Critically ill patients frequently develop gastrointestinal (GI) motility disorders resulting in feeding intolerance with increased morbidity and mortality. Sedatives and opioids per se have adverse effects on GI motility and can aggravate GI motility disorders.The aim of this study was to evaluate the inhibitory potency of propofol alone and in combination with remifentanil or sufentanil.Guinea pig´s small bowel segments of 8 cm length were set up in organ baths containing oxygenated Tyrode´s solution. Peristalsis was elicited by luminal perfusion (0.5 ml/min) against an aboral resistance of 400 Pascal (Pa). Perfusion of the segments resulted in an increase of the intraluminal pressure up to a pressure threshold , where peristaltic contractions were triggered. The pressure was recorded at the aboral end of the segments. An increase of the PT was interpreted as an inhibition of peristalsis, while a decrease of the PT was interpreted as a stimulation of peristalsis. A PT of 400 Pa was equated with a complete block of peristalsis. In a first setting increasing concentrations of propofol  were added to the organ bath and the PT was evaluated at each concentration. These results were compared to increasing concentrations of propofol after pretreatment with sufentanil (0.1 resp.0.3 nM) or remifentanil (3 resp. 10 nM). Statistic calculations were performed using the general linearlized model for repeated measures and the two sided t-test of IBM SPSS 21.0.2= 0.9; observed power= 1.00). Pretreatment with sufentanil 0.1 nM  and 0.3 nM  had an additional inhibitory effect at both tested concentrations (p= 0.041 resp. 0.029, figure Basic PT without any substances added to the organ bath was 73.83 ±5.3Pa. Propofol had a dose dependent inhibitory effect on peristalsis (p < 0.001, partial ŋPropofol and opioids affect neuronal activity via different points of action. While propofol acts on GABA receptors, opioids bind to µ, κ and δ receptors. In this experimental setting the combination of propofol with opioids shows a more pronounced inhibition of small bowel motility than propofol alone. Propofol combined with remifentanil at a high concentration had a pronounced inhibitory effect on peristalsis, while the difference between the two tested concentrations of sufentanil was minor.


Iron is in the trivalent state and is distributed over both type of sites.The synthetic spinel Li 0.64Fe2.15Ge0.21O4, lithium diiron(III) germanium tetraoxide, has been formed as a by-product during flux growth of an Li–Fe–Ge pyroxene-type material. In the title compound, lithium is ordered on the octahedral B sites, while Ge4+ orders onto the tetrahedral A sites, and iron distributes over both the octahedral and tetrahedral sites, and is in the trivalent state as determined from Mössbauer spectroscopy. The oxygen parameter u is 0.2543; thus, the spinel is close to having an ideal cubic closed packing of the O atoms. The title spinel is compared with other Li- and Ge-containing spinels.Spinel-type Li The perfect normal spinel is one in which the single A cation occupies the tetrahedral site and the two B cations reside at the two equivalent octahedral positions. Introducing parentheses, i.e. (…) and brackets, i.e. […], for tetrahedral and octahedral coordination, respectively, one may write the normal spinels in the form (A)[B2]O4. In contrast, the complete inverse spinel has a cationic distribution of (B)[AB]O4 . For LiMn2O4 and LiNi0.5Mn1.5O4, which represent excellent cathode materials, it was found that Li+ orders onto the tetrahedral site [Li+0.64Fe3+1.36]O4, with the valence state of iron determined from 57Fe Mössbauer spectroscopy (see below). This formula is balanced in charge and agrees very well with the chemical composition determined from electron microprobe analysis. Generally, the title compound is similar to the Li0.5Fe2.5O4 spinel-type materials. The shift of Li+ to the octahedral site, for example, in comparison with LiCrGeO4 or LiMn2O4, can be explained by the strong preference of Fe3+ for the tetrahedral site. Based on the concept of crystal field stabilization energy, Miller  Å; Fleet, 19814  is smaller in comparison with, for example, magnetite FeM is 2.0373 (11) Å, which is 1.07 times larger than the bond length involving the tetrahedrally coordinated site. The M—O bond length is somewhat larger than 2.025 (3) Å in Li0.5Fe2.5O4  being larger than that of Fe3+  Å, while in GeFe2O4, all the Fe atoms are in a divalent state and an M—O bond length of 2.132 (2) Å is observed.The bond length involving the octahedrally coordinated site 57Fe Mössbauer spectrum was recorded at 340 K. It shows a broad, slightly asymmetric, doublet, which can be evaluated with two Lorentzian-shaped doublets  of 0.57 (3) mm s−1, and can be assigned to the ferric iron on the tetrahedral site. The second doublet has a larger IS of 0.115 (14) mm s−1 and an almost identical QS of 0.58 (2) mm s−1, and is assigned to ferric iron at the octahedral site. No indications for ferrous iron are present. The QS values suggest low polyhedral distortion, which is almost identical in both sites. The relative area ratio of tetrahedral to octahedral sites is 38.6 (8) to 61.4 (9)%. Assuming a total amount of 2.15 formula units Fe3+, the results of Mössbauer spectroscopy give a cation distribution of (Fe3+0.83)[Fe3+1.32], which is in good agreement with that obtained from the site-occupation refinement of the X-ray data. At room temperature, the title compound is magnetically ordered, as revealed by its 57Fe Mössbauer spectrum.In order to quantify the valence state of iron in the title compound, a ts Fig. 2. The fir2O6 in flux-growth experiments  were mixed together, heated to 1473 K in a platinum crucible, covered with a lid, held at this temperature for 24 h and cooled afterwards at a rate of 1.5 K h−1 to 973 K. The experimental batch consisted of large pyroxene crystals and a distinct amount of black crystals with idiomorphic octahedral habit, up to 200 µm. Semi-quantitative EDX (energy-dispersive X-ray) analysis revealed iron and some germanium as the main elements; powder X-ray diffraction analysis revealed the crystals as a spinel-type material. An electron microprobe analysis on polished/embedded crystals (three different grains with five measurement points each) yielded a chemical composition of 84.86 (30) wt% Fe2O3, 10.52 (25) wt% GeO2 and 4.62 wt% Li2O, with the latter calculated from the difference to 100 oxide%. There is no evidence for Mo or V from the flux, nor for any other chemical elements. From the oxide percentage, a chemical formula of Li0.63 (2)Fe2.18 (1)Ge0.20 (2)O4 was calculated, which is in good agreement with that obtained from the structure refinement. Individual crystals are homogeneous in composition, with no significant systematic variation from rim-core; also, there is no systematic variation in composition from crystal to crystal.The spinel formed as a by-product during the synthesis of pyroxene-type LiFeGeA and B sites, thereby allowing unconstrained refinement of the site-occupation factors. This gave a surplus of electron density  at the tetrahedral site, while a lower occupation than possible was found for the octahedral site. From this it was concluded that Li enters the octahedral site and Ge enters the tetrahedral site. In the final refinements, it was assumed that both tetrahedral and octahedral sites are fully occupied, with Fe + Ge = 1 as a restraint for the tetrahedral site and Fe + Li = 1 for the octahedral site.Crystal data, data collection and structure refinement details are summarized in Table 110.1107/S205698901600414X/wm5279sup1.cifCrystal structure: contains datablock(s) global, I. DOI: 10.1107/S205698901600414X/wm5279Isup2.hklStructure factors: contains datablock(s) I. DOI: 1463892CCDC reference:  crystallographic information; 3D view; checkCIF reportAdditional supporting information:


Monocyte chemotactic protein 1 (MCP1) stimulates phosphorylation of cortactin on Y421 and Y446 residues in a time-dependent manner and phosphorylation at Y446 but not Y421 residue is required for MCP1-induced CDK-interacting protein 1 (p21Cip1) nuclear export and degradation in facilitating human aortic smooth muscle cell (HASMC) proliferation. In addition, MCP1-induced cortactin tyrosine phosphorylation, p21Cip1 degradation and HASMC proliferation are dependent on Fyn activation. Upstream to Fyn, MCP1 stimulated C-C chemokine receptor type 2 (CCR2) and Gi/o and inhibition of either one of these molecules using their specific antagonists or inhibitors attenuated MCP1-induced cortactin tyrosine phosphorylation, p21Cip1 degradation and HASMC proliferation. Cortactin phosphorylation at Y446 residue is also required for another G protein-coupled receptor (GPCR) agonist, thrombin-induced p21Cip1 nuclear export and its degradation in promoting HASMC proliferation. Quite interestingly, the receptor tyrosine kinase (RTK) agonist, platelet-derived growth factor-BB (PDGF-BB)-induced p21Cip1 degradation and HASMC proliferation do not require cortactin tyrosine phosphorylation. Together, these findings demonstrate that tyrosine phosphorylation of cortactin at Y446 residue is selective for only GPCR but not RTK agonist-induced nuclear export and proteolytic degradation of p21Cip1 in HASMC proliferation. Cell proliferation plays an essential role in the development of an organism and tissue repairing4710Post-translational modifications of cortactin especially acetylation and phosphorylation were shown to govern its interactions with other cytoskeletal proteins in the modulation of cell migration1213141517181414162124While the functional role of cortactin in cell migration and receptor endocytosis has been well studied, its role in cell proliferation is limited to a few studies. Overexpression of cortactin enhances serum- and epidermal growth factor-stimulated proliferation of head and neck squamous carcinoma cellsStudies from various laboratories including ours have demonstrated that cortactin plays an important role in cell migration915733Recently, we have reported that MCP1 induces serine phosphorylation of cortactin on S405 and S418 residues in enhancing F-actin stress fiber formation and HASMC migration17Since cortactin tyrosine phosphorylation is required for MCP1-induced p21Cip1 degradation and HASMC proliferation, we next wanted to identify the tyrosine residue(s) that are phosphorylated by MCP1. To gain insights into this, we mutated each of the five tyrosine residues to phenylalanine by site-directed mutagenesis and tested their effects on MCP1-induced cortactin tyrosine phosphorylation. While Y453F, Y470F, and Y486F cortactin mutants had no effect, the Y421F and Y446F cortactin mutants prevented MCP1-induced cortactin tyrosine phosphorylation, suggesting that MCP1 induces phosphorylation of cortactin at Y421 and Y446 residues . HoweverIt was reported that cortactin is a substrate for the Src family of the kinases (SFKs)12In our previous study we have demonstrated that Gαq/11 mediates MCP1-induced cortactin serine phosphorylation via PLCβ3-dependent PKCδ activationMCP1 mediates its effects via its receptors CCR2 and CCR4To find the specificity of cortactin tyrosine phosphorylation signaling in MCP1-induced HASMC proliferation, we first tested the effect of Y446F cortactin mutant on MCP1, thrombin and PDGF-BB-induced cortactin tyrosine phosphorylation. Surprisingly, overexpression of Y446F cortactin mutant prevented only MCP1 and thrombin but not PDGF-BB-induced cortactin tyrosine phosphorylation . These fMany studies have demonstrated a role for cortactin in the regulation of actin cytoskeleton remodeling and cell migration113840171814132425262713134547It is noteworthy that besides MCP1, thrombin but not PDGF-BB stimulates cortactin phosphorylation at Y446 residue and this event is required for thrombin-induced p21Cip1 degradation and HASMC proliferation. Although, PDGF-BB induced cortactin phosphorylation at other tyrosine residues, those sites appear to be not required for PDGF-BB-induced p21Cip1 degradation or HASMC proliferation. Thus, these findings indicate that the involvement of cortactin phosphorylation at Y446 residue in p21Cip1 degradation is selective to GPCR agonists. Previous studies have reported that PDGF-BB induces tyrosine phosphorylation of murine cortactin on Y421, Y466 and Y482 residues in the mediation of lamellipodium formation and cell migration1316In elucidating the upstream mechanisms of cortactin tyrosine phosphorylation by MCP1, we found that MCP1 activates Fyn more robustly than the other SFK members and depleting its levels inhibits MCP1-induced cortactin tyrosine phosphorylation, p21Cip1 degradation and HASMC proliferation. Fyn has also been shown to co-localize with cortactin in the mediation of metastatic murine melanoma cell migration16212291355In summary, as shown in 3H]-Thymidine (specific activity 20 Ci/mmol) was obtained from Perkin Elmer Life Sciences . Pertussis toxin from Bordetella pertussis was purchased from Sigma-Aldrich . PP1 (567809), PP2 (529573) and PP3 (529574) were bought from Calbiochem .CCR2 antagonist (SC-202525), CCR4 antagonist (SC-221406) HDAC6 inhibitor (SC-223877), HDAC8 inhibitor PCI-34051 (SC-364566), anti-cortactin (SC-11408), anti-Gαq (SC-393), anti-Gα11 , anti-GFP (SC-9996), anti-Fyn (SC-365913), anti-Lyn (SC-15), anti-p21Cip1 (SC-6246), anti-p27Kip1 (SC-528), anti-p57Kip2 (SC-1040), anti-pYes (SC-130182), anti-Yes (SC-8403) and anti-β-Tubulin (SC-9104) antibodies were obtained from Santa Cruz Biotechnology, Inc. . Anti-pSrc (2101) antibodies were purchased from Cell Signaling Technologies . Anti-phosphoserine/threonine antibody (ab17464), anti-proteasome 20Sα + β (ab22673), and pFyn (ab192172) were bought from Abcam . Anti-pLyn (04–375), anti-Src (05–184), anti-phosphotyrosine (05–777) antibodies were obtained from Millipore . Proteasome 20Sα1, 2, 3, 5, 6 & 7 subunits monoclonal antibody (BML-PW8195-0100) was obtained from Enzo Life Sciences . Goat anti-rabbit HRP (31460) and goat anti-mouse HRP (31437) antibodies, ambion silencer validated human Fyn siRNA (AM51331), BAPTA-AM (B6769), Lipofectamine 3000 transfection reagent (L3000-015), Hoechst 33342 (H3570), Prolong gold antifade mounting medium (P36930), Medium 231 (M231-500), smooth muscle growth supplements (S-007-25), and gentamicin/amphotericin solution (R-015-10) were obtained from Thermo Scientific . Protein A sepharose CL-4B (170780-01), Protein G sepharose Fast flow (17061801) and ECL Western blotting detection reagents (RPN2106) were received from GE Healthcare . Human cortactin siRNA (ON-TARGETplus SMARTpool L-010508-00-0005) and control non-targeting siRNA (D-001810-10) were purchased from Dharmacon RNAi Technologies . Anti-CCR2 antibody (NB110-55674) was purchased from Novus Biologicals . Rhodamine phalloidin (00027) was bought from Biotium . -thymidine incorporation as described previously and expressed as counts/min/dishDNA synthesis was measured by [After appropriate treatments, HASMC were fixed with 3.7% paraformaldehyde for 15 min, permeabilized in 0.3% Triton-X-100 for 15 min, blocked with 3% BSA for 1 hr and incubated with mouse anti-p21Cip1 (1:100) and rabbit anti-proteasome 20Sα + β (1:500) antibodies followed by incubation with Alexa Fluor 488-conjugated goat anti-mouse and Alexa Fluor 568-conjugated goat anti-rabbit secondary antibodies (1:500), respectively. After washing with PBS, cells were counterstained with Hoechst 33342 (1:2000) and mounted onto glass slides with Prolong Gold antifade mounting medium. Fluorescence images of cells were captured using an inverted Zeiss fluorescence microscope (AxioObserver Z1) with 40X/NA 0.6 objective and AxioCam MRm camera without any enhancements using the microscope operating and image analysis software AxioVision verstion 4.7.2 (Carl Zeiss Imaging Solutions GmbH).After appropriate treatments the cytoplasmic and nuclear fractions of HASMCs were prepared using NE-PER nuclear and cytolasmic extraction reagents as described in the manufacturer’s  instructions.An equal amount of protein from control and treatment samples was separated by SDS-PAGE and analyzed by Western blotting for the indicated molecules using their specific antibodies as described previously with minor modificationsHASMCs were transfected with non-targeted control or on-target siRNA at a final concentration of 100 nM using Lipofectamine 3000 transfection reagent according to the manufacturer’s instructions. Cells were transfected with plasmid DNAs at a final concentration of 2.5 μg/60 mm dish or 5 μg/100 mm dish using Lipofectamine 3000 transfection reagent according to the manufacturer’s instructions. After transfections, cells were growth-arrested for 36 hrs and used as required.All the experiments were repeated three times, and the data are presented as Mean ± S.D. One-way ANOVA was used to perform statistical analysis and the p values < 0.05 were considered statistically significant.How to cite this article: Janjanam, J. and Rao, G. N. Novel role of cortactin in G protein-coupled receptor agonist-induced nuclear export and degradation of p21Cip1. Sci. Rep. 6, 28687; doi: 10.1038/srep28687 (2016).


Choristoneura fumiferana AFP, including stereo-specific binding and consequential melting and freezing inhibition. The protein binds indirectly to the prism ice face through a linear array of ordered water molecules that are structurally distinct from the ice. Mutation of the ice binding surface disrupts water-ordering and abolishes activity. The adsorption is virtually irreversible, and we confirm the ice growth inhibition is consistent with the Gibbs–Thomson law.Antifreeze proteins (AFPs) protect certain cold-adapted organisms from freezing to death by selectively adsorbing to internal ice crystals and inhibiting ice propagation. The molecular details of AFP adsorption-inhibition is uncertain but is proposed to involve the Gibbs–Thomson effect. Here we show by using unbiased molecular dynamics simulations a protein structure-function mechanism for the spruce budworm DOI:http://dx.doi.org/10.7554/eLife.05142.001 Water expands as it freezes. If this happens to the water inside plants and animals, the resulting ice crystals can rupture cells. To prevent this, many plants and animals that live in cold climates have evolved ‘antifreeze proteins’. When a small particle of ice first starts to form, the antifreeze proteins bind to it and prevent the water around it freezing, hence preventing the growth of an ice crystal.There are many different types of antifreeze protein, and some are more active than others. For example, some insects including the spruce budworm are exposed to extremely cold temperatures—sometimes below −30°C—and these insects have antifreeze proteins that are highly active.It is not fully understood how different antifreeze proteins interact with ice and prevent the growth of ice crystals. This is largely because, as yet, there are no experimental techniques that make it possible to see how antifreeze proteins and water molecules arrange themselves at the surface of a growing particle of ice. Instead, scientists have developed computer simulations to investigate this process. While many of these studies have provided valuable information, the computational methods used have only recently become powerful enough to analyze how the antifreeze proteins approach the surface of the ice particle.Kuiper et al. carried out simulations involving a highly active antifreeze protein from the spruce budworm. The results of these simulations revealed that this antifreeze protein does not bind directly to ice; instead, water molecules at the surface of the protein act as a bridge between the protein and the ice. These water molecules are highly ordered and though they have similarities with how water is structured in the ice, they are distinct from the ice lattice itself. Furthermore, this arrangement appears to be important for allowing the spruce budworm antifreeze protein to interact with the ice.This study provides detailed insights as to how a highly active antifreeze protein helps to prevent ice crystals forming. In the future, the computational simulations used here may be extended to study the dynamics of other antifreeze proteins, and also how crystals of other materials form.DOI:http://dx.doi.org/10.7554/eLife.05142.002 Many species of fish, insects, plants and micro-organisms living in cold environments produce antifreeze proteins (AFPs) whose main function is to target and modify the growth of regular ice . By bindIh) has three equivalent a-axes (a1–a3) which are parallel to three respective prism planes and perpendicular to the c-axis. The ice basal plane lies perpendicular to the c-axis. Remarkably AFPs have evolved independently numerous times to bind stereo-selectively to ice in various orientations, resulting in a structurally diverse protein family  by Tenebrio molitor  provides an excellent model for antifreeze simulations and has been used previously in computational studies  of the TIP4P water model is known to be 230.5 K, . To simulate AFP-ice interactions more efficiently a specially arranged model of an ice-water interface was developed to provide a continual ice growth mechanism while mimicking an infinite ice plane by taking advantage of periodic boundary conditions (PBCs). The ice/water system employed here consisted of 17,434 water molecules in a 51.5 × 126.6 × 83.9 Å box with PBCs, of which 1692 water molecules were harmonically constrained to an ideal prism ice lattice. This served as the seed crystal while an additional 1049 water molecules below the seed were constrained in a 5 Å thick disordered layer to act as an ice barrier preventing downward ice growth, leaving a small wedge of mobile water between constrained layers as shown in m. This arrangement mimics step growth as might be produced by screw dislocations, as originally proposed by The TIP4P water model  was used230.5 K,  it is hic-axis of ice. Within this ice/water box the unconstrained sbwAFP model was initially placed 20 Å above the seed, with its ice binding face directed towards the seed approximately aligned in the expected binding orientation. In our preliminary work the seed ice crystal was arranged with the prism face parallel with the axis of the simulation boundaries. Crystals from these arrangements did not grow well, extending the ice front very slowly if at all. We realized that this was because one had to effectively wait for a two-dimensional nucleation event to occur on the prism plane and reach a critical size before the next layer of ice would grow.As the simulation proceeds, water molecules join the ice phase through hydrogen bonding and can be easily identified by their tetrahedral arrangement, which appears hexagonal when viewed directly down the c-axis which would normally be bridged by two water molecules in regular ice as shown in To characterize the binding mode of sbwAFP, 32 independent, unconstrained (apart from the waters constrained in the ice seed and ice-barrier) simulations of at least 150 nanoseconds (ns) to as long as 250 ns duration were carried out at 228 K. Definitive ice binding was observed 26 times with each binding event occurring with equivalent orientation with respect to the ice lattice, though adsorbing at a number of different step heights. The remaining 6 simulations either diffused away from the growing ice front (3 times), or had partial engagement at alternate angles (3 times). Of the partial engagements, the AFP still had significant rotational and translational movement with respect to the ice lattice so was not considered truly bound. Each of the definitive ice binding events incorporated a linear array of 6 water molecules bridging between the prism face and a row of conserved threonine residues  as shown in Dendroides canadensis, measured with terahertz spectroscopy  known to disrupt activity , were petroscopy . Represem). The sbwAFP adsorbed to the ice front within 40 ns and within 150 ns the ice front displayed marked convex curvature creating cylindrical ice fronts as shown in Three contiguous ∼1 μs MD simulations of sbwAFP adsorbed to an active ice front at temperatures of 225 K, 230 K and 232 K were run to test and simulate the adsorption-inhibition mechanism by observing subsequent ice growth as shown in m) the simulation showed a significant reduction of the ice fraction to an equilibrated value of 0.21 ± 0.02,  shown in When the temperature was raised to 230 K,  and showed further reduction in the ice fraction which equilibrated to an average of 0.15 ± 0.01 . The sbwAFP remained attached to the ice protrusion approximately 8.5 Å above the main ice plane which developed slight concave curvature with respect to the adsorbed protein . A contiTenebrio molitor with a similar ice binding surface to sbwAFP in water have also found equivalent arrangements of bound water molecules emphasizing the importance of the hydroxyl groups of the ice-binding threonine residues. Contrary to this, as mentioned previously, it is noteworthy that the putative ice-binding threonine residues of the alpha helical wfAFP can be substituted to structurally similar but hydrophobic valine residue with little effect on activity , bound waters exhibited an extensive ‘clathrate-like’ network that matched ice lattice dimensions ,R is the radius, Mw the molecular weight of water (0.018 kg/mol), L the latent heat of melting of water ice (6.02 × 103 J/mol) (σ is the ice-water surface energy 29.1 × 10−3 J/m2 (ρi is the density of ice (917 kg/m3), T0 the normal melting temperature (273.15 K), and Tr is the melting temperature depressed by curvature (272.15 K). These values give R = 518 Å. A sphere of this radius has a volume of 5.82 × 108 Å3 and would contain 1.8 × 107 water molecules.Using 3 J/mol) , σ is th0−3 J/m2 , ρi is tT0 of 230.5 K, σ of 23 × 10−3 J/m2, ρi of 944 kg/m3 and L of 4.4 × 103 J/mol. Using the cylindrical version of this Gibbs–Thomson equation (Ag = 1) and a freezing point depression of 5 K, we find the critical cylindrical radius to be 46 Å.Using parameters derived from the TIP4P water model , the cor 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 , a Reviewing editor, and two reviewers.Thank you for sending your work entitled “The biological function of an insect antifreeze protein simulated by molecular dynamics” 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.eLife.This paper explores the molecular mechanism of an antifreeze protein with the help of molecular dynamics simulations. The simulations show that spruce budworm antifreeze protein (sbwAFP) stalls ice growth at the ice prismatic plane in a manner consistent with the Gibbs-Thomson effect. While the study is clearly interesting, a number of serious criticisms have been raised, in particular concerning novelty (in light of earlier simulation studies of ice-growth inhibition by the same protein), the choice of the water model (with a TIP4P having a very low freezing point), the assumed ice growth mode, and the calculation of binding free energies. The points listed below would have to be convincingly addressed for the paper to be publishable in Major comments:1) Novelty. The present study is closely related to the earlier work of Furukawa and Nada. In Nada, 2001, the same protein was studied, and very similar conclusions were reached. In particular, the ice melting point depression due to the Gibbs-Thomson effect has already been reported by Nada and Furukawa . Specific questions to address are: What distinguishes the present study from these earlier simulations? Is the claim that “to the best of our knowledge [the Gibbs-Thomson phenomena] has not been observed directly” justified? What are the differences to these earlier studies? Does the broader temperature range, the consideration of mutant proteins, and the longer simulations lead to substantially different conclusions or a deeper understanding? It has to be clear that the present paper constitutes a major scientific advance.2) Water model. The simulations are performed with the TIP4P model. However, TIP4P gives a only a poor rendering of the water phase diagram. In particular, the ice melting point is far too low. As a result, the simulations were carried out at ∼230 K. However, the diffusion of water and protein at that temperature is very slow, resulting in poor sampling. There are several models in which the reproduction of the phase diagram is much better than in the TIP4P model, e.g., the six-site model and TIP5P/ice model. Could the authors comment about the dependence of the simulation results on the water potential model? It would be reassuring if simulations with a different water model gave similar results.3) Ice growth mode. The simulations are set up assuming a step growth mode on the ice prismatic plane. Is this realistic? At 1 atm pressure, ice crystals grown from water do not have prismatic plane facets. This means that prismatic planes should not grow by a step-growth mode. Prismatic plane facets appear on grown ice crystal shapes only when pressure is extremely high . The formation of hexagonal column ice crystals in the presence of sbwAFP suggests that sbwAFP binds to the prismatic plane and inhibit ice growth on the plane, but it does not necessarily mean that the prismatic plane growth occurs by a step growth mode. Would the results be affected if a different, more typical growth mechanism were considered?4) Estimate of binding free energy and enthalpy. The calculated binding free energies are questionable because of several issues: (i) The variance in the calculated work values is very large , which makes the application of perturbative methods problematic; (ii) Entropic effects  are probably not sampled correctly; (iii) It is not clear how the work is calculated; (iv) It is not clear if the free energy contains any contributions from the harmonic restraint that would have to be subtracted for a proper binding free energy ; (v) The enthalpy estimate in terms of hydrogen bond numbers does not seem to include hydrogen bonds of free water with ice. To get a more controlled estimate of the binding free energy, it would be important to include also the reverse process of binding and use (variants of) the Crooks fluctuation theorem. In its present form, the binding free energy calculations seem to be more confusing than helpful. They may be more suitable for a separate technical publication, in which details can be explored more carefully.eLife. Your revised article has been evaluated by John Kuriyan (Senior editor) and a member of the Board of Reviewing Editors, and the two reviewers who read your original submission. The manuscript has been improved but there are major remaining issues that need to be addressed before acceptance, as outlined below in the summary statement and review prepared by the Reviewing Editor.Thank you for resubmitting your work entitled “The biological function of an insect antifreeze protein simulated by molecular dynamics” for further consideration at It should be possible for you to address the issues raised by this round of review by editing your manuscript in response to the issues raised, without the need for any further calculations. Please pay particular attention to the fact that the Reviewing Editor asks for the elimination of the data and discussion concerning the calculation of ice-binding free energies. Our proceeding further with a revised manuscript is contingent on this being done, because the editor and the reviewers have identified gaps in this analysis.Summary statement from the Reviewing Editor:Molecular dynamics simulations are used to study the trapping of an advancing ice-water interface by antifreeze proteins. The simulations are insightful and interesting, and shed light on an important but poorly understood process at the interface of biology and physics. In their rebuttal and revision, the authors have made a good effort to address the concerns raised in the previous reviews. However, there are important issues that have not been adequately addressed.eLife will require (1) a considerably more nuanced discussion of preceding work, and (2) the elimination of the data and discussion of the calculation of ice-binding free energies. Detailed reasons are given below. In addition, (3) the discussion of the semi-quantitative agreement with the Gibbs-Thomson formalism, as given in To make the paper acceptable for publication in First, I find the discussion of earlier studies inadequate. I am particularly troubled by statements such as “previous AFP simulations have fallen short of complete, convincing molecular descriptions of an AFP-ice interaction” and “recent advances in computational science now make complete simulations feasible and we report them here.” What do the authors mean by their simulations being “complete”? Is there nothing to be done in this field after this study? This is certainly not the case, with many questions remaining open, e.g., concerning the water model with its freezing point of ∼240 K, the quantification of the phenomenological theory in Second, there are major issues with the calculation of the protein-ice binding free energy. Quoting Reviewer #2, in revising their manuscript, the authors have substantially increased the details of their simulation methodology and have now included a much-expanded discussion on the calculation of the binding free energy. Unfortunately these details now render these calculations and the conclusions derivable from them somewhat questionable. These calculations could be presented separately in a more technical journal to do justice to the many challenges involved in such demanding calculations.The instantaneous switching of the control parameters (either the spring constant or harmonic minima) is expected to yield reliable free energy estimates only when such changes result in dissipative work values on the order of kT . The accumulated work values in Figure 9 place this study far outside of this regime, and it is likely the poor sampling of exponentially rare work values that lead to systematic errors and ultimately the disagreement between various protocol estimates of the binding energy discussed in the appendix. Contrary to the authors' claims in their revision, simulating both forward and backward protocols to converge the binding energy would be hugely beneficial precisely because of the irreversibility of binding that the authors allude to. Only in instances of microscopically reversible dynamics do the two procedures yield redundant information. The authors may, e.g., consider Dellago and Hummer, 2013, for guidelines concerning the application of these equations : 41-61).The discussion of the binding free energy in terms of the number of broken hydrogen bonds is also too vague to be useful. As pointed out by Reviewer #2, the number listed for the hydrogen bond enthalpy seems to be judiciously picked to yield agreement with the calculated binding energy  and moreover hydrogen bonding energies are known to be rapidly varying functions of bonding geometry. The authors' recent correction of this estimate to reflect an activation energy rather than the binding energy presupposes that the binding is barrierless for the equivalence of these two quantities to be thermodynamically meaningful and that it is necessary to break hydrogen bonds along the reaction coordinate for this estimate to be kinetically meaningful. From the data provided and cited, it is not clear that either of those assumptions is valid.I want to add to the comments of Reviewer #2 that even the definition of the binding free energy is unclear. Binding equilibria from a bulk phase to a surface have dimensional contributions that require a choice of standard state. Furthermore, does the calculated free energy, fully or partially, include the entropic contribution from a substantial increase in rotational freedom? Probably not, since the time to sample full rotation in the dissociated state has been too small. On the technical side, I wonder how the work was calculated , in particular for the cases in which the spring constant was changed with time.http://www.its.caltech.edu/∼atomic/snowcrystals/ice/ice.htm) be used to explain the observations of the simulations? Such a validation procedure seems questionable. There are also technical issues with the application of Third, Reviewer #2 points out that the near quantitative agreement of the simulation results with the radius of curvature calculated from Finally, Reviewer #1 asks for further clarification in the paper concerning the ice-growth mode. In the rebuttal, the authors mention: “Our preliminary work using ‘perfect’ crystals did not grow well, extending the ice front very slowly if at all. We realised this was because one had to effectively wait for a 2 dimensional nucleation event on the prism plane to occur and reach a critical size before the next layer of ice would grow.” The authors should include this explanation in the paper to justify their assumption of a step-growth mode on the prismatic plane. 1) Novelty. The present study is closely related to the earlier work of Furukawa and Nada. In Nada, 2001, the same protein was studied, and very similar conclusions were reached. In particular, the ice melting point depression due to the Gibbs-Thomson effect has already been reported by Nada and Furukawa . Specific questions to address are: What distinguishes the present study from these earlier simulations?Our studies are the first to show reproducible ice binding of an AFP as it migrates freely from the aqueous phase to the ice surface and the first to show total ice inhibition consistent with the Gibbs-Thomson mechanism while also revealing surprising details of the water arrangements at the interface layer. It is also one of the first, by including mutant control proteins, to demonstrate how crucial the local water binding arrangement is for AFP activity.Nada and Furukawa performed superficially similar simulations in 2011, however their study has serious shortcomings. Firstly, their two AFPs initial conformations was pre-determined using a docking routine on an idealized ice prism surface without liquid water. As their simulations were also quite short, (only 2 simulations of 6 nanoseconds), the pre-positioning of AFP at the ice interface compounds the biases by limiting the sampling of interfacial water.Secondly, Nada and Furukawa have not clearly demonstrated total ice inhibition, but only a slowing of the ice growth rate (shown in their Nada and Furukawa based their conclusions on limited sampling of 2 simulations of only 6 nanoseconds length. Our study has the fortune to be based on sampling of a long 3000 nanosecond simulation plus 32 wildtype binding simulations (each of 150 to 250 nanoseconds in length) and 16 simulations of the mutant form (each of 250 nanoseconds in length) totalling over 12 microseconds for the binding/inhibition part of our experiments alone.We have modified our text to emphasise these advances over earlier studies (Introduction):“Impeded by computational constraints, previous AFP simulations have fallen short of complete, convincing molecular description of an AFP-ice interaction. […] Recent advances in computational science now make complete simulations feasible and we report them here.”Is the claim that “to the best of our knowledge [the Gibbs-Thomson phenomena] has not been observed directly” justified?Upon wider reading, we did find references that refer to observing the Gibbs-Thomson effect in nanomaterials research dealing with semiconductors. Consequently, strictly speaking the original wording would be invalid. We further consulted with an expert in the field, Dr. Charlie Knight, who agreed that in context of AFPs, resolving the ice/water interface at the scale of a few tens of nanometers is nearly impossible, even with atomic force microscopy.We have thus modified the subject text to be more specific (subsection titled “Adsorption inhibition”):“Though the Gibbs-Thomson effect is widely accepted as the basis of AFP activity, resolving these nanoscale features at the ice/water interface has so far not been attained experimentally.”2) Water model. The simulations are performed with the TIP4P model. However, TIP4P gives a only a poor rendering of the water phase diagram. In particular, the ice melting point is far too low. As a result, the simulations were carried out at ∼230 K. However, the diffusion of water and protein at that temperature is very slow, resulting in poor sampling. There are several models in which the reproduction of the phase diagram is much better than in the TIP4P model, e.g., the six-site model and TIP5P/ice model. Could the authors comment about the dependence of the simulation results on the water potential model? It would be reassuring if simulations with a different water model gave similar results.The TIP4P water model does indeed have a well-known discrepancy with regards to its freezing temperature. We would have preferred to use a more sophisticated water model such as the TIP5P/ice, however the only water models that were supported by the NAMD software we used were TIP3P and TIP4P. Other MD programs such as Amber can support more water models, but at significantly slower performance. NAMD was the only software we had that would scale efficiently on our BlueGene clusters to get the required timescales. Also the TIP4P model has been used in a number of notable ice simulation papers, namely Mochizuki et al., (Nature 498.7454 (2013): 350-354) and Matsumoto et al., (Nature 416.6879 (2002): 409-413).Also, we do agree the diffusion of water is slow at ∼230K but with our longer simulations we have seen the fluctuations in the ice front; observing the front grow and retreat. The fact that we show the molecules are able to reversibly arrange themselves into a crystal structure shows sufficient sampling the relevant regions of phase space. It is certainly a good point to be wary of, so we have added an additional note in the text (in the subsection “Adsorption inhibition”):“The extent of the inhibited ice front fluctuated, advancing and retreating in the range of 5 to 8 Å showing the reversible arrangement of water molecules to the crystal ice lattice, (this is an important indication of reaching equilibration and adequate sampling given the reduced water diffusion at these low temperatures).”Overall, we are confident that the TIP4P model can be used to give good representative behaviour of AFP activity, as long as sufficient sampling is done to compensate for slower water diffusion.3) Ice growth mode. The simulations are set up assuming a step growth mode on the ice prismatic plane. Is this realistic?Ice crystals are typically riddled with imperfections as shown by various vacuum etching studies, which serve as growth mechanisms such as screw dislocations . Dislocations in ice as revealed by etching. Philosophical Magazine, 36(6), 1385-1404).Our preliminary work using “perfect” crystals did not grow well, extending the ice front very slowly if at all. We realised this was because one had to effectively wait for a 2 dimensional nucleation event on the prism plane to occur and reach a critical size before the next layer of ice would grow. The tilt of the seed crystal and resultant step growth ensures a constant ice growth mechanism with similar effect to a screw dislocation. We believe this to be not only a realistic model of ice growth, but immensely practical to introduce as it greatly reduces the computational time necessary to achieve a certain rate of growth.At 1 atm pressure, ice crystals grown from water do not have prismatic plane facets. This means that prismatic planes should not grow by a step-growth mode. Prismatic plane facets appear on grown ice crystal shapes only when pressure is extremely high .Our model ice face is only centred on the prism ice face for practical purposes. It would be incorrect to take this as representative of a prism plane of macroscopic ice crystal.The formation of hexagonal column ice crystals in the presence of sbwAFP suggests that sbwAFP binds to the prismatic plane and inhibit ice growth on the plane, but it does not necessarily mean that the prismatic plane growth occurs by a step growth mode. Would the results be affected if a different, more typical growth mechanism were considered?The step growth model we have developed here is a convenient way to test the AFP activity in the presence of a growing ice front. With a much larger system, , one could create other crystal growth initiators conditions, such as a screw dislocation for similar layer-by-layer growth mechanism. We would expect similar results, as steps originating from screw dislocations are still essentially step growth. It would certainly be interesting to test AFP activity around such a dislocation in simulation, but that would currently require yet another massive increase in computational capability.4) Estimate of binding free energy and enthalpy. The calculated binding free energies are questionable because of several issues: (i) The variance in the calculated work values is very large , which makes the application of perturbative methods problematic... We are aware of no superior method of error estimation. We do not see that larger error and bias estimates raise the possibility of sources of error not captured by the estimates used, though we have carefully examined the literature on the use of the Jarzynski theorem.If the error bounds seem large, it is because we have been most careful and conservative in our estimation of the free energy. The estimates are as derived and analysed by Gore et alFurther to these error estimates, we have examined the effect of varying the speed of the protein, and checked that calculation of ΔG for the motion of the protein through pure water is appropriately close to 0. We used two independent sets of trajectories and identified their strengths and weaknesses. Our paper depends only on the demonstration that the binding is irreversible. We submit that this is clearly shown by our results. We have now also expanded and clarified the discussion of the Jarzynski method.(ii) Entropic effects  are probably not sampled correctly.In the dissociated state, the protein is surrounded by water on all sides, to a distance of at least the correlation length in pure water. This state on average is symmetrical, and so sampling more orientations would not alter the thermodynamics of this state. More broadly, and not only for the purposes of the Jarzynski calculation, equilibration is important. As discussed in the main paper, we see stabilisation of the ice front and protein in each structure examined.(iii) It is not clear how the work is calculated.We have greatly expanded and clarified the report of this calculation. There are too many changes for these to be set out in full here, but for example the relevant Methods section now opens with an outline of its components, and how they fit together.Other salient improvements are in the Methods section, where the role of the error and bias estimates is discussed, as well as the use of tests of the bias method.(iv) It is not clear if the free energy contains any contributions from the harmonic restraint that would have to be subtracted for a proper binding free energy .Fortunately the free energy of a harmonic oscillator is exactly and easily calculable, and the force constants we use correspond to free energies smaller than the random errors.The work by Hummer and Szabo pertains to the application of Jarzynski's theorem to single molecule force spectroscopy, rather than to a simulation. The important correction that they consider concerns the measurement of force as a function of time, when the work W in Jarzynski's theorem is the integral of force with respect to distance. This is a subtle issue, but one that we circumvent in a simulation because the exact value of the work W is available for each trajectory.(v) The enthalpy estimate in terms of hydrogen bond numbers does not seem to include hydrogen bonds of free water with ice.Estimating the hydrogen bonds of free water with ice is problematic as it is difficult to define which molecule is water and which is ice. We have clarified our original estimate and argument to consider the initial step of removing the protein from the ice as requiring simultaneously breaking the 12 hydrogen bonds we observe made from the ordered water to the ice and relating this to the activation free energy for the removal of the protein from the ice.To get a more controlled estimate of the binding free energy, it would be important to include also the reverse process of binding and use (variants of) the Crooks fluctuation theorem.The individual trajectories are done deterministically and so doing more trajectories is the same as studying also the reverse process. To use the Crooks theorem on top of Jarzynski would be redundant.In its present form, the binding free energy calculations seem to be more confusing than helpful. They may be more suitable for a separate technical publication, in which details can be explored more carefully.We have made improvements on the clarity of the binding free energy calculations as detailed above.To make the paper acceptable for publication in eLife will require (1) a considerably more nuanced discussion of preceding work, and (2) the elimination of the data and discussion of the calculation of ice-binding free energies. Detailed reasons are given below. In addition, (3) the discussion of the semi-quantitative agreement with the Gibbs-Thomson formalism, as given inshould better reflect evident uncertainties in this comparison. Also, (4) a clearer justification of the choice of ice-growth model should be included in the paper, not just in the rebuttal. In the following, I discuss these four points in detail.First, I find the discussion of earlier studies inadequate. I am particularly troubled by statements such as “previous AFP simulations have fallen short of complete, convincing molecular descriptions of an AFP-ice interaction” and “recent advances in computational science now make complete simulations feasible and we report them here.” What do the authors mean by their simulations being “complete”? Is there nothing to be done in this field after this study? This is certainly not the case, with many questions remaining open, e.g., concerning the water model with its freezing point of ∼240 K, the quantification of the phenomenological theory in, the mode of ice growth, or the calculation of a binding free energy. A fairer discussion of earlier results and a more modest presentation of the new results are in place. Such a discussion should explicitly mention the work of Nada and Furukawa on the use of MD simulations to study ice growth kinetics around AFPs, as influenced by the Gibbs-Thomson effect.We have greatly elaborated on prior work as suggested by the reviewers, including citing an additional 8 references and adding three paragraphs in relation to the historical context of computational simulation of AFPs, with particular mention of the work from Nada and Furukawa.Second, there are major issues with the calculation of the protein-ice binding free energy. Quoting Reviewer #2, in revising their manuscript, the authors have substantially increased the details of their simulation methodology and have now included a much-expanded discussion on the calculation of the binding free energy. Unfortunately these details now render these calculations and the conclusions derivable from them somewhat questionable. These calculations could be presented separately in a more technical journal to do justice to the many challenges involved in such demanding calculations.The instantaneous switching of the control parameters (either the spring constant or harmonic minima) is expected to yield reliable free energy estimates only when such changes result in dissipative work values on the order of kT . The accumulated work values in Figure 9 place this study far outside of this regime, and it is likely the poor sampling of exponentially rare work values that lead to systematic errors and ultimately the disagreement between various protocol estimates of the binding energy discussed in the appendix. Contrary to the authors' claims in their revision, simulating both forward and backward protocols to converge the binding energy would be hugely beneficial precisely because of the irreversibility of binding that the authors allude to. Only in instances of microscopically reversible dynamics do the two procedures yield redundant information. The authors may, e.g., consider Dellago and Hummer, 2013, for guidelines concerning the application of these equations : 41-61).The discussion of the binding free energy in terms of the number of broken hydrogen bonds is also too vague to be useful. As pointed out by Reviewer #2, the number listed for the hydrogen bond enthalpy seems to be judiciously picked to yield agreement with the calculated binding energy  and moreover hydrogen bonding energies are known to be rapidly varying functions of bonding geometry. The authors' recent correction of this estimate to reflect an activation energy rather than the binding energy presupposes that the binding is barrierless for the equivalence of these two quantities to be thermodynamically meaningful and that it is necessary to break hydrogen bonds along the reaction coordinate for this estimate to be kinetically meaningful. From the data provided and cited, it is not clear that either of those assumptions is valid.I want to add to the comments of Reviewer #2 that even the definition of the binding free energy is unclear. Binding equilibria from a bulk phase to a surface have dimensional contributions that require a choice of standard state. Furthermore, does the calculated free energy, fully or partially, include the entropic contribution from a substantial increase in rotational freedom? Probably not, since the time to sample full rotation in the dissociated state has been too small. On the technical side, I wonder how the work was calculated , in particular for the cases in which the spring constant was changed with time.We have removed all results and discussions regarding the free-energy calculations. We agree that this aspect of the work is difficult and perhaps better suited in a more technical journal where we can explore technical and theoretical details fully.We greatly appreciate the detailed critique of the free-energy methods we originally employed. Since we have removed the controversial components raised by the reviewers, specifically instantaneous switching of control parameters and binding energy in terms of broken hydrogen bonds from this manuscript, we shall address them and binding energy definitions in subsequent work with and not elaborate them here.Third, Reviewer #2 points out that the near quantitative agreement of the simulation results with the radius of curvature calculated fromseems fortuitous. Is there a reason to believe that the surface tension or enthalpy of fusion for the water model used are the same as in experiment? Can experimental parameters taken from a website  be used to explain the observations of the simulations? Such a validation procedure seems questionable. There are also technical issues with the application ofto a molecular simulation system. As discussed previously : 2651-2659), how exactly the interface and the radius of curvature are calculated is not unique. The implications on the calculated value of the radius would need to be discussed for this quantitative comparison to be meaningful.., leading to a smaller radius value of 46 Å compared to our original 59 Å figure. We agree the quantitative comparison is problematic and have dropped the direct reference in favour for a qualitative comparison and a brief mention of the difficulties in measuring curvature from a simulation. We have replaced the shaded 60 Å radius circular segment in We have recalculated the radius of curvature of ice using parameters derived from the TIP4P model from literature values of Vega et al., and Handel et alFinally, Reviewer #1 asks for further clarification in the paper concerning the ice-growth mode. In the rebuttal, the authors mention: “Our preliminary work using ‘perfect’ crystals did not grow well, extending the ice front very slowly if at all. We realised this was because one had to effectively wait for a 2 dimensional nucleation event on the prism plane to occur and reach a critical size before the next layer of ice would grow.” The authors should include this explanation in the paper to justify their assumption of a step-growth mode on the prismatic plane.We have further clarified the step growth mode of the ice model by mentioning our earlier experience of slow growth of ‘perfect’ crystals and referencing work of Frank and Hobbs in relation to step propagation as a mechanism crystal growth.


Although pedicle or lateral mass screws are usually chosen to fix atlantoaxial (C1-C2) instability, there is an increased risk for vertebral artery (VA) injury when used in patients with bone or arterial anomalies or osteoporotic bone. Here we report the C1 posterior arch screw as a new technique for upper cervical fixation.A 90-year-old man complained of upper cervical pain after falling in his house. The initial computed tomography (CT) scan showed C1-C2 posterior dislocation with a type II odontoid fracture. The patient underwent C2 fracture reduction and posterior C1-C2 fixation. On the right side of C1, because lateral mass screw placement could cause injury to the dominant VA considering a risk in oldest-old osteoporotic patients, a posterior arch screw was chosen instead as an auxiliary anchor. An intralaminar screw was placed on the right side of C2 because a high-riding VA was observed. A lateral mass screw and a pars interarticularis screw were placed on the left side of C1 and C2, respectively. Ten months later, the odontoid fracture had healed, with normal anatomical alignment. Although the patient experienced slight weakness when spreading his bilateral fingers, his overall condition was good.We have presented a novel technique using C1 posterior arch screws for the fixation of a C1-C2 dislocation. Such a screw is an alternative to the C1 lateral mass screw in patients who are at risk for a VA injury because of anomalous bone and arterial structures or poor bone quality.Although there have been few comparable studies, and the long-term outcome is unknown, fixation with a posterior arch screw could be a beneficial choice for surgeries involving the upper cervical region. Instability in the atlantoaxial (C1-C2) complex can arise from trauma, malignancy, inflammatory disease, or congenital malformation. To remedy this pathological condition, surgical intervention is often needed to achieve realignment and fixation of the vertebrae. The methods used for C1 posterior fixation have included posterior wiring, transarticular screws, and pedicle or lateral mass screws  scans revealed that C2 was fractured at the odontoid process, with 6 mm of displacement Figure B. This wThe patient was given general anesthesia and treated in the prone position. The odontoid fracture was reduced successfully back into the correct anatomic position through a combination of gentle manual traction and neck flexion, using a fluoroscope for guidance. Next, using standard anatomical landmarks, a unicortical lateral mass screw was placed via the posterior arch on the left side of C1, as advocated by Tan et al. Figure A and B . On the Postoperatively, the patient was immobilized in a hard cervical collar. Three months after the surgery, the odontoid fracture had healed into an anatomically correct alignment, and the cervical collar was removed Figure A-C. Ten Written informed consent was obtained from the patient for publication of this case report and any accompanying images.Recently, the preferred anchor screw for C1 stabilization has been the lateral mass screw. In this case, however, the patient was oldest-old, with low bone quality, which increased the chance of the screws loosening after surgical fixation. In fact, previous reports have demonstrated that a lower bone mineral density is strongly associated with a lower screw pullout strength (Savage et al. Some key techniques for placing the posterior arch screws are noteworthy. First, we used a 3.5-mm tap for a 3.5-mm screw. It is possible that a smaller tap would have provided an improved screw purchase. However, because the bone quality was weak, we believed that the excessive torque created during screw insertion could lead to splitting and fracturing of the posterior arch. Therefore, we used the same size tap as the screw diameter. The next consideration was the entry point of the screw, since the ease in securing the rods depends on the position of the connected screws. In this case, we took into account the position of the C2 intralaminar screw head, and placed the posterior arch screw about 10 mm from the midline of the posterior tubercle. The exit point for the screws is another important consideration, because a longer screw could increase the fixation strength. However, if the screw was inserted in the wrong direction, there still existed the risk of VA injury. To put the screw properly, we rigorously examined the VA courses by preoperative CT angiography, and purchased the screw with sufficient gap between the tip of the screw and the artery. Moreover, when calibrating in the CT images, height of the posterior arch in the inserted place was 9.0 mm. Since the diameter of the screw was 3.5 mm, we judged the screw purchase was safe if inserted in the middle of the arch. Thus, thorough preoperative planning and careful intraoperative technique are needed for the successful placement of screws.There are some risks associated with posterior arch screws. During surgery, the posterior arch of the atlas can be destroyed if its height or thickness is insufficient for the screw purchase. In addition, deep screw purchase can result in dural tear or spinal cord injury, requiring the use of a spatula to protect these tissues. After surgery, screw loosening can occur due to biomechanical weakness. Therefore, a posterior arch screw should be used only in a complementary role for C1-2 fixation, and the indication for this placement is quite limited.A review of the literature revealed only one report describing the use of posterior arch screws in the treatment of cervical fracture (Carmody et al. There have been a few reports describing the biomechanical testing of posterior arch screw fixations that are slightly different from the technique discussed here. Zarro et al. compared the pullout strength of C1 lateral mass screws versus unicortical posterior arch screws, and demonstrated that the posterior arch screws provided significantly stronger resistance to pullout via axial load than did the lateral mass screws (Zarro et al. Traumatic C1-C2 dislocation complicating an odontoid fracture is relatively rare, and most of these cases are probably fatal (Pissonnier et al. We have presented a novel technique using C1 posterior arch screws for the fixation of a C1-C2 dislocation. Such a screw is an alternative to the C1 lateral mass screw in patients who are at risk for a VA injury because of anomalous bone structures or poor bone quality. Although there have been few comparable studies, and long-term outcome or biomechanical testing research has yet to be described, fixation with a posterior arch screw could be a reasonable treatment option for surgeries of the upper cervical region.


The cap-dependent mRNA translation process that enables post-transcriptional control of gene expression and protein synthesis is tightly regulated in eukaryotes. Deregulation of this process, particularly in the formation of the eIF4F translation initiation complex—comprising the eIF4E mRNA cap-binding protein, the eIF4G scaffolding protein and the eIF4A RNA helicase—is associated with cancer development and progression. It is widely believed that translation of certain key oncogenic mRNAs is strongly dependent on the level of eIF4E. Consequently, expression of these oncogenic mRNAs is preferentially and dis-proportionately affected by eIF4E availability and is sensitive to alteration of its level. Free eIF4E level can be increased substantially in cancer cells by a number of mechanisms, including elevated eIF4E expression, decreased expression of eIF4E inhibitory binding proteins, 4E-BPs, and release of eIF4E from 4E-BPs by inactivating phosphorylation of 4E-BPs results from the oncogenic activation of PI3K/AKT and RAS/RAF/MEK/ERK signaling pathways.KRAS and PIK3CA mutation occur simultaneously in colorectal carcinoma; BRAF and PIK3CA mutation in thyroid carcinoma; and BRAF and PTEN mutation occur simultaneously in melanoma. A number of small molecule inhibitors targeting components of these two pathways have been tested for the treatment of cancer in preclinical and clinical studies, but have shown only limited activity as a single agent. Our work demonstrated that in tumors such as colorectal cancer (CRC) with coexistent activation of both PI3K/AKT and RAS/ERK pathways, drug resistance, following inhibition of either pathway, is associated with redundant activation of eIF4E-initiated cap-dependent translation through convergent phosphorylation of the translational repressor 4E-BP1 mediated by the AKT and ERK pathways. We found that combined inhibition of both pathways is required to maximally inhibit 4E-BP1 phosphorylation and subsequent induction of 4E-BP1 binding to eIF4E to prevent cap-dependent translation of oncogenic mRNAs (e.g. survivin) and ultimately achieve antitumor activity. Furthermore, we showed that a genetically-modified non-phosphorylated 4E-BP1 mutant that binds constitutively to eIF4E and represses eIF4E-dependent oncogene expression exerts similar inhibitory effects on tumor growth and metastasis as the combined pharmacological inhibition of AKT and ERK pathways, whereas loss of 4E-BP1 expression induces epithelial-mesenchymal transition through translational control of snail expression and its activity to promote metastatic progression of CRC and reduce CRC dependence on AKT/ERK signaling for translation, survival and motility. Collectively, these findings support the conclusion that deregulation of 4E-BP1 phosphorylation-mediated assembly of eIF4F translation initiation complex causes CRC resistance to its upstream kinase targeted therapies and thus maintain the transformed phenotype for CRC progression and metastasis. Of notice, several lines of evidence have also established incomplete inhibition of 4E-BP1 phosphorylation, amplification of eIF4E or increased eIF4E/4E-BP ratio as a mechanism of primary resistance to anti-PI3K/AKT/mTOR and anti-BRAF/MEK/ERK pathway inhibitors in a variety of cancers [Our recent studies have revealed that 4E-BP1 is a critical regulatory node that integrates oncogenic signals of the P3K/AKT and RAS/RAF/MEK/ERK pathways for cancer progression and metastasis [ cancers .Our data and others strongly suggest that 4E-BP1 phosphorylation is an important biomarker and therapeutic target in cancer. It is well know that mTOR kinase complex 1 (mTORC1) phosphorylates 4E-BP1, which in turn, activates cap-dependent translation and promotes protein synthesis. Our recent studies demonstrated that the effects of AKT and ERK activation on translational regulation of CRC progression are largely mediated by mTORC1 , suggestOur findings further suggest that directly targeting the convergence of multiple oncogenic signals on eIF4F translation initiation complex may provide a promising strategy for improving cancer therapy. Several translation initiation inhibitors, including eIF4E antisense-oligonucleotides and the eIF4A inhibitor silvestrol, have recently produced encouraging anti-tumor effects with limited toxicity. Future works require additional clinical correlative studies and await the development of clinically effective translation initiation inhibitors for cancer treatment, either as single agents or as part of combination therapy.


Drosophila) as a critical target and effector of cAMP in injured axons. DLK/Wnd is thought to function as an injury ‘sensor’, as it becomes activated after axonal damage. Our findings in both Drosophila and mammalian neurons indicate that the cAMP effector kinase PKA is a conserved and direct upstream activator of Wnd/DLK. PKA is required for the induction of Wnd signaling in injured axons, and DLK is essential for the regenerative effects of cAMP in mammalian DRG neurons. These findings link two important mediators of responses to axonal injury, DLK/Wnd and cAMP/PKA, into a unified and evolutionarily conserved molecular pathway for stimulating the regenerative potential of injured axons.A broadly known method to stimulate the growth potential of axons is to elevate intracellular levels of cAMP, however the cellular pathway(s) that mediate this are not known. Here we identify the Dual Leucine-zipper Kinase (DLK, Wnd in DOI:http://dx.doi.org/10.7554/eLife.14048.001 Adult mammals typically cannot repair damage to the nerve fibers in their brain or spinal cord. This is because these nerve cells cannot generally grow new nerve fibers. However this inability to regenerate nerve fibers is not set in stone. Instead, it can be unlocked by a second injury in nerves elsewhere in the body, the so-called “peripheral nervous system”. This process relies on an enzyme called DLK, which becomes activated in damaged nerve fibers.But how does DLK ‘sense’ damage to nerve fibers? Injuring the peripheral nervous system causes the levels of a molecule called cAMP to increase in the damaged nerve cells, and the elevated cAMP levels stimulate the nerve fibers to regenerate. However, it was not known if cAMP activates DLK, or if the two act independently of each other.By looking at the regeneration of damaged nerve fibers in fruit fly larvae, Hao et al. now show that the cAMP and DLK signaling pathways are clearly linked. Further experiments with nerve cells from mice and human cells revealed more detail about this link. Together the results showed that another enzyme called PKA activates DLK directly when cAMP levels are high. These findings reveal a unified pathway that is the key to unlocking the regenerative potential of injured nerve fibers, which has been conserved for hundreds of millions of years of evolution. Further work could now ask if the DLK enzyme is involved in the other known roles of cAMP signaling in nerve cells; or if cAMP and PKA activate DLK in other forms of nerve damage, including injuries where nerve fibers normally fail to regenerate.DOI:http://dx.doi.org/10.7554/eLife.14048.002 Repair of lost axonal connections generally fails to occur after neuronal injury in the adult mammalian central nervous system (CNS). This failure is not only a reflection of the growth inhibitory nature of CNS tissue , but alsin vivo , mediates protective effects of a conditioning lesion in Drosophila motoneurons  or activation of PKA by overexpression of the catalytic subunit (PKACA) (Drosophila motoneuron axons after nerve crush injury (3 to 200 µm3 15 hr after injury. PKA activation led to a 1.5 fold increase in this volume compared to control (WT) axons   led to ah injury . The newh injury . In contT) axons . The enhT) axons . These oignaling . In addiol axons . cAMP anDrosophila for monitoring the activation of Wnd and downstream nuclear signaling. Wnd signaling induces expression of the c-Jun N-terminal Kinase (JNK) phosphatase puckered (puc), which can be measured as lacZ expression using fly lines that contain the puc-lacZ enhancer trap reporter  version (GFP-WndKD) that contains a point mutation in the kinase domain , we have generated phospho-specific antibodies against a phosphorylated peptide corresponding to activation loop of mouse DLK (KELSDKpSTKMpSFAGTV). The phospho-DLK antibodies detected a strong band in HEK293 cells that overexpress WT mouse DLK, but show no reactivity for mutant DLKS302A . RemarkaS302 DLK , even whS302 DLK . SimilarS302 DLK .in vitro  conditional knockout (KO) mice and littermate controls. The effects of forskolin on neurite outgrowth were abolished in DLK conditional knockout DRG neurons . These fThe Wnd/DLK kinase is likely to function as a sensor of axonal damage. Depending upon the context, its activation can lead to either axonal regeneration or cell death and degeneration . The facC. elegans, PKA gain-of-function mutations can induce axonal outgrowth even in dlk mutants  and UAS-moody-RNAi (VDRC 100674) were from the Vienna RNAi center  and UAS-dnc-RNAi (27250) were acquired from Bloomington stock center.The following fly strains were used in this study: Canton-S (WT), m12-Gal4 , BG380-GUAS-Wnd, , UAS-GFPWnd,  was diluted 1:100. Anti-dsRed polyclonal antibody  was diluted 1:1000. For secondary antibodies, A488- or Cy3-conjucated goat anti-mouse or rabbit  were used at 1:1000.Confocal images were collected on an Improvision spinning disk confocal system, consisting of a Yokagawa Nipkow CSU10 scanner, and a Hamamatsu C1900-50 EMCCD camera, mounted on a Zeiss Axio Observer with 40X (1.3NA) oil objectives. Similar settings were used for imaging of all compared genotypes and conditions. Volocity software (Perkin Elmer) was used for intensity measurements and quantification of all confocal data.A quantification of the sprouting response in injured motoneuron axons was measurement of the change of total volume of regenerating axonal membrane, labeled by mCD8-GFP, within a 100 µm distance from the injured tip. Pixels within the most distal 100 µm of the injured proximal stump were selected based on mCD8-GFP intensity criteria of >3 standard deviation above the mean, and then summed to measure total membrane volume. Puc-lacZ expression was quantified by measurement of mean intensity for lacZ staining in the nuclei of motoneurons located along the dorsal midline (segments A3-A7) of the nerve cord of third instar larva. The mean intensity of GFP-WndKD within segmental nerves was quantified by measuring GFP intensity within 100 µm distance of each nerve at the site of exit from the ventral nerve cord.S302A (K185A (directly generated from Flag-DLK) and PKACA (HEK293 cells were cultured in DMEM/F12 (Gibco) supplemented with 10% fetal bovine serum (Gibco) and 1% penicillin-streptomycin (Gibco). For transient transfection, 1 µg of given vectors were transfected into 3.5 cm dish using Lipofectmine 2000 (Invitrogen). 20 hr after transfection, cells were processed for Western blotting. Plasmids used for transfection were Flag-DLK , Flag-DLS302A , Flag-DLnd PKACA .Cortical neurons were dissected from E18 rat embryos. The cortex was digested by incubating with 0.5% trypsin-EDTA (Gibco) and DNAse I (Roche) at 37°C for 10 min. Following digestion, neurons were washed twice in DMEM medium (Gibco) containing 10% FBS before resuspension in neuronal growth media which containing neurobasal (Gibco), Glucose (Sigma), Glutamax (Gibco), penicillin-streptomycin and B27 supplement (Gibco). All the plates were coated with 100 µg/mL ploy-D-lysine  for 2 hr. Neurons were then triturated and plated at a final concentration 400,000 cells/mL.in vitro (DIV)1, half of the media was removed and fresh media containing AraC  was added. Drug treatment and replating were performed as described previously (2). Trypsin was removed and fresh culture media was added to the cells which were then gently pipetted and transferred to culture slides. 18 hr after replating, neurons were fixed (4% PFA) and stained for βIII-tubulin . At least 100 neurons were imaged per group using either Leica DFC310 FX or DFC7000T color fluorescence cameras and longest neurite was traced using NeuronJ plugin for ImageJ. For replating experiments with PKA inhibitor , vehicle or inhibitor were added at the same time as DMSO and forskolin.For adult DRG experiments, DRG neurons were collected from either CD1(Charles River), or Map3k12 F/F; Advillin-Cre-/- (WT), or Map3k12 F/F; Advillin-Cre+/- (DLK KO) mice. WT and DLK KO mice were age-sex matched. Neurons were prepared as previously described . Brieflyeviously . On DIV4For detection of Wnd protein levels in larva nerve cords, the whole brains were carefully dissected from third instar larva. The two brain lobes were removed before they were frozen in liquid nitrogen and processed for western blotting (25 nerve cords per lane).For western blots using HEK293 cells or cortical neurons, cells are lysed by incubating on ice for 10 min with RIPA buffer  supplemented with Complete protease inhibitor cocktail (Roche) and PhosSTOP phosphatase inhibitor (Roche). Protein concentrations were measured by BCA assay kit (Thermo Scientific).Equal amount of protein samples were loaded on each lane of NuPAGE 4–12% Bis-Tris gels (Invitrogen) and subject to electrophoresis separation in MOPS buffer (Invitrogen). Blots were visualized with SuperSignal chemiluminescent substrate (Thermo Scientific) and exposure to either film or ChemiDoc (Bio-Rad). Bands intensities were determined using software ImageJ (NIH) using the gel analysis plug-in.The following antibodies were used for Western blotting: anti-Wnd A3-1,2 at 1:700 ; anti-DLS302 antibodies were raised by immunization of rabbits with the peptide KELSDKpSTKMpSFAGTV and affinity purified before use at dilution 1:100. While the antibodies were raised against a dually phosphorylated peptide , no difference in reactivity was noticed for DLK S298A mutants (data not shown).Anti-phospho-DLKForskolin  was applied to either HEK293 cells or cortical neurons at the final concentration of 30 µM for 6 hr. H-89  was used at the final concentration of 5 µM for cortical neurons for 6 hr. JNK inhibitor VIII  was used at the final concentration of 10 µM on HEK293 cells for 6 hr. Torin1  or Rapamycin (LC laboratories) was applied to the cells at 1 µM  for 2 hr before harvest.2 (NEB) and either 1000 U lambda protein phosphatase (NEB) or equal amount of glycerol (control) in RIPA buffer for 30 min at 30°C. Beads were then removed from the reaction buffer and Flag-DLK was eluted by boiling in SDS sample buffer for 10 min. Equal amount of samples were analyzed by western blot.24 hr after transfection of the given constructs, HEK293 cells in a 6 cm dish were washed by ice-cold PBS and harvested in ice-cold RIPA buffer supplemented with EDTA-free protease inhibitor (Roche). The cell lysates were incubated on ice for 30 min and centrifuged at 14,000 rpm at 4°C for 10 min. The soluble lysate was incubated with 3 µg anti-Flag antibody pre-bound with 15 µl Dynabeads Protein G (Novex) for 1 hr at 4°C. After removal of supernatants the beads were washed 3 times with RIPA buffer, and then incubated with 1X PMP buffer (NEB), 1X MnCl2, 0.01% Triton X-100, 2 mM DTT, and 30 µM ATP). 10,000 U recombinant human full length PKA catalytic subunit alpha (NED Millipore) was added to one of the tubes, while the control tube were added with glycerol. Both tubes were incubated at 30°C for 90 min. Flag-DLK was eluted from beads by boiling in the SDS sample buffer. Equal amounts of samples were analyzed by western blotting with anti-DLK and anti-pDLKS302 antibodies.HEK293 cells transfected with Flag-DLK were washed by ice-cold PBS and harvested using ice-cold cell lysis buffer  and 30 µM MG132 (Sigma)). Flag-tagged DLK was immunoprecipitated from HEK293 cell lysates using anti-Flag M2 antibody (Sigma) and Protein G Dynabeads (Novex). The DLK-bound beads were then washed with 3 times with lysis buffer and incubated with 2000 U lambda protein phosphatase (NEB) for 30 min at 30°C to remove all the phosphate groups. After incubation, the beads were washed with wash buffer containing cocktail phosphatase inhibitor (Roche) and split equally into two tubes containing kinase reaction buffer  has previously been implicated in invertebrate and mammalian systems to be essential for neuronal axon regeneration. DLK is enriched in axons after axonal injury, and DLK is required for injury-induced axonal regeneration in neurons of mammals, flies and worms. However, the specific mechanism by which DLK is activated is currently unknown, but multiple signaling pathways are likely to activate axonal regeneration. The authors focus on the cAMP-PKA signaling pathway, which stimulates axonal elaboration in multiple models systems of axon growth/regeneration. Based on an evolutionarily conserved PKA phosphorylation site residing in the kinase domain of DLK, the authors hypothesized that DLK phosphorylation lies downstream of the cAMP-PKA pathway to modulate DLK-dependent axonal regeneration. Consistent with this hypothesis, the authors demonstrate that DLK is subject to direct PKA phosphorylation at the proposed PKA phosphorylation site (S302). Additionally, the authors demonstrate in HEK293 mammalian cultured cells that DLK phosphorylation is enhanced (phospho-specific DLK antibody) when the PKA signaling pathway is activated either pharmacologically or by expression of constitutively activated PKA. The authors correlate DLK phosphorylation with enhanced DLK abundance in HEK293 cells and implicate a previously described JNK feedback pathway that affects DLK ubiquitination and degradation. These observations are consistent with their well-executed in vivo experiments demonstrating #1) a genetic interaction in Drosophila between PKA and Wnd in mediating axonal regeneration after injury, #2) enhanced levels of Wnd protein and activity after axonal injury or genetically activating the PKA pathway, and #3) failure to rescue axonal regeneration by expressing the phosphorylation incapable Wnd S302A in the wnd mutant compared to WT controls.Hao et al. describe an evolutionarily conserved mechanism mediating cAMP axonal regeneration via the phosphorylation of dual leucine zipper-bearing kinase (DLK). DLK  levels are reduced.I recommend that the authors perform a few experiments to assess whether elevating cAMP levels in neurons mimics the expression of PKACA is attenuated in the hiw mutants (by Westerns OR by imaging Wnd-GFP in axons). The results of this experiment will be interesting and relevant to this study regardless of the outcome! At the very least, the authors should discuss the stabilization of Wnd by PKA in the context of their previous findings regarding Hiw.Given that PKA activation leads to a strong upregulation of Wnd levels, I wonder whether Hiw and PKA function in the same pathway. I recommend that authors examine whether the increase in Wnd levels by PKADrosophila after expression of PKACA and/or axonal injury using westerns and/or immunostaining? Not sure if the antibody works across species, so this might not be an option. If not, has the antibody been tried in forskolin treated cultured cortical or DRG neurons? Would be a very nice positive addition to support the model, though not required given how difficult phospho-antibodies can be.Is endogenous DLK/Wnd phosphorylated by PKA? The authors generated a beautiful DLK S302 phospho-specific antibody and successfully used it in transfected HEK293 cells expressing DLK. Can this antibody be used to demonstrate enhanced S302 phosphorylation of endogenous DLK/Wnd in S302A mutant exhibited decreased overall DLK abundance by enhancing DLK degradation. Consistent with these previous findings, the current authors observed PKA-induced enhancement of DLK levels are blocked by JNK inhibitors without affecting S302 phosphorylation , why do they behave differently with JNK inhibitors? The authors' statement that "The induction of DLK activation loop phosphorylation independently of its own signaling abilities places PKA firmly upstream of DLK, as an upstream regulator/activator” (Results) seems true, but what does this data mean for the proposed model . That is, DLK downstream activation of the JNK pathway was proposed to result in a feedback loop in which JNK→DLK phosphorylation results in increased DLK abundance by reducing DLK degradation. There, the DLKrylation . Intereshibitors , suggestir model  and preved model ? Finallyeriments , as theyDrosophila rescue experiments in the supplemental figures, but I'm not sure why these are not in the main text. As mentioned in #2, failure of the DLKS302A rescues to enrich DLK in axons after PKACA expression or axon injury would strengthen the authors model that PKA-dependent phosphorylation of DLK modulates its local stability to promote regeneration programs. Finally, are the DRG experiments amenable to rescue experiments with WT and DLKS302A rescue experiments?The authors placed the WT and WndS302A wnd levels in axons than cell bodies. Does this mean PKA is activated by injury in axons but not cell bodies? This should be possible to test with a PKA activity reporter, and possibly also the phospho-specific wnd antibody.Axons vs. cell body. PKA activity has much more effect on  Essential revisions:CA with regard to Wnd-dependent axonal sprouting and puckered induction. For instance, the authors could utilize loss-of-function alleles of the gene encoding the cAMP phosphodiesterase, Dunce. It will also be worthwhile to evaluate whether axon sprouting and puckered induction in dunce mutants are sensitive to Wnd knockdown. Conversely, the authors could examine if injury dependent axon sprouting and puckered induction are diminished if adenylyl cyclase (for e.g. Rutabaga or AC78C) levels are reduced.I recommend that the authors perform a few experiments to assess whether elevating cAMP levels in neurons mimics the expression of PKADunce in Drosophila motoneurons, which leads to enhanced axonal sprouting response . Note that this previous study already showed that the enhanced regeneration in phosphodiesterase mutants required dlk-1 function.In our revision we have included new data with knockdown of phosphodiesterase response . These rDrosophila motoneurons cause increased puckered expression in uninjured animals . The results of this experiment will be interesting and relevant to this study regardless of the outcome! At the very least, the authors should discuss the stabilization of Wnd by PKA in the context of their previous findings regarding Hiw.Given that PKA activation leads to a strong upregulation of Wnd levels, I wonder whether Hiw and PKA function in the same pathway. I recommend that authors examine whether the increase in Wnd levels by PKAWe agree that the relationship between Hiw and PKA is very interesting to understand, particularly since some biochemical data in other species suggest that Hiw/Pam may regulate cAMP levels by inhibiting adenylate cyclase activity  . Our data thus far suggest that the relationship is complex, so including data/experiments on this issue would be both distracting to the current story, and would require many more experiments to address the relationship properly. So instead we are following the reviewer’s suggestion to discuss the regulation of Wnd by PKA in the context of what is previously known for Hiw, (in the Discussion section).Is endogenous DLK/Wnd phosphorylated by PKA? The authors generated a beautiful DLK S302 phosphospecific antibody and successfully used it in transfected HEK 293 cells expressing DLK. Can this antibody be used to demonstrate enhanced S302 phosphorylation of endogenous DLK/Wnd in Drosophila after expression of PKACA and/or axonal injury using westerns and/or immunostaining? Not sure if the antibody works across species, so this might not be an option. If not, has the antibody been tried in forskolin treated cultured cortical or DRG neurons? Would be a very nice positive addition to support the model, though not required given how difficult phospho-antibodies can be.We have tested the pS302-DLK antibody in flies and unfortunately it does not work across species. Over the past two months, we have tried hard to get a decent signal for endogenous DLK in cultured neurons, including scaled up cultures and immunoprecipitations to enrich for the protein. This has been very challenging technically – we think that the levels of endogenous DLK are very low, and that the antibodies may also not be very sensitive. We prefer to leave this experiment out of the paper. As the phosphorylation of S302 is important for DLK to function as a kinase, our functional data combined with our biochemical data in HEK293 cells strongly suggests that S302 is indeed phosphorylated on endogenous DLK.S302A mutant exhibited decreased overall DLK abundance by enhancing DLK degradation. Consistent with these previous findings, the current authors observed PKA-induced enhancement of DLK levels are blocked by JNK inhibitors without affecting S302 phosphorylation (K185A kinase dead version of DLK remains enhanced AND phosphorylated at S302 with PKA activation even in the presence of JNK inhibitors (K185A and DLKS302A mutants lack kinase activity and fail to activate downstream effector pathways (e.g. JNK), why do they behave differently with JNK inhibitors? The authors' statement that "The induction of DLK activation loop phosphorylation independently of its own signaling abilities places PKA firmly upstream of DLK, as an upstream regulator/activator” (Results) seems true, but what does this data mean for the proposed model . That is, DLK downstream activation of the JNK pathway was proposed to result in a feedback loop in which JNK→DLK phosphorylation results in increased DLK abundance by reducing DLK degradation. There, the DLKrylation . Intereshibitors , suggesthibitors  and preved model ?We thank the reviewers for these points. We have carried out and included some additional experiments  and still have a great deal of troubleshooting to carry out in order to implement the reporter in a new preparation and verify that it is working properly. The PKA reporter would require even further validation and controls. We think this is an interesting future project, but is not practical for this current story. An important additional caution is that even if any of these suggested assays work, a negative result in our injury assays could still have the caveat that the method of detection is not sensitive enough to detect functional changes that indeed occur.We agree that it is very interesting to know whether PKA regulates DLK locally in axons, however this is currently a very technically challenging question. The phospho-DLK antibodies do not work in flies, and our recent efforts lead us to conclude that they are also intractable for IHC in mammalian neurons (due to a high degree of non-specific staining). While a FRET reporter has been used for PKA in flies , it has never been used before in our larval motoneuron preparation. We have begun to work with a cAMP FRET reporter that is more widely used in


Anterior occiput-to-axis screw fixation is more suitable than a posterior approach for some patients with a history of posterior surgery. The complex osseous anatomy between the occiput and the axis causes a high risk of injury to neurological and vascular structures, and it is important to have an accurate screw trajectory to guide anterior occiput-to-axis screw fixation.Thirty computed tomography (CT) scans of upper cervical spines were obtained for three-dimensional (3D) reconstruction. Cylinders (1.75 mm radius) were drawn to simulate the trajectory of an anterior occiput-to-axis screw. The imitation screw was adjusted to 4 different angles and measured, as were the values of the maximized anteroposterior width and the left-right width of the occiput (C0) to the C1 and C1 to C2 joints. Then, the 3D models were printed, and an angle guide device was used to introduce the screws into the 3D models referring to the angles calculated from the 3D images.We found the screw angle ranged from α1  to α2 ; on the lateral view, the screw angle ranged from β1  to β2 . No statistically significant difference was found between the data of the left and right sides. On the 3D printed models, all of the anterior occiput-to-axis screws were successfully introduced, and none of them penetrated outside of the cortex; the mean α4 was 12.00 ± 4.11 (left) and 12.25 ± 4.05 (right), and the mean β4 was 23.44 ± 4.21 (left) and 22.75 ± 4.41 (right). No significant difference was found between α4 and β4 on the 3D printed models and α3 and β3 calculated from the 3D digital images of the left and right sides.Aided with the angle guide device, we could achieve an optimal screw trajectory for anterior occiput-to-axis screw fixation on 3D printed C0 to C2 models. Simultaneously, a biomechanical study showed that anterior occiput-to-axis screw fixation could achieve biomechanical stability comparable to that of posterior approaches.8In a number of clinical situations, such as in the case described by Dvorak et al9 which has the advantages of being minimally invasive, causing less blood loss, and resulting in shorter hospital stays.Compared to posterior surgery, which causes considerable damage to the extensor muscles as well as bleeding, anterior occiput-to-axis screw fixation could be performed by a percutaneous approach,The following additional advantages of occiput-to-axis screw fixation are reported: the anterior approach could be performed more conveniently than the posterior approach; the supine position is better for reduction and stable recovery and prevents having to change the position after anesthesia; and the anterior approach might be more suitable for patients with pressure from anterospinal compression.The complex osseous anatomy between the occiput and the axis causes a high risk of injury to the neurological and vascular structure; to avoid these complications, it is important to have an accurate screw trajectory to guide the anterior occiput-to-axis screw fixation.The research was performed following the Declaration of Helsinki principles and was approved by the Institutional Review Board of The Second Affiliated Hospital of Wenzhou Medical University. Informed consent was obtained from the participants.Thirty computed tomography (CT) scans of upper cervical spines were obtained from the Star PACS system  of our hospital; the participants were without spinal diseases, and had CT scans for health testing or had presented with oral diseases; patients with any spinal abnormality such as a fracture or tumor were excluded.The data from the CT scans (in DICOM format) were imported into Mimics software v10.01  for the three-dimensional (3D) reconstruction. We drew a cylinder (1.75 mm radius) to simulate the trajectory of the anterior occiput-to-axis screw.10 and the angle of the imitation screw was adjusted to 4 different angles with the following measurements: maximized interior angle (α1) and maximized lateral angle (α2) on the anteroposterior (AP) view, minimized dorsal angle (β1), and maximized dorsal angle (β2) to the referential line on the lateral view  to print the 3D model.After the 3D model was printed, it was fixed in a holding device, and an angle guide device was used . A paired On the AP view, the screw angle ranged from α1  to α2 ; on the lateral view, the screw angle ranged from β1  to β2 . The anteroposterior width of the C0 to C1 facet was 16.87 ± 1.34 (left) and 16.88 ± 1.38 (right), slightly larger than that at the C1 to C2 facet (left: 15.76 ± 1.53) and 15.94 ± 1.56 (right); the left-right width of the C0 to C1 facet was 12.43 ± 1.93 (left) and 12.04 ± 2.17 (right), shorter than that at the C1 to C2 facet (left: 15.92 ± 2.26) and 16.18 ± 2.00 (right); they were all wide enough for 3.5 mm or 4.0 mm diameter anterior occiput-to-axis screw fixation. No statistically significant difference was found between the data of the left side and right side  and is suitable for spinal fixation research. In our study, we used the α3 and β3 calculated from the 3D digital images as the reference and the angle guide device to introduce the anterior occiput-to-axis screws. We found that our method could guide an optimal screw trajectory, and none of the screws in this study penetrated outside of bone cortex.17 and its use could enable accurate screw placement; however, the navigation system is excessively expensive for most hospitals. A worldwide survey found that18: despite widespread distribution of the navigation system in North America and Europe, 11% of surgeons use these systems routinely; the lack of equipment, inadequate training, and high costs were the major reasons that the nonusers do not use the navigation systems. The rate of having and using navigation systems in developing countries was less than that in developed countries. Our novel angle guide device is much less expensive than the navigation system, with simple construction and methodology that is relatively easy to learn. The device has potential value for surgeons who do not have access to the expensive navigation system. Further study of this novel angle guide device in clinical practice is needed.CT-based navigation was developed in recent decades,There was enough space for anterior occiput-to-axis screw fixation, and by referring to the parameters of optimal screw trajectory and with the aid of the angle guide device, we could achieve an optimal screw trajectory on a 3D printed C0 to C2 model.