Yersinia sp. bacteria. Inactivation of YopH activity would lead to the loss of bacterial pathogenicity. We have studied the inhibitory properties of aurintricarboxylic acid (ATA) against YopH phosphatase and found that at nanomolar concentrations ATA reversibly decreases the activity of YopH. Computational docking studies indicated that in all binding poses ATA binds in the YopH active site. Molecular dynamics simulations showed that in the predicted binding pose, ATA binds to the essential Cys403 and Arg409 residues in the active site and has a stronger binding affinity than the natural substrate (pTyr). The cyclic voltammetry experiments suggest that ATA reacts remarkably strongly with molecular oxygen. Additionally, the electrochemical reduction of ATA in the presence of a negative potential from −2.0 to 2.5 V generates a current signal, which is observed for hydrogen peroxide. Here we showed that ATA indicates a unique mechanism of YopH inactivation due to a redox process. We proposed that the potent inhibitory properties of ATA are a result of its strong binding in the YopH active site and in situ generation of hydrogen peroxide near catalytic cysteine residue.YopH is a bacterial protein tyrosine phosphatase, which is essential for the viability and pathogenic virulence of the plague-causing  Yersinia genius contains three species of bacteria pathogenic to humans: plague-causing Yersinia pestis, septicemia-inducing Yesinia tuberculosis and Yersinia enterocolitica, which is responsible for a range of gastrointestinal disorders [Yersinia pestis is transmitted by fleas while Y. tuberculosis and Y. enterocolitica are transmitted by the fecal oral route [isorders . Yersinial route .Yersinia sp. utilizes a type III secretion system for translocation of virulence effectors into the host cell [Yersinia species contain a 70kb plasmid that encodes a type III complex system and effectors (Yops). During infection, Yersinia translocates Yops virulence effectors into a host cell leading to inhibition of the innate immune response [ost cell . All thrresponse .Yersinia's outer membrane protein effectors is a highly active YopH protein tyrosine phosphatase, which is essential for virulence since the YopH mutant plasmid is avirulent [One of virulent . YopH isvirulent . YopH divirulent .The catalytic center of the YopH contains an amino acid sequence similar to eukaryotic protein tyrosine phosphatases (PTPs). There is a cysteine residue located at the bottom of the active site, which is essential for catalysis and enzymatic activity . As for Yersinia pestis still causes several thousand human cases per year and the climate change is increasing the risk of plague outbreaks in new geographic areas as well as spreading of its natural reservoirs [Yersinia pestis is one of the most virulent infectious agents threatening humans, there is an ever increasing risk of the use of Y. pestis by unauthorized groups as a biological weapon of terror [servoirs . For hisf terror . The grof terror .There are libraries of chemical compounds that have been tested as YopH inhibitors and numerous of them were reported to inhibit YopH activity, mostly in micromolar concentrations . Example50 value around 10 nM, which was evaluated by the Liang group [One of the most effective YopH inhibitors is aurintricarboxylic acid Figure , with anng group . It is cng group . It has ng group . Becauseng group .Yersinia sp.Taking the above studies into consideration, it is readily concluded that ATA is a remarkable compound, which should be seriously considered as a drug for infectious diseases, including epidemics caused by We decided to evaluate the effectiveness of ATA as YopH inhibitor and to study possible mechanisms of ATA induced inactivation, as well as electrochemical properties of ATA. We performed computational docking and molecular dynamic studies to gain a molecular-level insight into the binding affinities and conformations of ATA in the YopH active site.Yersinia pestis and Yersinia eneterocolitica was treated with ATA and IC50 values were calculated. We also treated YopHs with peroctanoic acid, containing a peroxycarboxyl group with a higher oxidizing potency than that of a carboxyl group, studied by our group as a strong PTP inhibitor [YopH recombinant phosphatase from nhibitor , in orde50 values, ATA was more effective against YopH from Y. pestis and YopH from Y. enterocolitica than against CD45 phosphatase  to investigate the reversibility of ATA induced inhibition. We observed that ATA inactivates YopH reversibly and the ATA induced inhibition of YopH was completely reversed after a 20 minutes incubation process with DTT Figure .Due to the fact that YopH, as other PTPs, contains an oxidation-sensitive cysteine located in the active site, we decided to prepare an NBD-Cl assay to calculate the amount of thiol groups after treatment with ATA. We tested the amount of thiol groups modified by NBD-Cl forming Cys-S-NBD adducts. After treatment of recombinant YopH with 100 nM ATA, the quantity of Cys-S-NBD adducts was significantly decreased in comparison with control Figure . ApplicaWe also observed that catalase not only prevents a decrease of thiol adducts but also prevents an inhibition of enzyme activity. The catalase pretreatment almost completely protected from ATA induced YopH inactivation Figure , as well−4 M) the presence of ATA causes significant changes in the reduction and oxidation process. When the concentration of ATA increased from 5.0 · 10−5 to 3.7 · 10−4 M (dotted lines) the new reduction peak has appeared and the reoxidation process for oxygen disappeared.We performed electrochemical measurement in order to investigate the oxidizing potency of ATA. In this study the signals from the redox process of ATA preformed in the DMSO solution were not observed .A small addition of ATA caused significant changes in the cyclic voltammetric curve resulting in the appearance of a new cathodic peak at potential values form 0.74 V to 0.82 V Figure   in a wide potential range Figure  indicate−4 M (black dotted line) to 6.0 · 10−3 M (blue line)  Figure  measuredATA and natural substrate phosphotyrosine (pTyr) molecules were docked into the 3D structure of YopH in order to investigate the possible binding conformation and affinity. We performed blind flexible docking and retained top 30 conformations from docking runs. In all 30 conformations pTyr and ATA are bound to the active site of YopH, as shown in Figure To study the binding conformation of ATA in the YopH active site we performed molecular dynamics simulations using Amber12 and identified top scoring poses from docking studies. The interactions of ATA in the YopH binding site are presented as a PLIF diagram (protein ligand interaction fingerprints) in Figure vdwE = the Van der Waals contribution from MM.ΔeleE = Electrostatic energy as calculated by the MM force fieldΔpolarG = The electrostatic contribution to the solvation free energy calculated by PB or GB respectivelyΔnon-polarG = Nonpolar contribution to the solvation free energy calculated by an empirical modelΔbindG = final estimated binding free energy calculated from the terms above  are solvated far less efficiently than in water [In aprotic solvents, such as dimethyl sulfoxide (DMSO) electrochemically generated superoxide anions . The YopHs and CD45 enzymes were untreated (control) or treated with solution of ATA and peroctanoic acid. The assay was performed in 96-well microplates, and the final volume of each sample was 200 μL. The enzymatic activities of YopHs and CD45 were measured using 1 mM chromogenic substrate para-nitrophenyl phosphate (pNPP) in 10 mM HEPES buffer pH 7.4, at 37°C. Phosphatase hydrolyzed pNPP to para-nitrophenol and inorganic phosphate. Para-nitrophenol is an intensely yellow colored soluble product under alkaline conditions. The increase in absorbance (due to para-nitrophenol formation) is linearly proportional to enzymatic activity concentration  and was assessed at 405 nm on a microplate reader Jupiter (Biogenet) using DigiRead Communication Software (Asys Hitech GmbH).Bacterial recombinant YopH protein tyrosine phosphatase from Subsequently, recombinant phosphatase YopH that had been previously inactivated by ATA, was then treated with 10 mM dithiothreitol (DTT), and the samples were incubated at 37°C to reverse the inactivation, if possible. Restoration of YopH enzymatic activity was measured every minute as an increase of absorbance taken at 405 nm as previously described.The recombinant phosphatase YopH was inactivated by ATA and the amount of modified YopH thiol adduct with NBD (Cys-S-NBD adduct) was measured after 30 minutes incubation with NDB-Cl (0.6 mM in a 0.5 mL sample) as absorbance at 420 nm with a spectrophotometer.2O3 powder on a wet pad, then the electrode was washed with nanopure water, and dried under a stream of nitrogen. The oxygen-saturated DMSO solution used to perform measurement was obtained by the procedure described in the literature [−3 M [Cyclic voltammetry (CV) experiments were performed using an Autolab potentiostat/galvanostat (model PGSTAT30) supported by the GPES software. The experiments were performed at room temperature in DMSO solution with 0.1 M tetrabutylammonium tetrafluoroborate (TBAP) as the supporting electrolyte, at a scan rate of 100 mV/s under argon atmosphere or in the oxygen-saturated DMSO solution. A conventional three-electrode configuration consisting of a glassy carbon electrode (GC), a working electrode with a 3-mm diameter. The platinum wire used as the counter electrode and an Ag/AgCl in 3M KCl reference electrode were used, respectively. Each time before use, the working GC was polished with Alterature . The conre [−3 M . During http://www.pdb.org) with code 2YDU.pdb [The initial structure of YopH was imported from the RCSB protein data bank . Statistical analyses were performed using ANOVA combined with Tukey's test or T test combined with Wilcoxon test. The data were expressed as means ± SD. Differences between means were considered significant for


Introduction. The use of locking plate technology for anterior lumbar spinal fusion has increased stability of the vertebral fusion mass over traditional nonconstrained screw and plate systems. This case report outlines a complication due to the use of this construct. Case. A patient with a history of L2 corpectomy and anterior spinal fusion presented with discitis at the L4/5 level and underwent an anterior lumbar interbody fusion (ALIF) supplemented with a locking plate placed anterolaterally for stability. Fifteen months after the ALIF procedure, he returned with a hardware infection. He underwent debridement of the infection site and removal of hardware. Results. Once hardware was exposed, removal of the locking plate screws was only successful in one out of four screws using a reverse thread screw removal device. Three of the reverse thread screw removal devices broke in attempt to remove the subsequent screws. A metal cutting drill was then used to break hoop stresses associated with the locking device and the plate was removed. Conclusion. Anterior locking plates add significant stability to an anterior spinal fusion mass. However, removal of this hardware can be complicated by the inherent properties of the design with significant risk of major vascular injury. Successful techniques in limb fracture fixation and deformity correction are often expanded to other areas of the body. For plate fixation, these techniques have evolved from nonconstrained devices to the use of fixed or variable angle locking plate and screw constructs. The development of the LISS locking plate in the mid-1990s and the subsequent evolution of locked plating devices advanced care of extremity injuries by providing very stable constructs for challenging extremity fractures . As the The push for less invasive and muscle sparing approaches to the spine, along with efforts to save time while providing a stable fusion environment, gave impetus for the development of anterior plates for use in lumbar spine fusions. Stability of nonconstrained screw and plate systems for the lumbar spine is primarily in extension and does not provide significant stability for coronal and rotational moment arms. Utilizing locked plate technology creates a fixed angle construct that provides a strong constraint in all three planes of motion –8. The fA 44-year-old male involved in a motor vehicle collision presented with polytrauma that included L2 burst fracture. He underwent a corpectomy of L2, anterior vertebral reconstruction, and fusion utilizing a carbon-fiber cage and lateral spinal instrumentation (Kaneda device). Two months after his surgery, he developed increasing back pain and was found to have pyogenic discitis in his previously degenerated L4/5 disc between his fusion levels of L1 through L3 and his previous L5 to sacrum fusion. He underwent extensive debridement of the L4/5 disc and paraspinal tissues, as well as an L4/5 anterior discectomy and fusion with femoral allograft secured with a Synthes Spine titanium anterolateral locking plate . CultureOne year after his L4/5 fusion, the patient developed increasing pain in his lower back along with laboratory studies consistent with recurrent infection. Radiographic studies indicated changes at the L1–3 fusion mass, indicating the infection may not be isolated to the previous L4/5 disc. We planned a staged surgical debridement, hardware removal, and reconstruction, first at the L1–3 site, followed by debridement, hardware removal, and reconstruction at the L4-5 site. Exploration of the L2 corpectomy site confirmed a pyogenic infection extending into the psoas. After this was debrided aggressively, we reconstructed the L1–3 space with tibial allograft, followed by posterior segmental instrumentation. Cultures again grew methicillin sensitive staph aureus, and the patient was placed on IV vancomycin and gentamicin. Ten days later, a paramedian approach was used to access to the L4/5 locking plate, where purulence was again noted. Using the standard screw driver we were unsuccessful in our attempt to remove any of the screws and stripped the screw interfaces. We were successful in removing one screw with a reverse threaded screw driver, but we subsequently broke a total of 3 reverse threaded drivers attempting to remove the final three screws .Complete hardware removal was considered essential to treat this patient's infection. To protect the great vessels, they were mobilized and retracted medially as much as possible. A combination of various sized metal malleable retractors was secured between the vessel and area of burring. A high speed burr (Stryker TPS) was used with both an aggressive 4 mm round tip burr and a 3 mm neuro tipped cutting burr. Each tip was replaced frequently before they became dull. Cuts were started inside the plate and directed towards the screw head to break the screw plate hoop stresses. This inside-out technique avoided the vessel walls which were in contact with the outer edges of the plate. The final screw was drilled from the periphery to the screw head because it was located farthest away from the vessels with adequate space available for the drill bit. Once the plate threads were released the screw was easily removed . The L4/The difficulties encountered with the removal of locked plates in orthopaedic traumatology are well recognized. These problems have been universally reported to be with titanium implants usually due to the phenomenon of cold welding of the locking screw heads into the locking screw holes. Other problems reported with the removal of titanium locking implants are related to the relative malleability of the titanium screw heads. As the torque required to remove a jammed (cold welded) screw increases, often the screw head fails to maintain a competent interference fit with the screw driver. This can result in many potential problems including damage to the recesses (stripping) of the screw head or the screwdriver, jammed screws, broken screws, and shearing off the screw head due to rigid interface of bone ingrown into the screw threads. not been reported when stainless steel implants have been utilized.Another proposed mechanism for the difficulty of locked screw removal is related to the eccentric placement of the screw head into the locking screw hole, producing a cross thread mechanical lock which is not necessarily due to cold welding. Again, with the more malleable titanium implants, this phenomenon of cross threading is more likely to occur in an unrecognized fashion compared to a stainless implant, especially if the screw placement is off axis  with regard to the screw hole. Most of these removal issues haveMany techniques have been described to overcome these difficulties. Most frequently, in the case of a cold welded screw, the technique of radial side cut into the screw hole releases the hoop stresses about the screw . Other tThere are difficulties encountered when using a high speed burr to perform a radial side cut to burr down a screw head. Heat generation with subsequent bone and tissue necrosis may be an issue, especially when removing these devices around sensitive structures, such as nerves and vessels. The issue of metal debris is also a concern, especially in certain locations. Debris can be collected easily by spreading a sterile, water soluble gel (Doppler gel) to cover local soft tissues. The debris is then captured in the gel and held locally, allowing for easy removal at the end of the procedure.For the spine specifically, anterior lumbar plating with locking screw plate interfaces allow for the use of fixed angle constructs, improving stability and rigidity in all planes of motion –8. When In situations where an anterior spinal locking plate requires removal from the lumbar spine, we recommend the following techniques based on the described case: first, expect that cold welding of the screws may have occurred when the plate was placed and have a high speed burr ready with several drill bits in anticipation of having to drill through the plate. The great vessels will be in close proximity, including the aortic bifurcation around the level of L4, and the confluence of the vena cava at L5. An approach surgeon who is very comfortable with vascular surgery can assist with vessel mobilization and with any complications that may arise with the vessels. Malleable retractors can be used to lightly retract, but more to protect the vessels; they can simply be anchored on the bone between the vessel and the area to be drilled. Finally, a sterile ultrasound jelly or lube can be applied around the area to be drilled to catch the metal fragments so they are easier to remove. By anticipating the complication of cold welding that can require hardware drilling and the potential associated complication of vascular injury, these precautions should allow for a safe patient outcome.


To compare the von Mises stresses of the internal fixation devices among different short segment pedicle screw fixation techniques to treat thoracic 12 vertebral fractures, especially the mono-segment pedicle screw fixation and intermediate unilateral pedicle screw fixation techniques.Finite element methods were utilised to investigate the biomechanical comparison of the four posterior short segment pedicle screw fixation techniques .The range of motion (ROM) in flexion, axial rotation, and lateral bending was the smallest in the I6+2 fixation model, followed by the I5+2 and S4+2 fixation models, but lateral bending was the largest in the M4+2 fixation model. The maximal stress of the upper pedicle screw is larger than the lower pedicle screw in S4+2 and M4+2. The largest maximal von Mises stress was observed in the upper pedicle screw in the S4+2 and M4+2 fixation models and in the lower pedicle screw in the I6+2 and I5+2 fixation models. The values of the largest maximal von Mises stress of the pedicle screws and rods during all states of motion were 263.1 MPa and 304.5 MPa in the S4+2 fixation model, 291.6 MPa and 340.5 MPa in the M4+2 fixation model, 182.9 MPa and 263.2 MPa in the I6+2 fixation model, and 269.3 MPa and 383.7 MPa in the I5+2 fixation model, respectively. Comparing the stress between different spinal loadings, the maximal von Mises stress of the implants were observed in flexion in all implanted models.Additional bilateral pedicle screws at the level of the fracture to SPSF may result in a stiffer construct and less von Mises stress for pedicle screws and rods. The largest maximal von Mises stress of the pedicle screws during all states of motion were observed in the mono-segment pedicle screw fixation technique. Approximately 90% of spinal fractures are found in the thoracolumbar segment, and burst fractures comprise 10%–20% of such injuries In this study, we investigated the biomechanical comparison of the four posterior short segment pedicle screw fixation techniques  used in thoracolumbar burst fractures. We developed a finite element model of the intact thoracolumbar junction from T9 to L3 with thoracic 12 vertebral fracture, and various spinal fusion models were developed using the intact model. The range of motion (ROM), von Mises stress, and stress nephogram of the pedicle screws and rods were evaluated.The software Mimics 10.0 , Rapidform 2006 , and Abaqus 6.9  were used. A computed tomography scanner  was used to collect raw data in DICOM format with a scan slice of 0.625 mm.A finite element model of the T9-L3 spine, which included seven vertebras and six discs, was reconstructed. Geometrical details of T9-L3 spine vertebras were obtained from 64 spiral computed tomography (CT) images of a 40 years old healthy male (65kg and 175cm) without a history of spine injury, osteoporosis and radiographic evidence of degenerative sign. The CT images were scanned and imported into Mimics 10.0 . The surface model was then exported into Rapidform 2006  to generate and enhance the quality of the solid model. Eventually, it was imported to Abaqus 6.9  for meshing and analysis. Each vertebral body consisted of cortical bone and cancellous bone, and each vertebral disc was composed of nucleus pulposus, annulus fibrosus, and endplates. Posterior elements were built separately from the vertebral bodies. Based on Boolean operation, the lower half of the T12 segment was resected, and the structure of the posterior part was reserved to establish a finite element model of an unstable thoracolumbar fracture Fig. 1.Four fixation models were established using the unstable thoracolumbar fracture: the S4+2 model, M4+2 model, I6+2 model, and I5+2 model. In the current study, S4+2 means traditional short-segment pedicle screw fixation, M4+2 means mono-segment pedicle screw fixation, I6+2 means intermediate bilateral pedicle screw fixation, I5+2 means intermediate unilateral pedicle screw fixation. The implants and the vertebral structure were precisely engaged. The diameter of the screw was 6 mm, and the total length of the screw was 45 mm. The fixation models are shown in The inferior endplate of L5 was constrained in all degrees of freedom. A pure moment of 10 Nm combined with a pre-compressive load of 150 N To validate the rationalities of the models, including model simplification, material properties, boundary conditions, and loads, a moment of 10 Nm and a compressive load of 150 N were applied to the reference point. These loading conditions are adopted from the biomechanical experiments and published finite element analyses The range of motion (ROM) of T11-L2, von Mises stress, and stress nephogram of the pedicle screws and rods of the four fixation finite element models under six loading  conditions were analysed. No statistical analysis was performed in the manuscript because only one subject was modeled.Compared with the intact normal spine model, the four fixation models showed a decreased range of motion (ROM) in all states of motion, as shown in The largest maximal von Mises stress of pedicle screws all happened in flexion among all of the fixation models during all states of motion Fig. 3. The largest maximal von Mises stress of rods all happened in flexion among all of the fixation models during all states of motion Fig. 4.  and demonstrate that the stress was concentrated at the screw root under flexion conditions.The results of the pedicle screw stress distribution are shown in There are several posterior surgical techniques currently employed to treat thoracolumbar burst fractures, and a large number of biomechanical studies suggest that reinforcement with a fracture-level screw combination can help to improve the biomechanical stability The finite element model constructed in this study was based on data collected from a spinal CT scan of the patient's thoracolumbar region. The geometric structure of the thoracolumbar vertebrae was generated using medical three-dimensional reconstruction Mimics software. The geometric surface data were preprocessed through smoothing and grid reduction and optimised by Rapidform software to accurately represent the geometry of all models and to minimise the analytic error due to the loss of geometric data. The vertebrae were made up of a solid volume (cancellous bone) and a layer of shell  with a thickness of 0.4 mm. The intervertebral disc was constructed as a continuum structure that occupied the intervertebral space and was partitioned into annulus fibrosus and nucleus pulposus in approximately 6-to-4 ratio In the current study, we attempted to re-create a burst fracture model where the inferior half of T12 and the intervertebral disc between T12 and L1 were completely resected. The construct is highly artificial as depicted, with absence of significant osseous and discal anatomy. We think the spinal fracture model we have built is an extreme case which is similar to A3.1 type that can provide almost little support to the upper vertebrae, and is severe instability, and then we compare the von Mises stresses of the internal fixation devices among different short segment pedicle screw fixation techniques through the extreme spinal fracture model with severe instability. As is known to all, the most common pattern of burst fracture is fracture between the pedicles in the upper half of the body. The spinal fracture model we have built is more severe instability than the most common pattern of burst fracture, we chose the extreme case and carry out our biomechanical study, the conclusions draw from the study on the extreme spinal fracture model with more severe instability can also provide references for clinical treatment of A3.1 type spinal burst fractures.Compared with the intact normal spine model, the four fixation models showed a decreased range of motion (ROM) in all states of motion. The ROM in flexion, axial rotation, and lateral bending was the smallest in the I6+2 fixation model, followed by the I5+2 and S4+2 fixation models, but the ROM was largest in the M4+2 fixation model. In axial rotation, the ROM could be ranked in the following order from small to large: I6+2, I5+2, S4+2, and M4+2. The results were consistent with previous biomechanical studies that suggested that reinforcement with fracture-level screw combinations can help to improve biomechanical stability In the current study, the largest maximal von Mises stress of pedicle screws all happened in flexion among all of the fixation models during all states of motion. The value of largest maximal von Mises stress of pedicle screws during all states of motion was largest in the M4+2 fixation model. As shown in the stress distribution results, a maximum level of pedicle screw stress was apparent at the root of the screws under all loading conditions. In clinical practice, the majority of screw breaks occur at this site. These findings were also consistent with the stress features of pedicle screws described by Kim The value of the largest maximal von Mises stress on rods during all states of motion was the largest in the I5+2 fixation model. The value of the largest maximal von Mises stress of the pedicle screws and rods during nearly all states of motion was smallest in the I6+2 fixation model. The pedicle screws at the fracture site carry some loading stress, thus relieving stress from internal fixation. Therefore, this stress distribution may reduce the possibility of broken or loosened screws. Flexion, extension, right/left lateral bending, and right/left axial rotation tests were conducted to measure the loading forces on the screws. In the current study, we found that additional bilateral pedicle screws at the level of the fracture may result in a stiffer construct and less von Mises stress on pedicle screws compared with traditional short-segment 4 pedicle screw fixation. Index-level pedicle screw fixation to the SPSF decreased the von Mises stress of the pedicle screws; the von Mises stress of the internal fixation was scattered. With pedicle screw fixation at the level of the fracture, the T12 vertebral screw carried a portion of the loading stress. The intermediate screw is thought to function as a push point with an anterior vector, thus creating a lordotic force. The intermediate screw also provides improved “three point fixation”, thus decreasing the cantilever and parallelogram effects and correcting junction kyphosis.There were a few limitations to this study. The most common pattern of burst fracture is fracture between the pedicles in the upper half of the body. In the current study, we attempted to re-create a burst fracture model where only the inferior half of T12 was resected. In fact the fracture model re-created by the authors was not the most common clinical scenarios. We acknowledge that our model for simulating the fracture is done of for ease of computer simulation, and not a direct reflection of the reality of burst fractures. That's because the fracture model in the current study can be easily created through finite element analysis method and ensure the repeatability of the study, the type of the burst fracture in the current study is similar to A 3.1  according to Magerl's classification In theoretically, the spine should be a symmetric construct and the ROM and implant stress should be similar in left/right bending or rotation. However, we kept the reference model  in its natural configuration of the subject to respect the fact that it was not as symmetry as expected. Despite that, in term of the ROM in the symmetric fixed models , the differences between left and right lateral bending and rotation were less than 5.2% . In termAdditional bilateral pedicle screws at the level of the fracture may result in a stiffer construct and less von Mises stress on pedicle screws and rods compared with SPSF. Index-level pedicle screw fixation to the SPSF decreased the von Mises stress of the pedicle screws, and the von Mises stress of the internal fixation was scattered, thus reducing screw fatigue and breakage. The largest maximal von Mises stress of the pedicle screws during all states of motion were observed in the mono-segment pedicle screw fixation technique. The conclusions draw from the study can provide references for clinical treatment of A3.1 type spinal burst fractures, but we can't motivate surgeons to make clinical and surgical decision for a more broad spectrum of burst fractures.


Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterised by neuropathological deposits of amyloid plaques and neurofibrillary tangles comprised of β-amyloid and tau protein, respectively. In AD, tau becomes abnormally phosphorylated and aggregates to form intracellular deposits. However, the mechanisms by which tau exerts neurotoxicity in disease remain unclear. Recent studies have suggested that the presence of tau at synapses may indicate a role in neuronal signalling, which could be disrupted in pathological conditions. The non-receptor-associated tyrosine kinase fyn is located at the dendrite in neurons, where it was recently shown to interact with tau to stabilise receptor complexes at the post-synaptic density. Fyn also co-localises with tau in a proportion of neurons containing tau tangles in AD and fyn is also a tau kinase. Hence, tau-fyn interactions could play a pathogenic role in AD. Here we report the identification of critical proline residues, Pro213, Pro216, and Pro219, located within the fifth and sixth Pro-X-X-Pro motifs in the proline-rich region of tau, that are important for its binding to fyn. These residues in tau are flanked by numerous phosphorylation sites and therefore we investigated the relationship between fyn and the degree of tau phosphorylation in human post-mortem brain tissue. We found no difference in the amount of fyn present in control and AD brain. Notably, however, there was a significant correlation between fyn and phosphorylated tau at specific phospho-epitopes in control, but not in AD brain. Our results suggest that the pathological mechanisms underlying AD, that result in increased tau phosphorylation, may disrupt the physiological relationship between tau phosphorylation and fyn.The online version of this article (doi:10.1186/s40478-016-0317-4) contains supplementary material, which is available to authorized users. The microtubule-associated protein tau has long been recognised as having a role in stabilising microtubules in neurons, particularly in axons. Recent evidence however, suggests that tau is also present in dendrites where it may play a role in synaptic function –3. In suWe focused here on identifying the binding site(s) for the tau-fyn interaction. It is well known that SH3 domains of src family kinases, of which fyn is a member, bind to PXXP motifs . Tau hasIn this study, we extend the findings from Lee et al.  and UsarA plasmid expressing the longest isoform of human CNS tau containing two N-terminal inserts and four microtubule-binding repeats (2N4R) has been described previously . The gen2 atmosphere at 37 °C. Cells were plated at a density of 200,000 per well of a 6-well plate and plasmids were transfected 24 h after plating using jetPEI® (Polyplus Transfection), according to the manufacturer’s instructions. Cells were harvested 24 h after transfection.Chinese hamster ovary (CHO) cells were maintained in Ham’s F-12  supplemented with 10 % (v/v) foetal bovine serum (Biosera), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin, and incubated in a 5 % COE.coli containing GST- and GST-fyn-SH3-encoding plasmids were streaked across agar plates and incubated overnight at 37 °C. A single bacterial colony was used to inoculate 5 ml LB-amp broth and incubated overnight at 37 °C with shaking, before further inoculating 250 ml LB-amp broth and incubating for two hours at 37 °C with shaking until an optical density of 0.6–1.0 nm was reached. 1 mM isopropyl-1-thio-β-D-galactopyranoside (IPTG) was added to the cultures and incubated for a further 2 h with shaking. The culture was then centrifuged at 20,000 g(av) for 15 min at 4 °C to pellet the bacteria. The pellet was resuspended in 12 ml TNE buffer , and complete protease inhibitor cocktail without EDTA [Roche]) and homogenised by sonication  for six 15 s bursts on ice. Triton X-100 was added to a final concentration of 1 % (v/v), mixed, and the samples were centrifuged at 30,000 g(av) for 10 min at 4 °C. The supernatant, containing GST-fusion proteins, was collected and stored on ice. A 50 % (v/v) slurry of glutathione-4B beads  was washed three times in TNE buffer and added to supernatant containing GST-fusion proteins and rotated for 90 min at 4 °C, then centrifuged at 3300 g(av) for 1 min at 4 °C to pellet the beads. Beads coupled to GST-fusion proteins were washed three times with TNE buffer and resuspended as a 50 % (v/v) slurry.Frozen glycerol preparations of N,N,N’,N’-tetraacetic acid, 150 mM NaCl, complete protease inhibitor cocktail without EDTA [Roche], and phosphatase inhibitor cocktail 2 [Sigma]), and incubated on ice for 15 min. Lysates were centrifuged at 6000 g(av) for 5 min to pellet cell debris. GST-beads (50 % slurry) were washed twice in wash buffer  Triton X-100, 150 mM NaCl, complete protease inhibitor cocktail without EDTA [Roche], and phosphatase inhibitor cocktail 2 [Sigma]) and incubated with the cell lysates. The pre-cleared cell lysates were incubated overnight at 4 °C with washed GST-beads or GST-fyn-SH3-beads. Beads were pelleted at 6000 g(av) for 5 min and the supernatants were discarded. Beads were washed twice with wash buffer, then Laemmli sample buffer was added and the samples heated at 100 °C for 5 min to elute bound protein. Proteins were separated on 10 % (w/v) sodium dodecyl sulphate (SDS)-polyacrylamide gels, transferred to nitrocellulose membranes and western blots were probed with antibodies to total tau, as described below. The amount of tau bound to GST-fyn-SH3 beads was quantified using an Odyssey scanner (Li-Cor Biosciences) and standardised against the total amount of tau in the corresponding cell lysate.CHO cells transfected with WT or mutant tau were washed with phosphate-buffered saline, harvested into ice-cold lysis buffer  glycerol, 0.5 % (v/v) Triton X-100, 1 mM EDTA, 1 mM ethylene glycol-bis(2-aminoethylether)-(av) at 4 °C for 20 min. The supernatants were collected and stored at −80 °C.Samples of frozen temporal cortex from post-mortem human brain (15 AD and 11 controls) were obtained from the London Neurodegenerative Diseases Brain Bank at the Institute of Psychiatry, Psychology and Neuroscience, King’s College London (details in Table Proteins were separated on 10 % (w/v) SDS-polyacrylamide gels and transferred to nitrocellulose membranes. After blocking, membranes were incubated overnight at 4 °C with primary antibodies directed against total tau , GST , phosphorylated tau , dephosphorylated tau , fyn , β-actin , and neuron-specific enolase . Blotted membranes were incubated with secondary antibodies  and antigens were visualised and quantified using an Odyssey scanner (Li-Cor Biosciences).For analysis of GST proteins bound to glutathione beads, SDS-polyacrylamide gels were incubated in ProtoBlue Safe Working Solution  and protein bands were imaged using an Odyssey scanner (Li-Cor Biosciences).p < 0.05) using Student’s t-test or ANOVA. Data that were not normally distributed were analysed using Mann-Whitney or Kruskal-Wallis tests. Pearson’s or Spearman’s rank correlation was used to determine correlations between proteins, as appropriate. GraphPad Prism five was used for all statistical analyses.Data were tested for normal distribution using the D’Agostino-Pearson normality test. Normally distributed samples were analysed for statistical significance  in which individual proline residues were substituted by alanine Fig. . Analysip < 0.01). Interestingly, the amount of P213A tau bound to fyn-SH3 was significantly increased by 50 % compared to WT tau . There were no significant differences between the amounts of any of the other PXXP tau mutants tested , compared to WT tau bound to fyn-SH3 , in agreement with previous reports  or XXX[PXXP]XR respectively, depending on whether the flanking by positively charged residues are located N or C terminal to the PXXP motif . Class IThe sixth tau PXXP motif P216–P219) appears to be the preferred binding domain for fyn-SH3, although disruption to this region of tau could enable fyn to bind to alternative PXXP motifs in tau. The fact that none of the individual proline to alanine substitutions in tau examined here completely abolished fyn-SH3 binding, suggests that fyn-SH3 has the capacity to bind to different PXXP motifs under different physiological and pathological conditions. Further support for this hypothesis comes from a study by Ittner et al. [6–P219 apInterestingly, we found that the P213A tau mutation resulted in an increased amount of tau bound to fyn-SH3. One possibility is that the alanine substitution at this residue alters the conformation of tau to make it more favourable for binding to fyn-SH3, providing that the sixth PXXP motif is also present. It has been shown that specific residues outside the core PXXP ligand can contribute to the binding between PXXP motifs and SH3 domains , which cIn a related study, we showed previously that the phosphorylation status of residues S199/S202 in tau influences its binding to fyn-SH3 , but theHowever, the relationship of fyn with total tau or tau phosphorylation has not been previously investigated in AD. In agreement with previous reports, and despite the wide variation between individuals with AD, we detected increased tau in AD brain , 20.in vitro, dependent on the presence of the microtubule-binding repeats of tau [We found no significant correlation between the amount of fyn present and the degree of tau phosphorylation in AD brain tissue, suggesting that modification of tau phosphorylation, at least at the serine/threonine residues examined in this study, does not result or lead to increased fyn in AD. Interestingly, in control brain tissue, although the amount of fyn did not correlate with tau phosphorylation at residues S199 or S214, there was a significant positive correlation between fyn and tau phosphorylated at S202, S262, and S396/404. The absence of this correlation in AD brain suggests that, at least during moderate to late stages of AD (Braak IV–VI), underlying pathogenic mechanisms may disrupt the association between fyn and tau phosphorylated at S202, S262, and S396/404. Notably, inhibition of casein kinase 1 delta, which phosphorylates tau , in prims of tau . Phosphos of tau , 43. OurIn this study, identification of the binding site between tau and fyn-SH3 may facilitate the development of compounds that can inhibit tau-fyn interactions, which presents an alternative therapeutic strategy for AD. We also provide evidence that a physiological correlation between phosphorylated tau at S202, S262, and S396/404 and fyn is not present in AD brain, suggesting that progression of disease pathogenesis can influence the relationship between tau and fyn. Further investigation as to the relationship between fyn kinase activity and tau phosphorylation will be important in developing novel therapeutics for AD.


The influence of ultraviolet B (UVB) radiation on transglutaminase 1 (TGase 1), a major factor that regulates skin keratinization, has not been sufficiently characterized especially at the gene or protein level. Thus, we determined whether UVB affects the expression of TGase 1 in human keratinocytes and clarified the intracellular stress signaling mechanism(s) involved. Exposure of human keratinocytes to UVB significantly up-regulated the expression of TGase 1 at the gene and protein levels. Treatment with inhibitors of p38, MEK, JNK or NFκB significantly abolished the UVB-stimulated protein expression of TGase 1. Treatment with astaxanthin immediately after UVB irradiation did not attenuate the increased phosphorylation of Ser536/Ser468NFκBp65, c-Jun, ATK-2 and CK2, and did not abrogate the increased or diminished protein levels of c-Jun/c-Fos or I-κBα, respectively. However, the same treatment with astaxanthin significantly abolished the UVB-stimulated expression of TGase 1 protein, which was accompanied by the attenuated phosphorylation of Thr565/Ser376/Ser360MSK1, Ser276NFκBp65 and Ser133CREB. The MSK1 inhibitor H89 significantly down-regulated the increased protein expression of TGase 1 in UVB-exposed human keratinocytes, which was accompanied by an abrogating effect on the increased phosphorylation of Ser276NFκBp65 and Ser133CREB but not Thr565/Ser376/Ser360MSK1. Transfection of human keratinocytes with MSK1 siRNA suppressed the UVB-stimulated protein expression of TGase 1. These findings suggest that the UVB-stimulated expression of TGase 1 is mediated predominantly via the NFκB pathway and can be attenuated through a specific interruption of the p38/MSK1/NFκBp65Ser276 axis. Exposure of the skin to ultraviolet B (UVB) radiation causes inflammation and subsequent hyperkeratosis of the epidermis . Hyperke2+-dependent enzymes which catalyze ε-(γ-glutamyl)lysine cross-linking reactions. Four TGases  are constitutively expressed in epithelial tissues such as the epidermis [TGases are Caes 1, 2,  and X arpidermis , and TGapidermis , 6, 7. Tpidermis , 9 and spidermis , whose rpidermis , 12. Mutpidermis , 14. In pidermis . TGase 1pidermis , which apidermis . A recenpidermis . On the As for studies examining the effect of UVB on TGase 1 in the epidermis, Takahashi et al.  reportedSerum-free keratinocyte growth medium (Medium 154S) containing low calcium (0.2 mM), bovine pituitary extract (BPE) and epidermal growth factor (EGF) were obtained from Kurabo . Human primary keratinocytes (HPKs) were purchased from Biopredic Intl . Human HaCaT keratinocytes , 23 were2. Human HaCaT keratinocytes were maintained in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS), 100 μg/ml penicillin, 100 μg/ml streptomycin and 250 ng/ml amphotericin B at 37°C in a 95% air, 5% CO2 atmosphere. In the signaling experiments, HPKs and human HaCaT keratinocytes were cultured in Medium 154S without EGF and BPE and in DMEM without FCS, respectively, after UVB irradiation.HPKs were maintained in Medium 154S supplemented with 5 ng/ml EGF and 50 μg/ml BPE at 37°C with 5% CO2 [UVB irradiation was carried out using a SE fluorescent lamp  that provided an energy spectrum with a peak at 305 nm in the UVB region (280–320 nm) as previously reported . The cal2 , 24 as m2 .3VO4) and the lysates were then centrifuged at 13,000 rpm for 15 min. The sediments were solubilized in SDS sample buffer plus 50 mM dithiothreitol (DTT) and were boiled for 5 min. The boiled samples were separated on SDS-PAGE gels and then were transferred onto polyvinylidene difluoride (PVDF) membranes . After blocking with 5% skim milk in Tris/HCl buffer  containing 0.1% Tween for 1 h, the membranes were treated with monoclonal or polyclonal primary antibodies for 1 h to overnight at room temperature. This was followed by incubation with anti-mouse or anti-rabbit IgG conjugated to horseradish peroxidase for 1 h, and finally, membranes were developed with ECL or ECL plus reagents . Immunoblots were then exposed to X-ray film for the specified times to detect bands, and specific bands were quantitated with Quantity One (Bio Rad).Immunoblot analysis was performed as previously reported , 27. Bri5’-GAAGGTGAAGGTCGGAGTCAACG-3’, Reverse 5’-AGTCCTTCCACGATAACCAAAGTTG-3’, TGase 1: Forward 5’-GCTCGAAGGCTCTGGGTTACA-3’, Reverse 5’-TCGCACAGGCACAAACGAC-3’.After UVB exposure, the expression of TGase 1 in HPKs was examined using a real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay and was normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as previously detailed , 29. PrisiRNA transfection was performed with a predesigned siRNA against MSK1  as described previously . A negatProtein concentrations were determined using a BCA assay kit  and bovine serum albumin (Amersham Biosciences) as the standard.All data are expressed as means ± SD (n = 3). For multiple comparisons, data were tested by one-way ANOVA, and subsequently using the Tukey or Dunnet multiple comparison test. P values less than 0.05 are considered statistically significant.2. UVB induced a significant increase of TGase 1 at 24 and 48 h post-irradiation at the gene and protein levels, respectively  to fresh signaling medium without UVB radiation also increased the phosphorylation of ERK and JNK with a peak at 15 min post-medium exchange, to a similar or a slightly lesser extent compared to the increased level of ERK or JNK phosphorylation, respectively, at 15 min post-irradiation in UVB-exposed cells . This in2, the pre- (data not shown) but not post-irradiation treatment with AX distinctly abolished the increased phosphorylation of p38, JNK and ERK at 15 min post-irradiation in UVB-exposed human HaCaT keratinocytes  and the protein levels of I-κBα were distinctly increased and markedly decreased at 30 min and 6 h post-irradiation, respectively, (2), the slight shift of CK2 protein toward a higher molecular mass and the decreased level of I-κBα protein was not abrogated at 0.5–3.0 h and 6–48 h post-irradiation, respectively , the significant increase of the Ser276 but not the Ser468/Ser536 phosphorylation of NFκBp65 was significantly abolished at 0.5–6.0 h post-irradiation (When human HaCaT keratinocytes were exposed to UVB at a dose of 80 mJ/cmadiation . When AXadiation .2) of HPKs significantly stimulated the Thr581 phosphorylation of MSK1 at 0.5 h post-irradiation, which was significantly abrogated by treatment with AX at 4 or 8 μM following UVB irradiation , the significant increase of Ser133 phosphorylation of CREB was significantly abolished at 0.5 h post-irradiation, the abrogation of which was sustained until 3 h post-irradiation  at concentrations of 0.5 and 0.05–0.5 μM, respectively, at 0.5 h post-irradiation in UVB-exposed HPKs . TreatmeTransfection of MSK1 siRNA in HPKs significantly reduced the UVB-stimulated expression of TGase 1 protein without any changes in unexposed HPKs .The present study demonstrates for the first time that UVB exposure of human keratinocytes significantly up-regulates the expression of TGase 1 at both the transcriptional and translational levels. This suggests that the stimulated expression of TGase 1 is highly involved in the accentuated keratinization and desquamation which occurs in UVB-exposed human skin. Our study also shows for the first time the detailed evidence on multiple UVB-activated signaling cascades that are the front lines of stress-activated signaling, such as p38, ERK and JNK, and are distinctly activated (phosphorylated) with a peak at 15 min post-irradiation in UVB-exposed human keratinocytes. Further, the results show that those downstream signaling pathways, including NFκB, IKK, CK-2/I-κBα, AP-1(c-Fos/c-Jun), ATF-2 and MSK1, are also subsequently activated. Therefore, we used several inhibitors of those stress-activated signaling factors to elucidate the intracellular signaling mechanism(s) involved in the UVB-induced accentuation of TGase 1 expression. We found that the UVB-induced increase of TGase 1 is significantly abrogated by inhibitors of the front lines of stress-activated signaling, p38, ERK (to a lesser extent) and JNK, when added prior to the UVB irradiation of human keratinocytes. This suggests that those stress-activated signaling factors are involved in the UVB-induced accentuation of TGase 1 expression.UVB exposure of keratinocytes is well known to activate NFκB signaling by stimulating IKKkinase, which phosphorylates IκκBα, enabling NFκBp65 to release toward translocation into nuclei during the signaling pathway downstream of p38 or ERK activation  and/or vMany studies have reported that AX , 34 as wIn this approach, consistent with our previous study in HPKs , AX inteα, which is involved in NFκB activation during the UV response and CK2 activity is UV-inducible through a mechanism that depends on the activation of p38 MAPK [CK2 is a c-terminal I-κB kinase responsible for degradation of I-κBp38 MAPK . In thisp38 MAPK  is not ap38 MAPK  that shop38 MAPK , was notDuring a search for the interrupted signaling lineages by the post-irradiation treatment with AX, which is consistent with our previous study in HPKs , we finaIn epidermal keratinocytes, MSK1 is phosphorylated at either Ser360 or Thr581 by p38 MAPK or by ERK p44/42 MAPKs , 40, 41 Available evidence indicates that TGase 1 expression in keratinocytes is regulated primarily by activator protein 1 (AP-1), which consists of heterodimers between members of the Jun and Fos families of transcription factors, such as c-Jun and c-Fos , 56, 57,2, cholera toxin and dibutyryl cAMP, decrease the production of NFκB pathway-triggered proteins, such as IL-8 and COX2, in irradiated cells by down-regulating NFκB activation in response to UVB radiation [Our observations on the effects of AX on the UVB-induced activation of stress-activated signaling pathways strongly suggest that the abrogation of MSK1 activation is the only and initial inhibitory site affected by the post-irradiation treatment with AX, which leads to the attenuation of the up-regulated protein expression of TGase 1 by UVB irradiation. Therefore, to determine whether MSK1 inhibition or inactivation is substantially involved with the attenuated protein expression of TGase 1, as observed for AX, we next examined the effect of the MSK1 inhibitor H89  on the aadiation . This isA similar mechanism of action on MSK1 was seen in human keratinocytes and in dendritic cells by dimethyfumarate (DMF) as an anti-psoriatic agent  and a thIn conclusion, to our knowledge, this is the first detailed in vitro study of the signaling mechanisms involved in TGase 1 expression during epidermal differentiation subsequent to UVB exposure. Since MSK1 activation is essentially involved in the NFκB signaling pathway, which serves as a nuclear kinase for Ser276 NFκBp65 after its translocation into the nucleus , the sum


Twsg1), which is associated with a low penetrance of HPE, are sensitized to retinoic acid (RA) teratogenesis. Pregnant +/−Twsg1 dams were treated by gavage with a low dose of all-trans RA (3.75 mg/kg of body weight). Embryos were analyzed between embryonic day (E)9.5 and E11.5 by microscopy and geometric morphometric analysis by micro-computed tomography. P19 embryonal carcinoma cells were used to examine potential mechanisms mediating the combined effects of increased BMP and retinoid signaling. Although only 7% of wild-type embryos exposed to RA showed overt HPE or neural tube defects (NTDs), 100% of −/−Twsg1 mutants exposed to RA manifested severe HPE compared to 17% without RA. Remarkably, up to 30% of +/−Twsg1 mutants also showed HPE (23%) or NTDs (7%). The majority of shape variation among +/−Twsg1 mutants was associated with narrowing of the midface. In P19 cells, RA induced the expression of Bmp2, acted in concert with BMP2 to increase p53 expression, caspase activation and oxidative stress. This study provides direct evidence for modifying effects of the environment in a genetic mouse model carrying a predisposing mutation for HPE in the Twsg1 gene. Further study of the mechanisms underlying these gene-environment interactions in vivo will contribute to better understanding of the pathogenesis of birth defects and present an opportunity to explore potential preventive interventions.Holoprosencephaly (HPE) is a developmental anomaly characterized by inadequate or absent midline division of the embryonic forebrain and midline facial defects. It is believed that interactions between genes and the environment play a role in the widely variable penetrance and expressivity of HPE, although direct investigation of such effects has been limited. The goal of this study was to examine whether mice carrying a mutation in a gene encoding the bone morphogenetic protein (BMP) antagonist twisted gastrulation ( Holoprosencephaly (HPE) is a malformation characterized by inadequate or absent midline division of the embryonic forebrain. Incomplete brain septation is accompanied by corresponding midline facial defects in ~80% of cases  and, lesSHH  is the most common defect of the developing forebrain and has an incidence of 1 in 250 conceptuses and about 1 in every 10,000 at term. It is characterized by inadequate or absent midline division of the embryonic forebrain and midline facial defects. A perplexing feature of HPE, as well as of other craniofacial syndromes, in humans is their widely variable penetrance and expressivity even in the case of the same single gene mutation within the same family, with some individuals having severe defects, some mild defects and some being unaffected. It is currently unknown what causes manifestation of HPE in genetically at risk individuals, but it has been speculated that environmental factors might play a role. This work investigates the effects of environmental exposure to teratogens in a mouse model predisposed to HPE.ResultsTwsg1) mutant mice serve as a model of human HPE because they show incomplete penetrance and a range of defects among homozygotes. In this study, the authors demonstrated that Twsg1 mutants show increased susceptibility to the teratogenic effects of relatively low doses of retinoic acid (RA) that in control mice cause few, if any defects. The exposure to RA was performed at embryonic day 7.5, which is the most sensitive window for teratogen-induced HPE (corresponding to the 3rd to 4th week post-fertilization in humans). Remarkably, even Twsg1 haploinsufficiency exacerbated teratogenic effects of prenatal RA exposure. The majority of midfacial shape variation among +/Twsg1− mutants was associated with narrowing of the midface, as demonstrated by micro-computer tomography (microCT) analysis. Given that the only known action of TWSG1 is through binding bone morphogenetic proteins (BMPs) in the extracellular space, the authors hypothesized that BMP-RA interactions could contribute to the enhanced expressivity of HPE in this mouse model. To test this, P19 embryonic carcinoma cells were used as an in vitro model to elucidate the mechanisms mediating these gene-environment interactions. In P19 cells, RA induced the expression of Bmp2 and its downstream targets Msx1 and Msx2, and acted in concert with BMP2 to increase apoptosis, p53 target gene expression and oxidative stress, suggesting a role for these pathways in inducing cell death and exacerbating the disease outcome.Twisted gastrulation  to examine whether a mutation in a gene encoding the BMP-binding protein TWSG1 confers susceptibility to RA exposure, and (2) whether this effect can be quantified by micro-computed tomography (microCT) of the craniofacial region. We chose the poptosis . A seconTwsg1+/− and Twsg1−/− mice.Our first step in examining the sensitivity of TWSG1-deficient mice to RA was to establish an appropriate treatment dosage that would cause a low but observable incidence of defects in wild-type (WT) mice, which could then be used to assess the sensitivity of TWSG1-deficient mice. Given that a dose of all-trans RA (ATRA) of 7.5 mg/kg of body weight has been previously shown to cause significant HPE , we alsoTwsg1−/− mutants manifested HPE compared to 17% without exposure to ATRA (P=6×10−12). This rate of defects is far more than what would be expected based solely on adding the prevalence of defects from this dose in WT mice and untreated −/−Twsg1 mice. Remarkably, even 30% of heterozygous Twsg1 mutants, which are phenotypically normal without ATRA exposure, showed neural defects (predominantly HPE). This represents a statistically significant increase over the incidence seen in WT embryos with the same dosage (P=0.01) and likewise over the 0% incidence seen in untreated heterozygotes (P=1×10−6). Thus, TWSG1 deficiency increased the teratogenic effect of low dose ATRA in both the homozygous and heterozygous states.Although only 7% of WT embryos exposed to low dose ATRA showed overt HPE or NTDs, 100% of −/−Twsg1 embryos exhibited severe HPE phenotypes (cyclopia or proboscis) after in utero exposure to a low dose ATRA, +/−Twsg1 showed a range of defects of variable severity. To quantify these defects, geometric morphometric (GM) analysis was employed. The analysis included only +/−Twsg1 embryos because severe HPE phenotypes in homozygotes precluded landmark assignment. Principal component 1 (PC1), which reflects narrowing of the midface, was the only PC that discriminated between treatment groups and accounted for 49% of the total variance. As shown in +/−Twsg1 embryos clustered with WT embryos, those that were affected by low dose ATRA treatment could be clearly discriminated along PC1. Thus, ATRA treatment resulted in a continuum of midfacial narrowing in mice heterozygous for Twsg1 mutation.Although Twsg1 mutant mice to ATRA, we selected P19 mouse embryonic carcinoma cells as an experimental system because they resemble embryonic cells, represent a homogenous cell population that is amenable to quantitative assays and have been used by others as a model for BMP-retinoid signaling interactions (Rara and Rarb)  , the RA Twsg1 mutin vivo. Although there is some evidence that TWSG1 can promote BMP activity in some species, in mice it appears to act mostly as a BMP antagonist .Given that the only known mode of action for TWSG1 is through regulation of BMP signaling, it was essential that the P19 cells be competent to respond to BMPs to mimic what occurs tagonist . We examtagonist . We fountagonist , that Bmand Msx2  showed aTrp53inp1  of craniofacial and neural defects  was associated with a high risk of congenital malformations (relative risk 25.6) . However, vitamin A, when used in recommended doses, is needed for normal fetal growth  has also been proven to cause birth defects in humans, including central nervous system abnormalities such as HPE . The asssk 25.6) . All womsk 25.6) . Vitaminl growth  and is sIn summary, TWSG1-deficient mice represent a genetic mouse model of a mutation with low penetrance that sensitizes embryos to environmental influences. The mechanisms underlying these gene-environment interactions are poorly understood. Given that similar biological processes appear to be involved in the pathogenesis of a variety of birth defects, and in response to various teratogens, better understanding of these interactions will likely be applicable to other birth defects beyond craniofacial malformation.Twsg1 (tm1.1 MbocTwsg1) were as described previously . A detailed description of this technique and computation methods have been previously published . A set o−2 M. All cells in BMP and ATRA treatment experiments were adjusted with non-solute-containing vehicles to final concentrations of 0.0001% (w/v) BSA and 0.01% (v/v) DMSO. Antimycin-A  was prepared in propylene glycol at 6 mg/ml.P19 mouse embryonic carcinoma cells (ATCC CRL-1825)  were culBmp2, Crbp1, Cyp26a1, Gapdh, Hoxa1, Hoxb1, Msx1, Msx2, Rara, Rarb and Trp53inp1 are shown in supplementary material Table S1.P19 cells were plated into six-well plates with 20,000 cells per well and allowed to grow overnight. Medium was removed and replaced with treatment media containing test compounds. For RA induction experiments, cells were treated either with 1 μM ATRA or with DMSO vehicle in the same v/v dilution. For BMP and RA experiments, cells were treated with vehicle control, 10 ng/ml BMP2 with DMSO ATRA vehicle control, 1 μM ATRA with BSA BMP2 vehicle control, or BMP2 combined with ATRA. In both sets of experiments, media were removed after 24 hours and 1 ml of Trizol  was added for RNA isolation according to manufacturer’s instructions. cDNA was prepared from RNA samples by reverse transcription using the Thermoscript reverse transcription sysetem . Quantitative PCR (qPCR) was performed using 2× SYBR green mastermix with ROX from SABiosciences on an MX3000p thermocycler  and analyzed using the MxPro software (Stratagene). Primers used for the following genes 3 LB 960 Microplate Luminometer .Caspase 3 and 7 activity was measured using reagents from the Apotox-glo triplex assay kit  according to the manufacturer’s protocol. Briefly, cells were plated in a white-walled clear-bottomed 96-well plate with 2500 cells per well, then test compounds were added, diluted in growth medium. Cells were treated with vehicle control, 10 ng/ml BMP2 with DMSO ATRA vehicle control, 1 μM ATRA with BSA BMP2 vehicle control, or BMP2 combined with ATRA. After 24 hours, caspase activation was measured by addition of luminogenic caspase substrate and measured in a Centro XSP19 cells were assayed for ratio of reduced to oxidized glutathione using the GSH/GSSG-glo kit (Promega) according to the manufacturer’s instructions. Briefly, cells were plated in a white-walled clear-bottomed 96-well plate with 2500 cells per well, then test compounds were added, diluted in growth medium. Cells were treated with BMP and RA vehicle control, 10 ng/ml BMP2 with DMSO ATRA vehicle control, 1 μM ATRA with BSA BMP2 vehicle control, BMP2 combined with ATRA, propylene glycol AMA vehicle control or 70μM AMA. After 16 hours cells were washed once with HBSS then lysed and analyzed for GSH and GSSG content using kit-provided reagents for luminogenic reactions.t-tests and ANOVA combined with Tukey’s multiple comparison tests were performed using Prism4 . Significance was accepted at P<0.05. Fisher’s exact test was used to compare frequencies of embryonic phenotypes between different genotypes and with and without RA treatments.Chi-squared tests, Student’s


Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs), such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defense as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defense.Mitogen-activated protein kinase (MAPK) cascades are central signaling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened  Arabidopsis thaliana. FLS2 contains an extracellular leucine-rich repeat (LRR) domain for ligand binding, a transmembrane domain and a cytoplasmic serine/threonine kinase domain  production, activation of mitogen-activated protein kinases (MAPKs), transcriptional reprogramming like PR-1 and WRKY gene expression and production of antimicrobial compounds like phytoalexins  and ETI , phosphorylating a MAPK kinase (MAPKK), which phosphorylates a MAPK . Then, tpression . The secpression . A recenpression . A fourtpression  or otherpression .in vitro kinase assays. Microarray-based testing of 1690 Arabidopsis proteins identified 48 in vitro substrates for MPK3 and 39 for MPK6, with an overlapping set of 26 candidates . This domain is responsible for binding to the W-box element with the consensus “(C/T)TGAC(T/C)” nucleotide sequence, although flanking DNA sequences appear to contribute to binding specificities  and the arrangement of conserved cysteine/histidine residues of their zinc fingers , a key enzyme for the synthesis of antimicrobial camalexin  and 16 h dark (20°C). Arabidopsis thaliana ecotype Col-0 was used as wild type. The mpk3 and mpk6 mutants (in Col-0 background) were described previously  with a flanking BsaI site were used to amplify the entire pDONR220 vector harboring WRKY46. After DpnI digestion of the template plasmid, the amplified vector was gel-purified, digested with BsaI and ligated. The mutated WRKY46 variants were then transferred to destination vectors by standard GatewayTM LR- recombination-based cloning (Invitrogen). For the phosphomimetic S168D,S250DWRKY46 variant, two fragments were amplified by PCR using the primer pairs, W46-S168D_3/W46-S250D_5 and W46-S168D_5/W46-S250D_3, and pUGW15-WRKY46 as a template. Both fragments were gel-purified and mixed and incubated in the presence of LguI and T4-DNA ligase in a PCR cycler machine (10 cycles of 10 min 37°C and 10 min 22°C).The escribed . Briefly10-tagged recombinant proteins. The vectors were transformed into KRX competent cells (Promega). Cells were grown in 2xYT media  to OD600 of 0.5–0.6 and protein expression was induced at 24°C overnight by adding 1 mM rhamnose. Denaturing purification was performed by resuspending the pellet from 100 mL culture in 20 mL of lysis buffer  and incubated at room temperature for 30 min. After centrifugation the supernatant was incubated for 1 h with 100 μL of equilibrated Ni-NTA-Agarose beads (Thermo Scientific). The beads were washed thrice with wash buffer . The proteins were refolded on the beads as previously described  after drying the gel.escribed  with sliThe MPK:WRKY proteins were incubated at approximately 1:10 (w/w) ratio for 30 min as described above (except for the lack of radioactive ATP). After separation by SDS-PAGE, the gel was fixed with 50% methanol and 10% acetic acid overnight. The gel was washed with water for 30 min and stained with 3x diluted Pro-Q diamond stain (Invitrogen) in the dark for 2 h. The gel was destained four times for 30 min each with 20 % acetonitrile, 50 mM sodium-acetate (pH 4.2). The gel was washed again with water for 10 min and was scanned at 400 V using a Typhoon Scanner .4HCO3 and finally rinsed twice with water. After standard reduction and alkylation (4HCO3 with 3 ng μL-1 modified trypsin (Promega). Peptides were extracted into 35% acetonitrile with 0.4% TFA, dried down in a Speed-vac and finally resuspended in 20 μL of 0.1% TFA. After 5 min of sonication in a sonifier water bath, the peptides were either frozen for storage or immediately analyzed on an LC-MS system consisting of a split-free nano-LC  coupled to a hybrid-FT-mass spectrometer . Additional targeted LC-MS measurements of selected phosphopeptides were performed using an inclusion list in a data dependent acquisition (DDA) scan method. Peptide fragmentation was achieved using CID with MSA (multi-stage activation). Proteins were identified and phosphorylation sites were identified using Mascot V.2.3.02 and the Phospho RS module in Proteome Discoverer v1.3.Phosphorylated proteins were separated by 10% SDS-PAGE. The gel was stained with Colloidal Coomassie Blue Stain (Life Technologies) and washed with water. The corresponding gel bands were excised, destained by multiple washes with 30% acetonitrile, 100 mM NHkylation , the proIn silico functional prediction was performed with WRKY-family proteins. First, they were classified according to functional annotation clustering using DAVID 6.7 software1 . Transformation of Arabidopsis mesophyll protoplasts was performed as described  and pUBQ10-GUS . After overnight incubation in the dark at 22°C, 200 μM luciferin were added and mixed gently by inverting the tubes. Then, 100 μL of protoplasts were transferred into 96-well microtiter plates suitable for luminescence measurements (Greiner) and were elicited with 100 nM flg22/elf18, 200 μg/mL chitin or water as control. The luciferase activity kinetics were measured for 3 h with a 96-well plate luminescence reader . After the measurement, protoplast samples were directly lysed by adding 10 μL of 10-fold concentrated GUS extraction buffer  and vortexing. GUS-activity was measured upon incubation with 4-methylumbelliferyl glucuronide , based on the fluorometric detection of the reaction product 4-methyl umbelliferone (4-MU). Values are expressed as LUC/GUS ratios relative to the CFP-transfected controls (at timepoint 0 min).The promoter-LUC assay was performed as described earlier . BrieflyWRKY46 was cloned into pEXSG-YFP vector  as described P vector  to expreP vector  was used4.All statistical analyses were performed using GraphPad Prism 5DD; In vitro kinase assays were performed using the 48 purified WRKY proteins as substrates for MPK3 or MPK6 . Most of the WRKYs (or their truncated products) were phosphorylated by both kinases. The WRKYs showed differential phosphorylation intensities, possibly reflecting either the strength of their interaction with MPK3/MPK6 or the number of putative MAPK-targeted (SP/TP) sites present in them.We expressed 48 members out of the 74 WRKYs, covering all three WRKY groups, as recombinant proteins. MPK3 and MPK6 were also prepared and activated by incubating with a constitutively active MAPKK .To narrow down the candidate WRKYs possibly involved in plant defense responses, Arabidopsis WRKY46 is a group III WRKY of 295 amino acids (molecular weight of 33.6 kDa) and contains two consensus MAPK phosphorylation sites at positions S168 and S250. We performed site-directed mutagenesis to change these two sites from Ser to Ala. To determine which phosphorylation site of WRKY46 can be phosphorylated by MAPKs in vitro, His-tagged recombinant WRKY46 protein variants were purified and used for MAPK phosphorylation assays. Using MPK3, MPK4 and MPK6 with comparable kinase activities , MPK3 showed the strongest phosphorylation of WRKY46 . The observation was also confirmed by using a non-radioactive kinase assay with MPK3 and subsequent Pro-Q diamond phosphoprotein staining . The MPK3-phosphorylated WRKY46 bands were excised from the non-radioactive gel, trypsin-digested and subjected to mass spectrometry (MS) analysis. A phospho-peptide containing phosphorylated S168 was detected . Phosphorylation at S250, however, cannot be excluded since the peptide coverage of the MS analysis did not include this region of the protein. Nevertheless, MPK3-mediated phosphorylation of the WRKY46S168A variant was strongly reduced compared to WRKY46S250A , so that S168 may be the preferred in vitro phospho-site. The recombinant WRKY46S168A,S250A double mutant was, unfortunately, poorly expressed in bacteria and it was difficult to purify sufficient amounts for comparison in the in vitro kinase assay. Curiously, the phosphorylated WRKY46 band(s) often show a double band, except for the WRKY46S250A variant; this may mean that phosphorylation at S250 results in a mobility shift in SDS-PAGE .in vivo, WRKY46 was cloned into a vector expressing HA-tagged proteins under control of the Cauliflower Mosaic Virus 35S promoter and transfected into Arabidopsis protoplasts. After an overnight incubation (∼15 h) to enable protein expression, protoplasts were treated with flg22 (100 nM) or an equivalent volume of water (as control) for 15 min. Protoplasts were harvested, proteins extracted, and immunoblotted against an anti-HA antibody. An aliquot of proteins from the flg22-treated sample was incubated with lambda phosphatase to dephosphorylate the proteins. As reflected by a mobility shift in SDS-PAGE, which is abolished by the phosphatase treatment, WRKY46 is already phosphorylated before any PAMP elicitation . Since the MAPKs are typically not activated prior to PAMP treatment, it appears that there are other kinase(s) that target WRKY46. As there is no additional phospho-shift after PAMP treatment, it is also not possible to use this assay as an indicator for in vivo phosphorylation by PAMP-activated MAPKs, such as MPK3 or MPK6 (see below).To determine whether WRKY46 is phosphorylated Figure 3B). By contrast, flg22-induced reduction in protein levels was less pronounced for the WRKY46S168A, WRKY46S250A and WRKY46S168A,S250A mutant variants over the tested period of 3 h. In general, the highest level of protein expression was observed for the WRKY46S168A,S250A double phosphorylation site mutant . Taken together, phosphorylation of WRKY46 (at S168 and/or S250) is required for the flg22-induced degradation in vivo and the non-phosphorylatable WRKY46 is more stable.The half-life of proteins is critical for the regulation of various signaling pathways and stability of several MAPK substrates has been shown to be regulated through phosphorylation after PAMP treatment . Therefompk3 or mpk6 mutant backgrounds to specifically activate only MPK6 or MPK3, respectively. As seen in Figure 3C, specific activation of MPK3 and/or MPK6 led to reduced WRKY46 protein levels – thus pointing to a direct effect of MPK3/MPK6-mediated phosphorylation on WRKY46 stability. As mpk3mpk6 double mutants are embryo-lethal and even the so-called “rescued” mutants have severe growth defects . To exclude that WRKY46 might have been tethered to the ERF104 nuclear protein, WRKY46 was expressed alone where the WRKY46-YFP signals were also in the nucleus . Western blotting using an anti-GFP monoclonal antibody revealed predominantly protein bands with the expected molecular weights for intact WRKY46-YFP and ERF104-CFP fusion proteins , so that the nuclear fluorescence signals detected is not due to free CFP/YFP cleavage products. In addition, we also tested the non-phosphorylatable WRKY46S168A,S250A variant, which is also localized in the nucleus . These findings are in agreement with WRKY46 functioning as a putative transcription factor in the nucleus  is not due to its mis-localization.To examine WRKY46 subcellular localization, we transiently expressed WRKY46-YFP in ocalized . The res nucleus , 2015 anNDR1/HIN1-like 10 (NHL10), in Arabidopsis protoplasts co-expressing either different variants of WRKY46 or CFP (as a control for expression of a defense-unrelated protein). Protoplasts were treated with either water, bacterial (flg22/elf18) or fungal (chitin) elicitors and luciferase activity was measured. In protoplasts expressing any WRKY46 variant, an elevated basal promoter activity (time point 0 min) was observed for the water and also the PAMP-treated samples . Also at later time points, all treatments showed an overall elevation of NHL10 promoter activity in the WRKY46-expressing protoplasts. This suggests that WRKY46 is a positive regulator of the defense-related NHL10 gene. Interestingly, the S168A variant consistently showed a higher boost on the NHL10 promoter activity. Since western blotting showed comparable expression of the respective WRKY46 variants , the enhanced activity of WRKY46S168A is not simply due to enhanced protein stability/expression . To further test if phosphorylation of WRKY46 influences its effect on transcription controlled by the NHL10 promoter, we created a WRKY46S168D,S250D phospho-mimic. For simplicity, we tested only flg22 and concentrated only on the peak NHL10 promoter activity. As seen in Figure 4C, there was no difference in activities of the phospho-mimic  or the non-phosphorylatable  variants compared to wild type WRKY46. However, in line with the role of phosphorylation in regulating WRKY46 stability , the phospho-mimic variant is expressed at lower levels . We therefore compared the stability of WRKY46 phosphosite-mutated variants. While trace levels of wild type WRKY46 are expressed 2 h post flg22 treatment, the phospho-mimetic WRKY46 is hardly detectable . Thus, this serves as indirect evidence that mutated residues are functionally mimicking the phosphorylated serines. Taken together, transcriptional activity of WRKY46 does not seem to be directly controlled by its phosphorylation status.To investigate the role of WRKY46 in plant defense, we analyzed expression of a luciferase (LUC) reporter driven by a routinely used W-box-containing defense-related promoter, NHL10 reporter is often used as a reporter for MAPK impact on defense gene expression  were treated with water or flg22 for 10 min and MAPK activation was analyzed. Treatment with flg22 leads to strong activation of MPK6, MPK3, and MPK4/11 in both WRKY46 and CFP expressing samples without obvious differences . Thus, WRKY46 does not seem to have feedback regulatory activity on the MAPK pathway; and the enhanced NHL10 promoter activity is likely due to direct action of the overexpressed WRKY46.Since the pression , it is ppression . To inveArabidopsis plants have been shown to be more resistant to bacterial pathogens . Taken together, these observations suggest that WRKY46 is a positive regulator of plant defense.Transgenic WRKY46-overexpressing athogens . To valiin vivo PAMP-activated MAPK phosphorylation of WRKY46, which we routinely use for several other MAPK substrates  or flg22 treatments , indicating that both kinases are important for full WRKY46 activity. Notably, mpk3 showed a more dramatic effect than the mpk6 mutant, where the WRKY46-mediated boost on the basal NHL10 promoter activity was completely abolished . Furthermore, the flg22-activation of NHL10 promoter was also compromised compared to the CFP control  in the mpk3 background. Thus, despite functional redundancy between MPK3 and MPK6 , MPK3 may play a stronger in vivo role in regulating the transcriptional activity of WRKY46 (on the NHL10 promoter). This observation is in agreement to a preferential in vitro phosphorylation of WRKY46 by MPK3 than MPK6 .As mentioned above, we could not use the protein mobility phospho-shift in SDS-PAGE to demonstrate an bstrates . To alteIn order to adapt to new environmental conditions, plants have to reprogram transcription of the appropriate genes. Transcription factors play major roles in the control of gene expression. The WRKY family is among the ten largest families of transcription factors in higher plants and is found throughout the green lineage  phosphorylate S168, S250 and other positions to regulate its stability. In this respect, a WRKY46 promoter-based reporter assay revealed transcription activation of WRKY46 by the calcium-dependent protein kinases, CPK-3, -4, -5, -6, -10, -11, and -30 and several WRKYs were directly phosphorylated by CPKs  gene, which plays an important role in SA metabolism  also makes it a PTI marker gene. Since WRKY46 can contribute to both PTI and ETI signaling, it is plausible that it mediates interplay between PTI and ETI and this will be an interesting question to address in the future.WRKY46 regulates responses to several abiotic stresses in n stress . It is an stress , which mtabolism . WRKY46 mylovora . It alsosyringae . In geneeatments . WRKY46 egulator . The indin planta stability . Since there are WRKY DNA-binding elements in the NHL10 promoter  – where MAPKs as well as other signaling pathways are activated – is also compromised by mpk3 or mpk6 mutations. In summary, the mode of the WRKY46-MAPKs regulation of NHL10 promoter is currently unknown but it appears to be unrelated to WRKY46 protein stability or its phospho-status . Future experiments could reveal if the MPK3/MPK6 effect may be due to altered affinity of WRKY46 to the promoter or direct impact on its transcriptional activity. Alternatively, a more likely scenario would be that there may be other MPK3/MPK6 targets that influence WRKY46 activity. Furthermore, as already discussed, there may be additional kinase(s) that phosphorylate WRKY46. Likely candidates include members of the calcium-dependent protein kinase family – especially those that have been shown to regulate WRKY46 expression (Localization studies revealed that WRKY46 is localized in the nucleus. This result is consistent with the fact that it acts as a transcription factor, possibly regulating the transcription of defense-related genes. Accordingly, WRKY46 overexpression boosts the defense responsive promoter , which hpromoter , it is lpression . It willAS and LE designed and performed the experiments shown. PP cloned the WRKYs into the bacterial expression system and screened them for recombinant protein expression. WH performed the LC-MS/MS determination of the phosphorylation sites. DS, AS, and JL supervised the project and all authors contributed to writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.