of addressing these concerns. The authors might solidify the suggested mechanism by showing (1) scrambling rates in PC:PG with C22 saturated acyl chains, (2) the effect of 5% DEPC/DEPG in POPE:POPG liposomes, and (3) the effect of C22:1 ceramide. We realize that some of these experiments may be constrained by the commercial availability of lipids. Thus, if saturated C22 PC:PG is not available commercially, the authors could drop (1), but show the concentration dependence of DEPC/DEPG in a POPE:POPG background and try to relate this to membrane thickness . Alternatively, the authors could remove most of the functional data, and just show inhibition by ceramide and acknowledge that more work will need to be done to clarify its mechanism.As discussed above, we performed experiments in DOPC:DOPG to address the reviewers’ concerns and show that acyl chain saturation has no effect on lipid scrambling. This approach avoids concerns about the formation of microdomains that might arise in mixed chain length membranes.Unfortunately, neither the C22 PG lipids with one saturated chain nor the C22:1 ceramide lipids are commercially available, preventing us from carrying out the other suggested experiments.The cryo-EM structure of nhTMEM16 with ceramide is intriguing, but unfortunately does not provide any mechanistic insight into ceramide inhibition of the scrambling.2+-bound and ceramide inhibited afTMEM16/nanodisc complex”.We believe that the structure of afTMEM16 in the presence of ceramide provides limited but important mechanistic information. First, it rules out the possibility that this inhibitory lipid acts by preventing opening of the groove; second, it suggests that scrambling is determined by the balance between the protein’s ability to bend and thin the membrane and the chemico-physical properties of the membrane itself. These points are now clarified in subsection “Structure of the Ca4) Suggestions for improving presentation clarity:2+ 4.1) To benefit the reader who is unfamiliar with the other structures of TMEM16 proteins, it would be beneficial to provide additional context to the current structures. E.g.: They represent the first structures of TMEM16 scramblases determined in lipid nanodiscs – a previous structure of nhTMEM16 scramblase had been determined in detergent. They also represent first structural information for afTMEM16. It could be emphasized that a lipid environment is particularly important to understand the conformational changes +/- CaWe added emphasis to these points in the Abstract, Introduction, main text and Discussion section.2+-bound afTMEM16 scramblase in nanodisc", but the section goes on to discuss both the Ca2+-bound structure and conformational changes in the absence of Ca2+. Moreover, the first paragraph of this section is a bit confusing because it was difficult to discern the important information. Terms were introduced without context (e.g. the "hydrophobic dimer cavity"). The discussion of C1 vs. C2 might be better placed elsewhere.4.2) The first subsection is titled "Structure of the CaWe re-organized the first three paragraphs of the Results section to provide a better logical flow.2+ binding induces global rearrangements in afTMEM16" Ca2+-induced conformational changes are described partly as a movement going from the Ca2+-free to the Ca2+-bound state (sentences starting with "The cytosolic domains of afTMEM16 are translated…" and "In the absence of Ca2+…"), but partly as a movement going from the Ca2+-bound to the Ca2+-free state (sentence starting with "From the Ca2+-bound conformation…"). This is confusing to the reader. It might be better to describe conformational differences between the two structures consistently as going from one particular conformation to the other.4.3) In the subsection "Ca2+-free to Ca2+-bound conformation in the second and third paragraphs.We harmonized our descriptions as going from the Ca2+ structure). If mentioned (for the 0 Ca2+ or ceramide structures), it would be more accurate to refer to the "lipids" as "density consistent with the acyl chains of lipids" or something similar since densities for intact lipid molecules are not observed. The atomic coordinates of the 0 Ca2+ structure do not include lipids – they should either be included in the coordinates or removed altogether.4.4) It is questionable whether lipids  can be resolved at 3.9Å resolution (the 0 Ca2+-free structure.We removed the lipid chains from the figure and from the coordinates of the Ca


This increase is correlated with a larger store-operated calcium entry (SOCE) amplitude which is responsible for the Ca2+ ions influx. Furthermore, using a SOCE potentiating agent, we demonstrated that in the MCL Rec-1 cell line, the SOCE is already activated in resting conditions. Interestingly, this potentiating agent alone, by disturbing the SOCE, induced the apoptosis of Rec-1 cells with the same efficacy than rituximab. The use of the potentiating agent in addition to rituximab strengthens the rituximab-induced apoptosis of rituximab-sensitive Granta-519 and Rec-1 cells. However, this potentiating agent cannot convert the Jeko-1 rituximab-resistant to a rituximab-sensitive cell line. Our results confirm that the use of compound acting on the Ca2+ homeostasis could be a new target of interest in complement to existing therapies.Mantle Cell Lymphoma (MCL) is one of the worst lymphomas with a median overall survival of 3 to 4 years. Even if the use of rituximab was a great step in therapy, patients commonly develop resistance and relapse. New therapies or complement of existing therapies should be developed. Using spectrofluorimetry, we found that the resting cytosolic Ca Rituximab (RTX), a chimeric IgG1 anti-CD20 monoclonal antibody, allows the depletion of B cells by inducing their apoptosis by complex pathways . It has 2+ ion influx, known as the Store-Operated Calcium Entry (“SOCE”) and is crucial for apoptosis cyt of cells is near 100 nM in physiological conditions [2+ homeostasis of cells from four MCL patients.It is well admitted that the resting [Canditions . Using s2+]cyt of two healthy volunteers (HV) circulating B cells was ≈ 150 nM , the three others were largely and significantly increased: 390 ± 7, 347 ± 13 and 243 ± 12 nM respectively for MCL patients MCL1, MCL2 and MCL4 ,.The resting [Ca2+]cyt of leukocytes is mainly driven by the Ca2+ influx, known as Store-Operated Ca2+ Entry (SOCE) in lymphocytes, we next studied the variation of [Ca2+]cyt (Δ[Ca2+]cyt) under the stimulation by thapsigargin (TG). TG, by inhibiting the Ca2+ pumps responsible of Ca2+ ion accumulation in the endoplasmic reticulum (ER), induces the passive release of Ca2+ ions by the ER, and next the opening of the plasma membrane Orai1 channels resulting in a SOCE. No significant differences were observed between the four patient lymphoma cells and the healthy donor B cells . Thus, the MCL1 cell peak Δ[Ca2+]cyt reached 2339 ± 34 nM vs. 144 ± 7 nM for HV1 cells .As the resting [Ca B cells  in extra2+]cyt increase after Ca2+ ion readdition was totally blocked by 30 μM 2-APB  respectively for Jeko-1, Granta-519 and Rec-1 cells. Thus the resting [Ca2+]cyt of Granta-519 and Rec-1 cells is significantly higher than the one of Jeko-1 cells (p<0.05 and <0.01 respectively).As shown in 2+ homeostasis of MCL cell lines is not homogenous, and that the Rec-1 cell line presents a resting [Ca2+]cyt similar to what is observed with activated lymphocytes cyt, the three MCL cell lines responded differently to TG after Ca2+ readdition, especially Rec-1 cells  vs. 423 ± 16 nM  and 484 ± 14 nM  respectively. The Δ[Ca2+]cyt rise after Ca2+ readdition was inhibited by 30 μM 2-APB .As with the resting cyt and SOCE) we observed between the MCL cell lines are not directly linked to a difference of Ca2+ transporter expression.As the two main proteins responsible for the SOCE in B cells are STIM1 and Orai1, we next performed western-blotting to study their expression in the MCL cell lines  and 3C. 2+]cyt and SOCE amplitude without any significant difference in Orai1 and STIM1 expression compared to the other MCL cell lines. Thus, we wondered if the Ca2+ homeostasis changes could be due to an already activated SOCE in resting conditions allowing a passive extracellular Ca2+ influx. To assess if the SOCE is already opened, we next added 1 mM MDEB, a compound we recently characterized with only potentiating capacity on activated-SOCE to Rec-1 cells in presence of 1 mM extracellular CaCl2 [2+]cyt . On the opposite, when 10 mM EGTA was added to chelate the extracellular Ca2+ ions, the resting [Ca2+]cyt decreased instantaneously. As MDEB acts only on activated-SOCE [2+]cyt, our results showed that in resting conditions, Rec-1 cell SOCE is already activated and Rec-1 cells possess a “leaky SOCE”.Rec-1 cell line is the cell line with the largest values of resting cyt quickly fell.To confirm that MDEB targeted the SOCE, we also performed competition experiments with 2-APB as we previously done on Jurkat T cells . As clea2+]cyt 2 fragment (in presence or absence of 1 mM MDEB (Next the three MCL cell lines were stimulated in presence of extracellular 1 mM CaCl mM MDEB ).2+ (black curves) in the three cell lines. As shown previously on several Burkitt Lymphoma cell lines cyt rise.RTX alone was only able to slowly and weakly mobilize cytosolic Call lines , F(abʼ)22+]cyt in the different cell lines (red traces), especially in Jeko-1 cell lines which had a transient CL-RTX response Thus, in Jeko-1 cells, the CL-RTX-induced peak [Ca2+]cyt reached 510 ± 15 nM in presence of 3 mM MDEB . After 10 min of stimulation, the [Ca2+]cyt of Jeko-1 cells remained higher in presence of MDEB  but continuously decreased.MDEB addition in the same time as CL-RTX increased the Δ[Ca2+]cyt reached 214 ± 16 vs. 200 ± 4 nM in presence or absence of MDEB respectively (n = 3 for both).In Granta-519 cells, 1 mM MDEB had no statistical effects during the 10 min of the experiment even if a faint increase is viewable: [Ca2+]cyt was significantly increased by 1 mM MDEB: 669 ± 34 vs. 582 ± 17 nM . After 10 min of treatment, even if the MDEB-induced increase was weaker, it was still significant: 582 ± 15 vs. 541 ± 10 nM (n=3 for both). Interestingly, MDEB presence did not impair the constant increase of [Ca2+]cyt induced by CL-RTX.In Rec-1 cells, the CL-RTX – induced peak cyt after a stimulation cyt was recorded by a fluorimetric ratio technique as in cyt was calculated according to the equation [Ca2+]cyt = Kd (R-Rmin)/(Rmax-R), where Kd is the apparent dissociation constant of Indo-1-calcium complex (230 nM), and R is the ratio of fluorescence values recorded at 380 nm in absence and presence of 10 mM CaCl2 cyt subsequently to Ca2+ influx cyt), cells were resuspended in 1 mM CaCl2 – containing HBS. Then recordings of Indo-1 fluorescence were done in the same conditions, still with Ca2+-containing HBS.For resting cytosolic calcium concentration measurements (rest[Ca2) and MDEB 1 mM or left untreated. Then according to the manufacturer’s instructions, cells were spun, fixed, permeabilized and the Terminal transferase dUTP Nick End Labelling (TUNEL) reaction was performed during 60 min at 37°C in darkness. For visualization of results, an inverted epifluorescence microscope  was used at an excitation wavelength of 488 nm, and an emission wavelength of 545 nm. Cells were also loaded with 4ʼ, 6ʼ –diamidino-2-phenylindole (DAPI) to visualize the nuclei. Total cell number and TUNEL-positive cells were counted in several fields and the ratio of apoptotic cells was calculated. This experiment was repeated three times.We measured the apoptosis levels in the different cell lines with the “in situ cell death detection” kit . BrieflTM Mini Protease Inhibitor Cocktail Tablets, Roche Diagnostics, Meylan, France). SDS-PAGE electrophoresis (10%) was subsequently performed (50 μg proteins/lane). Proteins from the gels were electrotransferred onto Hybond ECL membrane . After blocking with 5% non-fat dry milk dissolved in Tris-buffered saline containing 0.1% Tween 20 (“Tris buffer”) for 1h at room temperature, blots were washed three times with Tris buffer and probed 3h with specific primary antibody in 5 % non-fat dry milk in Tris buffer. The primary antibodies used against Orai1 (1:1000), STIM1 (1:1000), cyclin D1 (1:500) and tubulin (1:3000) were from Sigma-Aldrich, Saint Quentin Fallavier, France). The anti-tubulin antibody was used to verify that equal amounts were loaded in each lane. Detection was performed using the enhanced chemiluminescence reagent . Quantification of the bands was performed with the ImageJ software.For cellular protein extraction, one million cells were lysed using RIPA buffer  supplemented with an antiprotease pellet .Methyl-diethylborinate (MDEB), thapsigargin (TG) and ionomycin were from Sigma-Aldrich . Rituximab  was a kindly gift of the Central Pharmacy of Hôpital Saint Louis . A goat anti-human F(abʼ)Given values are representative of at least 3 independent experiments and are given as mean ± SEM. When used, a t-test < 0.05 is considered as significant.


The selection of appropriate sites for miniscrew insertion is critical for clinical success. The aim of the present study was to evaluate how interradicular spaces measured on panoramic radiograph compare with Cone-Beam Computed Tomography (CBCT), and how crowding can influence the presence of available space for miniscrew insertion, in order to define a new “safe zones” map.  A total of 80 pre-treatment panoramic radiographs and 80 CBCT scans with corresponding digital models were selected from the archives of the department of Dentistry, Aarhus University. Crowding was measured on digital models, while interradicular spaces mesial to the second molars were measured on panoramic radiographs and CBCTs. For panoramic radiographs, a magnification factor was calculated using tooth widths measured on digital models. Statistical analyses were performed to investigate the correlation between the amount of crowding and the available interradicular space. Visual maps showing the amount of interradicular spaces measured were drawn. The most convenient interradicular spaces are those between the second molar and the first premolar in the mandible, and between the central incisors in the maxilla. However, some spaces were revealed to be influenced by crowding. Calibration of panoramic radiographs is of utmost importance. Generally, panoramic radiographs underestimate the available space. Preliminary assessment of miniscrew insertion feasibility and the related selection of required radiographs can be facilitated using the new “safe zone” maps presented in this article.  Different malocclusions show differences in bone availability between the roots due to dentoalveolar compensation of skeletal discrepancies.Landin et alA miniscrew insertion site evaluation method that would minimize or even discard the use of ionizing radiation would be very advantageous for both clinicians and patients in light of the ALARA (As Low As Reasonably Achievable) principle. For this reason, the present paper addresses the following question: Is there a way of increasing the amount of information extrapolated from a traditional radiograph by combining it with specific clinical observations to ensure safe miniscrew placement at low radiation cost? The goal is to help the clinician from the first step of his orthodontic treatment planning, choosing the appropriate clinical and/or radiological examination, in order to determine the possible insertion site(s) of miniscrews.Therefore, the aims of this study were to evaluate: 1) how the assessment of interradicular spaces on panoramic radiographs performs compared with CBCTs; and 2) how the presence of radiologically adequate interradicular spaces correlates with tooth size-arch length discrepancy, in order to define a new “safe zones” map. The hypothesis was that the amount of tooth size-arch length discrepancy can be used as a pre-treatment clinical tool to assess the possibility of miniscrew insertion.Permission to use the material for this retrospective study was granted by the Danish Data Protection Agency . Eighty pre-treatment panoramic radiographs (Panoramic group) and eighty CBCT scans (CBCT group) of patients previously treated at the Section of Orthodontics, Aarhus University were randomly selected (www.randomizer.org) from the archive according to the following inclusion criteria:» Permanent dentition, with second molars erupted.» No agenesis or missing teeth, except for third molars.» Patients under 35 years of age at the time of the radiographic examination.» Absence of signs of periodontitis and/or bone resorption on the radiographic examination.» Pre-treatment digital models available.The radiographic records were retrieved from the archive of previously treated patients; they had been taken in accordance with the radiological guidelines of the Department of Dentistry, Aarhus University.® software , which was used for the measurements. The tooth size-arch length discrepancy (i.e. crowding) was assessed according to the method described by Lundström.The digital models were imported into the O3DMMoreover, a value for tooth size-arch length discrepancy of the anterior region  and two values for the left and right posterior regions  were calculated as well.To measure the interradicular spaces, a specifically designed analysis from PorDios software  was used. Each image was calibrated using the correct DPI. For each interradicular space, one operator manually placed two points at the cemento-enamel junction (CEJ) of the two adjacent teeth and two points at the apex of the adjacent roots. Then, a line connecting the two CEJ points and a line connecting the two apex points were automatically drawn together with the two midpoints of these lines; a third line (midpoint line) connecting these midpoints was then drawn. Two lines were automatically generated perpendicular to the midpoint line, dividing the latter into three equal parts. The interradicular distance was measured along these two lines (i.e. at 1/3 and 2/3 of the root length) . This prTo account for the intrinsic horizontal magnification of the panoramic radiographs, the mesiodistal width of all first molars, first premolars and central incisors was measured both on the panoramic radiographs and on the digital models, to calculate three “magnification factors”: the magnification factor calculated for each first molar was used to adjust the measurement of the interradicular spaces mesial and distal to that first molar; the factor calculated for the first premolar was used for the interradicular spaces mesial and distal to that first premolar; the factor of the central incisor was used for all the interradicular spaces mesial to the canine.The vertical magnification, also present on the panoramic radiographs, was judged to be negligible since a ratio and not a linear measurement was used to divide the vertical height (1/3 and 2/3) used for the horizontal measurements.The volumetric data obtained from the NewTom 5G CBCT scanner  were opened with the NNT software  and used to generate custom images. First, the data-set was oriented parallel to the occlusal plane in both the sagittal and the transversal directions; then, using the “broken line” function with a thickness of 9 mm, a panoramic-like image was generated putting a point at the center of each tooth, likely at half the length of the root. Two panoramic-like images (one for the maxilla and one for the mandible) were created, with straight segments between each tooth, to avoid distortions and to ensure the repeatability of the measurements.The panoramic-like images that were created in this way were imported into the PorDios software, and the same protocol as described before was applied to measure the interradicular spaces.,The data averaged from the panoramic radiographs and CBCTs were used to define two visual maps of the interradicular spaces. In accordance with the literature,2)/2n, where d= difference between the first and second measurements) was used to calculate the standard error on the repeated sets of measurements. Bland-Altman plots were used to check for the intra-observer reliability between the two sets of measurements.To evaluate the error of the method, 30 panoramic radiographs, 30 CBCTs and 30 digital models were randomly selected from the whole sample using an online tool (www.randomizer.org), and the measurements were repeated by the same operator after at least one week. For all measurements, Dahlberg’s formula  or a Kendall’s tau test  was then performed. The significance level for all tests was set at 0.05.The mean age was 16 ± 5.2 years  for the Panoramic group, and 19 ± 6.1 years  for the CBCT group. The former included 29 males and 51 females, the latter 30 males and 50 females.The average error of the method for measuring tooth size-arch length discrepancy of the individual segments was between 0.18 mm and 0.27 mm in the maxilla and between 0.19 mm and 0.25 mm in the mandible; whilst for the measurement of whole arch tooth size-arch length discrepancy, the error was 0.52 mm for the maxilla and 0.5 mm for the mandible. The average error of the method in measuring interradicular spaces on panoramic radiographs was 0.28 mm (range 0.20-0.34 mm) in the maxilla and 0.48 mm (range 0.32-0.56 mm) in the mandible. The average error of the method in measuring interradicular spaces on CBCTs was 0.30 mm (range 0.21-0.49 mm) in the maxilla and 0.24 mm (range 0.15-0.34 mm) in the mandible. The Bland-Altman plots showed no systematic errors.In the maxilla, all data were normally distributed, except for tooth size-arch length discrepancy of the posterior right and left segments for patients from both CBCT and Panoramic groups. In the mandible, all data were not normally distributed, except for tooth size-arch length discrepancy of the left posterior segment for the CBCT group.The measurements of the left and right posterior segments were pooled since no statistically significant differences were found between the two sides both in the mandible and in the maxilla.p= 0.02) in anterior mandibular tooth size-arch length discrepancy was found between the CBCT group and the Panoramic group , it was decided to pool the data from the left and right sides for further analysis.In the maxilla, the only two places where the interradicular space exceeded 3 mm were the interradicular spaces at the apical third level between the canine and lateral incisor, as well as between the two central incisors . In the In the maxilla, a statistically significant positive correlation was found between the tooth size-arch length discrepancy and the presence of interradicular space between the canine and lateral incisor at the coronal and apical thirds, and between the lateral and central incisors at the coronal third . In the Descriptive statistics for interradicular spaces measured on CBCT images are reported in No differences between the interradicular spaces in the left and right sides in the maxilla as well as the mandible were statistically significant, except for the spaces between the maxillary second and first molars; however, since this difference was only 0.3 mm, a value lying in the same range of both the error of the method and of the voxel dimension, it was considered negligible, and all data from the left and right sides were therefore pooled.In the maxilla, an interradicular space exceeding 3 mm was present only between the two central incisors at the apical third. In the mandible, an interradicular space exceeding 3 mm was found at the apical third between the first and second molars as well as between the first molar and second premolar, and at both the apical and coronal levels between the first and second premolars . In the maxilla, a statistically significant positive correlation between tooth size-arch length discrepancy and interradicular space was detected both at the apical and coronal thirds between the first and second premolars, as well as between the canine and lateral incisor, and at the coronal third between the two central incisors. Furthermore, a statistically significant negative correlation was detected for anterior tooth size-arch length discrepancy and interradicular space between the central and lateral incisors at the apical third .In the mandible, a statistically significant positive correlation between tooth size-arch length discrepancy and interradicular space was detected at the coronal and apical thirds between the first molar and second premolar, as well as between the second and first premolars; at the coronal third between the first premolar and canine, and between the two central incisors at the coronal third. In addition, a statistically significant negative correlation was detected for anterior tooth size-arch length discrepancy and interradicular space between the central and lateral incisors at the apical third .All interradicular space measurements from panoramic radiographs and CBCTs and correlations are presented graphically in figures  and 4.The main aim of the present study was to assess the amount of mesiodistal interradicular space between adjacent roots for miniscrew insertion and to correlate these findings with the presence or absence of crowding, in order to develop a simple clinical diagnostic tool for preliminary planning of miniscrew placement. Overall, the error of the method for all measurements of interradicular spaces on both panoramic radiographs and CBCT was small and clinically non-relevant, with the Bland-Altman plots confirming good reliability of the measurements. The measurements of tooth size-arch length discrepancy also showed a small measurement error, confirming that digital models are a reliable method for assessing the presence of crowding and/or spacing in the arch.The horizontal magnification of the panoramic radiographs was overcome using a calibration method that involved digital models. Magnification assessed on panoramic radiographs was greatest in the lower molar region, whilst the smallest values were found in the upper incisors region . These vThe studied sample  presenteThe maps A and 3B In a previous study about “safe zones” for miniscrew placement based on panoramic radiographs,Poggio et alDespite the heterogeneity of the considered publications, a systematic reviewTo test the hypothesis that assessment of tooth size-arch length discrepancy can be used as a preliminary clinical tool for the evaluation of miniscrew insertion, the correlation between the amount of dental crowding and the presence of sufficient interradicular space for miniscrew insertion was assessed as well in the present study. In general, tooth size-arch length discrepancy measured at the crown level seems to be related to the amount of interradicular space. Schnelle et alIn their review, AlSamak et alThe last map provided , derivedFewer correlations between tooth size-arch length discrepancy and interradicular spaces were found on panoramic radiographs with respect to CBCTs. An explanation for this finding could be that smaller interradicular spaces were measured overall on panoramic radiographs, and therefore the smaller range of values could have limited the correlation. Although with Pearson correlation or Kendall’s tau tests a value of +1 means a perfect positive correlation and -1, a perfect negative correlation, in this particular case, a positive correlation coefficient means that when crowding increases, interradicular space decreases, since a positive (interradicular space) and a negative value  were correlated.Surprisingly, when correlating interradicular spaces measured on CBCTs and tooth size-arch length discrepancy, a negative correlation was found at the apical level of the interradicular space between the lateral and central incisors in both the maxilla and the mandible, which means that for these regions, more crowding results in more interradicular space. The reason for this finding may relate to the divergence of the roots where crowding is present; however, further investigations need to be performed to verify this finding.It is important to underline that single interradicular spaces were correlated with the tooth size-arch length discrepancy of the entire relative segment (anterior or posterior) and not with a value of tooth size-arch length discrepancy between the two adjacent teeth relative to that interradicular space. This procedure was chosen to reflect what is usually applied in clinical practice, where tooth-by-tooth assessment of crowding is meaningless. “Safe zone” maps should be used in combination with the map showing which interradicular spaces are correlated with dental crowding . This co The use of digital models to calibrate panoramic images constitutes a valuable tool, while direct horizontal measurements on non-calibrated panoramic radiographs lack precision. Overall, compared with CBCT, panoramic radiographs underestimate the actual interradicular space, hindering the use of miniscrews when in reality insertion would be possible, provided that the amount of crowding is the same. The findings of this study result in a new “safe zone” map. The best areas for miniscrew insertion are between the upper central incisors and the interradicular spaces from the mandibular second molar to the mandibular first premolar. The map correlating interradicular spaces to tooth size - arch length discrepancy may surpass previously published “safe zones” when crowding is present. Used in combination with the decision tree and the measured tooth size - arch length discrepancy, this map may help clinicians in the preliminary planning of miniscrew insertion and in choosing appropriate radiographs, when necessary.


It is well-known that the presence of the delamination in a plant fiber-reinforced composite is difficult to detect. However, the delamination introduces a local flexibility, which changes the dynamic characteristics of the composite structure. This paper presents a new methodology for composite laminate delamination detection, which is based on dynamic mechanical analysis. A noticeable delamination-induced storage modulus reduction and loss factor enhancement have been observed when the delaminated laminate was subjected to a forced oscillation compared to the intact composite laminate. For delamination area of 12.8% of the whole area of the composite laminate, loss factor of approximately 12% increase was observed. For near-to-surface delamination position, loss factor of approximately an 18% increment was observed. The results indicate that the delamination can be reliably detected with this method, and delamination position shows greater influence on the loss factor than that of the delamination size. Further investigations on different frequencies and amplitudes configurations show that the variation of loss factor is more apparently with low frequency as well as the low amplitude. Plant fiber reflects outstanding and comparable mechanical and dynamic mechanical properties to synthetic fiber such as fiber glass, this leading to extend its applications for special engineering materials such as aerospace and automotive interior parts, and construction structures ,2. AlthoDelamination is a common damage mode in laminated composites, which is occurring due to the low interlaminar strengths of the FRP laminates . It can E′), loss modulus (E″) and loss factor (tanδ) etc. are temperature dependent, and these parameters are usually influenced by dynamic loading conditions. For example, in aerospace and transportation fields, the engine noise, airflow and bump during turbulence will cause various vibration conditions. Moreover, the dynamic mechanical response of composites also depends on the physical or structural arrangement of phases such as interface, morphology and the nature of constituents. Researchers elaborated that the effect of the presence of compatibilizer, fiber content, fiber size, fiber orientation, stacking sequence, fiber/matrix interface, fiber breakage, matrix cracking, hybridization governed the dynamic mechanical properties of a composite material [Dynamic mechanical analysis (DMA) is a powerful technique on characterizing not only molecular motion, but also phase morphology of polymers and reinforcements, it provides a sensitive and nondestructive detection of the interfacial region within laminated composites ,18. The material ,25,26,27material  summarizAs mentioned previously, delamination could seriously influence the structural integrity of composite and cause fateful consequences, the impact of delamination on dynamic mechanical properties of composite need a more in-depth research. Since delamination within composite laminate could also have influence on damping properties which can affect the dynamic properties of structure, DMA could be used to characteristic delamination of the composite. However, this method has not been used in characterization delamination so far. Some papers mentioned above are regarding damping properties of delaminated composite plates, but most of them focus on the natural frequency behavior of such composites and do not assess the dynamic mechanical properties, which are the focus of this study. Hence, the effect of delamination with various size and transverse position on flax fiber-reinforced polymer (FFRP) composite laminate with regard to temperature, frequencies and amplitude are evaluated using DMA in this study. As such, this paper provides a starting step towards a better understanding of the viscoelastic behavior of delaminated natural fiber composites.2, respectively, and it was provided by Lineo Co. Ltd., MeulebekeBelgium. Epoxy resin (NPEL–128), amine curing agent (EH–6303), and accelerator (EH–6412) (100:26:2 weight ratio) were supplied by Nanya Electronic Materials Co., Ltd., Kunshan, China.Unidirectional (UD) flax fabric reinforced epoxy resin composite laminates were manufactured in this study. The unidirectional flax fabric has average thickness and areal density of 0.2 mm and 200 g/m2, 25 mm2 and 6.25 mm2. Moreover, delamination located in three different transverse positions was also investigated, including Position I (at middle thickness), position II (at 1/3 thickness) and position III (at 1/6 thickness). Square Teflon films were carefully stitched at two cross corners to one layer of the flax fabric at the correct location with a fine cotton thread in order to prevent moving positions during the manufacturing process. Prior to manufacturing, the fabrics were dried in an oven for 4 h at 120 °C in order to remove moisture. A single layer of PTFE (Teflon) film (15 µm in thickness) was placed at the pre-defined locations during layup to introduce delamination [Vacuum-Assisted Resin Infusion (VARI) technique was used to fabricate composite laminates as shown in E* is given by the ratio of the stress amplitude to the strain amplitude. The complex modulus is composed of the storage modulus (E′) and the loss modulus (E″). E′ represents the stiffness of a viscoelastic material and is proportional to the energy stored during one loading cycle. E″ is proportional to the energy dissipated in one loading cycle. These two moduli can be calculated according to Equations (1) and (2) [Dynamic Mechanical Analysis (DMA) is used to study the physical properties of a specimen subjecting to a small sinusoidal oscillating force as a function of time, temperature, and frequency. Due to the viscoelastic properties of polymer-based composite samples, the two signals (stress and strain) will be out-of-phase, and a phase shift δ can be measured. The complex modulus  and (2) :(1)E′=E*Then, the complex modulus can be calculated according to Equations (1) and (2):The complex shear modulus can be calculated according to the complex modulus, and given as:E′, E″, E* and G* are dependent on the sample dimension. Therefore, two geometry independent parameters tanδ and G″ are used to characterize the delamination within the composites. The loss factor (tanδ) is a dimensionless parameter representing the energy lost per cycle expressed in terms of recoverable energy, and gives a measure of the damping or internal friction in a viscoelastic system which can be obtained by Equation (3):It should be noticed that the G″, is related to the viscous dissipation as the energy dissipated per cycle per unit volume of the sample, and it gives better characterization on the effect of the fiber/matrix interface. The loss shear modulus, G″, can be expressed in terms of G* and δ as:It was indicated by Kennedy et al.  that theThe dynamic mechanical properties of the composites were obtained using the Q800 DMA machine  according to ISO 6721–1  in a duaAs for the temperature scans manner, firstly, the sample was clamped onto the fixture and a temperature scanning between 25 and 120 °C was realized with a ramp rate of 5 °C/min, the frequency and amplitude were set as 10 Hz and 10 μm, respectively. For the time scans, firstly, four different frequencies were tested for each sample, namely 1, 10, 50 and 10 Hz, and all tests were conducted at a fixed strain amplitude of 10 µm. Ambient temperature of 25 °C was chosen as the testing temperature for all samples and isothermally for 5 min. Secondly, five different strain amplitudes were tested for each sample, namely 10, 15, 20, 25 and 30 µm and all tests were conducted at a fixed frequency of 10 Hz. The samples were equilibrated at 25 °C for 5 min.®  in accordance with the following steps: Firstly, since the total recorded testing time for each sample was 5 min, the loss factor results were averaged to obtain significant results. Secondly, the average loss factor value in five minutes duration of three samples for each delamination area or position were averaged again. 2). A total of three signals were obtained from three samples produced with the same delamination area of 6.25 mm2. Dash lines with white markers are variations of loss factors against time for three samples, these signals were subsequently averaged in time, as represented by the dashed lines with black markers. The final loss factor of the specimen DEL_2.5_I was obtained by averaging the above black marker values and represented as a black line in The dynamic mechanical properties data of the composites obtained in frequency and amplitude scan modes were processed using the MatlabG″. The variation of tanδ and G″ were calculated according to Equations (5) and (6), these data were then plotted in G″ due to its integrated interlaminar interface. This seems reasonable, the interlaminar interface of the intact laminate are assumed to be well bonded, whereas, the delaminated zone of laminate is assumed to yield a very weak interlaminar interfacial bond, and this causes the extra energy dissipation during forced vibration of a DMA test. It can be seen from G″. The horizontal axis on each plot presents the delamination area both in SI units (mm2) and in terms of percentage area of the whole specimen. In another word, the 100 mm2 delamination area corresponds to a 12.8% delaminated area for the DEL_10_I specimen. It can be found from G″ increases with the proportion of delamination area to the whole area of the sample. In terms of the 3.2% (DEL_5_I) delaminated sample, both of these two values raise approximately 6% than that of the intact laminates. However, when the delamination area increased to 12.8% (DEL_10_I) of the whole area of the sample, the tanδ and G″ reduces to the same value of the intact laminate. This indicates that small delamination area can causes the enhancement of the loss factor and loss shear modulus due to the friction between adjacent separate layers in delaminated zone during forced vibration. However, if the delamination area is too big, the energy consumed through internal friction and molecule motion within the FFRP composite are seriously reduced, which cause a decrease in total energy dissipation.In dynamic mechanical testing, tanδ is also referred to damping and is an indicator of how efficiently the material loses energy to molecular rearrangements and internal friction. The magnitude of the loss shear modulus directly reflects the energy dissipated during testing. In a composite material both the matrix polymer and the interfacial contribute to these losses. In this study, the matrix and the fiber/matrix interfacial properties are the same. Therefore, the characterizing on the interlayer bonding or delamination performance depends on the relative magnitude of the interlayer contribution to the overall dynamic properties of the composite specimen. Therefore, when composite materials are subjected to dynamic mechanical testing, any decrease in the strength of the interlayer bonding should be reflected by an increase in the measured values of both tanδ and E′ and E″ with delamination areas tested at a fixed strain amplitude of 10 μm and frequencies of 1, 10, 50 and 100 Hz, respectively. It can be seen from the graph that the storage modulus of the FFRP samples decreases linearly (dash line) with the enlargement of the delamination area at four different test frequencies. The E′ of the largest delamination area sample (DEL_10_I) is approximately 15% lower than that of the intact one for all four test frequencies. This is due to the reason that bonds between fiber/resin and resin/resin in the composite provide energy storage capabilities during deformation. However, the introduced delamination prevents the resin/resin bonds adjacent this area from forming. The discontinuous interface in the delamination area can be assumed has a very week interfacial adhesion between two layers, this translates to a lower capacity of the laminate to store elastic deformation energy during vibratory oscillation. As a result, the specimen presents a lower storage modulus when compared to the intact one. Li et al. [E′ values at 10, 50 and 100 Hz test frequencies, which are slightly higher than that of the 1 Hz testing results. Similarly, the loss modulus was plotted as a function of the delamination area in E” for small delamination area, and the value decrease with the delamination area keep increase. However, there is an approximately 45% difference on loss modulus between test frequencies of 1 Hz and 100 Hz for all test sample. This indicates that the energy loss for this type of composite exhibits a dependence on the test frequency instead of delamination area. Dong and Gauvin et al. [E″ of a CFRP laminate during a DMA test, whereas, the effect on E′ is not observed.i et al.  also foun et al.  also fouE* and G* decrease with the enlargement of the delamination area. The reductions for both E* and G* of the largest delamination area composite (DEL_10_I) are approximately 10% compares to the intact laminates. The values of E* and G* are dependent on both of E′ and E″. Therefore, E* and G* also decrease linearly with the increase of delamination area.The variation of complex modulus and complex shear modulus with delamination area is shown in G″ value and it was 5% higher than that of the intact laminates tested at 1 Hz. After that, the G″ decreases steady with the increase of delamination area, and the value of the largest delaminated sample  was 5% lower than the intact laminates. There is no significant increase on the G″ for the small size delaminated laminates at other test frequencies. This result indicates that small size delamination at low test frequency causes slightly increase on G” but not for large size delamination samples and higher test frequencies. The average loss shear modulus of the specimens tested at low frequency is much higher than samples tested at higher frequencies, and this difference is as significant as 50%. The loss factor of the largest delamination area  is approximately 12% higher than that of the intact one for lower test frequencies of 1 Hz and 10 Hz. However, this difference become insignificant with the test frequency increase, and it was found the loss factor for the delaminated and intact sample are similar.The variation of the overall loss shear modulus and loss factor against delamination area is shown in E′ is not essential, all the storage modulus for the same laminate tested at different amplitudes falls to the same point in the graph. Again, the delamination area does not show great influence on the loss modulus. However, the loss modulus increases with the strain amplitudes, approximately 30% enhancement was observed for strain amplitudes of 30 μm compared to that of tested at 10 μm. The increase of E″ with amplitude observed for the composites was attributed to the Coulomb friction at the interfaces due to the reason that high amplitude would yield a more significant loss signal [G″ for all delaminated and intact laminates and a maximum reduction of 8% was observed on the largest delaminated laminates  tested at the 30 μm strain amplitude compares to the non-delaminated ones. In another hand, the loss factor also growth with the delamination area as stated in It can be found from 2) at different transverse positions are illustrated in E′, E″, E* and G* curves against temperature of FFRP with same delamination area but different transverse positions. The different delamination positions investigated in this study are schematically represented in E′, E* and G*, and the value of these three parameters decrease with the delamination far from the midplane. However, the E″ shows the opposite trend. Similarly, the typical G″ and tanδ against temperature curves in 2x/t, where x is measured from the mid-thickness of the specimen outwards (as indicated in the plot) and t is the specimen thickness. The bottom horizontal axis provides the correspondence between the nomenclature used and the transverse position of the preset delamination. It was found that the peak value of G″ for laminates with different delamination transverse positions are similar, which is 6% higher than that of the intact laminate. This indicates that transverse position of the delamination does not affect the total amount of energy dissipated caused by delamination and the value is consistent with the G″ value of middle-size delaminated sample. However, the loss factor tends to increase with the transverse position moves from mid-plane (2x/t = 0) to a near-surface delamination. In this study, the maximum value of 2x/t is 0.67, which is corresponded to Position III. In this transverse position, the delamination caused the highest enhancement on peak value of tanδ, which is 25%. This suggests that laminates with delamination in different transverse position exhibit distinct energy dissipation efficiency and damping properties.The dynamic properties of three different specimens with the same delamination area  have the lowest storage modulus and loss modulus, which are approximately 15% lower than that of the intact laminate. Compared to the storage modulus, the strain amplitude has a greater impact on the loss modulus, a 30% growth was observed between laminate tested at 30 μm and 10 μm strain amplitude. The same reduction trend was found for the E* and G*, the maximum decrement is 15% for laminate with delamination at transverse position of 2x/t = 0.34 (Position II). As for the G″ and tanδ, it was found that raise the amplitude lead to an enhancement of at least 20% on the loss shear modulus and loss factor of FFRP laminates. The reduction caused by delamination transverse position is approximately 15% and 3% for G″ and tanδ, respectively.The dynamic properties of FFRP laminates varied with delamination transverse position which can be tested under a fixed frequency of 10 Hz and five strain amplitudes of 10, 15, 20, 25 and 30 μm are shown in E″, G″ and tanδ, which means test frequency has great influence on energy dissipation of composite materials. Some researchers have reported that delamination induced modal frequency shifting and modal damping increasing due to modal stiffness reduction [The impact of delamination size and transverse location on the dynamic mechanical properties are the two main concerns in this study. Firstly, in terms of delamination size, the storage modulus decreases with the delamination size increase as expected. However, this does not mean the loss modulus goes the opposite way. Actually, the energy dissipation through friction and molecule motion will go down if the delamination size is too big. In fiber-reinforced composite material, viscoelastic damping appears to be the most dominant mechanism in undamaged polymer composites vibrating at a small amplitude. The interface phase is a region with a certain thickness on the surface and nearby layers of the composite material, it processes different properties to both fiber and matrix. As a bridge connecting adjacent layer, interlaminar interface phase has a great influence on the overall performance of composite materials. When stimulated by external excitation, the interface will produce a large shear strain with the deformation of the material, and these shear strains will cause friction within the material and dissipate the energy. The delamination equivalent to lower the interlaminar bonding and this leads to extra friction and local flexibility in the interlaminar debonding region, and this results in a higher loss factor. Moreover, it seems that the test frequency has remarkable effect on eduction ,16,31.Secondly, in terms of delamination transverse position, the dynamic mechanical properties are quite distinct in spite of all three laminates process same delamination size. It was found that among the three interlaminar position, mid-plane delamination laminate provides the highest dynamic mechanical properties, while other two delamination position laminate show similar properties. Also, it was found that the transverse position of the delamination has a greater effect on the damping parameter (tanδ). Mei et al.  also fouFor future work, it is desirable that the methodology should be extended to detect and quantify the size and axial/transverse location of the delamination in complex composite structures not just for unidirectional composite.In this paper, unidirectional flax fiber-reinforced composite laminate with different delamination areas and transverse positions were implemented using the dynamic mechanical analysis machine under temperature, frequency, and amplitude scanning manners. It was found that the delamination of different sizes and locations significantly affected the dynamic mechanical properties of the composite. The delamination could be reliably detected using test parameters with relatively low frequency and low amplitude, the increase on loss factor indicates the existence of delamination. Moreover, experimental results indicated that the effect of delamination transverse position on the detectability of the delamination was more notable than the size of the delamination. It was also found that the frequency has greater influence on energy dissipation of composite compares to the effect caused by amplitude. In summary, despite this, the DMA method requires more data obtained from a large number of delamination sizes and positions to get empirical formula, which might quantify the delamination parameters. This methodology has been proven useful for the detection of delamination in unidirectional composite.


Mycobacterium tuberculosis isolates of the Beijing-B0/W-148 sublineage that carry a 7-bp insertion within the pks15 gene, which leads to the synthesis of Pks15/1 fusion protein. Pks15/1 is involved in phenolglycolipid synthesis and biofilm formation, thus potentially contributing to the B0/W-148 lineage’s enhanced virulence and drug resistance.Here, we report the draft genome sequences of 15  Mycobacterium tuberculosis isolates of the Beijing-B0/W-148 sublineage that carry a 7-bp insertion within the pks15 gene, which leads to the synthesis of Pks15/1 fusion protein. Pks15/1 is involved in phenolglycolipid synthesis and biofilm formation, thus potentially contributing to the B0/W-148 lineage’s enhanced virulence and drug resistance.Here, we report the draft genome sequences of 15  Mycobacterium tuberculosis virulence and pathogenicity are determined by a set of genes which are polymorphic in different phylogenetic lineages  and highly virulent (4–pks15 gene (CCGCGGC) that leads to the synthesis of an active fused Pks15/1 protein  at 37°C for 4 weeks. Genomic DNA was isolated and purified by phenol-chloroform/isoamyl alcohol extraction after enzymatic cell lysis, as described by Belisle et al.  assemblies described here have been deposited in NCBI GenBank. The versions described in this paper are the first versions. The read archives have been deposited in NCBI SRA. The WGS (GenBank) and SRA accession numbers are listed in