In this work, ovotransferrin was digested into peptides by virtual enzymolysis. Subsequently, in vitro ACE inhibitory activity of potential tripeptides was conducted following the peptide score, toxicity, and water solubility prediction. Both pharmacophore study and flexible docking were applied to analyze ACE inhibition mechanism of tripeptides. Our results demonstrated that EWL was a potent ACE inhibitory tripeptide with IC50 value of 380 ± 10 μM. Besides, pharmacophore and flexible docking showed that the pi interaction and hydrogen bond were the key interactions in ACE-EWL complex. It appears that the in vitro ACE inhibitory activity of tripeptide EWL was consistent with its molecular modeling.Food-derived ACE inhibitory peptides have recently attracted increased attention. This work focused on a more efficient  Previous work has demonstrated that egg-derived peptides exerted antioxidant, antimicrobial, and angiotensin converting enzyme (ACE) inhibitory activities3. Particularly, ACE inhibitory peptides attracted more attention, which can block the active site of ACE and suppression angiotensin-I translate to angiotensin-II, therefore reduce blood pressure4. Regarded as an efficient strategy of anti-hypertension (AHT), ACE inhibitors have been studied for years. However, present clinical ACE inhibitors were almost obtained by chemical synthesis. Those synthetic ACE inhibitors were potent and widely used as an anti-hypertension drug, despite their adverse side effects including coughing, allergic reactions, taste disturbances and skin rashes5. Therefore, more attentions have been focused on finding less side effects and economically available substitute, like food-derived ACE inhibitory peptides6.Egg white is considered as a rich source of high quality proteins, which can release bioactive peptides after enzymatic hydrolysis9. However, conventional workflow of ACE inhibitory peptides discovering has several drawbacks, such as time and cost consuming10. Indeed, increasing attention has come to the idea of inventing a more efficient method to discover ACE inhibitory peptides from food proteins. As bioinformatics approaches emerged, bioinformatics e-tools and database were integrated to create an efficient and time saving method to discover ACE inhibitory peptides. In fact, some ACE inhibitory peptides have already been successfully discovered using in silico analysis11. Database information can be easily accessed, i.e., BIOPEP, NCBI, and UniProt databases. Subsequently, the potential peptides were analyzed using online services ExPASy and Bioware, aimed to predict properties of novel peptides. In particular, molecular docking can help to understand how molecules working together, and predict certain bio-activities.In recent years, more and more ACE inhibitory peptides have been found in food proteinsin silico approaches. Ovotransferrin from egg white was hydrolyzed via virtual digestion. Generated dipeptides and tripeptides were screened by peptide score, toxicity, and water solubility prediction. Subsequently, in vitro activity of peptides with the potential to inhibit ACE was assayed. Finally, the molecular mechanisms of potent ACE inhibitory tripeptides were investigated.This study was to discover novel ACE inhibitory peptides from ovotransferrin using As shown in Table 12. As so, three properties include high peptide score, non-toxin, and good water solubility. Peptide score was a score function given by PeptideRanker that predicts peptides bioactivity. In the results, a total of 1276 di- and tripeptides was produced by all 19 enzymes and 1 enzyme combination. All peptides with peptide score higher than 0.6 were predicted to be non-toxin, but peptides with good water solubility seem pretty limited. Various sequenced peptides may lead to more different potent ACE inhibitory peptides, Refer to the results, pancreat elastase and stem bromelain can produce most different kind and most quantity of qualified peptides, stem bromelain have 6 different kind of promising peptides; while pancreat elastase have 5 different kind of promising peptides. Sequence EWL and CDL had occurred in both digest result. All those peptides  have not been published as ACE inhibitory peptides and applied to later molecular docking project.In the current work, only di- and tripeptides were chosen to have selection with specific properties owing to they can be easily absorbed into the blood circulatory system from the digestive tract. However, the potency of peptides is not only depending on their human digestive tract absorption but also their bioactivity. And toxicity and water solubility were also important properties for peptide employment14.As shown in Table i.e., KDF and EWL, were performed by High Performance Liquid Chromatography (HPLC) method. It turned out that EWL can de-active ACE efficiently with IC50 value of 380 ± 10 μM. However, KDF can de-active ACE’s catalytic reaction for about 14.27% in concentration of 0.45 mM. After another two confirming test at concentration of 0.45 mM. We finally decided not to find out KDFs’ IC50 value owing to it’s extremely low suppressant activity. Moreover, the cytotoxicity of EWL was examined using MTT toward HepG2, results indicated that the high concentration EWL was low-toxicity , one hydrophobic region (Hyd), and one positive ionizable (PI) was the best model  was chosen to conduct further research. Even though, proline was well documented as the most favorable amino acid for peptides binding to ACE21. This indicate that ovotransferrin have theoretic ACE inhibitory after human digestion tract, but still too soon to say that egg can regard as an anti-hypertension strategy food. For human digestion of food include digestion by not only protease release from human body but also human gut microbiota, and digestions by human gut microbiota were not quite revealed. Hence, the yield of the two peptides after gastrointestinal digestion would still in myth.When properties of bioactive peptides limited in high peptide score, non-toxin, good water solubility and unpublished ACE inhibitory peptide, only one potent ACE inhibitory peptide showed up. However, after digestion by stem bromelain and pancreat elastase there are 7 published ACE inhibitory peptides and they also confirmed as peptide score over 0.6 and predict to be non-toxin. Those 7 peptides are MF, WL, WA, IF, MG, YG and FG. Stem bromelain digestion result comprise one MF; WL; IF; MG; KDF; EWL and two WA and YG. Pancreat elastase results in one WL; FG; MG EWL and two WA. It seems, digestion of egg white ovotransferrin by stem bromelain can lead to maximum non-toxin high scored peptides. Digestion of ovotransferrin by human gastrointestinal protease was also been investigated. Two proving ACE inhibitory peptides have been found in the results, GR and GF22. In this study, the mapping results showed peptide EWL could match HBA and PI features better than KDF, and the hydrogen bonds in EWL were more concentrated than those in KDF.To the number of all kinds of interaction, there are 8 conventional hydrogen bonds, 2 carbon hydrogen bond, 8 pi interaction, 2 attractive charge, and 3 unfavorable interactions in EWL-ACE complex; 6 conventional hydrogen bond, 2 carbon hydrogen bond, 1 pi interaction, and 5 attractive charge in KDF-ACE complex. Due to EWL’s potent ACE inhibitory activity, this result indicates that pi interaction also play an important role in peptide binding with ACE. There are previous paper had written about the importance of hydrogen bond in ligand complexes binding with ACEIn flexible docking results, even though, KDF can dock into three active pocket of ACE, it still cast low ACE inhibitory activity. It seems how many pocket ACE inhibitory peptides can bind with ACE isn’t ACE inhibitory activity determinant element. Except residues determined in the three active pocket, , the residues of ACE bind with EWL and KDF were different. Both lisinopril and EWL have complex binding results. It seems some residues would like to interact with different atoms at a certain distance. This may give ACE ability to immobilize peptides more stabilized which may lead to higher inhibitory ability.23. EWL can interact with ACE zinc atom by an attractive charge. Although KDF can form interaction with zinc ion but possess low ACE de-active activity. It seems interactions with zinc atom were not very important in inhibit ACE in this research, which against previous study24. Zinc ion has tetra-coordinate with three ACE residues . And the distortion of the tetra-herald geometry can cause ACE inhibitory activity25. However, this phenomenon was not detected in this study. In other words, the bond length between zinc ion and the three mentioned residues were not change, in other word, the bond length remain in 2.07086 Å with His387, 1.99577 Å with Glu411 and 2.03772 Å with His383. It seems that even zinc ion have well documented as an important factor for peptide interact with ACE, peptides ACE inhibitory activity can not been determined only by interaction with zinc ion.Interactions with zinc ion was always been mention for it playing key role in forming metal-acceptor interaction26. Different ovotransferrin sequences from egg white were published in UniProt database. As bioactivity of peptides are owing to their amino acid composition. Di- and tripeptides consisted of particular amino acids  was in favor of binding with ACE27. In the present work, those amino acids were applied to find the sole sequence with most possibility of producing potent ACE inhibitory peptides among the ovotransferrin sequences collected from UniProt database. Composition of all those sequences was analyzed in ProtParam, available at https://web.expasy.org/protparam/. A further amino acid sequence comparison was made by BLAST, available at https://blast.ncbi.nlm.nih.gov/Blast.cgi.Ovotransferrin make up about 12% of egg white protein content, which can be extracted in large quantities and is available to apply in food industry. Ovotransferrin was specially chosen in this study for it is the main ingredients in egg white and its reasonable sequence length28. This subject was completed by using webservers PeptideCutter (available at https://web.expasy.org/peptide_cutter/) and BIOPEP analysis ENZTME(S) ACTION (available at http://www.uwm.edu.pl/biochemia/index.php/en/biopep)28. Di- and tripeptides were chosen to have specific properties elimination for they can be absorbed directly into the blood circulatory system from the digestive tract29. Nineteen enzymes, i.e., cathepsin G, chymase, chymosin, clostripain, ficin, glutamyl endopeptidase II, leukocyte elastase, oligopeptidase B, oligopeptidase F, pancreat elastase, pancreat elastase II, papain, pepsin (pH = 1.3), pepsin (pH < 2), proteinase K, proteinase P1, stem bromelain, thermolysin, trypsin and V8 (pH 7.8) were utilized to digest ovotransferrin.Variety in proteases could lead to enormous differences in the bioactivity of peptides. Ovotransferrin was digested by different enzymes, aim to produce more di- and tripeptides for further screening, as well as select the enzyme which could produce maximum number of ACE inhibitory peptides. Total 19 enzymes and 1 enzyme combination  were utilized to accomplish this digital proteolysis. Enzymes were gathering for their using in food industry30. And toxicity would be another vital nature for drugs and bioactive peptides. In this part, bioactivity, toxin and solubility were employed to be the key parameters for choosing the qualified peptides to have ACE inhibitory activity prediction . Bioactivity prediction was made by Peptide Ranker (available at http://bioware.ucd.ie/compass/biowareweb/), and toxicity tests were finished by ToxinPred (available at http://crdd.osdd.net/raghava/toxinpred/multi_submit.php). PepCalc  was utilized to conduct water solubility prediction. Di- and tripeptides with peptide ranker score over 0.6, non-toxicity and good water solubility prediction were put into continued subjects. Meanwhile, chosen peptides were not identified in previous study from BIOPEP database.Some physiochemical properties were very important for bioactive peptides exhibiting biological activity in human body31. This means if peptides can bind with ACE tight, it could have ACE inhibitory activity. The ACE crystal structure (PDB ID: 1o86) was downloading from RCSB PDB, which bonded with lisinopril. The docking site was chosen from the five build-in active site in crystal structure, active site AC5, for it have docked lisinopril in its center and including zinc ion. However the radius of final docking active site was set at 9 Å. ACE crystal structure would been through two steps before docking started. Firstly, add hydrogen. Secondly, delete water and irrelevant atoms. Discovery Studio 2017 R2 was used to draw out the 3D structure of promising peptides as well as docking them to ACE crystal structure (1o86). Structures of these peptides were optimized by Dynamics simulation and prepare ligands protocol before docking into ACE. Docking protocol was calculated using CDOCKER algorithm, it would give out the values of CDOCKER ENERGY and CDOCKER INTERCTION ENERGY. The docking was carried out with coordinates x: 40.6183, y: 34.4676, and z: 44.7269 with a radius of 9 Å. The pose cluster radius were set to 0.1 Å. The random conformations were set to 10, the orientations to refine was set to 10, and the simulated annealing was set to true. Peptides with lower -CDOCKER ENERGY and -CDOCKER INTERACTION ENERGY values were selected for in vitro ACE inhibitory activity assay.It has been demonstrate that ACE inhibitors can competitively and unselectively block the active sites of ACE, therefore deactivate the course of angiotensin-II32.Potential peptides were synthesized by the solid phase procedure peptide using FMOC protected amino acids synthesis methods with an AAPPTEC 396 Automated Peptide Synthesizer . Detials have been written in our previous workIn vitro ACE inhibitory activity was measured followed by high-performance liquid chromatography 15 with some modification. More details seen in our previouslystudy7.To verify the accuracy of the toxicity prediction results, we used a cell assay to detect the cytotoxicity of EWL. After the HepG 2 cells were seeded and pre-incubated, the cells were treated with 10 μL EWL . Cell viability in this experiment was determined by the MTT  assay and absorption Value was measured at 490 nm.Ligand phamacophore mapping of Discovery Studio 2017 R2 Client was used to fits the tripeptides to the phamacophore generated in previous study. The conformation generation was set to fast, and the maximum omitted features was set at -1. All other parameters were left in their default configurations.i.e., Gln281,Tyr520, Lys511, His513, and His 353) and S2’ (Glu162)33.Ligand based pharmacophore study can help to understand ACE inhibitory mechanism in ligand level. Flexible docking, a more accurate docking protocol than CDOCKER, was employed to dock KDF and EWL into ACE crystal structure (1o86). Flexible docking is the protocol that allows the key residues in acceptor become movable; further lead to position changing of atoms in both ligand and acceptor to meet the best conformation. Key movable residues in ACE were selected as the residues in three ACE active sites: S1 (Ala354 Glu384 Tyr 523), S1’ (


Together, this review should provide a framework to help understand how a network of kinases and phosphatases cooperate to regulate two key mitotic processes.Multiple kinases and phosphatases act on the kinetochore to control chromosome segregation: Aurora B, Mps1, Bub1, Plk1, Cdk1, PP1, and PP2A-B56, have all been shown to regulate both kinetochore-microtubule attachments and the spindle assembly checkpoint. Given that so many kinases and phosphatases converge onto two key mitotic processes, it is perhaps not surprising to learn that they are, quite literally, entangled in cross-talk. Inhibition of any one of these enzymes produces secondary effects on all the others, which results in a complicated picture that is very difficult to interpret. This review aims to clarify this picture by first collating the direct effects of each enzyme into one overarching schematic of regulation at the Knl1/Mis12/Ndc80 (KMN) network . This schematic will then be used to discuss the implications of the cross-talk that connects these enzymes; both in terms of why it may be needed to produce the right type of kinetochore signals and why it nevertheless complicates our interpretations about which enzymes control what processes. Finally, some general experimental approaches will be discussed that could help to characterize kinetochore signaling by dissociating the direct from indirect effect of kinase or phosphatase inhibition  The kinetochore is a molecular complex of at least 100 different proteins that assembles on the centromeric region of chromosomes to allow their attachment to microtubules during mitosis , the Mis12 complex , and the Ndc80 complex   and two phosphatases (PP1 and PP2A-B56) . If this configuration is achieved for every single kinetochore pair, then upon mitotic exit, the sister chromatids will be split in opposing directions and each daughter cell will receive the correct complement of the genome. Since kinetochore-microtubule interactions form stochastically, this whole process must be regulated to ensure that improper attachments are continually removed, in a process termed error-correction , which also contains two regulatory subunits, borealin and survivin, that are tethered to Aurora B by INCENP   which can diffuse throughout the cell to inhibit the anaphase promoting complex/cyclosome (APC/C); an E3 ubiquitin ligase needed for mitotic exit  domains of Ndc80 and Nuf2  , and therefore mutations that abolish kinetochore PP2A-B56 also reduce PP1-Knl1  Figure . If bothWhen characterizing phosphatase inputs at the KMN network it is advisable to perform specific mutations that inhibit local phosphatase recruitment without affecting total phosphatase levels. This minimizes the indirect effects and may also produce more penetrant phenotypes, in comparison to siRNA-mediated depletion, for example. This may explain why Knl1-MELT dephosphorylation was strongly affected by targeting Knl1-PP1 directly, but not by depleting PP1 catalytic subunits . Does Mps1 need to be docked on Ndc80 to phosphorylate substrates or can it be released to form a gradient of activity around Ndc80? If it can be released, then what deactivates Mps1 and does Ndc80 binding promote clustering and reactivation . If rapid re-phosphorylation is observed under these conditions, then this would imply that activity of the inhibited kinase is not required for this phosphorylation , and whether Aurora B regulates Mps1 directly or indirectly (via PP1-Knl1 or PP1-Ska inhibition). When performing these experiments, it is important to measure the rates of (de)phosphorylation and to consider the fact that pharmacological inhibition is never absolute. Finally, when the indirect effects are known, it is possible to perform more elaborate experiments to determine their relative importance: for example, the downstream effects of Aurora B inhibition can be quantified in the context of Knl1 mutations that prevent activation of PP1-Knl1.in vitro kinase assays, however, there are still situations when it is difficult to distinguish between two kinases using this approach alone. For example, as discussed extensively above, Mps1 and Plk1 both phosphorylate the KMN network and have overlapping substrate specificities in vitro. Inhibiting these enzymes individually or in combination is one simple way to probe their relative contributions (Von Schubert et al., If kinase inhibition reduces target protein phosphorylation without increasing phosphatase activity, then this “direct” effect could still be mediated by another kinase. Often this can be ruled out by examining the kinase consensus motif and using in vivo. Therefore, care must be taken when implicating a phosphatase directly in substrate dephosphorylation. Even if removal of a specific phosphatase subcomplex prevents dephosphorylation without affecting kinase activity, this could still be mediated indirectly via a secondary phosphatase; as proposed previously for PP2A-B56 on KNL1-MELT phosphorylation (Nijenhuis et al., In general, determining specificity is more challenging for the phosphatases than it is for kinases, due to their relative weak substrate sequence preferences. That is not to say that there are no specificity determinants: PP2A-B55, for example, prefers to dephosphorylate phospho-threonine residues within motifs that are flanked by polybasic residues (Mccloy et al., This review highlights how both kinetochore-microtubule attachments and the SAC are coregulated at the KMN network. It is important to stress that both of these processes can also be regulated from other areas of the kinetochore as well. This is exemplified by the outermost “fibrous corona” in human cells, which is an expanded region that forms around kinetochores during prometaphase to aid the capture of microtubules (Cassimeris et al., A critical feature of KMN regulation, that may be important for corona regulation too, is the interplay between kinases and phosphatases. Whilst it is very easy to adopt a kinase centric view of signaling, the danger is that this could lead to important regulatory inputs being missed or misinterpreted. A good example is the widespread use of FRET reporters to monitor “kinase” activity Morris, , which iThe discussion about Aurora B nicely illustrates one important final point: the regulatory processes at the kinetochore could never be fully explained by considering either kinase or phosphatase inputs in isolation. These antagonistic enzymes work together in many different ways to define a signaling response (Gelens et al., The author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Spinopelvic fixations involving the S2-alar-iliac (S2AI) and iliac screws are commonly used in various spinal fusion surgeries. This study aimed to compare the biomechanical characteristics, specifically the risk of screw and adjacent bone failures of S2AI screw fixation with those of iliac screw fixation using a finite element analysis (FEA).A three-dimensional finite element (FE) model of a healthy spinopelvis was generated. The pedicle screws were placed on the L3-S1 with three different lengths of the S2AI and iliac screws . In particular, two types of the S2AI screw, 15°- and 30°-angled polyaxial screw, were adopted. Physiological loads, such as a combination of compression, torsion, and flexion/extension loads, were applied to the spinopelvic FE model, and the stress distribution as well as the maximum von Mises equivalent stress values were calculated.For the iliac screw, the highest stress on the screw was observed with the 75-mm screw, rather than the 60-mm screw. The bones around the iliac screw indicated that the maximum equivalent stress decreased as the screw length increased. For the S2AI screw, the lowest stress was observed in the 90-mm screw length with a 30° head angle. The bones around the S2AI screw indicated that the lowest stress was observed in the 90-mm screw length and a 15° head angle.It was found that the S2AI screw, rather than the iliac screw, reduced the risk of implant failure for the spinopelvic fixation technique, and the 90-mm screw length with a 15° head angle for the S2AI screw could be biomechanically advantageous. Degenerative spinal diseases are one of the most frequently reported chronic health problems affecting the adult population owing to aging. In addition, adult spinal deformities have increased owing to the growing elderly population. These spinal conditions lead to an imbalance in the structural support of the spine. With the development of surgical techniques and supporting surgical skills, spinal deformity surgeries have become more frequent. However, because of the poor bone quality of most elderly patients, implantation-related problems are frequent, especially in the lumbosacral area. Kim et al.  reportedTo overcome the complications associated with fusions ending at S1, sacropelvic fixation has been introduced as a safe alternative –7. SpinoThe S2-alar-iliac (S2AI) technique, first proposed in 2007, uses a starting point in the sacral ala, midway between the S1 and S2 dorsal foramina along a line that connects the lateral edge of the two foramina . This poAs previously mentioned, many studies have focused on the anatomy, surgical technique, and risk evaluation during iliac and S2AI screw fixations. However, there are few studies that have performed a biomechanical assessment and comparative study of these two fixation types. Few studies have evaluated the material internal stress in the screw and the bone around the screw, under various fixations and spine motions. To evaluate the biomechanical and physical factors, such as the von Mises equivalent stress, cadaver or computational approaches to the analysis are required.The finite element method (FEM) of computational analysis is widely used in the study of many biological systems, especially the musculoskeletal system. Through the FEM, many complex geometrical and material properties of the biological system can be effectively evaluated, and many physical variables, such as stress, strain, damage, and fracture, can be quantitatively analyzed.In a few studies, the biomechanical ability and stability of the spinopelvic system have been numerically investigated. Garcia et al.  carried As previously mentioned, many computational studies have focused on the spinopelvic fixation analysis. However, there is no comparative study regarding the iliac and S2AI screw fixation technique. Hence, in this study, the biomechanical characteristics of the iliac and S2AI screw fixed spinopelvic system under various implant configurations were computationally evaluated, and the simulation results, such as the equivalent stress values in the surrounding bone and screw, were investigated. Based on the calculated results, an optimal implant condition was proposed.This research was approved by the Institutional Review Board (IRB) of the Pusan National University Hospital (PNUH). The reference number is PNUH-IRB-E-2016068. An informed consent statement was signed after receiving oral description of the simulation prior to the start of simulation.The software programs Mimics 19.0 , SolidWorks 2016 , and ANSYS 16.1  were used. A computed tomography (CT) scanner  was used to collect raw data in the digital imaging and communication in medicine (DICOM) format with a scan slice of 0.75 mm.An FE model of the spinopelvis (L3-Pelvis), which included three vertebrae, three discs, and a pelvis, was reconstructed. The geometrical specifications of the spinopelvis were obtained from 64 spiral CT images of a 27-year-old healthy male without a history of spine injury and osteoporosis or radiographic evidence of degeneration. The patient underwent a CT examination for a health checkup at our hospital, and the CT images were used with the patient’s consent. The date of patient recruitment for imaging was August 15, 2016, and the time period for this research was June 28, 2016 to May 31, 2017.The CT images were scanned and imported into Mimics 19.0 to construct the three-dimensional (3D) surface of the spinopelvis. To avoid unexpected stress concentration, the surface of the spinopelvis was smoothed. The FE model was generated using the ANSYS meshing tool. A 10-node tetrahedral solid element was adopted to express the cancellous bone, and an 8-node hexahedral element with a 1-mm thickness was used to represent the cortical bone, surrounding the cancellous bone. To describe the intervertebral disc in the FE model, the nucleus pulposus and annulus fibrosus were adopted using an 8-node hexahedral solid element. The model without implants had a total of 223833 elements and 341569 nodes.Owing to their important roles in pelvic biomechanics, the anterior/posterior longitudinal, interspinous, sacroiliac, sacrospinous, sacrotuberous, and pubic ligaments were incorporated and modeled as the spring elements. The attachment points were ascertained by mimicking the anatomy as closely as possible. The final FE model of the normal spinopelvis is shown in This 3D geometry of the spinopelvic model was then exported into 2016 SolidWorks software to generate the fixation models. The polyaxial pedicle screw instrumentation (7.0 × 35 mm) was placed bilaterally from L3 to S1, and two different screws were adopted for spinopelvic fixation: iliac and S2AI screws. The diameters of the iliac and S2AI screws were 7.0 mm each, and the length of the three screws varied: 60 mm, 75 mm, and 90 mm. Two types of the S2AI screw, 15°- and 30°-angled polyaxial screws, were used.The iliac and S2AI screws were implanted into the spinopelvic model using the standard surgical technique. The iliac screws were inserted from the PSIS to the anterior-inferior iliac spine (AIIS) at each ilium, and the lateral connectors were placed in the iliac screws. The S2AI screws were placed 1-mm inferior and 1-mm lateral to the S1 dorsal foramen. Angulation of the screw was directed just above the sciatic notch in the coronal plane.The threads of the screws were eliminated to simplify the FE models. Based on the simplified model, the computational time as well as the analysis risk owing to an unrealistic high stress could be reduced, and a smooth transfer of stress between the screw and bone could be realized . The eleThe material properties of the bones, intervertebral disc, implants, and various ligaments are listed in Tables This study also evaluated the sensitivity of the elements that affected the accuracy of the results prior to the regular FE analysis (FEA). The coarse, medium, and fine densities of three representative meshes were selected to determine the number of elements, as shown in An analysis was performed using the three densities, and the results converged from the medium- to fine-mesh densities. The ANSYS 16.1 FEA software program was used to calculate the stress distribution in each model. As listed in Tables The bilateral acetabulum of the FE model was fixed. The interface condition between the screw and bone plays a vital role in determining the stress distribution . AccordiA pure moment of 10 Nm combined with a pre-compressive load of 700 N was applied to the top surface of L3 and the superior articular processes. 60% (500 N) and 40% (100 N) of the total load were separately applied to the upper vertebral body and superior articular processes, respectively, as the facet joints can commonly carry 10% to 40% of the compressive load of the total force subjected to the vertebrae –29.Through the combination of the moment and compression, four types of loads, compression, flexion, extension, and rotation, were generated and adopted to the FE model. The loading and boundary conditions are shown in The proposed FE model was validated by simulating the experimental cadaveric study of O’Brien et al. . The norAs shown in Figs In addition, Conversely, Figs Moreover, All simulation results are listed quantitatively in The simulation results according to the screw length, head angle, and screw type are listed below. For the iliac screw, the maximum equivalent stress decreased in the order of 75-mm, 90-mm, and 60-mm screw lengths in all loading conditions, as shown in Conversely, for the S2AI screw, the highest and lowest maximum equivalent stresses occurred in the 60-mm screw length with a 15° head angle and the 90-mm screw length with a 30° head angle, respectively, in all loading conditions, as shown in The stress ranges of the iliac screw and surrounding iliac bone were approximately 111–178 MPa and 40–51 MPa, respectively. In addition, the stress ranges of the S2AI screw and surrounding sacrum/iliac bones were approximately 54–110 MPa and 28–63 MPa, respectively.The aim of this study was to evaluate the biomechanical characteristics of two types of spinopelvic fixation systems under various implant configurations and loading conditions. To obtain the stress contour of spinopelvic fixation using FEM, 3D FE models for the cortical and cancellous bones and the intervertebral disc, including the nucleus pulposus and annulus fibrosus, which have a significant influence on the mechanical behavior of the spinopelvis, were fabricated. Moreover, 11 types of lumbar and pelvic ligaments were considered to accurately represent the human spinopelvic state. Two types of implants for iliac and S2AI fixation were also generated and inserted into the spinopelvic FE model. Based on a series of simulations under compression, flexion, extension, and axial rotation, the von Mises equivalent stress values in the screws and bony structures were calculated, and the simulation results were compared.An ideal internal fixation method should provide a maximum rigidity between the bone segments and a minimum stress on the surrounding tissues for healing. Excessive stress around the fixation devices can cause gradual resorption of the surrounding bone and loosening of the screws. This is an important clinical aspect that must be considered when choosing the appropriate rigid fixation system . The simAccording to previous studies on the stability of spinopelvic fixation, S2AI screw fixation can lead to a more reliable stability compared to iliac screw fixation owing to its longer screw length , 33. ConHowever, in this study, the difference in the maximum equivalent stress on the screw and the bone around the screw owing to the screw length was confirmed. For the S2AI screw, as the screw length increased, the magnitude of the stress on the screw decreased, and the maximum equivalent stress value on the ilium around the screw decreased as well. There was no large variation in the stress on the sacrum based on the screw length. Conversely, for the iliac screw, there was no proportional relationship between the screw length and the maximum equivalent stress on the screw. As described in the Results section, the maximum equivalent stress occurred in the 75-mm screw length. Nevertheless, as the screw length increased, the maximum equivalent stress on the ilium decreased, except in the flexion state.Guler et al.  reportedFor the accuracy and verification of the simulation results, it was necessary to specify the maximum von Mises equivalent stress point of each case. In the iliac screw, the maximum equivalent stresses of the screws were at the point where the screw and iliac cortical bone met, and the maximum equivalent stresses of the bones were in the iliac cortical bone at the SIJ. The maximum equivalent stresses occurred at the head portion of the screws in a few cases; however, these were beyond the scope of this study. Therefore, the point measured was the one at which the SIJ was not in contact with the head. When the S2AI screw was inserted, it was passed through the inside of the sacrum and ilium. At this time, the SIJ was penetrated through the cortical bone, and the maximum equivalent stress point was in the sacral and iliac cortical bone.The limitations of this study are addressed below. First, the threads of the pedicle, iliac, and S2AI screws in the spinopelvic FE model were simplified to reduce the computational time and analysis error owing to the stress concentration. However, to obtain an accurate stress distribution on the screw and the bone around the screw, the threads of the screw should be considered in the FE model. Specifically, the contact condition between the bone and implant should be precisely considered during the FEA.Second, the spinopelvic FE model in this study was asymmetrical. Accordingly, the stress distribution as well as the occurrence position of the maximum equivalent stress were not even. To acquire precise simulation results, this problem should be solved.Third, 11 types of ligaments were modeled as a spring element, and the intervertebral disc, including the nucleus pulposus and annulus fibrosus, was simply fabricated using a solid element. In addition, the endplate of the vertebra was ignored. Since the ligaments, intervertebral disc, and endplate play a vital role in the biomechanics of the spinopelvis, it is necessary to model these tissues in detail. For example, the ligaments could be modeled as a shell/solid element, and the endplate and annulus ground substance should be considered.Fourth, the pubic symphysis was modeled with 24 spring elements that could match the equivalent stiffness of the symphysis owing to the convenience of FE modeling . HoweverFifth, this study required various parametric analyses to obtain highly accurate results. A wide range of parametric analyses and numerical experiments using an advanced FE model and FEA should be helped comparative analysis and improvement, provided to further the optimal surgical plan and implant configuration.In conclusion, by comparing the biomechanical characteristics of the S2AI screw and Iliac screw, the S2AI screw showed a lower risk of implant failure than that of the iliac screw for the spinopelvic fixation technique. From analyzing the length and angle characteristics, the 90-mm screw length as well as the 15° head angle for the S2AI screw were considered biomechanically advantageous.


Activation of nicotinic acetylcholine receptors containing α4 and β2 subunits (α4/β2* nAChRs) in the mammalian brain is necessary for nicotine reinforcement and addiction. We previously identified interactions between α4/β2* nAChRs and calcium/calmodulin-dependent protein kinase II (CaMKII) in mouse and human brain tissue. Following co-expression of α4/β2 nAChR subunits with CaMKII in HEK cells, mass spectrometry identified 8 phosphorylation sites in the α4 subunit. One of these sites and an additional site were identified when isolated α4/β2* nAChRs were dephosphorylated and subsequently incubated with CaMKII in vitro, while 3 phosphorylation sites were identified following incubation with protein kinase A (PKA) in vitro. We then isolated native α4/β2* nAChRs from mouse brain following acute or chronic exposure to nicotine. Two CaMKII sites identified in HEK cells were phosphorylated, and 1 PKA site was dephosphorylated following acute nicotine administration in vivo, whereas phosphorylation of the PKA site was increased back to baseline levels following repeated nicotine exposure. Significant changes in β2 nAChR subunit phosphorylation were not observed under these conditions, but 2 novel sites were identified on this subunit, 1 in HEK cells and 1 in vitro. These experiments identified putative CaMKII and PKA sites on α4/β2* nAChRs and novel nicotine-induced phosphorylation sites in mouse brain that can be explored for their consequences on receptor function. High-affinity nicotinic acetylcholine receptors containing the α4 and β2 subunits  are essential for the rewarding and reinforcing properties of nicotine in the mouse ,2,3,4. αIn addition to initiating calcium signaling, nicotine also increases the number of nAChRs and can alter the associated proteome of α4/β2* nAChRs in mouse and human brain . BiochemSeveral studies have now evaluated the α4/β2* nAChR-associated proteome, and these studies have identified several proteins that co-immunoprecipitate with the receptor from mouse and human brain ,8,16. InIn the current set of experiments, we used mass spectrometry to identify the residues phosphorylated on the α4 and β2 nAChR subunits when co-expressed with CaMKIIα in HEK cells, when dephosphorylated and subjected to phosphorylation in vitro with CaMKIIα or PKA, and when isolated from mouse brain at baseline, or following exposure to acute or repeated nicotine in vivo. These studies were designed to determine whether CaMKII can phosphorylate the α4 and β2 nAChR subunits in cells that do not normally express these proteins, and whether sites identified in the cellular assay were recapitulated when purified nAChRs were incubated with CaMKIIα or a kinase that is endogenously expressed in HEK cells (PKA) in vitro. The in vitro study also allowed us to determine whether previously identified PKA sites ,10,11,12Adult male C3H mice  were obtained from Jackson laboratories and housed in groups of no more than 5 individuals per cage, maintained on a 12:12 h light/dark cycle, and given ad libitum access to food and water. All procedures involving animals were approved by the Yale University Institutional Animal Care and Use Committee and conformed to the standards for animal care and use set by the National Institutes of Health.2. DMEM cell culture medium supplemented with 10% fetal bovine serum  and antibiotic/antimycotic (Gibco), according to established ATCC protocols. Prior to transient transfection, HEK cells were split and plated at medium-high density on plastic 10 cm dishes which were pre-treated with 0.05 mg/mL poly-d-lysine (PDL) in water.HEK-293 (HEK) cells (ATCC) were grown and maintained in a humidified incubator at 37 °C and 5% COTransient expression of nAChR subunits, mRuby, and CaMKII-mRuby were performed in serum-free medium (SFM) using Lipofectamine 2000  at 2.5 μL per μg DNA. For cell-based phosphorylation assays, the following combinations of plasmids were used, each in triplicate: (1) untagged α4-nAChR + β2-nAChR-YFP + mRuby, (2) untagged α4-nAChR + β2-nAChR-YFP + CaMKII-mRuby, (3) α4-nAChR-YFP + Untagged β2-nAChR + mRuby, and (4) α4-nAChR-YFP + Untagged β2-nAChR + CaMKII. For in vitro phosphorylation assays, HEK cells were transfected with either untagged α4-nAChR + β2-nAChR-YFP or with α4-nAChR-YFP + Untagged β2-nAChR. For all experiments, transfection suspensions were prepared by combining DNA and Lipofectamine in a small volume of SFM and incubating at room temperature for 30 min. Transfection suspensions were added to cells with an additional volume of SFM, and returned to the incubator for 24 h prior to harvesting.Nhe1/Apa1 cloning site. mRuby2-C1 was purchased from Addgene .2) containing 2% Triton X-100, protease inhibitors (PMSF and Sigma protease inhibitor cocktail), and phosphatase inhibitors . For in vitro phosphorylation assays, only the baseline samples were collected in the presence of phosphatase inhibitors; separate samples for the lambda phosphatase, PKA, and CaMKIIα conditions were collected in ice-cold MEB without phosphatase inhibitors. Harvested cells underwent 2 rounds of sonication/vortex cycles on ice, and were then allowed to incubate on ice for ~30 min to facilitate solubilization. Insoluble material was removed by brief centrifugation, and the resulting supernatants were used for further experiments.24 h after transfection, HEK transients were harvested by scraping cells into ice-cold membrane extraction buffer , 120 mM NaCl, 5 mM KCl, 1 mM MgClw/v) saccharin (chronic condition only) or 2% saccharin with molar-matched tartaric acid  for 14 days. Animals were housed 3–4 to a cage, and pairings were set up at least 5 days prior to introduction of experimental water to allow for acclimation. Water was stored in darkened bottles to protect from light.The nicotine treatment paradigm used was essentially as we have used previously ,24. AdulAt the end of two weeks, animals were treated with a single, subcutaneous dose of either nicotine , or saline (chronic and control conditions). Experimental drinking water was removed from cages 1 h prior to dosing, and animals were sacrificed by cervical dislocation 15 min after dosing. Whole brains were immediately removed on ice, then flash-frozen and stored at −80 °C until processing.g for 10 min. Pellets were discarded, and “S1” supernatants were transferred to clean ultracentrifuge tubes and spun for 1 h at 100,000× g and 4 °C using a Beckman 70.1 Ti rotor. The resulting supernatants were removed and the pellets (“P2”) were resuspended in 0.3× their initial volume in membrane extraction buffer , 120 mM NaCl, 5 mM KCl, 1 mM MgCl2) containing 2% Triton X-100, protease inhibitors (PMSF and Sigma protease inhibitor cocktail), and phosphatase inhibitors . Samples were vortexed thoroughly, and allowed to incubate on ice for ~2 h to facilitate solubilization. P2 homogenates were transferred to microfuge tubes and spun at 1000× g and 4 °C in for 10 min. The supernatant  was used for immunoprecipitation of nAChRs.On the day of tissue preparation, frozen brains were thawed on ice and homogenized in 10 volumes of ice-cold tissue homogenization buffer  containing protease inhibitors (PMSF and Sigma protease inhibitor cocktail) and a panel of phosphatase inhibitors . Lysates were subjected to two rounds of sonication and vortexing, incubated on ice for 30 min, then spun at 1000× v/v). Wash buffer was completely removed from resin using a magnetic stand, and diluted HEK lysates were added to washed resin. IP was conducted by tumbling overnight at 4 °C.Immunoprecipitation (IP) of HEK transients was conducted using a magnetic GFP-nAb resin from Allele, essentially according to manufacturer protocols. Briefly, GFP-nAb resin was washed 3× with binding buffer , then resuspended and aliquot into microfuge tubes (20 μL resin/IP). While preparing resin, HEK cell lysates were thawed on ice and diluted in binding buffer such that the detergent concentration was not higher than 0.6% . Bound fractions of HEK lysates from co-expression experiments (co-expression of nAChRs with CaMKII or mRuby in HEKs) and baseline samples from in vitro phosphorylation experiments were eluted by boiling resin in in 60 μL 1× Laemmli sample buffer  with SDS (prepared in ultrapure water for MS). Bound fractions of HEK lysates, intended for in vitro phosphorylation experiments, were treated according to the dephosphorylation/phosphorylation protocol(s) detailed below, prior to eluting.w/v), then resuspended in PBS and stored at 4 °C until use. On the day of IP, linked resin was rinsed once in PBS and then equally divided into each of 15 clearly labeled microfuge tubes (5 samples × 3 treatment groups). Processed whole brain samples (S3 fraction) were added to the prepared resin, and IP was conducted by tumbling overnight at 4 °C. The next day, beads and bound proteins were separated on a magnetic stand and rinsed 3× with PBS and 1× with PBS containing 0.1% BSA (w/v) and 0.1% Triton X-100 (v/v). The final wash was then removed, and the bound fractions were eluted by boiling resin in 50 μL 1× Laemmli sample buffer (Bio-Rad) with SDS. Once cooled, the eluate was immediately removed from the resin and stored at −20 °C until gels were run.Immunoprecipitation of α4/β2-containing nAChRs from mouse brain lysates was done using M-270 Epoxy magnetic Dynabeads  linked to purified nAChR antiserum. Approximately 600 μg of rat-anti-α4  was combined with 175 μg of rat-anti-beta2  and linked to surface-activated M-270 Epoxy Dynabeads  according to the manufacturer’s instructions. mAb299 and mAb270 antisera were a generous gift from Jon Lindstrom, and were characterized previously . AntibodTransfected HEK cells for in vitro phosphorylation experiments were separated into four groups: baseline, lambda phosphatase, PKA, and CaMKII. Baseline nAChR transients were collected in the presence of phosphatase inhibitors, and eluted immediately following IP, as described above. The remaining three groups were harvested in the absence of phosphatase inhibitors, and immunoprecipitated receptors were subject to in vitro dephosphorylation with purified lambda phosphatase .2 (manufacturer supplied), 1 μL each of aprotinin and PMSF, and 2 μL of lambda phosphatase. Dephosphorylation continued for 1 h at 30 °C with gentle agitation. After incubation, resins were separated from dephosphorylation mixture by placing on a magnetic stand, washed with PMP, and dephosphorylation was conducted a second time. Following the second treatment, 50 μL of 500 mM EDTA was added to each sample and suspensions incubated on ice for 5 min to stop phosphatase activity. Supernatants were then removed, and resin was washed 2× in wash buffer. Bound receptors in the lambda phosphatase group were eluted here in 40 μL 1× Laemli Sample Buffer (LSB) prepared in ultrapure water. Remaining samples continued on to rephosphorylation with PKA or CaMKIIα.Immunoprecipitated receptors were dephosphorylated in vitro using recombinant lambda phosphatase (New England Biolabs), according to manufacturer instructions. Briefly, immunoprecipitated receptors (still bound to nAb-GFP resin) were rinsed once with 200 μL PMP phosphatase buffer (manufacturer supplied) to equilibrate. The following phosphatase mixture was then added to each tube: 100 μL PMP buffer, 10 μL MnClFollowing dephosphorylation, samples in the PKA group were phosphorylated with purified PKA (New England Biolabs) according to the manufacturer’s instructions. Briefly, bound receptors were rinsed in 1× PK buffer (manufacturer supplied), and then incubated in the following PKA phosphorylation mixture: 100 μL 1× PK buffer, 1 μL of sigma protease inhibitor cocktail, 2 μL of 10 mM ATP stock , and 2 μL of PKA enzyme. Phosphorylation continued for 1–2 h at 30 °C with gentle agitation. Following incubation, supernatants were removed, and resin was washed 2× in wash buffer. Bound receptors were then eluted in 40 μL 1× LSB prepared in ultrapure water.2, all provided by the manufacturer. The activation solution incubated for 10 min at 30 °C. Meanwhile, bound receptors were washed 2× in supplied kinase buffer and then incubated with activated CaMKII phosphorylation mix (+A/PMSF) for 45 min at 30 °C with gentle agitation. After incubation, the kinase suspension was removed, and resins were washed 2× in wash buffer. Bound receptors were then eluted in 40 μL 1× LSB prepared in ultrapure water.Following dephosphorylation, samples in the CaMKIIα group were phosphorylated with purified CaMKIIα (New England Biolabs) according to manufacturer instructions. CaMKIIα enzyme was activated by combining 2 μL of the purified kinase with 100 μL buffer, 200 μM ATP, 1.2 μL calmodulin, and 2 mM CaClAll IP samples were separated on Bio-Rad Mini-Protean TGX precast gels  using established protocols. Separated protein eluates were then stained using Simply Blue Safe Stain (Invitrogen). Samples were run alongside a protein molecular weight marker, and bands of the appropriate molecular weights were excised from the gel, transferred to microfuge tubes, and stored at −20 °C until processing for proteomics. Approximate molecular weights for the nAChR subunits and variants are as follows: α4-nAChR ~75 kDa (HEK transients and from mouse brain), β2-nAChR-YFP ~75 kDa, α4-nAChR-YFP ~100 kDa, and β2-nAChR ~55 kDa (HEK transients and from mouse brain). All processing was done in a clean environment and ultrapure water was used to prepare all buffers and reagents.v/v) acetonitrile (5 min), 50% (v/v) acetonitrile/10 mM NH4HCO3 (30 min). Gel pieces were dried with a speed vacuum, resuspended in 30 µL of 10 mM NH4HCO3/0.2 µg digestion grade trypsin (Promega), and incubated for 16 h at 37 °C. Peptides were acidified with 0.1% (v/v) trifluoroacetic acid (TFA) prior to mass spectrometry analysis.Gel bands were first cut into small pieces and subjected to the following washes with agitation: 50% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile, respectively. Peptides were eluted over 120 min with a mobile phase B gradient (6–20%) at a column temperature of 37 °C and a flow rate of 300 nL/min. Precursor mass scans  were acquired and followed by HCD-based fragmentation . A resolution of 70,000 at m/z 200 was used for MS1 scans, and up to 20 dynamically chosen, most abundant precursor ions were fragmented (isolation window: 1.7 m/z). The tandem MS/MS scans were acquired at a resolution of 17,500 at m/z 200 . Mass spectrometry raw spectra were searched against the Mascot algorithm  using Proteome Discoverer software . The search criteria were the following: precursor mass tolerance, 10.0 ppm; fragment mass tolerance, 0.020 Da; enzyme, trypsin; maximum missed cleavage sites, 2; variable modifications, carbamidomethyl (C), oxidation (M), phosphorylation (STY), propionamide (C).Reverse phase liquid chromatography tandem mass spectrometry (RP-LC-MS/MS) was performed using a NanoACQUITY  ultra-performance liquid chromatography (UPLC) coupled to a Q Exactive Plus Hybrid Quadrupole-Orbitrap  mass spectrometer. Peptides were loaded onto a nanoACQUITY  UPLC Symmetry C18 trapping column (180 µm × 20 mm) at a flowrate of 5 µL/min prior to separation on a nanoACQUITY  Peptide BEH C18 column (75 µm × 250 mm). Mobile phase A and B compositions were 0.1%  Normalization Preference; Calculation Type, Median; Blocking Level, Unique Peptides; Use Inter Experiment Normalization, true; Use Intra Sample Normalization, false; Use Peptide Normalization, false; Use Protein Average As Reference, true; Use Iterative Normalization, true; Spectrum Quality Filter, no filter. For annotation of protein PostTranslational Modification (PTM) sites, Scaffold PTM , which integrated the MS/MS results exported from Scaffold/Scaffold Q+. For differential phosphorylation analysis, phosphorylated peptide spectral counts were first normalized to the total spectral counts for each protein. This value was then normalized to the total spectral counts for the entire sample.Searched data was imported into Scaffold  software for validation of peptide and protein identifications. Peptide and protein identifications were accepted above a 95% and 99% probability threshold, respectively. Proteins containing less than two peptides per protein were filtered out, and proteins sharing redundant peptides were grouped. Peptide and protein probabilities were calculated by the Scaffold Local FDR algorithm and Protein Prophet algorithm , respectt-tests. The level for significance was set at p < 0.05.For all replicates, normalized quantitative values for the “treated” group were compared to those of the “control” group. Statistical analyses were performed in GraphPad Prism v7.01  using two-tailed Student Trafficking and activity of high-affinity nAChRs can be regulated by phosphorylation ,14,15. TFollowing transfection, nAChRs were immunoprecipitated and subunits were separated by gel electrophoresis. Gels were Coomassie-stained, bands of the appropriate size for the untagged and tagged α4 and β2 nAChR subunits were excised, and proteins were digested and subjected to mass spectrometry. We identified 8 serine residues on the α4 subunit with significantly increased phosphorylation following co-expression with CaMKIIα-mRuby, compared to those co-expressing mRuby alone  and inclThe coverage of the α4 subunit intracellular M3/M4 loop was ~80%, suggesting that the majority of physiologically relevant sites of phosphorylation were likely identified, and only 6 serine or threonine residues in the intracellular loop were uncovered . HoweverSince multiple kinases are found in all cell types, phosphorylation of the nAChR subunits may have resulted directly through CaMKII activity, or indirectly through activation of other kinases expressed in HEK cells. We, therefore, performed an in vitro phosphorylation experiment using α4/β2 nAChRs immunoprecipitated from HEK cells following dephosphorylation using lambda phosphatase. Following immunoprecipitation and dephosphorylation, tagged or untagged nAChRs were incubated with CaMKIIα in the presence of calcium and calmodulin or PKA. nAChRs were then separated by gel electrophoresis, and bands were excised for evaluation by mass spectrometry. As above, parallel experiments were performed using α4-YFP with untagged β2 and untagged α4 with β2-YFP, and no differences were found, so data for tagged and untagged subunits were pooled. At baseline, four highly phosphorylated serine residues were identified on the α4 subunit, S470, S530, S540, S543 a. IncubaWe next determined whether the phosphorylation sites identified as CaMKII or PKA targets in vitro were also phosphorylated in vivo under conditions in which nAChRs could be activated by nicotine. Using monoclonal antibodies raised against the α4 subunit, we immunoprecipitated native α4/β2* nAChRs from mouse brain following saline administration, a single nicotine dose in a novel environment (0.5 mg/kg), or chronic nicotine in the drinking water, a regimen known to increase locomotor activity in a dopamine-dependent manner . In miceThese experiments identify previously described and novel sites on the mouse α4 nAChR subunit, and the first report of specific residues on the β2 nAChR subunit, that can be phosphorylated in cells after heterologous expression with CaMKIIα, or by CaMKIIα or PKA in vitro  that have not been described as substrates for this kinase previously, along with highly significant phosphorylation of S470. A number of studies have identified S470 as an important site of phosphorylation on the α4 nAChR subunit ,28,29,30The decrease in phosphorylation of the S470 site on the mouse α4 subunit following acute nicotine treatment in vivo suggests that stimulation of nAChRs may result in activation of a protein phosphatase that dephosphorylates this residue. This observation is consistent with experiments showing that nicotine acting through nAChRs can activate the calcium-dependent phosphatase calcineurin in cultured cortical neurons . In addiThe consistent association between α4/β2 nAChRs and several CaMKII isoforms in mouse and human brain prompted us to investigate whether these receptors were a substrate for phosphorylation by this kinase. Neither of the phosphorylation sites identified following in vitro phosphorylation of α4/β2 nAChRs with CaMKIIα were identified in vivo at baseline or following nicotine exposure, suggesting that the nAChR may not be a major substrate for CaMKII in the mouse brain at baseline or under that conditions of nicotine exposure tested here. However, S444, S448, and S563 were phosphorylated both when CaMKIIα was co-expressed with the α4 and β2 nAChR subunits in HEK cells and in mouse brain. The physical interaction between α4/β2 nAChRs and CaMKII could result in activation of the kinase and phosphorylation of other protein targets. Therefore, the increased phosphorylation of α4/β2 nAChRs, when co-transfected with CaMKIIα in HEK cells, could be the result of a protein kinase cascade that indirectly results in phosphorylation of the nAChR by PKA and other unidentified kinases. Alternatively, the association between α4/β2 nAChRs and CaMKII may be important for other cell biological functions, such as localization of the receptor to particular intracellular compartments or the plasma membrane. Interaction with scaffolding proteins, such as 14-3-3, contributes to trafficking of nAChRs in a PKA-dependent manner , and CaMIn summary, this phosphoproteomic study has identified novel phosphorylation sites on the mouse α4 nAChR and β2 subunits, and is the first instance of identification of a subset of α4 nAChR residues phosphorylated, in vivo, in mouse brain tissue. Further, acute nicotine exposure increases the phosphorylation of two residues on the α4 subunit (S444 and S448), but decreases phosphorylation of a very well-characterized residue S470) that contributes to surface trafficking and resistance to desensitization of α4/β2 nAChRs in cultured neurons 0 that co,11,12. T


Tackling malnutrition is a global health priority, helping children both survive and thrive. Acute malnutrition (wasting) in infants aged under 6 months (u6m) is often neglected. Worldwide, some 8.5 million infants u6m are affected yet recent World Health Organization malnutrition guidelines highlight numerous evidence gaps on how to best manage them. To inform future research, policy and programming, we aimed to identify risk factors associated with infant u6m wasting.z-scores) vs. non-wasted (weight-for-length ≥−2 z-score). We used simple and adjusted  logistic regression to calculate odds of wasting associated with risk factors spanning three broad categories: household-related; maternal-related; infant-related.We did secondary data analysis of nationally representative, cross sectional Demographic and Health Surveys conducted in the last 10 years. We compared wasted infants u6m (weight-for-length <−2 p = 0.04)); low maternal body mass index ; small infant size at birth ); delayed start of breastfeeding ); prelacteal feed ); recent history of diarrhoea ); mother disempowered . ‘Protective’ factors associated with significantly decreased odds of infant u6m wasting included: educated mother ); mother in work ); currently breastfed ), exclusively breastfed .We analysed 16,123 infants u6m from 20 countries. Multiple risk factors were statistically associated with wasting. These included: poverty (Odds ratio, OR 1.22 (95% CI [1.01–1.48], Infant u6m wasting is a complex, multifactorial problem associated with many risk factors; knowing them will help shape international and national management strategies. Whilst our observational study cannot prove causation, many factors identified are biologically plausible and/or socially important. They should be considered when assessing and managing infants u6m. Although supporting breastfeeding is core to future interventions, this alone is unlikely to be sufficient; strategies should involve multiple sectors, beyond just health and nutrition. By noting our results, future intervention studies could focus resources and maximise chances of achieving impact. Tackling malnutrition plays a major role in child survival  and alsoby 2030, end all forms of malnutrition, including achieving by 2025 the internationally agreed targets on stunting and wasting [acute malnutrition] in children under five years of age’,  datasets. DHS are large, nationally representative household surveys regularly conducted in over 90 developing countries. They follow a common, standardised methodology (two stage cluster sampling) and are extensively used in global health and nutrition to support ‘policy formation, program planning and monitoring and evaluation’ . QuestioFollowing the approach from a previous paper exploring the prevalence of acute malnutrition in infants u6m , our samz-score, WLZ <−2) with those who were not wasted (WLZ ≥−2). We used WHO growth standards for our analysis  in each country . VariablPotential explanatory variables were grouped under: household characteristics; maternal characteristics; infant characteristics. Our rationale for these three categories was that they all plausibly affect infant nutrition and might be amenable to change in future intervention programmes. Since this was a hypothesis-generating exploratory study and we did not know exactly which aspects of household environment, which maternal characteristics and which infant characteristics are most closely associated with infant malnutrition, we deliberately chose all available variables and excluded none.Detailed variable definitions are given on DHS website and the exact phrasing of questions can be seen in country questionnaires . Among tz-score (WAZ)<−6.0 or WAZ>+5.0, length-for-age z-score (LAZ)<−6.0 or LAZ>+6.0, WLZ<−5.0 or WLZ>+5.0 SD. These more likely represent measurement or data processing errors rather than a truly very large or small infant.Using Stata version 13.1  we merged individual country files into one large dataset. This was then cleaned by excluding records with missing or extreme values. Infants u6m were excluded if any of the following were missing: age; sex; weight; length. Those with biologically unlikely extreme values (‘flags’) were also excluded from analysis following standard WHO ‘cleaning rules’ : weight-Z-scores were generated from raw age, weight and length variables using the WHO’s ‘Child Growth Standards’ macro  commands for our main analysis to account for the DHS survey design.www.populationpyramid.net, United Nations, Department of Economic and Social Affairs, Population Division. World Population Prospects).Firstly, we computed the prevalence and 95% confidence intervals of severe, moderate and overall wasting based on WLZ for each country and for all countries combined. For the overall figure and for logistic regressions (described below) we accounted for different population size using population estimates from the World Bank . All these may plausibly contribute to infant wasting, hence are important confounders and need to be taken into account when assessing other variables.p < 0.05. Stata  was used for all analyses.Odds ratios and 95% confidence intervals were calculated and are presented. Statistical significance was taken as Demographic and Health Survey are a well-established and well-respected global initiative conducted with all appropriate in-country permissions and informed consents. For inclusion of infants u6m, their main carers  would have given informed, usually written, consent. We registered our project on the DHS website  and wereOf the 36 ‘high burden’ countries, 20 had relevant data. The flow chart in We found 22 eligible DHS datasets with data on 29,854 infants u6m. Excluding those with no length data, the number dropped to 19,075. Eligible infants further dropped to 18,478 after excluding those with no weight data. Of the 18,478 infants u6m remaining (now from 20 countries), 2,265 had out-of-range WLZ, WAZ or LAZ and were thus excluded from further analysis. Of the 16,213 infants u6m included in the final analysis, 8,206 (51%) were boys and 11,352 (70%) lived in a rural area. There were fewest infants in the 0 to <1 month age bracket ); 2,706 (17%) in the 1 to <2 month age bracket; then just over 3,000 (19%) in each of the other four age bands u6m. Prevalence of severe and moderate wasting and wasting overall is shown in p < 0.05) were only observed at the two extremes: poorest and richest vs. middle quintile. A test of trend for wealth index was also statistically significant: for each unit increase in wealth, the odds of infant wasting decreased by 9% (95% CI [13–5%] reduction), p < 0.001.The same analyses subdividing the outcome into moderate and severe wasting is shown in p-value of 0.05. Compared to mothers with no education, those who have attended school are less likely to have a wasted infant u6m compared to those with no education. A test of trend of wasting vs. level of education is statistically significant (p < 0.001) on unadjusted analysis but not on adjusted analysis (p = 0.32). After adjusting, only primary education vs. no education is statistically associated.Compared with non-working mothers, those in work are significantly less likely to have wasted infants u6m; this association remains statistically significant even after adjusting.p = 0.06) when it is the husband deciding and strongly significant (p = 0.02) when anyone else in the household decides. Reported experience of violence is also a risk factor with statistically significant associations with wasting. Physical violence is the subtype of domestic violence with statistically significant associations.A mother being in a relationship (‘in union’) is also associated with statistically greater odds of having a wasted infant u6m: both on unadjusted and adjusted analysis. Whenever it is someone other than the mother/carer who decides about health issues in the household, the odds of having a wasted infant u6m are increased; the association is of borderline statistical significance .Numerous breastfeeding (BF) related indicators are statistically associated with infant u6m wasting. Late initiation of BF and a history of a prelacteal feed are both statistically associated with greater odds and adjusted odds of infant u6m wasting; ever breastfed, currently BF and exclusively BF are associated with significantly decreased odds of wasting.A history of following vaccine-related recommendations is associated with statistically significantly decreased odds of infant u6m wasting. This includes having a vaccine card and having recorded timely DTP  and polio vaccine.Finally, a 2-week illness history of fever and cough were not associated with infant u6m wasting, but those with a reported history of diarrhoea within the last 2 weeks were significantly more likely to be wasted.In this paper we explored a large number of factors which could plausibly increase or decrease the risk of acute wasting in infants u6m and found numerous statistically significant associations. These spanned all three of our conceptual categories: household factors; maternal factors; infant factors. For the majority of these, the direction of association is biologically or socially plausible and a probable mechanism can easily be understood. Noteworthy factors associated with increased odds of infant u6m wasting included: poverty; maternal malnutrition ; uneducated mother; mother disempowered ; reported small infant size at birth ; poor eWe are fully aware of the limitations of our work:Firstly, we emphasise that any associations observed in a cross-sectional study such as ours cannot prove causation, however plausible they may be. Special care must be taken regarding possible directions of association . Bradford-Hill criteria can help when considering possible causality of individual factors . These iStrength of association is another important Bradford-Hill criterion for causality. Even when statistically significant, most of our ORs are not far from 1. No one factor alone has a very high OR, as might be the case if it played a dominant causal role. Many weak associations as in our paper are not uncommon in nutritional epidemiology .Second, we are conscious of having conducted multiple analyses and therefore expect some statistically significant associations by chance alone. Again, Bradford-Hill criteria can help interpret both expected and some unexpected observations .Third, we acknowledge that our dataset was not designed for looking at infants u6m alone. Consistent with other studies  we foundFourth, we are conscious of an active debate around case definitions of acute malnutrition in infants u6m. In this paper, we focus on wasting as defined by low weight-for-length because this is currently recommended by For these reasons, we emphasise that our data is hypothesis generating. We intended to inform and advance current programmes and practices in this area, especially to give background for future research which can more definitively answer key questions arising. Balancing the limitations, we believe that there are many strengths of our analysis and several messages arising which are important and relevant to both practitioners and researchers.First, we found many factors associated with infant u6m wasting. This supports calls for broader and more integrated prevention and treatment packages . The treSecond, we also found that not all variables examined were in fact associated with wasting . Excluding some variables in this way thus provides greater focus for future research: rather than investigate all possible risk factors as we have done, others can investigate a smaller number of risk factors in more detail, to better understand how they impact infant u6m malnutrition and how they might play a role in future intervention strategies.Third, our findings are helpful in shaping clinical assessment tools. One such tool is the ‘C-MAMI’  tool which takes a similarly broad approach to assessing and managing infants <6 m . At coreFinally, our focus on infants u6m raises their profile. Other papers have looked at infant malnutrition in general but ours adds to the literature by focusing just on wasting and just on infants u6m, a very specific subgroup who, according to There is much scope for future research. Although our results are based on a large number of countries and thus broadly generalizable, individual countries planning their services may want to consider more local data from their own DHS or similar. Above all, however, there is a need for prospective research and especially for intervention studies to address infant u6m malnutrition. Our results could inform sample size calculations for such projects, giving investigators an idea of possible effect sizes. Most importantly, targeting single risk factors alone is unlikely to have the optimal impact, intervention ‘packages’ delivering multiple strands of care that consider infant, maternal and household factors are likely to be more successful, potentially even synergistic in their effects. For instance, breastfeeding support should be combined with programmes addressing gender inequalities and empowering women and girls . ‘PositiInfant u6m wasting is a complex, multifactorial problem associated with numerous risk factors encompassing household, maternal and infant characteristics. Many of those which we identified in our dataset are biologically plausible and/or socially important and could be included in assessment tools and intervention strategies to help nutritionally vulnerable infants u6m. Evidence-based breastfeeding support is vital and needs to be further strengthened in many settings . However, this alone may not be sufficient. Packages of care should also seek to support and empower mothers. Our data relating to antenatal care and early breastfeeding practices point to possible benefits of more proactive/early interventions that seek to prevent as well as treat acute malnutrition, and the need for collaboration between sectors  and service delivery providers.There is an urgent need to test these insights from observational work in future intervention studies. Our results will help design such studies, focus resources and maximise chances of successful identification and management of nutritionally vulnerable infants u6m.10.7717/peerj.5848/supp-1Supplemental Information 1Click here for additional data file.10.7717/peerj.5848/supp-2Supplemental Information 2Click here for additional data file.10.7717/peerj.5848/supp-3Supplemental Information 3Click here for additional data file.10.7717/peerj.5848/supp-4Supplemental Information 4Click here for additional data file.10.7717/peerj.5848/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.5848/supp-6Supplemental Information 6P<0.01, **P<0.001.*†Adjusted for infant age group, sex and socio-economic status.Click here for additional data file.10.7717/peerj.5848/supp-7Supplemental Information 7P<0.01, **P<0.001.*‡This is size as reported by the carer rather than measured size.†Adjusted for infant age group, sex and socio-economic status.Click here for additional data file.


ITAM-activated Syk stimulates PKC-dependent Syk S297 phosphorylation, which is reduced by SFK/Syk/PKC inhibition and cAMP. Inhibition of Syk S297 phosphorylation coincides with enhanced Syk activation, suggesting that S297 phosphorylation represents a mechanism for feedback inhibition in human platelets.The spleen tyrosine kinase (Syk) is essential for immunoreceptor tyrosine-based activation motif (ITAM)-dependent platelet activation, and it is stimulated by Src-family kinase (SFK)-/Syk-mediated phosphorylation of Y352 (interdomain-B) and Y525/526 (kinase domain). Additional sites for Syk phosphorylation and protein interactions are known but remain elusive. Since Syk S297 phosphorylation (interdomain-B) was detected in platelets, we hypothesized that this phosphorylation site regulates Syk activity via protein kinase C (PKC)-and cyclic adenosine monophosphate (cAMP)-dependent pathways. ADP, the GPVI-agonist convulxin, and the GPIbα-agonist echicetin beads (EB) were used to stimulate human platelets with/without effectors. Platelet aggregation and intracellular messengers were analyzed, along with phosphoproteins, by immunoblotting using phosphosite-specific antibodies or phos-tags. ADP, convulxin, and EB upregulated Syk S297 phosphorylation, which was inhibited by iloprost (cAMP pathway). Convulxin-stimulated Syk S297 phosphorylation was stoichiometric, transient, abolished by the PKC inhibitor GF109203X, and mimicked by the PKC activator PDBu. Convulxin/EB stimulated Syk S297, Y352, and Y525/526 phosphorylation, which was inhibited by SFK and Syk inhibitors. GFX and iloprost inhibited convulxin/EB-induced Syk S297 phosphorylation but enhanced Syk tyrosine (Y352/Y525/526) and substrate (linker adaptor for T cells (LAT), phospholipase γ2 (PLC γ2)) phosphorylation. GFX enhanced convulxin/EB-increases of inositol monophosphate/Ca IIbβ3), degranulation, synthesis/release of thromboxane A2 (TxA2), and surface exposure of anionic phospholipids, leading to platelet shape change, adhesiveness, aggregation, and coagulant activity to form a protective thrombus [2+ concentration, and activate Ca2+-dependent protein kinases, such as protein kinase C (PKC), thereby inducing platelet activation [Human platelets are small circulating blood cells, which control, monitor, and preserve the integrity of the vessel wall. These anucleate cells prevent blood loss during vascular injury and, on the other hand, have prominent roles in thrombotic, inflammatory, and immune pathologies ,2. Afterthrombus ,5 and cethrombus ,7,8. GPCtivation ,5.IIbβ3 [Syk gene (by deletion of one exon on Syk gene encoding for 41 residues in the Syk kinase domain in embryonic stem cells) die from severe hemorrhages before birth [2+/calmodulin-dependent protein kinases such as PKC, but not iloprost, stimulated Syk S297 phosphorylation. Very recently, we established methods for the selective quantitative assessment of GPVI-and GPIbα-mediated activation and function of human platelet Syk [The adhesion molecules collagen, vWF, podoplanin, fibrinogen, and fibrin bind to and activate platelet membrane receptors such as glycoprotein (GP) VI (GPVI), GPIb-V-IX, CLEC-2, integrin αIIbβ3 ,7, and sIIbβ3 . The SFKIIbβ3 ,8,9, whiIIbβ3 ,11, whilIIbβ3 ,13,14,15re birth , and micre birth , highligre birth ,19,20. Tre birth ,19. Howere birth ,22,23. Sre birth . While Sre birth , Syk S29re birth ,26. Inteelet Syk ,28. We oelet Syk .Based on previously published data and our own findings on Syk S297 phosphorylation in human platelets, and considering the crucial Syk interdomain location of S297 , we hypo12 , and on Syk Y352 (important for Syk activity enhancement and for binding other proteins). Syk S297 showed a transient phosphorylation, which increased within 1 min of stimulation and then decreased both with convulxin a,ai and EB-/cvx-mediated Syk tyrosine phosphorylation is mediated by SFKs and Syk autophosphorylation ,19,28. TUpon BCR stimulation in murine B-cells, Syk S291, in addition to several tyrosine sites, was a major PKC phosphorylation site in the interdomain-B of Syk which corresponds to S297 in human Syk . TherefoTo analyze quantitative aspects of Syk phosphorylation, we used the phos-tag SDS- polyacrylamide gel-electrophoresis (PAGE) method developed by the group of Koike ,32. ThisIn parallel, we analyzed total Syk with the standard SDS-PAGE method and detected only one constant major Syk band in all samples but a row of very minor bands (higher than 72 kDa), especially GFX-treated samples bii, whicThese samples from cvx-treated platelets were also analyzed by standard SDS-PAGE blots using phosphoantibodies. The phosphorylation of Syk S297 was strongly stimulated by cvx and completely inhibited by the PKC inhibitor GFX biii, whiTo obtain further evidence for PKC-mediated phosphorylation of Syk S297 in human platelets, we tested the effects of a global PKC activator, the phorbol ester phorbol 12, 13-dibutyrate (PDBu). PDBu induced strong platelet aggregation  and a vekinase substrate (MARCKS) phosphorylation, a well-established PKC substrate, also in platelets [We then investigated the role of Syk kinase activity on the phosphorylation of S297 mediated by PKC activation. Firstly, we validated the effects of PDBu and GFX by analyzing myristoylated alanine-rich C-latelets ,35. MARCNext, we investigated the role of PKC on Syk activation in GPVI-and GPIbα-mediated platelet activation. PKC inhibition by GFX completely prevented the S297 phosphorylation induced by cvx a,ai and To validate the possible functional consequence of Syk tyrosine hyperphosphorylation, we investigated the phosphorylation of Syk downstream substrates linker adaptor for T cells (LAT) and PLCγ2 on Y191 and Y759, respectively. Cvx-and EB-induced LAT phosphorylation was remarkably increased in the presence of the PKC inhibitor compared to the control a,b,ai,bi2+ mobilization. In the presence of GFX, cvx-and EB-evoked InsP3 production was significantly enhanced compared to the control in the absence of the PKC inhibitor  production (via inositol monophosphate (InsP1) accumulation) and intracellular Canhibitor a. Moreove of GFX b. These In this study, we show (1) that activation of human platelets via GPVI and GPIbα, as well as by pharmacological activation of PKC, caused phosphorylation of Syk S297, and (2) that pharmacological PKC inhibition or receptor-linked inhibition of PKC by cAMP/PKA abolished Syk S297 phosphorylation, but enhanced Syk tyrosine phosphorylation/activity and phosphorylation of Syk substrates. These data suggest that Syk S297 phosphorylation is mediated by PKC and represents a mechanism of feedback inhibition of Syk in human platelets.Several lines of evidence show that a classical, diacylgycerol/phorbolester-responsive protein kinase C (PKC) phosphorylates Syk S297 in human platelets:Increased Syk S297 phosphorylation is observed when ADP receptors, GPVI, and GPIbα are stimulated by selective agonists, which also stimulate PLC and PKC activation.GPVI-and GPIbα-caused Syk S297 phosphorylation is Syk-dependent and requires, at least predominantly, the release of secondary mediators ADP and TxA2.GPVI-/GPIbα-induced Syk S297 phosphorylation is blocked by a selective PKC inhibitor (GFX) and mimicked by an established, membrane permeable PKC activator (PDBu).+ responses and PKC activation [This agonist-induced Syk S297 phosphorylation is also inhibited by the cAMP/PKA pathway, which is known to strongly inhibit receptor-stimulated Ca2tivation ,36,37.The peptide sequence around Syk S297* 9ISRIKSYS*FPKPGHR) in humans  agrees well with the PKC phosphosite consensus sequence   activated by diacylglycerol alone, and atypical forms, which are diacylglycerol-independent. Human and mouse platelets highly express the conventional PKC isoforms α/β and the novel isoforms δ and θ [While the evidence for a conventional PKC as a responsible Syk S297 kinase in intact human platelets is quite strong, we do not know which PKC isoform (or perhaps more than one) is involved. PKC isoforms consist of conventional forms  activated by Ca δ and θ ,38, whic δ and θ ,39. Ther δ and θ . An Src/ δ and θ , indicatIn our study, we observed a rapid receptor-mediated Syk S297 phosphorylation (onset within 15 s of stimulation), which reached stoichiometry and then declined back to the basal status. This conclusion was possible due to the combination of regular Western blot/pSyk S297 phosphoantibody analysis with phos-tag analysis. The initial major Syk band in phos-tag gels was almost completely shifted to another major band, which was completely shifted back to the basal state when platelets where pre-treated with the PKC inhibitor GFX. This major band was the only Syk band with this property, a pattern also observed by regular Syk pS297 phosphoantibody analysis. The conclusion that these effects are PKC-mediated is strongly supported by observations made with the direct PKC activator PDBu. Treatment of platelets with PDBu caused, as detected in phos-tag gels, a time-dependent, complete shift of this major Syk band, which was also completely prevented by the PKC inhibitor. Compared to cvx treatment, no minor additional shifted band appeared in the PDBu treatment detected in phos-tag gels. The phos-tag data with PDBu additionally suggest that PKC phosphorylates only one major site (S297) in Syk, although Syk contains a number of serine/threonine protein kinase phosphosites . The othWhile the phosphorylation of Syk S297 can reach stoichiometry in our studies with human platelets, the corresponding stoichiometry of Syk tyrosine phosphorylation appears to be much smaller. Interestingly, Syk phosphorylation data obtained with murine B-cells suggested that phosphorylation of Syk at tyrosines is restricted to a subset of the kinase physically associated with ITAM-containing membrane proteins, whereas the interdomain serine sites are also phosphorylated in Syk, when Syk is not membrane-bound .Another feature of GPVI/GPIbα-mediated Syk phosphorylation and activation in platelets is the transient nature of both tyrosine  and serine S297) phosphorylation. Onset and decline of Cvx/EB stimulated SykS297 phosphorylation resemble the time course of Syk Y352/Y525/526 phosphorylation, which indicates Syk activation ,19. The 7 phosphoA remarkable result reported here is that the specific inhibition of Syk S297 phosphorylation by the PKC inhibitor GFX closely coincided with enhanced cvx/EB-stimulated Syk Y352/Y525/526 phosphorylation (indicating enhanced Syk activity), enhanced Syk substrate phosphorylation , and enhanced cvx/EB-induced InsP1/InsP3 increase. Considering the dynamic aspects of Syk activity by phosphorylation/dephosphorylation observed in other cells , the effThere is another line of evidence for the conclusion that PKC inhibition enhances Syk and Syk substrate tyrosine phosphorylation with the possible involvement of Syk S297 phosphorylation. We recently showed that both cAMP- and cGMP-elevating platelet inhibitors , enhance Syk tyrosine phosphorylation/Syk activity . Now, weOur study demonstrates the regulation of Syk S297 phosphorylation in a primary human cell, the platelet, in response to several agonists such as ADP, cvx, and EB . IncreasITAM-and/or tyrosine phosphorylation-dependent Syk activation occurs at the membrane and alters the interaction of the various Syk domains, releasing the autoinhibition and producing an active kinase. Although there is functional and structural information that the interdomain-B with its phosphorylation and various interacting proteins is essential for the transition of inactive to activated Syk, the exact molecular mechanism(s) for this transition inside a cell are unknown. A major part of the crucial interdomain-B (amino acids 262–337) was unfortunately not defined in the otherwise impressive crystal structure of full-length inactive Syk , perhapsThe role of Syk phosphorylation including serine 297 (S291 in murine Syk) was extensively investigated by two groups in the chicken B-cell model system DT40, but discrepant functional data were obtained ,24. WithEnhanced Syk tyrosine phosphorylation mediated by downregulation of Syk S297 phosphorylation through PKC inhibition is very likely due to decreased tyrosine dephosphorylation. There are several possible mechanisms via which Syk S297 phosphorylation could regulate this.(a)Syk S297 phosphorylation could alter the structure/properties of Syk, which could make Syk a better substrate for tyrosine phosphatases .(b)Syk S297 phosphorylation could recruit adapter proteins to this site , which could decrease Syk translocation to ITAM proteins and, therefore, diminish Syk tyrosine phosphorylation/activation.(c)PKC could phosphorylate and activate TULA-2/other tyrosine phosphatases resulting in enhanced Syk tyrosine dephosphorylation. However, there is no evidence for PKC-mediated phosphorylation and activation of TULA-2 .(d)PKC could regulate other proteins, which affect Syk phosphorylation/dephosphorylation.It is also possible that there are cell-specific mechanisms of Syk regulation, perhaps due to different binding/adapter proteins. These aspects should be investigated with platelets in the future. Our data with human platelets suggest that ITAM-dependent activation of Syk tyrosine phosphorylation is limited by Syk-dependent, PKC-mediated increased S297 phosphorylation, which can be prevented by PKC inhibition. This may represent a feedback inhibition important to prevent hyperactivation of Syk. Feedback inhibition is often observed with receptor-dependent signaling including tyrosine kinases. For example, PKC-stimulated phosphorylation of the epidermal growth factor receptor (EGFR) was reported to represent negative feedback inhibition of this signaling by converting the active EGFR dimer back to the inactive monomer . Of specConsidering the central role of Syk in immune cells and platelets, its established role in many disease processes including myeloid malignancies ,49,50, aClinically used Syk inhibitors not only target Syk but also other tyrosine protein kinases. A clear direction of further development would be to find more specific Syk inhibitors, perhaps inhibitors, which do not directly target the kinase domain but a Syk-specific regulatory/allosteric domain such as the interdomain-B. Another problem with clinically used protein kinase inhibitors and also Syk inhibitors is the emergence of resistance, which was very recently addressed in a large genomic screen in acute myloid leukemia (AML) . This scAll healthy donors gave their informed consent for inclusion before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the local Ethics Committee of the University Medical Center Mainz .g for 10 min at room temperature (RT). The platelet pellet was resuspended directly with HEPES buffer . Human washed platelets were adjusted to 3 × 108 platelets/mL and kept at 37 °C prior to stimulation under stirring conditions with the corresponding platelet agonist or activator in the presence or absence of vehicle control or effectors.Whole blood was collected in citrate tubes from healthy volunteers who did not take any medication at least 10 days before the experiments. Platelet washing and the isolation procedure were performed as previously described . A modif−1) at 37 °C. Platelets were pre-treated with different effectors prior to stimulation: Src family kinase inhibitor PP2 , Syk inhibitors OXSI-2  and PRT-060318 , PKC pan-inhibitor GF109203X , or cAMP-elevating agent iloprost .Aggregation of washed human platelets was monitored by light transmission aggregometry (LTA) using an Apact4S Plus aggregometer . Platelets were stimulated by ADP , convulxin , echicetin beads (EB) , or v/v) glycerol, 6% (w/v) SDS, 0.06% (w/v) bromphenol blue, 1:10 β-mercaptoethanol) directly in the aggregation cuvettes to stop platelet responses, then boiled at 95 °C for 10 min under gentle shaking. Platelet proteins were separated by electrophoresis using 8% SDS-polyacrylamide gels followed by immunoblotting as previously described [Platelet samples for Western blot analysis were prepared by adding 3× Lämmli buffer  were used diluted 1:1000 in 5% BSA or 1:700 for MARCKS. Blots probed with phospho-antibodies were stripped and reprobed with a corresponding antibody detecting the total protein, anti-Syk, or anti-PLCγ2 , or other loading controls anti-β-actin or anti-α-actinin  diluted 1:1000 in 5% BSA. After incubation with the appropriate secondary antibody, horseradish peroxidase (HRP)-conjugated goat anti-rabbit and anti-mouse IgG  enhanced chemiluminescence (ECL) detection was performed using Fusion FX7 .v/v) glycerol, 6% (w/v) SDS, 2% (w/v) bromphenol blue, 1:10 β-mercaptoethanol) in the absence of EDTA. Samples were boiled at 95 °C for 10 min under gentle shaking. Platelet proteins were separated by electrophoresis using gels containing 6% w/v polyacrylamide, 35 µM phos-tagTM compound, and 69 µM ZnCl2 in the separating gel. Then, 1× phos-tag running buffer was used for gel electrophoresis  SDS, 5 mM sodium bisulfite, pH 7.8). Prior to protein membrane transfer, gels were washed twice for 10 min with 1× transfer buffer containing 1 mM EDTA to remove the Zn2+ ions. A third washing step was performed with 1× transfer buffer without EDTA before the proteins were transferred to polyvinylidene difluoride (PVDF) membranes using a transfer buffer for phos-tag  methanol, 5% (w/v) SDS, pH 8.4).The phos-tag method was developed and described by the group of Koike ,32 and uImmunoblotting was performed as previously described . Mouse m8 platelets/mL) were stimulated with convulxin or EB in the absence or presence of effectors at 37 °C under stirring conditions, lysed after 5 min of activation, and centrifuged at 16,000× g for 10 min at 4 °C. InsP1–HRP conjugate and anti-InsP1 monoclonal antibody were pre-incubated with platelet lysates for 3 h prior to measurement, and InsP1 was quantified according to the manufacturer’s instructions.Accumulated inositol monophosphate (InsP1), reflecting the produced inositol triphosphate (InsP3) upon platelet stimulation, was measured by using the IP-One ELISA kit  in the presence of LiCl (1 mM), which prevents the degradation of InsP1 into myo-inositol. Washed platelets (3 × 108 platelets/mL) were loaded for 30 min at 37 °C with 5 µM Fluo-3 acetoxymethyl (AM) esters , a Ca2+ indicator dye with excitation/emission of 506 nm/526 nm for the Ca2+-bound form. Intracellular Ca2+ was monitored as mean fluorescence intensity for 2 min detected by flow cytometry  after stimulation with cvx or EB, without supplementation of extracellular Ca2+ and in the absence or presence of effectors. Data were analyzed by FACSDiva software v6.1.3  expressing the ratio of mean fluorescence intensity over time of treated platelets vs. basal platelet condition.Washed platelets . Statistical analysis was performed using GraphPad Prism 8 . One-way or two-way ANOVA, followed by Tukey’s multiple comparison test for comparison of more than two groups, or the two-tailed Student’s