Na) play an important role in the pathogenesis of cardiac arrhythmias and may also contribute to the development of cardiomyopathies. We have recently demonstrated a critical role for the regulation of the voltage-gated sodium channel NaV1.5 in the heart by the serum and glucocorticoid regulated kinase-1 (SGK1). Activation of SGK1 in the heart causes a marked increase in both the peak and late sodium currents leading to prolongation of the action potential duration and an increased propensity to arrhythmia. Here we show that SGK1 directly regulates NaV1.5 channel function, and genetic inhibition of SGK1 in a zebrafish model of inherited long QT syndrome rescues the long QT phenotype. Using computer-aided drug discovery coupled with in vitro kinase assays, we identified a novel class of SGK1 inhibitors. Our lead SGK1 inhibitor (5377051) selectively inhibits SGK1 in cultured cardiomyocytes, and inhibits phosphorylation of an SGK1-specific target as well as proliferation in the prostate cancer cell line, LNCaP. Finally, 5377051 can reverse SGK1’s effects on NaV1.5 and shorten the action potential duration in induced pluripotent stem cell (iPSC)-derived cardiomyocytes from a patient with a gain-of-function mutation in Nav 1.5 (Long QT3 syndrome). Our data suggests that SGK1 inhibitors warrant further investigation in the treatment of cardiac arrhythmias.Alterations in sodium flux (I Disorders of sodium flux (INa) have recently been shown to play an important role in the pathogenesis of cardiac arrhythmias in both acquired and inherited arrhythmia syndromes2. Notably, mutations in the SCN5a gene that encodes the primary cardiac voltage-gated sodium channel, NaV1.5, cause multiple inherited arrhythmia syndromes, including long QT syndrome 3 (LQT3), atrial fibrillation, and conduction disorders3. Interestingly, alterations in INa have also been observed in heart failure (HF), and may contribute to HF progression5.Sudden cardiac death (SCD) is a leading contributor to mortality in the United States. With a current incidence of 180,000 to 450,000 per yearNa may therefore have a role in the treatment of arrhythmias associated with primary inherited channelopathies, as well as acquired heart diseases. Despite some success in using anti-arrhythmic drugs to modulate SCD risk in either animal or small human studies7, attempts to target INa have not effectively been translated to the wider population, in part due to the pro-arrhythmic and negative inotropic side effects of many anti-arrhythmic agents. Consequently, treatment of SCD in high risk patients relies on the implantation of internal cardiac defibrillators (ICDs), an invasive procedure associated with significant cost and potential morbidity8. Therefore, there is a clear unmet need for novel therapeutic approaches to the treatment of arrhythmias in these disease populations.Medications that reverse abnormalities in INa in the heart4. Of the three known isoforms, SGK1 and SGK3 are predominately expressed in the heart9. Unlike the related kinase Akt1, SGK1 is an important regulator of Na+ and K+ channels11. Prior studies have implicated SGK1 in the regulation of the epithelial Na+ channel, voltage-gated K+ and Na+ channels, and other ionic transporters mostly by regulating their trafficking to the cell membrane15. Notably, SGK1 appears to be activated predominantly in pathological conditions16, and inhibition of SGK1 either by germ-line ablation17 or by dominant-negative genetic inhibition does not cause a noticeable phenotype under basal conditions, but appears to be protective against pathological stress4. In contrast, chronic SGK1 activation in cardiomyocytes leads to a marked alteration in the sodium flux (but not of K+ or Ca2+ fluxes), prolongation of action potential duration (APD) in cardiomyocytes (CMs) and a markedly increased propensity for lethal ventricular arrhythmias4. These studies raise the possibility that small molecule inhibitors would be anti-arrhythmic in cardiac diseases through correction of abnormal INa while having little or no adverse consequences in normal hearts.We have recently identified the serum and glucocorticoid-regulated kinase-1 (SGK1) as an important regulator of I18. Because of increased survival there has been a growing recognition that many traditional and advanced chemotherapeutics can adversely affect the cardiovascular system resulting in significant clinical complications19. More recently, the specific inhibition of key protein kinases has ushered in a new era of targeted therapy for oncology as dysregulation of protein signaling pathways are often associated with cancer progression. However, these therapies also have specific cardiac toxicities because many of signaling pathways targeted in cancer also are important in cardiac function and growth20. There now exists an enhanced awareness of the negative effects of chemotherapeutics on the heart, and the need for therapies that treat cancer while limiting cardiac side effects. Notably, SGK1 is in the rare category of kinases, whose inhibition may be of benefit both in heart disease and cancer.Over the past two decades, significant advances in targeted therapies have greatly changed the natural history of patient outcomes in many different cancersin silico screening, we identified a novel class of inhibitors for SGK1 to use as pharmacological tools to test the hypothesis that SGK1 inhibition can decrease INa, shorten action potential duration and thereby rescue phenotypes associated with prolonged repolarization in CMs. Our results demonstrate proof of concept for SGK1 inhibition as a therapeutic target for cardiac arrhythmias and identify a small molecule lead, that with further medicinal chemistry can form the basis for novel and specific SGK1 inhibitors.Using computational design and Na4, we sought to identify small molecule SGK1 inhibitors for use as pharmacological probes to validate SGK1 as a therapeutic target in the treatment of cardiac arrhythmias. Using a combination of structure-based and ligand-based virtual screens (SBVS and LBVS) to query a reference library for small molecule inhibitors of SGK1, we clustered compounds based on previously validated metrics of activity (using a filtered library for Lipinski properties (rule-of-5)21). From a collection of ~500,000 small molecules meeting our initial activity requirements, we identified ~1000 compounds using requisite structure-function details extracted from other benchmarked kinase inhibitors.Based on previous data that SGK1 regulates Iactive SGK1 determined by X-ray crystallography or NMR spectroscopy at the time of the screening, we used comparative protein modeling to build a three-dimensional model for SGK1 based on known crystal structures of related proteins with acceptable sequence homology and protein folds. The model was later updated with the SGK-1 crystal structure (PDB code: 2R5T) and conformations of ‘activation loop’ sampled. This model -dione) for the second generation screen.After validating the reproducibility and dynamic range of the nd generation screen yielded an additional 64 compounds that were again assessed using the in vitro kinase assay to exclude chemicals with poor SGK1 inhibitory activity. 9-point dose response curves on selected inhibitors that showed SGK1 inhibition at 20 μM dosage, identified several potential lead compounds  to be 2.1 μM  and false-positive  ligand-based pharmacophore models in an iterative discovery process. The chemotypes identified using the LVBS in our second-generation screen were filtered as detailed in the methods section to exclude chemicals that do not dock within the ATP-binding site of the SGK1 model. This 2nds Fig. . Based o μM Fig. .22. Cells were infected with either an adenovirus expressing a constitutively active form of SGK1 (Ad.SGK1-CA) or a control adenovirus (Ad.GFP). At the time of infection, cells were also treated with various concentrations of SGK1 inhibitors 5377051 and 6410136, two of the molecules identified from the second-generation CADD-generated screen and validated as SGK1 inhibitors in the in vitro kinase assay. Cell lysates were subjected to immunoblotting with an antibody against phospho (ser38)-glycogen synthase kinase beta (p-GSK3β), a well-established SGK1 substrate 48 hours after adenoviral infection. As expected, Ad.SGK1-CA caused an increase in p-GSK3β, which was inhibited by both 5377051 and 6410136 at sub-micromolar concentrations, suggesting inhibition of SGK1 activity in cardiomyocytes 23, we next wanted to determine whether our putative SGK1 inhibitors would affect Akt signaling. NRVMs were infected with an adenovirus expressing constitutively active Akt124 (Ad.myr-Akt) or a control adenovirus, and treated with the inhibitors as above. We again assessed levels of p-GSK3β, which is a common substrate for Akt and SGK1. While infection of NRVMs with Ad.myr-Akt caused the expected increase in phosphorylated GSK3β, incubation with either 5377051 or 6410136 failed to significantly inhibit Akt activity even at concentrations of 50 μM , which is involved in stress responses and cell growth28. As expected, addition of R1881 led to a significant increase in phosphorylated NDRG1 (1.7 ± 0.2 fold p < 0.05). LNCaP cells transfected with SGK1-DN plasmid showed no increase in phosphorylation of NRDG1 after R1881, confirming that this effect is SGK1-dependent. The R1881-mediated increase in NRDG1 phosphorylation was also abrogated by treatment with 5377051 at concentrations as low as 500 nM , which was inhibited with increasing concentrations of compound 5377051  activity in a heterologous expression system.We had previously shown that transgenic mice with chronic activation of SGK1 in the heart had an increased propensity to ventricular arrhythmias, and that this was likely due to a marked alteration in I29, encoding NaV1.5, (referred to as HEK-NaV1.5) were transiently transfected with a plasmid containing either a constitutively-active form of SGK1 , a dominant negative form of SGK1  or a vector expressing GFP only. Cells were studied by whole-cell voltage clamp 24–48 hours after transfection.HEK 293 cells stably expressing the SCN5a geneNa, while expression of SGK1-DN led to a decrease in sodium current density  shift in steady state activation and inactivation  that drives sodium flux in these cells  also abolished the hyperpolarization shifts in the activation and inactivation curves described above (data not shown). Interestingly, mutation of the C-terminal residue threonine to alanine (T1590A), effectively preventing the phosphorylation by SGK1 at that site, dramatically reduced the SGK1-induced increase in sodium flux   Fig. .Figure 5Na. There was no significant reduction in peak current of cells expressing the T1590A mutant channels when measured at a time point where there was more than 70% inhibition of wild-type NaV1.5  and ventricular arrhythmias32. Importantly, the increased arrhythmogenesis in the transgenic mice with SGK1 activation in the heart, could be attributed to an increase in INaL4. However, the HEK-NaV1.5 cells do not demonstrate appreciable INaL, likely due to the fact that the full voltage-gated sodium channel macro-molecular complex is not effectively reconstituted in this heterologous expression system. We therefore assessed the effect of pharmacological inhibition of SGK1 on native INaL in cultured primary cardiomyocytes (NRVMs).Multiple lines of evidence have demonstrated that the late sodium current INa  fish is a unique model of action potential prolongation due to a mutation in the zebrafish homologue of the KCNH2 channel recapitulating human LQT2 syndrome33. The phenotypic manifestation of the prolonged QT interval in the bkd−/− fish is the presence of functional 2:1 atrioventricular (AV) block (as is sometimes seen at in human neonates with LQTS as a consequence of prolonged repolarization and effective refractory period34), and previous high throughput experiments have taken advantage of this easily quantifiable phenotype to screen for compounds that shorten the action potential duration (APD) and ‘rescue’ the 2:1 AV block  at the 1–2 cell stage and scored for the presence or absence of 2:1 block at 99 hours post fertilization. The SGK-1 morpholino rescued 58% of mutants, compared with 5% spontaneous reversion in control-scrambled morpholino injections  for 24 hours with compound 5377051 resulted in effective rescue of 2:1 AV block in a dose-dependent fashion  of hiPSC-derived cardiomyocytes (hiPSC-CMs) from a normal patient as measured by the fluorescent voltage sensor Arclight39  that offer a unique model to study inherited arrhythmia disorderst39 Fig. . In contype Fig. . These dNa has been implicated in both inherited and acquired arrhythmia syndromes, and plays a causative role in the etiology of some ventricular arrhythmias. Here we show that SGK1 is an important regulator of INa, and that SGK1 inhibition represents a therapeutic strategy for the treatment of certain cardiac arrhythmias.Alteration of Iin vivo as an anti-hypertensive25. However, SGK1 inhibition as a novel therapeutic approach to modulating INa has not been previously explored to our knowledge. Using rational drug design along with directed screening, we have identified a novel scaffold for SGK1 inhibition that retains specificity for SGK1 over the closely related kinase Akt1. The compound 5377051 appears to inhibit SGK1 effectively and specifically in two different cell types, making it unlikely that our results are due to off-target effects. We anticipate that derivatives of this compound or other SGK1 small molecule inhibitors may have potential not only as a treatment for arrhythmias in inherited and acquired cardiac disease with abnormalities in INa flux, but may also protect against adverse cardiac remodeling as has been seen with genetic SGK1 inhibition in the heart4. Finally, as most kinase inhibitors used in oncological diseases have adverse cardiovascular effects, SGK1 inhibitors have the unique property of being potentially beneficial in both classes of disease.SGK1 inhibitors have previously been developed in the context of treatment for prostate cancer and one of these inhibitors has also been tested in vitro was less than the potency of other kinase inhibitors, the IC50 in cell-based assays was as little as 1–5 μM with a minimal effect on the related kinase Akt at the same concentrations. While additional refinement through medicinal chemistry will undoubtedly be necessary (in addition to testing against other kinases to gauge off-target kinase inhibition), our lead compound represents a useful tool to validate SGK1 as a relevant target for sodium channel modulation.While the potency of 5377051 V1.5 channel activity, recapitulating many of the effects of persistently active SGK1 on NaV1.5 we had previously reported. These effects were further replicated by acute application of the lead compound 5377051 during whole cell patch-clamp recordings. The time course of inhibition was on the order of several minutes, which would be slow for channel inhibition by a typical pore blocker, but consistent with a post-translational mechanism. In addition, mutation of the putative SGK1 phosphorylation T1590 to alanine significantly altered the sensitivity of the channel to SGK1 inhibition by 5377051, further demonstrating that the effect is not due to direct channel blockade. In contrast to CMs, the dramatic degree of inhibition of INa in HEK cells by either genetic or pharmacological inhibition of SGK1 is likely secondary to a high level of activity of SGK1 in HEK cells because of their epithelial lineage.In this study, our single cell electrophysiology experiments demonstrated the potency, biophysical effects and kinetics of the SGK1 inhibition on Na4, we did not observe the increase in late sodium current in our heterologous expression system in comparison to isolated cardiomyocytes from transgenic mice expressing constitutively active SGK1, likely due to the absence of the associated subunits such as the beta-subunits which can significantly modulate late current levels in HEK cells41 or related to reductions in external sodium necessary to fully clamp the peak current. However, in primary cardiomyocytes, our lead compound selectively inhibited the late sodium current over the peak current. Taken together our data strongly suggest that SGK1 is a potent modulator of the sodium channel and pharmacologic inhibition of SGK1 activity inhibits the late sodium current that is often associated with pathologic conditions4. Our studies provide ‘proof-of-concept’ of SGK1 inhibition as a therapeutic target for treating cardiac arrhythmias.Unlike our prior studiesV1.5 is modulated by multiple post-translational modifications including phosphorylation42 and acetylation43. These modifications can directly affect the biophysical properties of the channel, change the number of available protein complexes at the plasma membrane or modulate associated proteins that can further affect channel function. While traditional ion channel blockers have efficacy in treating certain types of arrhythmias, modulation of ion channel post-translational modification may provide more fine-tuned therapy without the pro-arrhythmic side effects commonly seen in typical ion channel blockers. In support of this, there appears to be a ‘plateau’ effect for SGK1 inhibition in vivo, both in mice with chronic SGK1 inhibition in cardiomyocytes (which do not have shorter repolarization4), and in human iPSC-derived cardiomyocytes from normal patients , pre-filtered for the Lipinski rule-of-five for drug-like properties. Ligand based virtual screen (LBVS) was performed using Sybyl, in which we used 2D and/or 3D conformations of known “ligands” including many of the identified kinase inhibitors (independent of target) on the basis of kinase inhibitory properties to construct multiple 3D pharmacophoric fingerprints to rapidly screen small molecule libraries in silico. The 3D-pharmacophore fingerprint/key we used is an extension of the 2D-pharmacophore models that are commonly used by others44. Briefly, these keys are comprised of functional groups responsible for mediating critical ligand receptor interactions and are represented as potential pharmacophore points (PPPs) in three-dimensional space. These PPPs are comprised of triplets or quartets with a triplet PPP using three pharmacophore points that include requisite distance data and/or quartets that are comprised of four pharmacophore points along with six distance and/or chirality constraints45. High-throughput docking (HTD)-based virtual screening was performed using FlexX and FlexX-ensemble, which consider the conformational flexibility within the target interface, in combination with a powerful incremental construction algorithm that allows the complexity and size of the ligand to be iteratively constructed from a predefined base fragment46. The algorithm used by FlexX is based on the model of molecular interactions defined by Bohm47 and Klebe and is divided into three segments: core or base selection, core placement, and incremental complex construction48. After docking, a variety of scoring functions are used to energetically assess each calculated pose, which take into account forces involved in this interaction as well as the affinity between the interacting atoms. Importantly, we performed cross docking; pose generation validation studies using additional HTD algorithms including: Surflex-Dock (TRIPOS) and/or Glide (Schrödinger) to ensure our hit list was not biased towards a single HTD algorithm/approach.All molecular modeling operations were performed in the SYBYL-X (Certara/Tripos) and/or BIOSOLVEIT  molecular modeling packages, running on Dell Precision 690 workstations with 8 CPUs, 10 GB memory and RHE Linux 5 OS. The chemical libraries employed in the virtual screening primarily included Chembridge (in vitro kinase assay (Upstate). Briefly, this assay utilizes a phosphorylated peptide that is labeled with a green fluorescent dye and increases its fluorescence polarization value upon binding with a phospho-specific antibody. We first validated the reproducibility and dynamic range of the assay using different concentrations of a recombinant GST-purified SGK1 and determined the EC50 of the recombinant kinase to be 1 ng. This assay was then used to evaluate the 1st and 2nd generation small molecule inhibitors identified using our CADD platform. Initial activity screening was done at a concentration of 50 μM to ensure we did not miss compounds with activity and several compounds had demonstrable SGK1 inhibitory potential. These inhibitors were re-screened using an 8 point dosing curve to obtain IC50s for several of the inhibitors, and used for the second round of CADD to generate an additional 64 compounds for further testing in this kinase assay.To screen CADD-generated SGK1 inhibitor candidates, we used a fluorescence polarization-based 6 neonatal rat ventricular myocytes (NRVMs) were plated on 60 mm plates. 24 h after plating, serum was removed and infected with SGK1-CA virus and treated with SGK1 inhibitors. Twelve hours after infection and treatment, the media was changed and cells were treated again. Twelve hours following the second treatment NRVMs were harvested and separated by gel electrophoresis.All experiments and protocols including animal procedures for isolation of cardiomyocytes, were approved by the Institutional Biosafety Committees (IBCs) and institutional IACUCs at both Beth Israel Deaconess Medical Center and Massachusetts General Hospital in accordance with institutional and national guidelines. To investigate the activity of SGK1 in cardiomyocytes, 2 × 10For Western Blotting, 2 million LNCaP (ATTC) cells were plated on 60 mm dishes in RPMI-1640 containing 10% FBS and 1% pen-strep antibiotic. After 24 h plating cell media was replaced with RPMI-1640 containing 10% charcoal stripped fetal bovine serum to decrease androgen interference from the media. Cells were either transfected with SGK-DN or treated with drug . Cells were treated at 0, 12 and 24 h and harvested 32 h after first treatment. Proteins were separated by gel electrophoresis using 50 μg of protein per well. P-NDRG1  was normalized to GAPDH and data presented as fold change from control. For CyQUANT cell proliferation assay (Life Technologies), LNCaP cells were plated at 25,000 cells/well in a 96 well plate in plating media. 24 h after plating, media was changed to RPMI-1640 containing 10% charcoal stripped fetal bovine serum. Cells were treated with either 5377051, R1881 (1 nM) or both every 24 h for 72 h. Cell proliferation was measured using the manufacturer’s protocol.29. To determine the effects of either constitutively (SGK1-CA) active or dominant negative (SGK1-DN) SGK1, respective plasmids were transfected for 4–6 hours with Lipofectamine 3000 (Life Technologies) and recordings were performed the following day. To improve the selection of transfected cells SGK1-CA was fused to mCherry by standard molecular biology techniques. All control cells were transfected with constructs expressing GFP or mCherry alone. All INa recordings (Axon Instruments) were performed at room temperature with patch pipettes of 1.5–2.5 MΩ tip resistance when filled with pipette solution containing (in mM): NaCl 5, CsCl 40, glutamate 80, CsOH 80, Mg-ATP 5, EGTA 5, HEPES 10, CaCl2 1.5 . The bath solution contained (in mM) NaCl 50, MgCl2 2, CaCl2 1.2, NMDG 80, HEPES 10, Glucose 10 (pH 7.4). In all experiments, recordings started 5 min after establishment of the whole-cell mode to permit stabilization of current amplitude, voltage dependence and kinetics of gating. Standard voltage protocols were used for assessment of the voltage-dependence of activation/inactivation, recovery from inactivation. To determine the membrane potential for V1/2 and the slope factor k, steady state inactivation data were fit with a Boltzmann function of the form: I/Imax = {1 + exp[(V − V1/2)/k]} − 1. For late sodium channel recordings, NRVMs were isolated and cultured on laminin-coated glass coverslips. Whole-cell recordings were performed 5 minutes after break-in and the external solution was similar to those used in the recordings of HEK293 cells, 50 mmol NaCl but without NMDG. We performed at least five current recordings per cell with an interval of 30 seconds between recordings, without drug and after bath exchange with the inhibitor (50 μmol/L). Currents before and after drug administration were averaged (n = 7 cells).HEK-293 cells with stably-transfected SCN5a were voltage clamped using the whole-cell patch-clamp configuration as previously described40. Human iPSCs were maintained in Essential 8 media and passaged every 3–5 days as single cells on Geltrex-coated plastic dishes (Invitrogen). Differentiation was performed as described elsewhere by sequential inhibition of GSK3 and the Wnt signaling pathways50. Spontaneously beating CMs between days 20 and 30 were dissociated with 0.25% Trypsin-EDTA (GIBCO), re-plated on Geltrex-coated 96-well plates and maintained in RPMI 1640 medium with B27 complete supplement (Invitrogen) as described previously50.Skin fibroblasts were obtained from a healthy volunteer and reprogrammed into pluripotent stem cells by standard methods. HiPSCs from a patient with LQT3 were previously described50. 5 days after the onset of transduction, CMs were incubated with DMSO or 5377051 (10 μmol/L) for 24 hrs. Cells were then imaged with a Nikon Eclipse Ti-U inverted microscope, a Nikon CFI S Fluor 20X objective, an ORCA-Flash2.8 C11440-10C digital CMOS camera (Hamamatsu) and the MetaMorph Microscopy Automation and Image Analysis Software at 37 °C. ArcLight fluorescence was obtained with a FITC filter set and a SOLA SE light engine for illumination (Lumencor). Images were recorded at 1000 frames per second. ArcLight recordings were analyzed using a custom-written Matlab program. Action potential duration at 80% repolarization (APD80) was calculated to be the time interval between when signals reached 50% of the difference in fluorescence between the initiation and maximal height of an upstroke, and 80% of the return to diastolic baseline. The peak-to-peak interval was used to determine beating rate and all APD80 analyzed was corrected to reflect ADP80 at 1 Hz. Optical AP tracings are presented as −ΔF/F0, where F0 is the resting diastolic fluorescence.Dissociated hiPSC-CMs were transduced with lentivirus carrying pLenti-CMV-ArcLight for 72 hrSupplemental Information


Training beginners of the pedicle screw instrumentation technique in the operating room is limited because of issues related to patient safety and surgical efficiency. Three-dimensional (3D) printing enables training or simulation surgery on a real-size replica of deformed spine, which is difficult to perform in the usual cadaver or surrogate plastic models. The purpose of this study was to evaluate the educational effect of using a real-size 3D-printed spine model for training beginners of the free-hand pedicle screw instrumentation technique. We asked whether the use of a 3D spine model can improve (1) screw instrumentation accuracy and (2) length of procedure.Twenty life-size 3D-printed lumbar spine models were made from 10 volunteers (two models for each volunteer). Two novice surgeons who had no experience of free-hand pedicle screw instrumentation technique were instructed by an experienced surgeon, and each surgeon inserted 10 pedicle screws for each lumbar spine model. Computed tomography scans of the spine models were obtained to evaluate screw instrumentation accuracy. The length of time in completing the procedure was recorded. The results of the latter 10 spine models were compared with those of the former 10 models to evaluate learning effect.p < 0.001). The mean length of time to complete 10 pedicle screw instrumentations in a spine model was 42.8 ± 5.3 min for the former 10 spine models and 35.6 ± 2.9 min for the latter 10 spine models. The latter 10 spine models had significantly less time than the former 10 models (p < 0.001).A total of 37/200 screws (18.5%) perforated the pedicle cortex with a mean of 1.7 mm . However, the latter half of the models had significantly less violation than the former half (10/100 vs. 27/100, A life-size 3D-printed spine model can be an excellent tool for training beginners of the free-hand pedicle screw instrumentation. Pedicle screws are frequently used in spine surgeries, and their use is expected to increase as the number of spinal fusion surgeries is rapidly increasing –4. PedicTraining beginners of this technique through surgical procedures of patients in the operating room is limited because of issues related to patient safety and surgical efficiency . Trainin3D printing technologies are common in product design industries, and their use is increasing in all fields , 20. RecTo our knowledge, no reports have described surgical skill training of the pedicle screw instrumentation technique using a life-size 3D-printed spine model. The purpose of this study was to evaluate the educational effect of using a life-size 3D-printed spine model for training beginners of the free-hand pedicle screw instrumentation technique in improving screw instrumentation accuracy and procedure time.Ten adult patients  with low back pain were enrolled. The mean age of patients was 35.2 years . They were confirmed not to have any congenital abnormalities, other deformities, and instability on the lumbosacral spine by using plain radiographs and CT scan. Patients underwent CT scan from the lower endplate of the 12th thoracic vertebra (T12) and below the 1st sacral vertebra (S1) with a 1-mm thickness slice  that can be used by the 3D printing machine  to produce a life-size lumbar spine model with polypropylene Fig. . Twenty Two residents who had no experience of pedicle screw instrumentation were selected to participate in this study. First, the residents were instructed by an experienced spine surgeon (KJC) on the instrumentation technique of pedicle localization and the method of pedicle screw fixation. Subsequently, each 3D-printed spine model was mounted on the lumbar spine holder  to secure each vertebral body with the L3 vertebra placed most ventrally Fig. . SynthetThe spinal models underwent CT scan immediately after the pedicle screws were inserted to evaluate their instrumentation accuracy Fig. . Screw mU test was used to compare the length of time to complete the screw instrumentation between the former and latter 10 spine models. Logistic regression analysis was performed to evaluate improvement in error rates through the 20 spinal models with respect to the total error rates. Bivariate associations between the vertebral level and the pedicle screw violation were examined by using Spearman’s correlation analysis. Statistical significance was accepted for p values < 0.05.Statistical analysis was performed using SPSS version 23.0  software. Data normality was assessed by the Kolmogorov–Smirnov test. Chi-square test was used to compare the incidence of screw violation of the pedicle between the former 10 (100 pedicles) and latter 10 spine models (100 pedicles). Mann–Whitney p > 0.05).Two-hundred pedicles in 100 vertebral bodies were inserted with 5-mm cylindrical pedicle screws. A total of 37/200 screws (18.5%) perforated the outer cortex of the pedicles with a mean of 1.7-mm violation . Of the 37 perforating screws, 36 (97%) violated the medial side of the pedicle. The first and second residents made 18 (49%) and 19 (51%) violations, respectively, and no significant difference was found between the two . However, blinded CT evaluations of screw placement indicated that 5.5% 11/200), 13% (26/200), and 81.5% (163/200) of screws were in categories C (2- to 4-mm breach), B, and A, respectively. When 20 spine models were divided into two groups (the former and latter 10 spine models), the former 10 spine models had 11% (11/100), 16% (16/100), and 73% (73/100) of screws in categories C, B, and A, respectively. The latter 10 spine models had 0% (0/100), 10% (10/100), and 90% (90/100) of screws in categories C, B, and A, respectively   compared with the former 10 spine models (27/100 pedicle screws) (p < 0.01). Based on the lumbar spine level, violations above and below L3 were 86% (32/37) and 14% (5/37), respectively, and a significant difference was found (p < 0.05).Violations occurred in all levels of the lumbar spine except at L5 Table . The mosp < 0.01). Violations more than 4 mm of the pedicles did not occur.Critical violation (> 2 mm) did not occur in L4 and L5. The most common level of critical violation occurred at L1 with 12.5% (5/40). L2 and L3 had 10% (4/40) and 5% (2/40) violations, respectively. Pedicles in the lower vertebral level had less percent violation compared with higher vertebral level . Later instrumentation of the pedicle screws required less time compared with earlier instrumentation  . Moreover, the length of time required to complete screw instrumentation decreased as residents continued to practice the skills on the 3D-printed models. The mean length of time to complete 10 pedicle screw instrumentations in one spine model was 42.8 ± 5.3 min and 35.6 ± 2.9 min for the former (100 pedicle screws) and latter 10 spine models (100 pedicle screws), respectively. The latter 10 spine models required significantly less time than the former 10 models (p < 0.001). A strong negative relationship was observed between repetitive time of screw instrumentation and length of procedure .Our study has shown that the use of the life-size 3D-printed spine model improves accuracy and length of time to complete pedicle screw instrumentation. Practicing instrumentation of pedicle screws on the 3D-printed spine model has shown a learning effect Fig. . Less vip < 0.01). Pedicles of the lower vertebral level are known to be wider than the higher vertebral level, and this can be the reason for the lower rate of screw violation in the lower vertebral level pedicles. Furthermore, total violation rate (18.5%) in this study on the lumbar vertebra was lower than that (29%) on the thoracic vertebra in different cadaveric studies [In the study, L5 did not show any screw violation compare with 32.5% of total violation in L1. Pedicles in the lower vertebral level showed less percent violation compared with the higher vertebral level (Spearman’s correlation coefficient, − 0.28; 3D printing has a number of applications in medicine, and we propose that this technique can be used for training of pedicle screw instrumentation , 20. Wu Limitation of using 3D-printed models for pedicle screw instrumentation is that the osseous feel may be different from the real pedicle. Various materials can be used for 3D printing, and further evaluation to mimic the osseous feel of the real pedicle may enable replication of the real osseous feeling replicas in the near future.A life-size 3D-printed spine model can be an excellent tool for training beginners of the free-hand pedicle screw instrumentation.


Neutrophils play a critical role in innate immunity and also influence adaptive immune responses. This occurs in good part through their production of inflammatory and immunomodulatory cytokines, in conjunction with their prolonged survival at inflamed foci. While a picture of the signaling machinery underlying these neutrophil responses is now emerging, much remains to be uncovered. In this study, we report that neutrophils constitutively express various Src family isoforms (STKs), as well as Syk, and that inhibition of these protein tyrosine kinases selectively hinders inflammatory cytokine generation by acting posttranscriptionally. Accordingly, STK or Syk inhibition decreases the phosphorylation of signaling intermediates  involved in translational control. By contrast, delayed apoptosis appears to be independent of either STKs or Syk. Our data therefore significantly extend our understanding of which neutrophil responses are governed by STKs and Syk and pinpoint some signaling intermediates that are likely involved. In view of the foremost role of neutrophils in several chronic inflammatory conditions, our findings identify potential molecular targets that could be exploited for future therapeutic intervention. However, the contribution of neutrophils to host immunity extends well beyond their traditional depiction as professional phagocytes specialized in pathogen clearance. It is now widely recognized that neutrophils and their products condition the onset and evolution of both innate immunity and the ensuing immune response , 2. This factors , 12–14.  factors , 10, 15.Whether other kinases also participate in controlling the aforementioned neutrophil responses remains to be explored. In this regard, one of the most immediate events occurring following neutrophil activation is the phosphorylation of cellular proteins by nonreceptor tyrosine kinases such as members of the Src family , as well as Syk –22. AccoWe now report that inhibition of STKs or Syk selectively hinders inflammatory cytokine generation by acting posttranscriptionally. Accordingly, STK or Syk inhibition decreases the phosphorylation of signaling intermediates  involved in translational control. By contrast, delayed apoptosis appeared to be independent of either STKs or Syk. Our data therefore significantly extend our understanding of which neutrophil responses are governed by STKs and Syk and pinpoint some of the likely mechanisms involved.β-actin were from Santa Cruz Biotechnology ; antibodies against Syk, as well as all phospho antibodies, were from Cell Signaling . Ficoll-Paque Plus was from GE Biosciences ; endotoxin-free (<2 pg/ml) RPMI 1640 was from Wisent . Recombinant human cytokines were from R&D Systems , and UltraPure LPS (from E. coli 0111:B4) was from InvivoGen . Dimethyl sulfoxide (DMSO), N-formyl-methionyl-phenylalanine (fMLP), and phenylmethanesulphonyl fluoride (PMSF) were from Sigma-Aldrich . Diisopropyl fluorophosphate (DFP) was from Bioshop Inc. . The protease inhibitors, aprotinin, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF), leupeptin, and pepstatin A, were all from Roche . Kinase inhibitors were all purchased through Cedarlane Labs . All other reagents were of the highest available grade, and all buffers and solutions were prepared using pyrogen-free clinical grade water.Antibodies raised against STK isoforms and 6 cells/ml (unless otherwise stated). As determined by Wright staining and FACS analysis, the final neutrophil suspensions contained less than 0.1% monocytes or lymphocytes; neutrophil viability exceeded 98% after up to 4 h in culture, as determined by trypan blue exclusion and by annexin V/propidium iodide FACS analysis.Neutrophils were isolated from the peripheral blood of healthy donors, following a protocol that was duly approved by an institutional ethics committee. The entire procedure was carried out at room temperature and under endotoxin-free conditions, as described previously . Purifie3VO4, and 10 mM Na4P2O7). For whole-cell samples, boiling 2X sample buffer was added directly to cell pellets, which were briefly vortexed and placed in boiling water for a further 5 min. Samples thus prepared were sonicated to disrupt chromatin and stored at −20°C prior to analysis. For subcellular fractions, cells were resuspended in relaxation buffer prior to disruption by nitrogen cavitation, as described previously [Cells were incubated at 37°C in the presence or absence of stimuli. Incubations were stopped by adding equivalent volumes of ice-cold PBS supplemented with DFP  and phosphatase inhibitors .Neutrophils were cultured in 24-well plates at 37°C under a 5% CO5 cells) were washed twice in ice-cold PBS containing 5 mM EDTA, then once more in cold PBS, and incubated on ice for 15 min with FITC-conjugated annexin V. Cells were then counterstained with propidium iodide and analyzed  on a Cytoflex instrument (Beckman Coulter) using the CytExpert software.After the desired culture period, neutrophils .All data are represented as the mean ± SEM of at least three independent experiments. Statistical differences were analyzed by Student's Previous studies have established that at least three STK isoforms are present in neutrophils , as well as Syk –22. To gα, fMLP, or GM-CSF, with the notable exception of TNF-elicited CXCL8 release, which was consistently unaffected by Syk inhibition. The release of another chemokine, CCL4, was likewise hindered by all inhibitors tested in fMLP- and GM-CSF-stimulated cells; in LPS- or TNF-stimulated neutrophils, however, CCL4 release was only modestly affected by Syk or STK inhibitors .Whereas Src family and Syk tyrosine kinases play an important role in various classical functions of neutrophils, such as phagocytosis, ROS production, and leukotriene synthesis , the potα, fMLP, or GM-CSF , whereas both GM-CSF and LTB4 potently countered the spontaneous apoptotic rate, as expected (p = 0.04) but not that of GM-CSF , with the exception of TNF-induced CXCL8 release, which appeared to occur independently of Syk. Similarly, CCL4 secretion was hindered by STK and Syk inhibitors in fMLP- and GM-CSF-activated neutrophils but was only moderately unaffected by the inhibitors in LPS- or TNF-stimulated cells. Thus, depending on the cytokine and the stimulus, inflammatory cytokine generation can be controlled by STKs or Syk in neutrophils. These tyrosine kinases appeared to act posttranscriptionally, since the induction of inflammatory cytokine gene expression was unaffected by STK or Syk inhibition. Accordingly, several signaling intermediates known to affect cytokine translation were found to be under the control of STKs and Syk. Among these downstream targets, MNK1 is particularly relevant, as we recently showed it to participate in the translational regulation of cytokine generation in neutrophils [We first found that STK or Syk inhibition strongly represses the secretion of CXCL8 elicited by physiological neutrophil agonists . More importantly, we uncovered some of the likely mechanisms involved in the translational control of cytokine generation by STKs and Syk. In view of the foremost role of neutrophils in several chronic inflammatory conditions, our findings identify potential molecular targets that could be exploited for future therapeutic intervention.Figure S1. Densitometric analyses of Figure 4 data. §, p≤0.01 vs unstimulated cells; ∗, p<0.05 vs matched condition without inhibitor; ∗∗, p<0.01 vs matched condition without inhibitor.


We found that gabapentin significantly reduced PKCε translocation induced by the pronociceptive peptides bradykinin and prokineticin 2, involved in both inflammatory and chronic pain. We recently showed that paracetamol (acetaminophen), a very commonly used analgesic drug, also produces inhibition of PKCε. We tested the effect of the combined use of paracetamol and gabapentin, and we found that the inhibition of translocation adds up. Our study provides a novel mechanism of action for gabapentin in sensory neurons and suggests a mechanism of action for the combined use of paracetamol and gabapentin, which has recently been shown to be effective, with a cumulative behavior, in the control of postoperative pain in human patients. Gabapentin is a well-established anticonvulsant drug which is also effective for the treatment of neuropathic pain. Although the exact mechanism leading to relief of allodynia and hyperalgesia caused by neuropathy is not known, the blocking effect of gabapentin on voltage-dependent calcium channels has been proposed to be involved. In order to further evaluate its analgesic mechanisms, we tested the efficacy of gabapentin on protein kinase C epsilon (PKC Gabapentin, a structural analogue of the inhibitory neurotransmitter gamma-aminobutyric acid, was originally developed in the early nineties as a third-generation antiepileptic drug. Besides epilepsy, gabapentin has been reported to be effective for the treatment of other pathological states such psychiatric and movement disorders, alcohol addiction, and restless leg syndrome . In partParacetamol is a nonprescription analgesic/antipyretic drug with little anti-inflammatory activity and partial inhibition of COX-1 and COX-2 , 5, of wε) is a very important noxious effector [ε is involved in both inflammatory and neuropathic pain states [ε of specific cellular targets located in the plasma membrane in nociceptive neurons [ε specific inhibitors at the site of nociceptive testing significantly inhibited pain in several models of peripheral hyperalgesia [ε induced in cultured sensory neurons by bradykinin and thrombin, inflammatory mediators that sensitize TRPV1 to heat stimuli and to capsaicin [ε inhibition of translocation in DRG neurons.The epsilon isoform of protein kinase C . Cells were cultured in DMEM in the presence of 1.5 μg/mL cytosine 1-D-arabinofuranoside , 10% fetal bovine serum, 1% penicillin/streptomycin, and 1% L-glutamine , and 100 ng/mL NGF  was also added as described previously, in order to improve neuronal survival and bradykinin and PK2 receptor expression, as it is common practice with this preparation [Sprague-Dawley rats (2–6 weeks old) were sacrificed under total anaesthesia according to Italian and European legislation, with protocols in agreement with the guidelines of the Committee for Research and Ethical Issues of IASP published in PAIN®, 16 (1983) pp. 109-110. Experimental protocols were also approved by local institutional animal care and use committee. Dorsal root ganglia (DRGs) were collected, incubated for 1 h at 37°C with 0.125% collagenase , mechanically dissociated, and then plated onto glass coverslips precoated with 10 paration , 26, 27.ε from the cytoplasm to the plasma membrane was visualized as previously described [μM concentration or to PK2 (100 nM) for 30 s and fixed for 10 min at room temperature with paraformaldehyde . Cells were pretreated for different times and different concentrations of gabapentin (see Results) alone or in combination with paracetamol at different concentrations (see Results). Water was used to prepare stock solutions for gabapentin, while DMSO was used for paracetamol. Final concentration of DMSO applied to cells was 1 : 1000, equal in all experiments. The stimulation with PK2 or bradykinin contained the same concentrations of drugs preapplied. Fixed cells were washed three times in PBS (with 0.1% fish skin gelatin to block nonspecific sites), permeabilized for 30 min at room temperature with Triton X-100 , and incubated overnight at 4°C with a polyclonal anti-PKCε antibody [ε following activation of bradykinin or prokineticin receptors (PKRs) resulted in translocation from the cytoplasm to the neuronal cell membrane, as shown several times before [Translocation of PKCescribed , 19, 23.antibody  diluted s before . Translos before ) using st-test for multiple comparison. An effect was determined to be significant if the P value was below 0.05.Data were analyzed by one-way analysis of variance (ANOVA), followed by Bonferroni's ε activation was quantified in an all-or-nothing fashion as the number of neurons in which translocation is observed , as previously observed [As described in observed , 20, inhCombined application of gabapentin and paracetamol resulted in summation of the respective effects, with a reduction from the control value to 16.2 ± 0.3% and to 13.2 ± 0.4%, respectively, in experiments with bradykinin and with PK2.In order to further investigate the nature of the apparently additive behavior of gabapentin and paracetamol effect, we performed a full isobolographic analysis, which confirmed an additive behavior, with no sign of synergistic interaction .ε and γ PKC isoforms in a visceral pain model [ε inhibition of translocation occurs, but a contribution of presynaptic PKCε, present in the central terminal of the sensory neuron, consistent with the one we report here, is largely possible. The γ isoform of PKC (PKCγ) is not expressed in dorsal root ganglia [γ can only occur downstream of the sensory neuron, or in surrounding nonneuronal cells. In this context, our paper contributes the first demonstration of PKC-involving antinociceptive functions of gabapentin in the therapeutic range of concentrations in the peripheral nervous system, by providing the first direct evidence that a dose-dependent, significant effect is produced by gabapentin with a mechanism that involves specific inhibition of translocation of the ε PKC isoform in sensory neurons. This mechanism is likely a relevant part of gabapentin antinociceptive action, as the lack of translocation will prevent sensitization of membrane targets in the significant percentage of sensory neurons where complete inhibition occurs.Gabapentin, paracetamol, nonsteroid anti-inflammatory drugs (NSAIDs), selective COX-2 inhibitors, and glucocorticoids are nonopioid drugs which display clinically relevant analgesic properties. In postoperative pain management, patients are very often treated with combinations of the above nonopioid analgesics to reduce opioid-related adverse effects. Although a variety of combinations have recently been tested and although some of them are currently employed in clinical practice , the ratin model . The wes ganglia ; therefoε translocation by gabapentin and by paracetamol appears to be highly relevant for a proper understanding of the multifactorial pharmacological actions of these drugs. Inhibitory effects by gabapentin and paracetamol were largely similar both in bradykinin- and in PK2-induced translocation, suggesting that these drugs do not specifically interact with bradykinin and PK2 membrane receptors but rather affect mechanisms of translocation intracellular pathway, possibly directly on PKCε itself, or on the interaction of PKCε with the receptors for the specific activated protein kinase C isoform, RACKs [ε are additive suggests that the overall inhibition of translocation is likely obtained with one or perhaps two different molecular mechanisms activated by each drug, converging on the same downstream action on PKCε, which are present in specific subsets of neurons. Sensory neurons are well known to represent a highly heterogeneous population, with different morphological, neurochemical, and biochemical properties [The present study shows also that the effect of gabapentin combined with paracetamol, in comparison to each drug alone, adds to the effect of each other. This observation is consistent with a previous study on acute postoperative pain, demonstrating that the combination of gabapentin and paracetamol reduced morphine self-administration in a randomized and double-blinded manner in human patients suffering from postoperative pain . Althougm, RACKs . The facoperties . The obsoperties  and the operties , 39, 40)operties .ε translocation and whether their combined effects are additive or possibly synergistic are currently in progress in our labs.More work is necessary to determine the nature of mechanisms preventing translocation in these specific subsets of neurons. Further experiments that will determine whether other novel or classic antinociceptive drugs currently in use, as well as paracetamol and gabapentin, produce part of their effect via inhibition of PKCIn conclusion, our results help to clarify the pharmacological action of largely used drugs such as gabapentin and paracetamol and of their combined use. From these results, in combination with data in the literature, gabapentin emerges as a drug with multifactorial modes of action on the pain pathway, with an increasing number of novel, interesting mechanisms not only in the central but also in the peripheral nervous system.


We have recently reported that, unlike IL-5 and GM-CSF, IL-3 induces increased translation of a subset of mRNAs. In addition, we have demonstrated that Pin1 controls the activity of mRNA binding proteins, leading to enhanced mRNA stability, GM-CSF protein production and prolonged eosinophil (EOS) survival. In this review, discussion will include an overview of cap-dependent protein translation and its regulation by intracellular signaling pathways. We will address the more general process of mRNA post-transcriptional regulation, especially regarding mRNA binding proteins, which are critical effectors of protein translation. Furthermore, we will focus on (1) the roles of IL-3-driven sustained signaling on enhanced protein translation in EOS, (2) the mechanisms regulating mRNA binding proteins activity in EOS, and (3) the potential targeting of IL-3 signaling and the signaling leading to mRNA binding activity changes to identify therapeutic targets to treat EOS-associated diseases. Control of protein production is critical for the maintenance of cell and tissue homeostasis. Excessive protein production may lead to hypertrophy and an unnecessary use of energy and other resources. However, inadequate protein synthesis antagonizes cell growth, proliferation, adaptation to environmental changes, and the implementation of new cell functions. Overproduction of transcription factors or cytokines contributes to or causes transformation and cancer. Thus, a carefully controlled balance within metabolic constraints but responsive to environmental and signaling cues is essential for optimal cellular function.via cell surface and intracellular receptors leading to the propagation of intracellular signals control each of these steps [reviewed in Ref. (Circulating eosinophils (EOS) are differentiated, non-proliferative cells, which become apoptotic within 2–3 days if lacking contact with pro-survival cytokines, such as IL-5, GM-CSF, and IL-3 . TherefoEosinophils have the ability to differentially regulate translation. As shown in Figure initiation begins with the binding of eukaryotic translation initiation factor 4E (eIF4E) to the mRNA 5′-cap. Next, eIF4E binds to eIF4G, which interacts with the other eIF4 proteins, eIF4A and eIF4B. The helicase activity of eIF4A is amplified by eIF4B, and most likely unwinds secondary GC-rich structures of the 5′-UTR, thus facilitating initiation of mRNAs possessing these structures. The interaction of eIF4G with the poly-A binding protein (PABP), which circularizes the mRNA, also increases mRNA translatability. The binding of eIF4B and eIF4G to the 43S preinitiation complex (PIC) via eIF3 links the mRNA to the ribosome. The 43S PIC is composed of the ribosomal 40S subunit, eIF3, eIF5 eIF1, eIF1A, and the complex eIF2/Met-tRNA. EIF2 binds Met-tRNA in its GTP-bound state (eIF2-GTP). The complex Met-tRNA/eIF2-GTP along with the initiation factors/40S complex scans the 5′UTR until the start codon (AUG) is recognized by complementarity with the anticodon of Met-tRNA (Elongation is predominantly controlled by eukaryotic elongation factor 1 (eEF1) and eEF2. Next, eEF1A-GTP recruits the second aminoacyl (aa)-tRNA complementary to the adjacent, C-terminal codon (A-site). After the peptide bound formation between Met and the second aa at the P-site, eEF2-GTP pushes (translocates) the mRNA and allows the third aa-tRNA to become positioned on the third codon at the A-site. Simultaneously, the first Met-tRNA is removed from the P-site and is replaced by the second aa-tRNA previously on the A-site. When the ribosome reaches a stop codon, no complementary tRNA exists to fill the A-site. At that point, the release factor ERF1 (ETF1) takes position in the A-site, and along with ERF3A-B (GSPT1–2) hydrolyzes the peptide chain (protein) attached to the last t-RNA to terminate translation.In eukaryotic cells, initiation, elongation, and termination are the three fundamental steps of protein translation. Some of the main proteins/mRNA interactions involved in the initiation and elongation of translation are shown in Figure Met-tRNA . ElongatIn general, in eukaryotic cells, initiation can be controlled at multiple levels. The eIF4BP proteins (4E-BP) interact with eIF4E, preventing its interaction with eIF4G and, therefore, inhibiting translation initiation . 4E-BP aMKNK1 and 2), which are downstream targets of the MAPK (ERK and p38) (Eukaryotic translation initiation factor 4E (eIF4E) can be phosphorylated on Ser209, which decreases its affinity for the 5′-cap structure and, therefore, inhibits translation , 11. eIFand p38) , 11. Alsand p38) .When the inactive form of eIF2, eIF2-GDP, leaves the 40S during initiation, it must be recharged with GTP for continuous translation initiation. Then, eIF2-GDP is converted to eIF2-GTP by eIF2B, which is a guanine-nucleotide-exchange factor (GEF). eIF2B is phosphorylated at multiple sites that includes two residues phosphorylated by casein kinase 2 (CSNK2A1) that are required for eIF2B/eIF2 interactions, eIF2 recycling and translation initiation . eIF2 iselongation is driven by the hydrolysis of eEF1A-GTP to eEF1A-GDP. Thus, the GEF eEF1B acts on eEF1A-GDP as eIF2B does on eIF2-GDP. eEF1 is also targeted by a variety of kinases, including PKC, CSNK2, and cyclin-dependent kinase 1 (CDK1), but the role of the phosphorylation states of these elongation factors remains uncertain . In addition, 4,802 site-specific phosphorylation events were simultaneously identified /Akt/mTOR and the MAPK pathways. These two pathways are generally triggered by extracellular stimuli 2) into phosphatidylinositol-3,4,5-triphosphate (PIP3), by the class I lipid kinase PI3K. This transformation into PIP3 is reversed by phosphatases such as the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and the SH2-domain containing inositol phosphatase (SHIP) (via 3-phosphoinositol-dependent kinase 1 (PDK1) (Ligation of growth factors with tyrosine kinase or G-protein coupled (GPC) receptors typically leads to phosphorylation of the membrane phospholipid, phosphatidylinositol-4,5-biphosphate  . PIP3 dr1 (PDK1) . Akt act1 (PDK1) . Akt can1 (PDK1) . In addi1 (PDK1) , 33. TheDownstream mTORC1, the TOS -containing 4E-BP and p70S6K are phosphorylated. As seen above, phosphorylated 4E-BP is inactive and allows eIF4E to bind eIF4G to initiate translation. In dividing cells, mTOR phosphorylates p70S6K at Thr389, which in turn can phosphorylate ribosomal S6 protein (RPS6), eIF4B and programmed cell death 4 (PDCD4). While the function of phosphorylated RPS6 remains largely unknown, eIF4B and PDCD4 are positive and negative regulators, respectively, of the RNA helicase, eIF4A . mTORC1 in vitro and in vivo. As shown in Table Surprisingly, mTOR has not been studied in EOS, although its inhibitor, rapamycin has shown effects on EOS 2 (PGE2) via EP4 induces PI3K/PDK1-dependent increase in Akt phosphorylation, which consequently inhibits eotaxin-induced EOS shape changes and chemotaxis  signaling pathways are involved in most of cellular functions, including differentiation and proliferation. ERK1, ERK2, p38α, and p38β are coded by four different genes . Following intracellular or extracellular activation, the MAP kinase kinase kinases (MEKK) are activated, leading to phosphorylation of MAP kinase kinases (MEK) and, finally, MAPK are phosphorylated . ERK1/2 via eIF4E phosphorylation , chemokines, fMLP, the PAF, and matrix proteins , 64–71. n Figure ; and desmRNA translation is clearly not an all or nothing event. Agonists may increase or decrease ribosomal mobilization of all, the majority or subsets of mRNAs. This may occur through a slowdown of global elongation by phosphorylated eEF2 allowing poorly translated mRNAs to enter initiation and to be translated when elongation becomes derepressed . AlternaSelective regulation requires the recognition of unique cis-elements within the mRNA by sequence-specific mRNA binding proteins. In this way, subsets of mRNAs can be selectively identified and regulated for differential translation and mRNA decay. One well-studied example is the pyrimidine-rich domain termed terminal oligopyrimidine (TOP). mRNA containing TOP usually code for elongation factors and ribosomal proteins  and theiSemaphorin-7A mRNA level is relatively high in resting cells and changes only slightly in activated blood EOS. However, its translation remains almost undetectable despite GM-CSF activation . SurprisFreshly purified blood EOSs possess surface semaphorin-7A, which tends to decrease overtime during the first 20 h of cell culture (unpublished data). Activation with IL-5 or GM-CSF maintains or slightly increases surface semaphorin-7A over this same time span . On the Along with RL13A , RPS6 isWe found that all β-chain cytokines strongly induced RPS6 phosphorylation at Ser235 and Ser236. However, while RPS6 phosphorylation persisted for only 1–4 h in EOS culture with IL-5 or GM-CSF, IL-3 induced continuous RPS6 phosphorylation for as long as IL-3 remained present in the culture medium . Of noteXenopus oocytes  were upregulated at the translational level by IL-3, in a p90S6K-dependent manner . Therefovia structure, sequence or structure, and sequence elements. A simple example is 5′-cap binding protein eIF4E. A more complex example is PABP, which interacts with poly A tails, a combination of sequence (Poly A) and structure. The iron-response binding protein (IRE-BP) interacts with a sequence presented on a stem-loop and bulge (RNA-binding proteins (RBP) regulate all aspects of RNA metabolism, including biogenesis, cellular localization and transport, stability, and translation. With the emergence of high throughput screening and quantitative proteomics, several hundred (approximately 500) potential RBP have been identified . Given tnd bulge . Alteratvia a variety of domains, including the so-called RNA-recognition motif (RRM), zinc finger motives, K-homology domains (KH), RGG boxes, and DEAD/DEAH boxes  in the cytoplasm. In response to extrinsic and intrinsic stimuli, free and bound RBP are subject to post-translational modifications (PTM)  that may induce conformational changes and alter the association between RBP and target mRNA , 98. DepAH boxes . Often mAH boxes .cis-acting sequences into their 3′-UTR  is one of the best-characterized AUBPs and has a multiplicity of functions. It is a positive regulator of mRNA translation  but can In EOS, an increase in YB-1 content led to the stabilization of GM-CSF mRNA. Binding was mediated through 3′ UTR ARE and resulted in increased GM-CSF translation and release with subsequent pro-survival signaling , 102. YBHeterogeneous nuclear ribonucleoprotein C has been predominantly associated with the regulation of mRNA stability although several reports describe translational regulation through 5′ UTR interactions –135. ThiHuR (stabilizer of ARE mRNA) and TIA-1 (U-rich binding protein) bind to GM-CSF and TGF-β mRNA and are associated with transcript stability in EOS. While the role of HuR in mRNA translation has not been reported, TIA-1 is believed to repress the translation of TNF-α , COX-2 , cytochrIn vitro, purified peripheral blood EOS synthesize small amounts (~1 pg/ml) of anti-apoptotic GM-CSF    and qPCRsue EOS)  analysest1/2 < 6 min) in resting EOS but show significantly increased stability (increased by fourfold to sixfold) after cell activation. This conversion likely reflects alterations in the composition of interacting AUBP , GM-CSF mRNA is extremely labile , other alternative therapeutic targets more specific to each of the 3 cytokine should be developed.TPI ASM8 is a drug in development, targeting the common β-chain receptor for all IL-5, GM-CSF, and IL-3, in the form of RNA-targeted inhaled oligonucleotide antisense phosphorotioates . Althougvia prolonged ERK/p90S6K signaling. It is probable that additional genes are similarly regulated at a translation level by IL-3/ERK/p90S6K. Likely other candidate genes may share specific mRNA cis-elements whose identity may be inferred by homology searches among IL-3 upregulated mRNA. We started analyzing how semaphorin-7A or FCGR2 affect EOS function. We found that IL-3-activated EOS adhere to the only known semaphorin-7A ligand, plexin-C1  as specific genes exclusively responding to IL-3 activation lexin-C1 . Plexin-lexin-C1 , and haslexin-C1 , which clexin-C1 . Migratilexin-C1 , 188. Selexin-C1 , 190, allexin-C1 .see other article by Nair in this issue. However, airway EOS are still present and active despite treatment (The upregulation of CD32 by IL-3 on EOS has a profound impact on EOS function. EOS-driven pathology in tissue requires both EOS migration from circulating blood to the site of inflammation and the release (degranulation) of preformed toxic proteins and mediators of the inflammation. IL-3-activated EOS strongly degranulate on heat-aggregated (HA)-IgG, with extrusion of ~25% of their total cellular eosinophil-derived neurotoxin (EDN) in 6 h  comparedreatment , 195. Threatment , 197. Coreatment , 198. Thin vivo and in embryonic fibroblasts , downstream of IL-3/ERK/P90S6K signaling, is unclear. If so, phospho-RPS6 would be a possible therapeutic target to reduce EOS-related pathology. On the positive side, knock-in mice lacking the ability to phosphorylate RPS6 have modest deficits  and showroblasts . Howeverroblasts . RPS6 phroblasts . Howeverroblasts , 201. ThThe three β-chain cytokines, IL-3, IL-5, and GM-CSF are all present in human eosinophilic diseases and have both highly redundant and yet critically unique roles in the EOS biology. Their signaling affects differentiation, maturation, survival, migration, piecemeal release of immune-mediators, and degranulation. IL-3 is unique among the β-chain cytokines in generating prolonged intracellular signaling leading to the translation of a subset of EOS mRNA. Signaling requires ERK and p90S6K activation and culminates in the phosphorylation of RPS6. The control of both translation and decay of cytokine mRNA ultimately involves an interplay between mRNA-BP, especially those that target ARE. The AUBP in turn are often regulated by the action of Pin1, leading to multi-level control over cytokine gene expression. Critical, unanswered questions include the identification of RPS6-dependent mRNA as well as additional Pin1 RBP interactors and whether drugs can be developed to target these important pathways.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.The authors declare no conflict of interest other than an issued patent on the use of Pin1 as a drug target to treat eosinophilia. The reviewer KWG and the handling editor declared their shared affiliation, and the handling editor states that the process nevertheless met the standards of a fair and objective review.


Transforaminal lumbar interbody fusion (TLIF) is an effective surgery for lumbar degenerative disease. However, this fusion technique requires resection of inferior facet joint to provide access for superior facet joint resection, which results in reduced lumbar spinal stability and unnecessary trauma. We have previously developed extraforaminal lumbar interbody fusion (ELIF) that can avoid back muscle injury with direct nerve root decompression. This study aims to show that ELIF enhances lumbar spinal stability in comparison to TLIF by comparing lumbar spinal stability of L4–L5 range of motion (ROM) on 12 cadaveric spine specimens after performing TLIF or ELIF.12 cadaveric spine specimens were randomly divided and treated in accordance with the different internal fixations, including ELIF with a unilateral pedicle screw (ELIF+UPS), TLIF with a unilateral pedicle screw (TLIF+UPS), TLIF with a bilateral pedicle screw (TLIF+BPS), ELIF with a unilateral pedicle screw and translaminar facet screw (ELIF+UPS+TLFS) and ELIF with a bilateral pedicle screw (ELIF+BPS). The treatment groups were exposed to a 400-N load and 6 N·m movement force to calculate the angular displacement of L4-L5 during anterior flexion, posterior extension, lateral flexion and rotation operation conditions.The ROM in ELIF+UPS group was smaller than that of TLIF+UPS group under all operating conditions, with the significant differences in left lateral flexion and right rotation by 36.15% and 25.97% respectively. The ROM in ELIF+UPS group was higher than that in TLIF+BPS group. The ROM in the ELIF+UPS+TLFS group was much smaller than that in the ELIF+UPS group, but was not significantly different than that in the TLIF+BPS group.Despite that TLIF+BPS has great stability, which can be comparable by that of ELIF+UPS. Additionally, ELIF stability can be further improved by using translaminar facet screws without causing more tissue damage to patient. Howeet. al. ,14. A nuet. al. , 16. In et. al. –19. HoweThis study was reviewed and approved by the ethics committee at Shanghai East Hospital, Tongji University School of Medicine. The experiment was performed in accordance with Declaration of Helsinki related to research carried out on human subjects. All donors and the next of kin were fully legally competent and consented to the use of spine for research. Written informed consent from the donor or the next of kin was obtained. None of the donors were from a vulnerable population and all donors or next of kin had provided written informed consent that was freely given. All family dependents were notified and written informed consents were obtained before lumbar spines harvested. The privacy rights of the family dependents were always observed. The next of kin of the individual in this manuscript has given written informed consent to publish case details.Twelve fresh-frozen cadavers were collected from April to August, 2015 . All thePedicle screws (Moss Miami SI series) and translaminar facet screws were both obtained from Depuy Spine, Inc. . The pedicle screw dimensions were 6.0 mm*45 mm, and the translaminar facet screw dimensions were 3.5 mm* 45 mm. The dimensions of the interbody fusion cage (Concorde series) were 9mm*11 mm*27 mm. A ZwickBZ2.5/TS1S universal testing machine , CCD camera , the MATFOLT CoDigital imaging software system (developed by the Solid Mechanics Laboratory Center of Shanghai University) and 3D printer  were used.Biomechanical tests were conducted on the 12 samples under anterior flexion and posterior extension, left and right lateral flexion, and left and right rotation. Before surgery, the L4-L5 range of motions (ROMs) of all intact samples were obtained, and recorded as the control group. The 12 samples were then randomly divided into two groups: TLIF group and ELIF group. We treated the two groups in accordance with the different internal fixations, including ELIF with a unilateral pedicle screw (ELIF+UPS), TLIF with a unilateral pedicle screw (TLIF+UPS), TLIF with a bilateral pedicle screw (TLIF+BPS), ELIF with a unilateral pedicle screw and translaminar facet screw (ELIF+UPS+TLFS) and ELIF with a bilateral pedicle screw (ELIF+BPS). TLIF surgery was conducted on the samples in the TLIF group based on the classical approach: The right L4 inferior facet joint and L5 superior facet joint were removed. The entire nucleus pulposus in the L4-L5 disc and the right posterior 2/3rds of the fibrous ring were resected, and a cage was implanted. The posterior supraspinous ligament, interspinous ligament, spinous process, and left structures were retained. A screw each was placed in the L4 and L5 pedicles on the right side  . SurfaceSPSS  software was used for statistical analysis. Count data were expressed as mean±standard deviation. All the data were tested for normality with Shapiro-Wilk test. Comparisons between multiple sets of sample means were conducted using one way analysis of variance (ANOVA). P<0.05 was considered statistically significant. If the ROMs were statistically significant, the ROMs for each two group were compared by using post-hoc Tukey’s-multiple-comparisons-test.Internal fixation was stable and reliable in all the lumbar spine samples. There were no cases of fracture displacement, subsidence deformation, etc., at the screw–bone interface and the implantation site of the intervertebral fusion cage. During the entire test, the joint capsule, ligaments, and retained fibrous ring tissues were kept moist and naturally elastic. There were no cases of tearing, breakage, etc.The L4–L5 ROM in different groups was showed in , Fig 4. Lumbar interbody fusion technique includes three main parts: decompression, fixation, and fusion –22. DecoThe implantation angle of intervertebral fusion cage in ELIF is different with TLIF. The cage is implanted at an angle of 45° with the sagittal plane using ELIF. As compared with conventional TLIF, the direction of intervertebral fusion cage implantation is more oblique; hence, theoretically a longer cage can be placed into intervertebral space. In addition, the entry point of pedicle screw is moved outward and entry angle is more inclined in ELIF. Therefore, it may be feasible to apply a longer pedicle screw fixation in ELIF surgery. It will be tested in the further study in terms of these two points described above. As is known to us, the longer cage placement and longer pedicle screw fixation can both affect the biomechanical stability. In this study, we use the same size of cage and pedicle screw in ELIF group and TLIF group to exclude the effect of different cage and pedicle screw size on biomechanical stability.In this study, the posterior lumbar stability of ELIF and TLIF under different internal fixation conditions was assessed and compared using biomechanical testing. The analyses indicated that spinal stability was better in the case of ELIF with a unilateral pedicle screw fixation than in the case of TLIF with a unilateral pedicle screw fixation, under various operating conditions, especially, left lateral flexion and right rotation. The reason for this finding may be that in ELIF+UPS group, part of superior facet joint retained on the decompression side could associate with inferior facet joint to limit left lateral flexion and right rotation of the lumbar spine, and improve spinal stability. Additionally, the screws in the ELIF+UPS group had greater extraversion, and the direction in which the cage was implanted was closer to the coronal plane. Both were also beneficial to improve lumbar spine stability. However, the stability of ELIF with unilateral pedicle screw fixation was significantly weaker than that of the TLIF with bilateral pedicle screw fixation, indicating that ELIF with a unilateral pedicle screw fixation still could not reach the stability achieved by TLIF with a bilateral pedicle screw fixation.Translaminar facet screw (TLFS) fixation was first introduced in 1948 and developed to improve the lumbar stability . RecentlIn summary, ELIF with a unilateral pedicle screw and translaminar facet screw fixation could meet the stability requirements for fusion surgeries , 33. FurAlthough this study has achieved primary success, more work needs to be done. The cadaver samples proved the feasibility of the surgery, but there are many differences between human bodies and cadavers. Many venous vascular plexuses exist in the intervertebral foraminal region, and injuries to these structures during operation may easily cause bleeding. The ELIF surgery approach is deep, and the surgery directly focuses on the intervertebral foramen. Therefore, attention should be paid to the vascular structures in this area, and hemostasis after bleeding must be achieved during the surgery. What’s more, since the approach differs from the conventional anatomical approach, a learning curve exists for this surgery. Additionally, this surgery involves an inclined direction and a deep approach, and thus, operation with conventional surgical instruments is not convenient. Special surgical instruments need to be designed and tested before the clinical application of ELIF. Finally, this fusion sugrey needs to be further tested in lumbar spondylolisthesis.Despite that TLIF+BPS has great stability, which can be comparable by that of ELIF+UPS. Additionally, ELIF stability can be further improved by using translaminar facet screws without causing more tissue damage to patient.S1 Table(DOC)Click here for additional data file.S2 Table(DOC)Click here for additional data file.S1 Fig(JPG)Click here for additional data file.


Previous biochemical data have shown that mammalian NAD-IDHs can be inhibited by NADH; however, the molecular mechanism is unclear. In this work, we show that the αβ, αγ and α2βγ enzymes of human NAD-IDH can be inhibited by NADH, and further determine the crystal structure of the αγ heterodimer bound with an Mg2+ and an NADH at the active site and an NADH at the allosteric site, which resembles that of the inactive αMgγ heterodimer. The NADH at the active site occupies the binding site for NAD+ and prevents the binding of the cofactor. The NADH at the allosteric site occupies the binding sites for ADP and citrate and blocks the binding of the activators. The biochemical data confirm that the NADH binding competes with the binding of NAD+ and the binding of citrate and ADP, and the two effects together contribute to the NADH inhibition on the activity. These findings provide insights into the inhibitory mechanisms of the αγ heterodimer by NADH.Human NAD-dependent isocitrate dehydrogenase (NAD-IDH) catalyzes the oxidative decarboxylation of isocitrate in the citric acid cycle. In the α In mammals, the mitochondria localized NAD-dependent IDHs (NAD-IDHs) are deemed to exert the catalytic role in the citric acid cycle. Mammalian NAD-IDH functions as a heterotetramer consisting of two α subunits (37 kDa), one β subunit (39 kDa), and one γ subunit (39 kDa). The α and β subunits share about 40% sequence identity, the α and γ subunits about 42% sequence identity, and the β and γ subunits about 52% sequence identity2. The α and β subunits form a heterodimer (αβ) and the α and γ subunits form another (αγ), and the two heterodimers are assembled into the heterotetramer (α2βγ), which can be further assembled into a heterooctamer4.Isocitrate dehydrogenases (IDHs) are the enzymes that catalyze the oxidative decarboxylation of isocitrate (ICT) into α-ketoglutarate (α-KG) and CO2βγ heterotetramer, the α subunit exerts the catalytic role, and the β and γ subunits play the regulatory roles11. In our previous biochemical studies, we demonstrated that the α2βγ heterotetramer and the αγ heterodimer of human NAD-IDH could be activated by CIT and ADP, whereas the αβ heterodimer cannot; and the β subunit plays the structural role in the assembly and the γ subunit the regulatory role in the function of the heterotetramer12. Due to the lack of structural information about the α2βγ heterotetramer, the molecular basis for the assembly of the α2βγ heterotetramer and the molecular mechanisms of the allosteric regulation of the α2βγ heterotetramer are still elusive. Although there is no evidence so far showing that the αβ and αγ heterodimers can exist alone in the cells and play any physiological roles, we have carried out detailed structural and functional studies of the αβ and αγ heterodimers, hoping that these studies could provide insights into the structure, function and allosteric regulation of the α2βγ heterotetramer. Our previous structural studies of the αγ heterodimer reveal that the binding of CIT and ADP to the allosteric site in the γ subunit causes conformational changes of the allosteric site, the heterodimer interface, and the active site in the α subunit in a concerted way, leading to decrease of the S0.5,ICT and thus activation of the enzyme13. The functional roles of some key residues participating in the ligand binding and structural changes in the αγ heterodimer and the α2βγ heterotetramer have been confirmed by mutagenesis and enzymatic studies13. The biochemical and structural data together provide insights into the molecular mechanism of the activation of the αγ heterodimer by CIT and ADP, and additionally indicate that the structural information derived from the αγ heterodimer might be applicable to the α2βγ heterotetramer.Previous biochemical studies have shown that the enzymatic activity of mammalian NAD-IDH could be positively regulated by citrate (CIT) and ADP; and in the α14; however, the inhibition mechanism is unclear. In this work, we examined the inhibition of the αβ and αγ heterodimers and the α2βγ heterotetramer of human NAD-IDH by NADH and found that all these enzymes can be substantially inhibited by NADH. To understand the molecular mechanism of the NADH inhibition, we solved the crystal structure of the αγ heterodimer bound with an Mg2+ and an NADH at the active site and an NADH at the allosteric site. The overall structure of the αMg+NADHγNADH heterodimer resembles that of the αMgγ heterodimer, representing the inactive state of the enzyme. Structure analysis shows that the bound NADH at the active site occupies the binding site for NAD+, thus preventing the binding of the cofactor; and the bound NADH at the allosteric site occupies a large portion of the binding sites for ADP and CIT, thus blocking the binding of the activators. The kinetic data confirm that the NADH binding competes with the binding of NAD+ to the active site and the binding of CIT and ADP to the allosteric site, and the two effects together contribute to the inhibition of NADH on the αγ heterodimer. These findings provide insights into the inhibitory mechanism of the αγ heterodimer by NADH.Previous biochemical studies have also shown that the enzymatic activity of mammalian NAD-IDH could be inhibited by NADH14. In this study, we first examined the inhibitory effect of NADH on the activities of the αβ and αγ heterodimers and the α2βγ heterotetramer of human NAD-IDH , the α2βγ heterotetramer and the αγ heterodimer exhibit a specific activity of 24.0 ± 0.5 and 6.75 ± 0.07 μmol/min/mg in the absence of NADH, and NADH can substantially inhibit the activity of both enzymes with an IC50,NADH of 78.5 ± 5.4 μM and 82.7 ± 6.0 μM, respectively. As the αβ heterodimer shows a very low activity at the standard conditions (1.15 ± 0.26 μmol/min/mg) and has a much higher S0.5,Mn (5.3 mM) than the α2βγ and αγ enzymes 12, we measured the inhibitory effect of NADH on the αβ heterodimer at a higher Mn2+ concentration (50 mM). At that conditions, the αβ heterodimer exhibits an activity of 7.22 ± 0.31 μmol/min/mg in the absence of NADH, which can also be inhibited by NADH with an IC50,NADH of 94.9 ± 7.8 μM. These results indicate that NADH can bind to and inhibit the activities of the αβ, αγ and α2βγ enzymes of human NAD-IDH.The previous biochemical studies have shown that the enzymatic activity of NAD-IDH purified from bovine heart can be inhibited by NADH2βγ heterotetramer using the isothermal titration calorimetry (ITC) method. The ITC results show that all three enzymes can bind NADH, and the αβ heterodimer has the highest binding affinity (Kd = 4.6 ± 0.7 μM) followed by the α2βγ heterotetramer (Kd = 46.3 ± 6.5 μM) and then the αγ heterodimer (Kd = 109.0 ± 19.5 μM) , whereas the α2βγ heterotetramer has a very weak binding and the αγ heterodimer has no detectable binding . As the αβ heterodimer contains only one NADH-binding site, it is very likely that NADH binds to the active site in the α subunit and hence prevents the binding of the cofactor and inhibits the activity of the enzyme. Nevertheless, it is unclear why the αβ heterodimer exhibits stronger binding affinities for both NADH and NAD+ than the αγ heterodimer and the α2βγ heterotetramer. One possibility is that the ability of the γ subunit and/or the inability of the β subunit to bind NADH might have different effects on the binding of NADH or NAD+ at the active site in the α subunit. Further structural and functional studies of the αβ heterodimer and the α2βγ heterotetramer will resolve this issue and uncover the inhibitory mechanisms of the αβ heterodimer and the α2βγ heterotetramer by NADH.In addition, the thermodynamic data reveal the n-value of the αβ and αγ heterodimers and the α2+ and an NADH at the active site and an NADH at the allosteric site (αMg+NADHγNADH) at 2.4 Å resolution using the molecular replacement method  at the active site and an NADH (or NAD+) at the allosteric site 13, with an RMSD of 0.5 Å. Both the α and γ subunits comprise of 10 α-helices (α1-α10) and 12 β-strands (β1–β12), which fold into a large domain, a small domain, and a clasp domain  form the Mg2+-binding site, and Thr74A, Ser82A, Arg88A, Arg98A, Arg119A, Tyr126A and Asp230A form the ICT-binding site13. The residues of the α subunit are unvaried in the corresponding subunits of all NAD-IDHs  but different conformations from those in the active αMgγMg+CIT+ADP heterodimer (PDB code 5GRE)13 13.The active site resides in the deep cleft formed largely by the small and large domains of the α subunit and the small domain of the γ subunit, which consists of the binding sites for ICT, Mgtly Fig. . PreviouDHs Fig. . In the )13 Fig. . SpecifiMg+NADHγNADH structure, the NAD+-binding site is bound with an NADH: the adenine moiety of the NADH is located at the deep pocket of the active site and the nicotinamide moiety positioned in close to the ICT-binding site. The NADH makes hydrogen-bonding interactions with a number of residues from the large domain of the α subunit, including Lys69A, Thr74A, Asn84A, Glu260A, His263A, Gly264A, Thr265A, Asp268A, and Asn276A , the equivalent residue Asp357 forms two hydrogen-bonding interactions with the 2′- and 3′- hydroxyl groups of the adenosine ribose of NAD+ 15. Sequence alignment shows that this Asp is invariable in the corresponding subunits of human, Xenopus laevis, and Danio rerio NAD-IDHs  is the specificity determinant for NAD+ against NADP+, and Arg314 and His315 of human cytosolic NADP-IDH  are the specificity determinants for NADP+ against NAD+.Comparison with the structures of DHs Fig. . However2+, and ADP adjacently  is substantially increased from 0.267 ± 0.002 mM to 0.690 ± 0.080 mM (by 2.6 folds) and 1.13 ± 0.12 mM (by 4.2 folds), respectively; however, the Vmax,NAD at the saturation concentration of NAD+ is only slightly decreased  conditions with varied concentrations of CIT , where “Vmax,CIT” is the maximal velocity in the presence of saturated CIT concentration and “V0” is the initial velocity in the absence of CIT] and CIT has an evident activation effect on the enzyme with a Vmax,CIT of 6.58 ± 0.14 μmol/min/mg. In the presence of 50 μM and 100 μM NADH, the S0.5,CIT of the enzyme is slightly increased to 2.07 ± 0.07 mM and 2.19 ± 0.05 mM, but the Vmax,CIT at the saturated CIT concentration is markedly decreased to about 66% and 40% of that in the absence of NADH, respectively. These results indicate that the NADH binding slightly decreases the binding of CIT at the allosteric site and thus increases the CIT concentration for activation, but the activation of CIT alone cannot fully reverse the inhibitory effect of NADH, which could be attributed to the binding of NADH to the active site. As the ADP activation is dependent on and synergistic with the binding of CIT13, we examined the effect of NADH on the ADP activation in the presence of 1 mM CIT , where “Vmax,ADP” is the maximal velocity in the presence of saturated ADP concentration and “V0” is the initial velocity in the absence of ADP] and a Vmax,ADP of 14.1 ± 0.2 μmol/min/mg. In the presence of 50 and 100 μM NADH, the S0.5,ADP is increased to 0.113 ± 0.008 mM (by 2.0 folds) and 0.206 ± 0.014 mM (by 3.7 folds), respectively, whereas the Vmax,ADP is only slightly decreased to 13.0 ± 0.1 μmol/min/mg and 12.7 ± 0.2 μmol/min/mg, respectively. Moreover, the inhibitory effect of NADH is almost completely reversed at the saturation concentration of ADP and 1 mM CIT. These results indicate that the binding of NADH to the allosteric site also decreases the binding of ADP and increases the ADP concentration for activation. These data together demonstrate that NADH can compete with the binding of CIT and ADP at the allosteric site and thus impedes the activation effect of CIT and ADP, and the inhibitory effect of NADH can be almost completely reversed by high concentrations of ADP and CIT.Our biochemical data show that the αγ heterodimer contains at least two binding sites for NADH Table . Consistons Fig. . Furthersed Fig. . These rCIT Fig. . In the CIT Fig. . As expe14; however, the molecular mechanism is unclear. It was suggested that the inhibitory effect of NADH is through its binding to the active site, which impedes the binding of NAD+ and thus impairs the enzymatic activity5. In this work, we demonstrate that the αβ, αγ and α2βγ enzymes of human NAD-IDH can also be inhibited by NADH and that all three enzymes have much higher binding affinity for NADH than NAD+, and the αβ heterodimer has the highest binding affinity for both NADH and NAD+ followed by the α2βγ heterotetramer and then the αγ heterodimer. We further solved the structure of the αγ heterodimer bound with an Mg2+ and an NADH at the active site and an NADH at the allosteric site and performed the kinetic studies. The structural and biochemical data together reveal the inhibitory mechanisms of NADH on the αγ heterodimer.The previous biochemical studies have shown that the activity of the purified bovine heart NAD-IDH can be inhibited by NADH+ at the active site. Consistently, our biochemical data show that the NADH binding markedly increases the S0.5,NAD of the enzyme, and hence inhibits the activity of the enzyme. In addition, our structural data show that NADH can also bind to the allosteric site to occupy a large portion of the binding sites for the activators CIT and ADP, and this binding competes with the binding of CIT and ADP and thus impedes the activation of the enzyme. Indeed, our biochemical data show that the NADH binding increases the concentrations of the activators for activation, and hence inhibits the activity of the enzyme. These two effects together contribute to the inhibition of NADH on the enzymatic activity of the αγ heterodimer. This mechanism is very likely applicable to the α2βγ holoenzyme of human and other mammalian NAD-IDHs. Nonetheless, we cannot rule out the possibility that the active site and/or allosteric site in the α2βγ heterotetramer might have different conformations from these in the αγ heterodimer and thus the ligands might have different binding modes. Further structural and functional studies of the α2βγ heterotetramer will resolve this issue and provide a complete understanding of the molecular basis for the assembly and the allosteric regulation mechanism of the α2βγ heterotetramer.Our structural data show that NADH can bind to the NAD-binding site, which competes with the binding of the cofactor NAD2βγ heterotetramer of human NAD-IDH followed the method described previously13. In particular, to obtain the αγ heterodimer, the human DNA fragments corresponding to the α and γ subunits of human NAD-IDH were amplified by PCR and cloned into the pQlinkN co-expression plasmid following the standard procedure18. For purification purpose, the C-terminal end of the γ subunit was attached with a TEV protease cleavage site followed by a His6 tag. The constructed pQlinkN-α-γ-tev-His6 plasmid was transformed into E. coli BL21(DE3) Codon-Plus strain (Novagen), and the bacterial cells were cultured in LB medium supplemented with 100 μg/ml ampicillin at 37 °C until OD600 reached 0.5. The protein expression was induced by adding 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 23 °C for 20 hr. The bacterial cells were harvested by centrifugation and resuspended in a lysis buffer , and then lysed by sonication on ice. Protein purification was first carried out by affinity chromatography using an Ni-NTA column (Qiagen). The C-terminal His6-tag of the γ subunit was cleaved with the TEV protease by dialysis overnight against the lysis buffer. The protein mixture was reloaded on an Ni-NTA column and washed with the lysis buffer containing 10 mM imidazole, and the eluent was further purified by gel filtration using a Superdex 200 10/300 GL column . The purified protein was of high purity (above 95%) as analyzed by SDS-PAGE (12%), and stored in the storage buffer  for the structural and biochemical studies.Preparations of the αβ and αγ heterodimers and the αMg+NADHγNADH heterodimer were grown at 20 °C from drops consisting of 1 μl each of the protein solution and the reservoir solution . Diffraction data were collected from a flashed-cooled crystal at 100 K at beamline 19U1 of NFPSS, China, and processed, integrated and scaled together using HKL300019. The flashed-cooled crystal was prepared by soaking the crystal into the cryo-protectant containing the reservoir solution mixed with 20% ethylene glycol and then quickly dipping into liquid N2. Crystallographic statistics are listed in Table To obtain the αγ heterodimer bound with NADH, the protein solution (10 mg/ml) was incubated with NADH  at 4 °C before crystallization. Crystallization screening was carried out using the hanging drop vapor diffusion method, and crystals of the αMg+NADHγNADH structure was solved with the molecular replacement (MR) method as implemented in program Phaser20 using the αMgγ structure (PDB code 5GRH)13 as the search model. Structure refinement was performed using programs Phenix21 and REFMAC522, and manual model building with program Coot23. Structural analyses were performed using programs in the CCP4 suite24 and the PISA server25. The structure figures were generated using PyMol26 and the structure-based sequence alignment figure using ESPpript 3.027. Statistics of the structure refinement and the quality of the structure model are also listed in Table The α13. The activity of the enzyme was determined by monitoring the reduction of NAD+ to NADH over time at the specific absorption wavelength of NADH (340 nm) using a Beckman Coulter DU 800 spectrophotometer. The standard reaction solution consisted of 33 mM Tris-acetate (pH 7.4), 20 mM DL-ICT, 2 mM Mn2+, and 1 mM NAD+ in a total volume of 1 ml. The catalytic reaction was initiated by addition of NAD+. The production of NADH follows pseudo-first-order kinetics and the time course of the product appearance is well modeled by a linear function up to the time when about 10% of the total substrate was converted to the product. The initial velocity (V) was determined from the slope of a linear fit of the early time point data. The activity of the enzyme is defined as the velocity at the specific conditions, which is expressed as μmol of NADH produced per min per mg of enzyme (μmol/min/mg). All experiments were carried out at 25 °C and repeated at least twice under the same conditions.The enzymatic activity assay was carried out using a method as described previously2βγ enzymes in the presence of NADH were determined at the standard conditions with varied NADH concentrations, and the activity of the αβ enzyme was determined at the same conditions with a higher concentration of MnCl2 (50 mM). The inhibitory effect of NADH on the activity of the enzyme is presented as percent of inhibition (I) as a function of NADH concentration. I = (V0 − V)/V0 * 100, where V0 and V were the initial velocity in the absence and presence of NADH, respectively.The activities of the αγ and α+, the apparent kinetic parameters of the enzyme for NAD+ in the presence of 0, 50 and 100 μM NADH were determined at the standard conditions with varied NAD+ concentrations (0–20 mM). The values of the Vmax,NAD and the S0.5,NAD were determined by fitting the experimental data into the Michaelis-Menten equation “V = Vmax * [S]/(S0.5 + [S])” using program Graphpad Prism (Graphpad Software), where “S0.5” is the NAD+ concentration at the velocity of 0.5 * Vmax, and “[S]” is the concentration of NAD+.To analyze the effect of NADH on the binding of the cofactor NADDL-ICT, 2 mM Mn2+, and 3 mM NAD+) with varied CIT concentrations (0–10 mM), and the activation curve for ADP in the presence of 0, 50, and 100 μM NADH were determined at the same conditions with varied ADP concentrations (0–2 mM) and the presence of 1 mM CIT. The apparent kinetic parameters for CIT (or ADP) were obtained by fitting the experimental data into the non-Michaelis-Menten equation “V − V0 = (Vmax − V0) * [S]^h/(S0.5^h + [S]^h)”, where “h” is the Hill coefficient, “S0.5” is the CIT (or ADP) concentration when (V − V0) equals to 0.5 * (Vmax − V0), “[S]” is the concentration of CIT (or ADP), “V0” is the initial velocity in the absence of CIT (or ADP), and “Vmax” is the maximal velocity in the presence of saturated CIT (or ADP) concentration. “Vmax−V0” reflects the maximal activation effect caused by addition of CIT (or ADP). The h value for CIT activation in the presence of 0, 50, and 100 μM NADH is about 2.5, 2.6, and 3.4, respectively, indicating the CIT binding exhibits significant cooperativity. The h value for ADP activation in the presence of 0, 50, and 100 μM NADH is all about 1, indicating that the ADP binding in the presence of 1 mM CIT exhibits no cooperativity. All of the values are the averages of three independent experiments with standard errors.To analyze the effects of NADH on the binding of the activators CIT and ADP, the activation curve for CIT in the presence of 0, 50, and 100 μM NADH were determined at subsaturating substrate (ICT) conditions  was discarded for each dataset to remove the effect of titrant diffusion across the syringe tip during the equilibration process. The αβ, αγ or α2βγ protein (30–200 μM) (titrand) was first dialyzed against the buffer  and then placed in the sample cell with a reaction volume of 200 μl. The titrant (0.8–8 mM) was prepared in the same buffer as the titrand, and aliquots of the titrant (2 μl each) were injected incrementally into the sample cell at an interval of 120 sec (or 130 sec or 150 sec) with constant stirring at 750 rpm. The exact concentrations of titrand and titrant used in the measurements are listed in Table Kd) equals to reciprocal of the association constant (Ka).Isothermal titration calorimetry (ITC) measurements were performed at 20 °C using an ITC200 Micro-Calorimeter Q6 . An initial injection of 0.4 μl titrant (NADH or NADMg+NADHγNADH structure has been deposited with the RCSB Protein Data Bank under accession code 5YVT.The α