Spinal cord injury (SCI) causes a high rate of morbidity and disability. The clinical features of SCI are divided into acute, subacute, and chronic phases according to its pathophysiological events. The mammalian target of rapamycin (mTOR) signaling pathway plays an important role in cell death and inflammation in the acute phase and neuroregeneration in the subacute/chronic phases at different times. Resveratrol has the potential of regulating cell growth, proliferation, metabolism, and angiogenesis through the mTOR signaling pathway. Herein, we explicate the role of resveratrol in the repair of SCI through the inhibition of the mTOR signaling pathway. The inhibition of the mTOR pathway by resveratrol has the potential of serving as a neuronal restorative mechanism following SCI. Presently, more than 2.5 million people suffer from SCI, with annually reported new cases being about 12,000 in the United States . Disrupt+ . SCI cre+ . Additio+ . Oligode+ . It is w+ . In recee (MAPK) –12. The As a member of the phosphatidylinositol-3-kinase-related kinase superfamily, mTOR is a serine/threonine protein kinase. The combination of mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) protein complexes forms the mTORC signaling pathway. mTORC1 phosphorylates downstream effectors such as p70 ribosomal S6 protein kinase (p70S6K) and further regulates mRNA translation. Thus, mTORC1 is important in stimulating protein synthesis. mTORC2, on the other hand, has been demonstrated to phosphorylate members of the AGC kinase family including protein kinase B (Akt), which is linked to several pathological conditions. mTORC2 is regarded as a regulator of the actin cytoskeleton . PhosphaRegarding the acute phase in the wake of SCI, the mTOR signaling pathway participates in the regulation of cell death, activation of macrophage/microglia, and inflammation .β (GSK-3β) and reducing protein 27 kinase inhibition protein 1 (p27). The inhibition of Akt phosphorylation leads to G1 arrest, which in turn induces apoptosis [Neuronal death includes neuronal apoptosis, autophagy, and necrosis. As mTOR's inhibitor, rapamycin could block the activation of the Akt/mTOR pathway, thereby preventing apoptosis of nerve cells . Akt coupoptosis . Also, tpoptosis . TORC1 ipoptosis . Moreovepoptosis . A studypoptosis . Autophapoptosis . Autophapoptosis . The inhpoptosis . Rapamycpoptosis . Autophapoptosis , 33. Howpoptosis . Autophapoptosis . Autophapoptosis . Again, poptosis . This, upoptosis . Both aupoptosis . The cropoptosis . Hypoxiaβ/IκB-α/NF-κB, and results in inflammation [The traumatic spinal cord induces local inflammatory response by activated microglia, infiltrating neutrophils, and macrophages. Moreover, the upregulation of associated proinflammatory cytokines' expression has been reported to be induced by the traumatic spinal cord . mTOR reammation , 43. mTOammation . Again, ammation . mTOR inammation . Furtherammation . The insammation . Thus, iDuring the chronic phase following SCI, mTOR participates in regulating neuroregeneration and glial scar formation.Injured neurons of the CNS undergo normal cell apoptosis rather than regeneration. The chief cause of central nerve regeneration failure has to do with inhibitory factors in the myelin as well as with the formation of glial scar and weak growth capability of mature neurons . There aGlial scar is another major barrier for regeneration; thus, overcoming this barrier would be significant to axonal regeneration . Glial sPolygonum cuspidatum, red grape skins, red wine, blueberries, and some nuts. Resveratrol has a number of biological activities and pharmacological actions, which include anti-inflammation, antioxidation, inhibition of platelet aggregation, and improvement of microcirculation [β, IL-10, and TNF-α [κB's activity. mTOR and NF-κB pathways are tied in many aspects. For instance, the activation of mTOR by Akt culminates in the activation of NF-κB, which is associated with inhibitor of NF-κB kinase (IKK) and mTORC1 complex's Raptor [κB and inflammatory molecules through the mTOR pathway [Each herb consists of numerous chemical constituents from different categories. Different active ingredients show therapeutic functions in a number of disease treatments. In recent years, traditional Chinese medicine (TCM) has been drawing attention in SCI treatment . Resveraculation –75. Follculation . In the culation . Resveraculation , 79 (Figculation . Moreoveculation . Interesculation . The apoculation . In a stculation . In anotculation . The detculation . Again, culation . More sond TNF-α . Resveras Raptor  (Figure  pathway .mTOR is involved in the regulation of several diseases, cellular functions, and trauma in the CNS. The mTOR signaling pathway plays an important role at different times. As such, the perspicacious knowledge concerning how the mTOR signaling pathway works in the process of neural protection is of great significance in SCI. TCM is an important supplementary treatment for SCI, which may offer therapeutic and reparative benefits in SCI. TCM might replace the application of nonsteroidal anti-inflammatory drugs, neurotrophic factors, or even methylprednisolone. Resveratrol participates in apoptosis, formation of pathological scar, and proliferation of fibroblasts as well as in anti-inflammation via the inhibition of mTOR in repairing SCI. Resveratrol, through the mTOR pathway, has the tendency of serving as an SCI therapeutic. However, further research pertaining to the effect and specific molecular mechanisms in the different phases of SCI still needs to be elucidated.


Collapsin response mediator protein-2 (CRMP2) regulates axonal growth cone extension, and increased CRMP2 phosphorylation may lead to axonal degeneration. Axonal and synaptic pathology is an important feature of Lewy body dementias (LBD), but the state of CRMP2 phosphorylation (pCRMP2) as well as its correlations with markers of neurodegeneration have not been studied in these dementias. Hence, we measured CRMP2 phosphorylation at Thr509, Thr514 and Ser522, as well as markers of β-amyloid (Aβ), tau-phosphorylation, α-synuclein and synaptic function in the postmortem neocortex of a longitudinally assessed cohort of LBD patients characterized by low  and high  burden of Alzheimer type pathology. We found specific increases of pCRMP2 at Thr514 in DLB, but not PDD. The increased CRMP2 phosphorylation correlated with fibrillogenic Aβ as well as with losses of markers for axon regeneration (β-III-tubulin) and synaptic integrity (synaptophysin) in LBD. In contrast, pCRMP2 alterations did not correlate with tau-phosphorylation or α-synuclein, and also appear unrelated to immunoreactivities of putative upstream kinases glycogen synthase kinase 3β and cyclin-dependent kinase 5, as well as to protein phosphatase 2A. In conclusion, increased pCRMP2 may underlie the axonal pathology of DLB, and may be a novel therapeutic target. However, antecedent signaling events as well as the nature of pCRMP2 association with Aβ and other neuropathologic markers require further study.The online version of this article (doi:10.1186/s13041-016-0264-9) contains supplementary material, which is available to authorized users. Collapsin response mediator protein-2 (CRMP2) is the first identified member of the collapsin response mediator protein (CRMP) family . There aTable CRMP2 is known to be abundantly localized to the cytosol as a cytoskeletal associated protein , which hBecause previous studies on AD have reported increased CRMP2 phosphorylation which may be related to amyloid burden , 17, we 42) is more fibrillogenic and prone to forming toxic oligomers compared to Aβ40 [42 to Aβ40 ratio (Aβ42 : Aβ40) is therefore used as an indicator of neurotoxicity and disease severity in AD [42 : Aβ40 is also a biomarker for AD clinical assessment and has been shown to be more consistently associated with cognitive impairments than only Aβ42 or Aβ40 [42 : Aβ40 in BA40 was significantly increased in DLB, but not PDD  group  and inactivation (pSer9 GSK3β). Furthermore, given that PP2A can dephosphorylate CRMP2 at Thr514 [42 : Aβ40, see Fig. The CRMP2 member of the collapsin response mediator protein family has well-established roles in various synaptic functions, including regulation of neurotransmitter release, axonogenesis and axon guidance –7. It isN’s may be needed to have sufficient power to study potential correlations between different pCRMP2 species and tau phosphorylation, especially since certain pCRMP2, e.g., 3 F4 epitopes, are known to be associated with neurofibrillary tangles [While the current study investigated a comprehensive range of CRMP2 neurochemical markers and neuropathological correlates, there are several limitations and questions which require follow-up investigations. First, due to issues of tissue availability, we limited our measurements to the parietal cortex BA40). Although previous studies have shown similar involvement of plaques, tangles and α-synuclein pathology between BA40 and other neocortical regions like the frontal or temporal cortex , it is u0. Althou tangles . With re tangles , 49, and tangles , and pat tangles , LBD, si tangles   validate the observed pCRMP2 changes in other brain regions in a larger cohort; (2) elucidate the mechanisms linking Aβ to pCRMP2 in LBD; and (3) investigate potential associations between CRMP2 function and other pathological features, including various different α-synuclein species.N values are listed in the table and figure legends.All subjects for this study were selected based on clinicopathological consensus diagnoses, including the “one-year rule”  and the 3) were thawed on ice and dissected free of meninges and white matter with disposable surgical scalpels. After dissection, samples were homogenized (50 mg tissue wet weight/mL) with an Ultra-Turrax homogenizer  in ice-cold homogenizing buffer , then aliquoted and stored at −75 °C until use. To obtain insoluble PHF fractions and soluble cytosolic fractions, dissected brain pieces were dispersed in a glass tissue grinder at 50 mg tissue wet weight/mL in ice-cold mild extraction buffer . Dispersed lysates were passed through a 26G needle 10 times using 1 mL syringes. Lysates were first centrifuged at 800 × g for 5 min at 4 °C, then at 10,000 × g for 10 min at 4 °C, with the resulting supernatant designated as the cytosolic soluble fraction  and stored at −75 °C until use. Pellets resulting from the 800 × g centrifugation step (see above) were resuspended in 1 % sarkosyl Tris-buffer, centrifuged  with further suspension in 5 M guanidine buffer for 3 h at 25 °C, then centrifuged again  before the final supernatant was designated as the insoluble sarkosyl PHD fraction [All reagent grade chemicals were purchased from Sigma-Aldrich (USA) unless otherwise stated. Blocks of frozen brain tissues . Membranes were blocked with 5 % bovine serum albumin (BSA) in 20 mM Tris-buffered saline, pH 7.6 with 0.1 % Tween® 20 (TBST) at 25 °C for 1 h, then incubated with primary antibodies overnight at 4 °C degree in 5 % BSA in TBST at specified dilutions  according to manufacturer’s instructions. Ratios of Aβ42 to Aβ40 were calculated by dividing values of Aβ42 by Aβ40. Similarly, pSer396 tau and total tau were assayed using ELISA kits , and ratios of pSer396 tau to total tau were calculated.Aβpost-hoc corrections) or non-parametric  based on normality of the data, as assessed by Kolmogorov-Smirnov tests. Due to the ordinal nature of some of the variables, all correlations were based on non-parametric Spearman rank coefficients. A p-value < 0.05 is considered statistically significant. As the study is exploratory in nature, no adjustment for multiple correlations were made.Data was analyzed using SPSS 20.0  or Prism 6.0  software. Comparisons between groups were assessed with parametric (one way analyses of variance, ANOVA with Bonferroni


Clostridium difficile (EtfAB/Bcd)4 complex in the dehydrogenase-conducting D-state, α-FAD (bound to domain II of EtfA) and δ-FAD (bound to Bcd) being 8 Å apart. Superimposing Acidaminococcus fermentans EtfAB onto C. difficile EtfAB/Bcd reveals a rotation of domain II of nearly 80°. Further rotation by 10° brings EtfAB into the bifurcating B-state, α-FAD and β-FAD (bound to EtfB) being 14 Å apart. This dual binding mode of domain II, substantiated by mutational studies, resembles findings in non-bifurcating EtfAB/acyl-CoA dehydrogenase complexes. In our proposed mechanism, NADH reduces β-FAD, which bifurcates. One electron goes to ferredoxin and one to α-FAD, which swings over to reduce δ-FAD to the semiquinone. Repetition affords a second reduced ferredoxin and δ-FADH−, which reduces crotonyl-CoA.The electron transferring flavoprotein/butyryl-CoA dehydrogenase (EtfAB/Bcd) catalyzes the reduction of one crotonyl-CoA and two ferredoxins by two NADH within a flavin-based electron-bifurcating process. Here we report on the X-ray structure of the  Clostridium difficile EtfAB/Bcd and discuss the bifurcation mechanism for electron flow.The electron-transferring flavoprotein / butyryl-CoA dehydrogenase (EtfAB/Bcd) complex catalyzes the reduction of crotonyl-CoA and ferredoxins by NADH in anaerobic microbes. Here, the authors present the crystal structure of  In the applied enzyme machineries, a flavin hydroquinone (HQ or FH−) acts as the bifurcating donor; one electron flows to a high-potential acceptor, whereas the other electron reduces the low potential ferredoxin or flavodoxin. Thus, the exergonic reduction of such an acceptor drives the endergonic electron transfer (ET) to ferredoxin or flavodoxin; in other words: an exergonic reaction is coupled to an endergonic reaction. Reduced ferredoxin and flavodoxin can be regarded as “energy rich” compounds, which in many fermenting anaerobes form H2 and generate an electrochemical Na+ gradient for ATP synthesis via ferredoxin-NAD+ reductase (Rnf)6. In addition, they reduce CO2 to CO in acetogens8 and to a formyl group in methanogens10. Flavins, FMN or FAD, are suitable coupling agents as they can adopt a two-electron redox state, quinone/hydroquinone (Q/HQ or FAD/FADH−) as well as two one-electron redox states, Q/SQ and SQ/HQ , one with a strong and a second with a weak electron donor potential and vice versa. An analogous process known as Q-cycle was reported for the membrane-spanning cytochrome bc1 complex where ubihydroquinone is the site of quinone-based electron bifurcation12.Flavin-based electron bifurcation (FBEB) has been recognized as a new mode of energy coupling in strictly anaerobic bacteria and archaea18. Besides NADH-dependent ferredoxin: NADP+ oxidoreductase (Nfn)21, the best studied FBEB system is the electron transferring flavoprotein/butyryl-CoA dehydrogenase (EtfAB/Bcd) complex, which is a key enzyme in butyrate producing anaerobes13. Various biochemical, kinetic, spectroscopic, structural and mechanistic data are already reported24. The EtfAB/Bcd complex is composed of subunits EtfA, EtfB and Bcd; each subunit contains one FAD as prosthetic group, termed α-FAD, β-FAD and δ-FAD, respectively. The electron-bifurcating EtfAB/Bcd complex couples the exergonic reduction of crotonyl-CoA to butyryl-CoA (E0’ = −10 mV)25 with the endergonic reduction of 2 ferredoxins (E0’ = −405 mV)26 by oxidation of 2 NADH to 2 NAD+ (E0’ = −320 mV) 4 are separate proteins, which form an (EtfAB)2-(Bcd)4 complex during catalysis24. In contrast, the complexes present in the Gram-positive Clostridia, a main class of the Firmicutes, do not dissociate during purification. Examples are the tight complexes from Clostridium kluyveri2, Clostridium tetanomorphum27 and Clostridium difficile13, the latter studied in this work. Recently, the crystal structure of EtfAB from A. fermentans has been solved24. It closely resembles that of the non-bifurcating EtfABs from aerobes and respiring anaerobes but contains β-FAD at the place of AMP in addition to the α-FAD30. All EtfABs are composed of three domains; domains I and II are components of the A- or α-subunit, and domain III of the B- or β-subunit. α-FAD is located in the mobile domain II and β-FAD sits between the rigid domains I and III. The distance between α-FAD and β-FAD amounts to 18 Å, which can be reduced to 14 Å by moving domain II towards domains I + III without causing serious steric clashes24. Soaking the EtfAB crystal with NADH revealed a binding close to β-FAD, though only the ADP part of NADH could be structurally resolved. Since the known structures of EtfAB and Bcd from M. elsdenii31 and A. fermentans24 cannot tell how EtfAB is bound relative to Bcd, we solved the structure of the C. difficile complex as shown in this work. Surprisingly, in this structure the position of α-FAD is close to δ-FAD of one Bcd subunit (8.5 Å distance) ready for electron donation . By combining the structures of A. fermentans Etf and C. difficile EtfAB/Bcd, we built a FBEB-like state of the whole complex  and modeled the FBEB state . We also explored conformational changes by site-directed mutagenesis and found that the α-FAD on domain II acts like an electron seesaw swinging between the B and D states.There are two types of bifurcating EtfAB-Bcd complexes. In the Gram-negative Firmicutes, 4 state with a molecular mass of ca. 440 kDa 2/(TMADH)2 complexes30.The EtfAB/Bcd complex was found in a heterododecameric (EtfAB/Bcd)kDa Fig. . Archite30, EtfAB is built up of three domains, of which I (1–199) and II (200–331) belong to subunit A and III (5–265) to subunit B. The structurally related domains I and III are tightly associated  that are placed around 50 Å apart from each other  touching Bcd only form negligible interactions. The covalent linkers between domains I and II and between domain III and the EtfB arm  and the rmsd is 1.3 Å essentially indicating a rigid-body movement  were produced in Escherichia coli and purified to homogeneity with yields varying between 2 to 20 mg from 4 L cultures. The variants were analyzed by measuring kinetic activities with respect to FBEB, NADH oxidase, and butyryl-CoA dehydrogenase  bind either spatially far away from β-FAD or only transiently and unspecific, respectively, domain II swinging is presumably triggered by NADH binding or subsequent β-FADH− formation . (3) In the structure of the EtfAB-NAD+ complex of A. fermentans, the ribose-nicotinamide moiety is disordered24. Obviously, NAD+ but also NADH cannot push aside the anchor of the EtfB arm and occupy its natural binding site parallel to the isoalloxazine. One reason might be that domain II of A. fermentans EtfAB is arrested in the B-like state and not located in the resting D state in the absence of Bcd. (4) Multiple attempts to crystallize EtfAB/Bcd in the presence of NADH failed although crystallization in its absence is well reproducible. This finding argues for a significant conformational rearrangement within the EtfAB/Bcd complex dependent on NADH binding. (5) Site-specific mutagenesis experiments support important functions of the EtfB arm and the EtfB protrusion in the catalytic process  Domain II can be considered as rigid body connected by two conformationally mobile, solvent-exposed linkers to the EtfAB base (domain I + III). The flavodoxin-like fold of domain II is strongly attached to the EtfB arm such that a pulling force on the latter affects its location Fig. . The sup34. This behavior makes sure that after the high-potential ET from HQ to a high-potential acceptor, the remaining semiquinone has a very low potential able to reduce low potential acceptors, such as ferredoxin (E0’ = −405 mV)26 or the SQ of flavodoxin. Interestingly, the one-electron midpoint potentials of free flavin are also crossed; Q/SQ, E0’ = −314 mV; SQ/HQ, E0’ = −124 mV36 and their separation (ΔE = −124 − (−314) = + 200 mV) is increased by interaction with the protein. In flavodoxin, however, the potentials are not crossed; Q/SQ, E0’ = −60 mV; SQ/HQ, E0’ = −420 mV37  transition must be much higher than that of the quinone/ semiquinone (Q/SQ) transitionMegasphaera elsdenii, the midpoint potential values at pH 7 have been measured for the flavodoxin-like α-FAD: Q/SQ, E0’ = + 81 mV, SQ/HQ, E0’ = −136 mV and for β-FAD: Q/HQ, E0’ = –279 mV22. The two-electron potential of δ-FAD of Bcd is assumed to be close to that of crotonyl-CoA/butyryl-CoA, E0’ = −10 mV25. The one-electron potentials of β-FAD cannot be determined by the standard redox titration method, because the concentration of the semiquinone SQ of the bifurcating β-FAD is expected to be extremely low; the stability constant Ks = [SQ]2 × {[Q] × [HQ]}−112 for the quinones in the Rieske/cytochrome b complexes of the Q-cycle was estimated as 10−14 to 10−15 39. In a recent paper, transient absorption spectroscopy (TAS) of the bifurcating FAD of Nfn from Pyrococcus furiosus was used to determine the half-life of an artificial generated anionic SQ of FAD as 10 ps (10−11 s) from which the one-electron redox potential of FAD, Q/SQ was calculated as −911 mV21. This potential is 635 mV lower than the measured two-electron redox potential for FAD: Q/HQ, E0’ = −276 mV. Hence the one-electron redox potential of FAD, SQ/HQ amounts to –276 + 635 mV = + 359 mV. Thus the two one-electron redox potentials are separated by ΔE = 2 × 635 = 1270 mV21, which at 25 °C (298 K) is converted to log Ks = ΔE × F × (2.3 RT)−1 = −21.5, Ks = 10−21.5 12. For β-FAD of EtfAB, however, such a large separation of the one-electron redox potentials appears not necessary because the low potential acceptor ferredoxin has a 306 mV higher potential than that of Nfn, the proximal [4Fe-4S] cluster (E0’ = –711 mV). If we assume a Ks = 10−16, close to that of ubiquinone, the one-electron redox potentials become separated by ΔE ≈ 900 mV leading for β-FAD, to SQ/HQ, E0’ = –279 mV + 450 mV = + 171 mV and to Q/SQ, E0’ = –279 mV – 450 mV = –729 mV.To establish an energy scheme of the EtfAB/Bcd complex, the two- and one-electron redox potentials of each of the reactants have to be known Fig. . In caseE0’ = + 81 mV) or α-FAD•−  acts as high-potential acceptor. In earlier experiments fully oxidized EtfAB (10 µM) was titrated with increasing concentrations of NADH, showing the reduction of EtfAB to the stable red anionic semiquinone of α-FAD at 4–6 µM NADH. At 20 µM NADH both α- and β-FAD became reduced to the HQ24. This certainly happens also in vivo, where NADH is present. Similarly, the neutral blue semiquinone of flavodoxin  is the dominant form in vivo6. Hence, most likely α-FAD•− serves as high-potential acceptor for the bifurcation and delivers as α-FADH− one electron to δ-FAD of Bcd, because the redox potential of the SQ/HQ pair is too high.Due to the high redox potential of α-FAD the question arises whether α-FAD  = +307 mV), the extremely low potential semiquinone of β-FAD  reduces ferredoxin (E0’ = −405 mV)26, making the EtfAB partial reaction slightly exergonic as indicated by the green dashed line in Fig. − ensures a tight coupling with the exergonic reduction of ferredoxin, as observed experimentally2. The formed α-FADH− bound to domain II of EtfAB swings over from the β-FAD (B state) to the δ-FAD (D state) ET mode, which enables a smooth one-electron transfer to δ-FAD, ΔE = −10 − (−136) = +126 mV. Binding of a new molecule of NADH close to β-FAD most likely triggers the return of α-FAD•− to the B-state, where it accepts another electron and swings over to Bcd to generate δ-FADH−, which reduces crotonyl-CoA to butyryl-CoA (E0’ = −10 mV).FBEB starts with a two-electron reduction of β-FAD to β-FADH+171 −−13 = +307 m− (forward) and α-FAD•− (backward) together with the associated domain II over 30 Å which first ensures the shuttling of two reducing equivalents from NADH to crotonyl-CoA. Second, the high redox potential of the α-FAD/α-FAD•− pair of domain II resulting in a α-FAD•− resting state might be also exploited to gate the second electron released by β-FADH• to the weak electron acceptor ferredoxin because α-FADH− cannot accept a further electron. The substantially reduced enzymatic activities of enzyme variants, specifically modified in the two contact regions, substantiate the existence of the two distinct domain II orientations during the catalytic process in solution  are clustered in the genome as bcd (CD 1054), etfB (CD 1055) and etfA (CD 1056). They were cloned in this order into the pASG IBA-3 vector according to manufacturer’s instruction 13. Both applied primers are listed in the Supplementary Table E. coli BL21(DE3),  using anhydrotetracycline . Cells were grown in 4 L culture of Luria Bertani broth containing ampicillin (100 µg ml−1). EtfAB/Bcd production was induced at an optical density (OD600nm) of 0.5. After growing overnight at room temperature, cells were harvested (15 g wet mass), suspended in 50 mM Tris/HCl buffer containing 150 mM NaCl, pH 7.5 (buffer A) and disrupted by a passage through a French press at 120 MPa. Cell lysate was centrifuged at 30,000 × g for 1 h at 4° C. The supernatant was loaded onto a 10 ml Strep-Tag II (IBA) column and was washed with 150 ml buffer A. The recombinant protein was eluted with 15 ml of 2.5 mM D-desthiobiotin in buffer A and concentrated using a Centricon filter tube . After incubation with an excess of FAD at 4° C overnight, protein and unbound FAD was separated using a Superdex 200 pg column ; only peak fractions were used for crystallization. The protein homogeneity was checked by SDS-PAGE after purification.The EtfAB/Bcd genes from E coli DH5α  and plated on to LB agar containing ampicillin (100 µg ml−1). The plasmid was isolated and sequenced for verifying the correctness of the mutation. Finally, the corresponding plasmid was transformed into E. coli BL21(DE3), and the EtfAB/Bcd variant produced and purified as described for the wildtype protein. The EtfA R136Q/K/M variant showed little or no overproduction, perhaps due to instability.Multiple variants of the EtfAB/Bcd complex were generated by site-directed mutagenesis using overlapping primers (see Supplementary Table d = 1 cm) containing 50 mM potassium phosphate, pH 6.8, 250 µM NADH, 100 µM crotonyl-CoA, 1.5 µM EtfAB/Bcd, 5 µM ferredoxin from Clostridium tetanomorphum24 and crude [FeFe] hydrogenase (30 µg ml−1) from Clostridium pasteurianum24. The decrease in NADH concentration was monitored at 340 nm, ε = 6.3 mM−1 cm−1 42. The Bcd activity was measured under aerobic conditions using 0.35 µM EtfAB/Bcd, 0.2 mM ferricenium hexafluorophosphate (Fc+) and 50 µM butyryl-CoA in 50 mM potassium phosphate, pH 6.8. The decrease of absorbance was followed at 310 nm, ε = 2 × 4.3 mM−1 cm−1, because 2 mol of Fc+ are required to oxidize 1 mol of butyryl-CoA43. The NADH oxidase activity of EtfAB/Bcd was measured in the presence of oxygen and in the absence of crotonyl-CoA using 0.2 µM EtfAB/Bcd and 150 µM NADH in 50 mM potassium phosphate, pH 6.8. Prior to the reaction, the phosphate buffer was bubbled with oxygen gas. The assays were repeated with the EtfAB/Bcd variants. The data were plotted in bar graphs using Graphpad Prism 5.The anaerobic bifurcation activity of EtfAB/Bcd was measured in a 500 µl cuvette . The final R/Rfree values were 20.8/25.9 %. Data quality was checked by MOLPROBITY49 and refinement statistics were listed in Table EtfAB/Bcd was concentrated to 20 mg mlAtomic coordinates and structure factors for the crystal structure of the EtfAB/Bcd domplex are deposited in the protein databank under the accession code 5OL2. Other data are available from the corresponding authors upon reasonable request.Supplementary InformationPeer Review File


Altered inhibitory control has been implicated in the development and maintenance of eating disorders (ED), however it is unclear how different types of inhibitory control are affected across the EDs. We explored whether individuals with bulimia nervosa (BN), binge eating disorder (BED) and anorexia nervosa (AN) differed from healthy individuals (HC) on two types of motor inhibitory control: proactive inhibition (related to the preparation/initiation of a response) and reactive inhibition . Ninety-four women  completed two neuropsychological tasks , and questionnaires assessing clinical variables, mood, anxiety, and inhibitory control. Self-reported inhibitory control was poorer in women with BN compared to the HC and AN groups, but greater in women with AN compared to all other groups. However, no group differences in reactive inhibition were observed. Proactive inhibition was augmented in women with AN compared to HC, and this was related to self-reported intolerance of uncertainty. The findings suggest that proactive inhibition may be a relevant target for behavioural interventions for AN, and call for further research into the relationship between intolerance of uncertainty and proactive inhibition. •Women with eating disorders self-report altered motor inhibition.•No behavioural differences in reactive inhibition on the stop signal task were observed.•Proactive inhibition is elevated in adult women with anorexia nervosa.•Group differences in proactive inhibition are related to intolerance of uncertainty. HoweverThese types of motor inhibitory control may be relevant to the way in which symptoms are manifested in EDs and how decisions are made across contexts. For example, poor reactive inhibitory control may contribute to impulsive behaviours that reflect an inability to resist temptation or overcome urges, e.g., binge eating . In contThis study compares proactive and reactive motor inhibitory control in individuals with AN, BN and BED to healthy controls (HC) and explores how proactive and reactive inhibition are related to ED pathology, anxiety and intolerance of uncertainty. We predict that participants with BN and BED will show less motor inhibitory control and participants with AN will show greater inhibitory control compared to HC. Poorer inhibitory control will be reflected by faster reaction times (RTs) in the context of uncertainty, poorer stop accuracy, and less benefit from warning cues. We also predict that poorer inhibitory control will be related to frequency of impulsive ED symptoms, i.e., binge eating and purging, and that inhibitory control (particularly proactive inhibition) will be related to intolerance of uncertainty.22.1www.callforparticipants.com). ED diagnoses were confirmed using the Eating Disorder Diagnostic Scale  DSM-5 diagnosis of BN and a BMI > 18.5 kg/m2, or (c) DSM-5 diagnosis of BED and a BMI > 18.5 kg/m2. Participants taking psychotropic medication were included provided their medication had been stable for ≥ 14 days prior to the study. HC were required to be of healthy BMI (18.5–25 kg/m2) and never had an ED or any current/previous neurological/psychiatric disorder. Participants were excluded if they were using illicit drugs or were pregnant.Ninety-four adult women participated: 28 with AN, 27 with BN, 11 with BED, and 30 healthy normal-weight women (HC). Healthy participants were recruited via online and poster advertisements at King's College London (KCL). Participants with an ED were recruited via online and poster advertisements at KCL, via the Eating Disorder Unit at the South London and Maudsley NHS Foundation Trust, via online adverts on the Beat website , and a research recruitment website . The practice block included 18 trials , 3 trials at 100 ms SOA, 4 trials at 300 ms SOA and 4 trials at 500 ms SOA). One experimental block consisted of only non-cued trials (“pure” block) whereas the other two included a mixture of cued and non-cued trials (“mixed” block), presented in a pseudorandomised order . The order in which the experimental blocks were completed was randomised and counterbalanced across participants. This task assessed proactive inhibition in two ways. (1) “Preparation cost” reflects the extent to which one's responses are slowed when not all stimuli are targets for response . This is2.2.2Participants were asked to provide rapid responses to a stimulus but to withhold their response in the presence of a stop signal. On each trial, participants were presented with a blue arrow (target) pointing left or right, and were asked to indicate the arrow's direction as quickly as possible using the keyboard's arrow keys. On a specified number of trials, a stop signal (red dot) was presented at a variable delay after the target. Participants were instructed to inhibit their response to the target if the stop signal appeared, but to not wait for the stop signal. The stop signal was presented at irregular intervals within each block to minimise predictability. The delay between the go signal and stop signal is termed the stop signal delay (SSD). The ability to inhibit a response is dependent on the length of the SSD: the longer the delay, the harder it is to inhibit a response. The SSD was varied from trial to trial in a staircase procedure  that converged subjects toward an overall performance of 50% for each run.  ) to calculate the SSRT.A blocked design was used, including a practice block  and 4 experimental blocks that varied in the proportion of stop signal trials , 15% , 25% , 35% . The block comprised of 25% stop trials was used to assess reactive inhibition, consistent with previous literature . Differe|signal) . Therefo2.3Diagnosis and eligibility was confirmed through a telephone screen, including the EDDS, SCID, and a study-specific inclusion and exclusion screening questionnaire developed by us. The EDDS  is a bri2.4® SPSS® software (Version 22). All tests were two-tailed and the level of significance was set at α=0.05. On the stop signal task, SSRT and mean RT (assessing reactive inhibition) were not normally distributed. Difference scores  for stop accuracy and post-error slowing were normally distributed, but not for go accuracy or mean RT. Logarithmic transformations normalised the distribution of the SSRT data, but not the mean RT data. Scores on the BIS-11 Attention and Motor subscales and DASS-21 Stress subscale were normally distributed, but those for all other subscales were not. Age, ED pathology , binge frequency and hunger were not normally distributed.Data were collected using in-house software and analysed using IBMt-tests and Mann-Whitney U tests. For ease of readability, we have reported all statistical results from post-hoc tests in a supplementary file  were conducted to explore whether the main ANOVA results were influenced by confounding effects of intolerance of uncertainty. Group differences in specific clinical variables  were compared across only the ED groups, whereas group comparisons on all other variables  included all 4 groups. Finally, Pearson and Spearman's correlations were conducted to explore the relationship between inhibitory control during the tasks, with respect to (a) task-based measures of proactive and reactive inhibitory control, (b) mood and anxiety ; (c) self-reported impulsivity and (d) clinical variables , BMI, symptom frequency).One-way analyses of variance (ANOVAs) were conducted to assess the effect of group on self-reported and task-based inhibitory control. Where data were not normally distributed, Kruskal-Wallis tests were employed to explore the effect of group, and Friedman's tests were conducted to explore the effects of within-subject variables. Significant main effects were further explored using Bonferroni-corrected post-hoc ary file . Given t2.5Ethical approval was obtained from the London Bloomsbury NHS Research Ethics Committee (REC ref: 15/LO/0251). All participants gave written consent after the procedures were explained and were debriefed after the experiment.3Ninety-four participants were included in the analysis. Due to computer malfunctions, incomplete data were collected from 3 participants  on the SST though all completed the playlist with 25% stop trial and thus, these participants were excluded from comparisons across playlists (to explore proactive inhibition) but were included in assessments of reactive inhibition . A further 4 participants  had very low levels of stop accuracy across all 4 blocks of the SST, indicating that the task was not completed correctly. These participants were excluded from all analyses of SST data. All participants completed the proactive inhibition task. Demographic and clinical information for each group is reported in 3.1p < 0.001). This was driven by lower anxiety, stress, depression and intolerance of uncertainty in the HC group compared to all ED groups, with no differences on any measure observed between the ED groups  = 177.627, p < 0.001), whereby participants responded significantly faster on non-cued trials in the pure block compared to the mixed block  = 0.792, p = 0.501) or interaction between group and condition  = 1.535, p = 0.211) was observed.Preparation cost was assessed by comparing mean RTs on all of the trials in the pure block to the non-cued trials in the mixed blocks . A mixed-effects ANOVA assessing the effects of group and condition revealed a main effect of condition =162.919, p<0.001), but no significant effect of group  = 1.823, p = 0.149) nor group by SOA interaction  = 1.074, p = 0.382). Post-hoc t-tests revealed significant differences between all SOAs, with fastest RTs observed at 500 ms SOAs, and slowest at 0 ms SOA .A mixed-effects ANOVA was conducted to assess the effects of and interaction between SOA  and group on proactive inhibition. This revealed a significant effect of SOA3.2.3F = 3.130, p = 0.030; IUS Self-Implications subscale: F = 2.911, p = 0.039). Post-hoc t-tests comparing RTs between groups (in the absence of covariates) revealed these group differences to be predominantly due to greater RTs in the AN group compared to HCs at all SOAs in the mixed block but not the pure block, though these findings did not survive correction for multiple comparisons. This suggest that group differences in proactive inhibition exist between AN and HC, but are masked by individual differences in intolerance of uncertainty.The main effect of SOA on RT was unaffected after inclusion of covariates for both preparation cost and warning benefit, however, a main effect of group on warning benefit emerged after covarying for intolerance of uncertainty , but no other relationships were observed . However, these findings did not survive Bonferroni correction for multiple comparisons.Uncorrected exploratory correlational analyses exploring the relationship between proactive inhibition outcomes on the cued RT task and intolerance of uncertainty  across all of the groups revealed a positive association between intolerance of uncertainty and preparation cost .χ2(3) = 97.690, p < 0.001) and stop accuracy (F(2166) = 22.672, p < 0.001) increased with the degree of stop signal probability, whereas go accuracy decreased (trend: χ2(3) = 6.957, p = 0.073). Participants reacted more quickly and with greater go accuracy during the block with no stop trials compared to blocks during which stop trials occurred, and showed the greatest RTs and stop accuracy during the block with the greatest stop trials. Post-error slowing also increased as stop signal probability increased, though this was not significant (F(2166) = 0.653, p=0.522).Mean RT on go trials  = 4.440, p = 0.218; 15% stop trials: χ2(3) = 2.511, p = 0.473; 25% stop trials: χ2(3) = 3.152, p = 0.369; 35% stop trials: χ2(3) = 5.049, p = 0.168) or on go accuracy on any of the blocks  = 5.459, p = 0.141; 15% stop trials: χ2(3) = 2.980, p = 0.395; 25% stop trials: χ2(3) = 2.707, p = 0.439; 35% stop trials: χ2(3) = 2.972, p = 0.396).As the distribution of mean RT and go accuracy data could not be normalised, Kruskal-Wallis tests were conducted to explore the main effect of group separately for each block. These did not find any significant effect of group on mean RT on any of the blocks  = 1.312, p = 0.276) or post-error slowing  = 0.893, p = 0.449), or interactions between group and block on either measure  = 1.028, p = 0.409; post-error slowing: F = 0.726, p = 0.629).Mixed-design ANOVAs similarly revealed no effect of group on stop accuracy (3.3.1.3F(2164)=3.016, p=0.052; IUS Self-Implications subscale scores: F = 2.886, p = 0.059), suggesting that intolerance of uncertainty contributed substantially to the variability of stop accuracy across participants, reducing the sensitivity of our initial analysis.The main effect of block on stop accuracy reached trend-level significance after controlling for intolerance of uncertainty .Post-hoc exploratory correlational analysis did not reveal any relationships between proactive inhibition outcomes on the SST and intolerance of uncertainty . As many participants did not converge at 50% inhibition accuracy as intended by the task, the mean SSD and inhibition (stop) accuracy (%) were analysed as secondary outcome measures to aid interpretation.F = 1.609, p = 0.193; stop accuracy: F = 1.355, p = 0.262; mean RT: χ2(3) = 3.152, p = 0.369; mean SSD: χ2(3) = 4.820, p = 0.185). However, visual inspection of the group differences did suggest that the (non-significant) differences were in the direction we had predicted, i.e., the AN group showed the greatest stop accuracies, mean stop signal delays and mean RTs (suggesting a more inhibited approach), whereas the BN group showed the poorest accuracies, smallest stop signal delays and fastest RTs  as a covariate did not affect the initial observations: no group differences were observed on measures of reactive inhibition (stop accuracy (%), log-transformed SSRT).3.4Self-reported impulsivity was explored using the BIS-11 and the behavioural inhibition subscale (BIS) of the BIS-BAS questionnaire.U tests revealed that while the AN and BN groups reported greater impulsivity in some domains compared to HC (BIS-11 Attention (both AN and BN), BIS (AN only) and BIS-11 Total (BN only)), the BN group reported greater overall impulsivity compared to the AN group  and the AN group reported less motor impulsivity (BIS-11 Motor) compared to all other groups. No other comparisons remained significant after correction for multiple comparisons.As some of the subscales of the BIS-11 and the BIS subscale were non-normally distributed, Kruskal-Wallis analyses were conducted to explore differences between group means. These revealed a significant difference between groups on the BIS subscale of the BIS-BAS, the BIS-11 Attention and Motor subscale scores and BIS-11 total scores, whereas no group differences were observed on the BIS-11 Non-planning subscale . Post-ho3.5Reactive inhibition outcomes showed negative associations with BMI  and binge frequency (SSRT [trend]), but no other relationships were observed . This inProactive inhibition outcomes also showed a relationship with clinical variables across the ED groups, with go accuracy change negatively associated with length of illness and change in post-error slowing negatively associated with purge frequency. This suggests that individuals reporting a longer illness duration and more frequent purging episodes show less adjustment of their motor responses as uncertainty changes, suggesting poorer proactive inhibition. However, none of these findings survived correction for multiple comparisons, therefore the findings must be interpreted with caution.4This study investigated whether proactive and reactive motor inhibitory control differed across ED. The self-report data indicated that individuals with BN considered themselves to have less overall inhibitory control compared to those with AN and HCs, whereas individuals with AN reported the greatest motor inhibitory control compared to all other groups see . In termWe did not observe any differences in reactive inhibitory control between the groups. This is in contrast to the literature supporting impaired reactive inhibitory control in individuals with binge-type eating disorders  for this disorder, possibly due to the relatively short illness duration. This may have contributed to the lack of statistically significant results in the behavioural tasks, as a trend towards a positive relationship between BMI and SSRT has previously been reported in obese participants with and without BED , and difThis study is the first to explicitly explore proactive inhibition in EDs using multiple neurocognitive methods for assessing proactive inhibition, and provides early empirical support for altered proactive inhibition as a correlate of AN. Our findings suggest that motor inhibitory control is not uniformly implicated across EDs. Studies of proactive inhibition in larger samples (particularly in the BED group) are required to provide more definitive evidence on the involvement of proactive and reactive inhibition in EDs.The authors report no conflicts of interest


S275LPDL), lacking basic residues that are otherwise critical for SIK/MARK recognition as well as 14-3-3 binding. Correspondingly, the activity of these motifs differs between CRTC family members. As the variant (SLPDL) motif is present and apparently phosphorylated in other mammalian proteins, our studies suggest that the regulation of cellular targets by AMPK family members is more extensive than previously appreciated.The second messenger cAMP stimulates cellular gene expression via the PKA-mediated phosphorylation of the transcription factor CREB and through dephosphorylation of the cAMP-responsive transcriptional coactivators (CRTCs). Under basal conditions, CRTCs are phosphorylated by members of the AMPK family of Ser/Thr kinases and sequestered in the cytoplasm via a phosphorylation-dependent association with 14-3-3 proteins. Increases in cAMP promote the dephosphorylation and nuclear translocation of CRTCs, where they bind to CREB and stimulate relevant target genes. Although they share considerable sequence homology, members of the CRTC family exert non-overlapping effects on cellular gene expression through as yet unidentified mechanisms. Here we show that the three CRTCs exhibit distinct patterns of 14-3-3 binding at three conserved sites corresponding to S70, S171, and S275 (in CRTC2). S171 functions as the gatekeeper site for 14-3-3 binding; it acts cooperatively with S275 in stabilizing this interaction following its phosphorylation by the cAMP-responsive SIK and the cAMP-nonresponsive MARK kinases. Although S171 contains a consensus recognition site for phosphorylation by AMPK family members, S70 and S275 carry variant motifs (MNTGG The three members of the cyclic AMP (cAMP)-regulated transcriptional coactivator (CRTC) family share a common domain structure consisting of a conserved N-terminal cAMP response element-binding protein (CREB) binding domain (CBD), a central regulatory domain, and a C-terminal transactivation domain (TA) . Both thHormonal signals that stimulate intracellular cAMP concentrations increase gene expression via the phosphorylation of CREB and via the dephosphorylation of the CRTC family . CREB ph14-3-3 proteins bind as monomers or dimers to phosphorylated substrates that contain either MODE I (R[S/Ar][+/Ar]pS[L/E/A/M]P) or MODE II  recognition sites . In keepCRTCs contain three principal phosphorylation sites— that are regulated by cAMP and calcium signals, respectively–although the extent to which they and perhaps others modulate the activity of individual family members is unclear , 9, 20. Small molecules were solubilized in DMSO  at the indicated concentrations and stored until usage at -80°C (long term storage) or -20°C (working dilution): 20 mM Forskolin (Sigma-Aldrich) and 2 mM Carfilzomib (PR-171) .The antibodies used in Western blot analysis were purchased from Santa Cruz Biotechnology , EMD Millipore (α-tubulin), Sigma-Aldrich (FLAG M2), Roche (Anti-HA-Peroxidase), Cell Signaling Technology , and Covance . See Antiserum Production for the P-CRTC3 S273 antiserum (PBL #7378).All animal procedures were approved by the Institutional Animal Care and Use Committee of the Salk Institute and were conducted in accordance with the PHS Policy on Humane Care and Use of Laboratory Animals , the U.S. Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research and Training, the NRC Guide for Care and Use of Laboratory Animals (8th edition) and the USDA Animal Welfare Act and Regulations. Three 10 to 12-week old, female New Zealand white rabbits, weighing 3.0 to 3.2 kg at beginning of the study, were procured from Irish Farms . All animals were housed in an AAALAC accredited facility in a climate controlled environment  under 12-hour light:12-hour dark cycles. Rabbits were provided with ad libitum feed (5326 Lab Diet High Fiber), micro-filtered water and weekly fruits, vegetables and alfalfa hay for enrichment. Upon arrival, animals were physically examined by veterinary staff for good health and acclimated for two weeks prior to initiation of antiserum production. Each animal was monitored daily by the veterinary staff for signs of complications and weighed every two weeks. Routine physical exams were also performed by the veterinarian quarterly on all rabbits.268pSer273 CRTC3(268–282) coupled to keyhole limpet hemocyanin via maleimide. The peptide, <Hnt>CNTGGpSLPDLTNLHY<NH2>, was synthesized and purified by Jean E. Rivier . The antigen was delivered to host animals using multiple intradermal injections of peptide-KLH conjugate in Complete Freund's Adjuvant  or incomplete Freund's adjuvant (booster inoculations) every three weeks. Rabbits were bled, <10% total blood volume, one week following booster injections and bleeds screened for titer and specificity. Animals were administered 1–2 mg/kg Acepromazine IM prior to injections of antigen or blood withdrawal. At the termination of study, animals were exsanguinated under anesthesia  and euthanized with an overdose of pentobarbital sodium and phenytoin sodium (1 ml/4.5 kg of body weight IC to effect). After blood was collected death was confirmed. All animal procedures were conducted by experienced veterinary technicians, under the supervision of Salk Institute veterinarians.The rabbits were injected with a peptide fragment encoding Cys268CRTC3(268–282)-agarose resin. Covalent attachment of peptide to resin  was per manufacturer's instructions.The antiserum obtained from the rabbit (code PBL #7378) with the best characteristics of titer and specificity was used for all experiments. So that the same batch of serum could be used for this and future studies, a large volume of serum from a single bleed was depleted of antibodies recognizing the non-phosphorylated form of CRTC by passing over a column containing CysHomo sapiens Ubiquitin C promoter (pUbC), whose activity is unaffected by cAMP signaling. pUbC was incorporated into three vectors (MCS = multiple cloning site): pUbC-3xFLAG-TEVsite-His6-MCS-IRESeGFP as well as pUbC-MCS-His6-TEVsite-3xFLAG-STOP-IRESeGFP , and pUbC-3xHA-MCS (pCHA backbone). Additional fragments integrated into the final vectors originated from the following sources: p3xFLAG-CMV™-14 (Sigma-Aldrich), pCeMM-CTAP(SG) , 60  ng of emAmino acid sequences were aligned using MegAlign and Clustal W method (DNASTAR v7).In mass spectrometry (MS) studies to identify proteins that modulate CREB-dependent transcription, we recovered large quantities of 14-3-3 proteins from immunoprecipitates (IPs) of epitope-tagged CRTCs prepared from HEK293T cells, even following exposure to the adenylyl cyclase activator Forskolin (Fsk) . In keepBinding of CRTCs to 14-3-3s sequesters them in the cytoplasm; and increases in cAMP promote the dephosphorylation and release of CRTCs from 14-3-3 proteins leading to their nuclear localization , 20, 31.We performed Ser/Ala mutagenesis of individual phospho-acceptor sites within each conserved region to determine their effects on CRTC transcriptional activity by transient assay with a luciferase reporter containing cAMP responsive elements (CREs) . For eac146RR) and CRTC3 (L157NR)  decreased CRTC3 activity more than 50% and increased CRTC1 more modestly (Having seen that mutation of region II in CRTC1 (S151A) has a stronger effect on 14-3-3 binding and CRE reporter activity compared to the corresponding mutant in CRTC3 (S162A), we examined the potential role of residues flanking this phospho-acceptor site in modulating the transcriptional activity of individual family members. S151 in CRTC1 is preceded by two arginine residues at positions , whereas CRTC2 and CRTC3 contain only a single arginine . Becausemodestly . These rAlthough members of the AMPK family have been shown to modulate CREB target gene expression through inhibitory phosphorylation of the CRTCs, their relative activities have not been examined , 8, 9. B2+-dependent Ser/Thr phosphatase calcineurin . In. InSLPDLDL motif . Correspbination , 42. Notbination , 44, Our


Staphylococcus aureus (S. aureus) (MRSA). Importantly, MRSA lacking Stk1 become sensitive to β-lactam antibiotics, implying that Stk1 could be an alternative target for combination therapy. However, the autophosphorylation mechanism of Stk1 remains elusive. Using a phosphoproteomic study, we identified six in vivo phosphorylated activation loop residues  of Stk1, which are also phosphorylated in vitro. We further showed that cis autophosphorylation of Thr172 in the GT/S motif is essential for self-activation and kinase activity of Stk1 kinase domain (Stk1-KD), whereas the trans autophosphorylation of other activation loop serines/threonines are required for the optimal kinase activity of Stk1-KD. Moreover, substitution of the activation loop serines/threonines impaired in vivo autophosphorylation activity of kinase variants, while T172A and T172D variants were unable to autophosphorylate in the cellular content, underlining the essential role of Thr172 for Stk1 activity in vivo. This study provides insights into molecular basis for regulation of Stk1 activity from S. aureus.The eukaryotic-like Ser/Thr kinase Stk1 is crucial for virulence, cell wall biosynthesis, and drug susceptibility in methicillin-resistant  Staphylococcus aureus (S. aureus) is one of the major human pathogens that greatly impacts individuals and causes a variety of illnesses ranging from minor skin infections to life-threatening diseases, such as endocarditis, pneumonia, septicemia, and toxic shock syndrome (S. aureus strains (MRSA) and vancomycin-resistant S. aureus strains . PASTA domains are composed of ∼65 amino acids and are thought to be a sensor motif to bind beta-lactam compounds as well as cell wall fragments  (stk1 renders MRSA to become susceptible to β-lactam antibiotics (sistance . Based osistance . Stk1 isglycans) . Intriguibiotics , indicatibiotics . Howeverin vivo and in vitro. Mutagenesis and biochemical studies revealed the involvement of these residues in autophosphorylation and kinase activities. We demonstrated that Thr172 is crucial for activity of Stk1 both in vitro and in vivo. Our findings comprise a step toward understanding the activation of RD-family kinases of eSTKs.In this study, we focus on investigating the autophosphorylation mechanism of Stk1. Using mass spectrometry studies, we found six residues  located at the activation loop are phosphorylated both stk1  was amplified from a previous plasmid pET22b-stk1 carrying full length of stk1  with desired point mutation(s). All of the constructs were sequenced to rule out unwanted mutations.The primers used in current study are listed in Supplementary Table  of stk1 . The pur600 reached 0.6–0.8, the cells were induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 16°C for overnight. Bacteria were collected by centrifugation and re-suspended in 30 mL of buffer A  with protease inhibitor phenylmethanesulfonyl fluoride (PMSF). After loaded to a HisTrap HP column, the column was washed with appropriate volume of 0 and 5% (v/v) buffer B . Then the protein was eluted with a 50% (v/v) buffer B. The fractions were collected, concentrated, and further purified by a HiTrap desalting column with desalting buffer I .The expression and purification of kinases and Stp1 have been described previously , 2016. BTo prepare His-tag-free T172A (fT172A), the His-tag of T172A was removed by thrombin (Novagen) cleavage for 20 h at room temperature (RT). The protein solution was passed through a HisTrap HP column to remove the His-tag, and was further purified by a Superdex 200 10/300 GL column with desalting buffer I. All the purified proteins were >90% pure as estimated by the 12% (wt/vol) SDS-PAGE gel  at 37°C for 30 min, followed by the addition of 8 μg MBP (Sigma) and another 30-min incubation at 37°C. To examine the trans-autophosphorylation activity of WT, 5M and T172D toward fT172A, 0.2 μg WT or 5M or T172D was incubated with 1.5 μg fT172A at RT for 30 min, then 8 μg MBP was added subsequently for additional 15-min incubation at 37°C. All reactions were quenched with the addition of 4× SDS-PAGE loading buffer. Samples were separated by electrophoresis on 12% SDS-PAGE gel and transferred to a PVDF membrane. After equilibrating with TBST buffer for 1–2 h, the membrane was probed with Phos-tag-bound Streptavidin-conjugated HRP [prepared from Phos-tag Biotin (Wako)] at RT for 30 min. After incubation, the membrane was washed twice for 5 min each time in TBST buffer and detected by chemiluminescent reaction with ChemiDoc XRS System (Bio-Rad).To investigate the effect of single-point mutation on autophosphorylation activity and kinase activity of Stk1, 2 μg kinase was first incubated in 20 μL of buffer P .Phosphorylation levels of Stk1-KD variants were determined by western blotting analysis. BL21 (DE3) pLysS cells harboring pET28a-in vitro phosphorylation with non-radioactive ATP, the mixture containing purified Stk1-KD was subjected to electrophoresis on a 12% SDS-PAGE gel, which was stained by Coomassie R250. The gel band corresponding to Stk1-KD was cut into pieces, subjected to reduction with 10 mM DTT at 56°C for 50 min, and subsequent alkylation with 55 mM iodoacetamide (IAA) for 30 min at RT in dark. Then in-gel digestion was performed with trypsin  at 37°C for 18 h in 25 mM ammonium bicarbonate buffer. The lyophilized tryptic digested samples were re-dissolved in 2% acetonitrile, 0.1% formic acid, and loaded on ChromXP C18  nanoLC trap column. The online trapping, desalting procedure was carried out at a flow rate of 2 μL/min for 10 min with 100% solvent A (Solvent A: water/acetonitrile/formic acid = 98/2/0.1% solvent B: 2/98/0.1%). Then, an 60-min gradient elution ranging from 5 to 35% acetonitrile (0.1% formic acid) was used on an analytical column . LC–MS/MS analysis was performed with a Triple TOF 5600 System  fitted with a Nanospray III source . Data was acquired using an ion spray voltage of 2.5 kV, curtain gas of 30 PSI, nebulizer gas of 5 PSI, and an interface heater temperature of 150°C. The MS was operated with TOF-MS scans. For IDA, survey scans were acquired in 250 ms and as many as 25 product ion scans (90 ms) were collected if exceeding a threshold of 150 counts per second (counts/s) and with a +2 to +4 charge-state. A Rolling collision energy setting was applied to all precursor ions for collision-induced dissociation. Dynamic exclusion was set for ½ of peak width (∼12 s). For data analysis, the.wiff file was processed by ProteinPilot 5.0. Searches were performed against the local database including the protein sequences for Stk1-KD and decoys. The search effort was set to ‘Thorough ID’ and the False Discovery Rate Analysis was involved with the default setting for ‘Detected Protein Threshold [Unused ProtScore (Conf)]’ at 0.05. Only phosphopeptides with 99% confidence were accepted and further checked manually.Liquid chromatography–mass spectrometry/mass spectrometry (LC–MS/MS) analysis was performed as previously described with modifications . After iStaphylococcus aureus Newman cells were grown at 37°C overnight in tryptic soy broth (TSB) containing 5 μg/mL nalidixic acid. The overnight culture was diluted 100-fold in fresh 100 mL of TSB, and incubated at 37°C, 250 rpm for 5 h. 10 μM Mn2+ was added at 1 h before cell harvest. Bacteria were collected by centrifugation, washed quickly once with 20 mL of ice-cold Tris-bufer (pH 8.0) and re-suspended in 5 mL of ice-cold lysis buffer . After the addition of 1.2 g glass beads, the mixture was subjected to vortex for 20 min . Then the lysate was centrifuged at 4°C, 14,000 rpm for 30 min. 0.8 mg protein sample was transferred to a 10K Nanosep® Centrifugal Devices , and centrifuged at 12,000 rpm for 30 min. Reduction was conducted with 100 μL of buffer1  containing 10 mM DTT for 1 h at 37°C. Subsequent alkylation was performed in buffer2 (buffer1+ 50 mM IAA) for 15 min at RT in dark. After centrifugation at 12,000 rpm for 20 min (remaining volume ∼30 μL), buffer3 (50 mM ammonium bicarbonate) was added for exchanging buffer for three times. The proteolytic digestion was conducted in 200 μL of buffer3 with trypsin [enzyme: protein = 1:50 (wt/wt)] at 37°C for 18 h. After the digested sample was centrifuged at 12,000 rpm for 10 min and washed with 100 μL of buffer3 for twice, all the filtrates were pooled and lyophilized. The trypsin-digested sample was enriched by titanium dioxide beads (GL Sciences). Enriched sample was analyzed by LC–MS/MS as described above. Acquired data were processed by ProteinPilot 5.0 against the local protein database of S. aureus Newman (GenBank Accession No.: AP009351.1). Dataset were filtered with the following criteria: for proteins, Unused ProtScore ≥ 2.0, which corresponds to a protein confidence cut-off threshold of 99%; for phosphopeptides, Conf ≥ 99%. Final manual inspection of spectrums was engaged. The mass spectrometry proteomics data have been deposited in the PRIDE Archive1 with the project accession number PXD006285 .Circular dichroism (CD) experiments were conducted as previously described . BrieflyThe common regulatory mechanism for activity of Ser/Thr kinases is the phosphorylation of a canonical kinase segment known as the activation loop, which is flanked by the conserved N-terminal DFG and C-terminal APE motifs. Autophosphorylation of activation loop serine, threonine, or tyrosine residues will bring a conformational change that stabilizes the kinase in an active conformation . AlthougS. aureus, we conducted a phosphoproteomic study of S. aureus Newman strain. The protein extract was first digested with sequencing grade trypsin and enriched by titanium dioxide beads. The isolated phosphopeptides were then subjected to LC–MS/MS analysis. With this approach, we identified 76 peptides with 50 mapped phosphorylation sites belonging to 29 distinct proteins . Purified Stk1-KD was incubated with ATP/Mn2+ and subjected to in-gel trypsin digestion. Subsequent LC–MS/MS analysis yielded mono-/di-/triphosphorylated activation loop peptides with same phosphorylation sites as identified in vivo . Altogether, these data imply a similar autophosphorylated pattern of activation loop for Stk1 both in vivo and in vitro. And all the phosphorylated residues of Stk1-KD are the dephosphorylated targets of Stp1.In order to identify the phosphorylation sites on activation loop of Stk1 in  of Stk1 . We furtFigures 2A,B, S159A, T161A, S162A, and T166A variants exhibited decreased autophosphorylation activity, while T172A variant barely showed any autophosphorylation activity.The activation loop serines/threonines are all autophosphorylated, implying that the differential phosphorylation of activation loop may regulates the kinase activity of Stk1. To investigate this possibility, we first explored the contributions of phosphorylation sites of activation loop to kinase activity of Stk1. We generated six Ala substitution variants: S159A, T161A, S162A, T164A, T166A, and T172A. We first examined the autophosphorylation activity of all Stk1-KD variants. As shown in in vitro kinase assay. Compared to wild type (WT) Stk1-KD, all Stk1-KD variants showed differentially reduced substrate phosphorylation activity, among which T172A variant was not able to phosphorylate MBP . We then replaced Ser159 and Thr172 with phosphomimic residue Asp (S159D and T172D) to see if activity of these kinase variants could be rescued. As expected, S159D variant has similar autophosphorylation and substrate phosphorylation activities as the WT kinase, whereas Asp substitution of Thr172 did not restore any activity of the kinase variant despite similar loading of samples in the gel . The marginal phosphorylation of T172A and T172D variants may be caused by unknown reasons, which did not activate the autophosphorylation and kinase activities of these variants in the absence of intact Thr172 in vitro . Collectively, these results suggest that different phosphorylation level of Stk1 activation loop regulates the extent of autophosphorylation and substrate phosphorylation activities of Stk1. In particular, phosphorylation of Thr172 is essential for the functionality of Stk1.To evaluate the substrate phosphorylation activity of Stk1-KD activation loop variants, we used myelin basic protein (MBP) as a surrogate substrate in the As phosphorylation of activation loop residues will bring conformational changes in Ser/Thr kinase, we next investigated the effect of Ala substitutions of Stk1 activation loop serines/threonines on the overall folding of the proteins. Far-UV (195–250 nm) CD spectroscopy was used to monitor changes in protein secondary structure of Stk1-KD variants. As expected, the CD spectra of WT Stk1-KD was consistent with a mixed α/β fold of structure of Ser/Thr kinases. Ala replacement of Stk1 activation loop serines/threonines caused minor changes in secondary structure content of the kinase variants  (Mycobacterium tuberculosis (M. tuberculosis) and Ser173 of Ba-Stk1 from Bacillus anthracis (B. anthracis) are also crucial for kinase activity (2+ into the active site of Stk1-KD model by using Molecular Operating Environment (MOE). After measurement, Thr172 is the nearest residue to the γ-phosphate group of ATP (7.6 Å) compared to other activation loop serines/threonines (14.5–21.5 Å)  . Indeed,activity . Mtb-Pknactivity . To compactivity  . However, despite the marginal phosphorylation background, fT172A and T172D did not show autophosphorylation and substrate phosphorylation activities, in line with the aforementioned results . In addition, these two variants could not trans-autophosphorylate each other . Although 5M shares similar overall folding with WT Stk1-KD , which may be due to that substitution of Ser/Thr with five alanines hinders the allosteric regulation of Stk1 activation loop during autophosphorylation. However, 5M could still significantly trans-autophosphorylate fT172A, although less efficiently than WT . Interaction between 5M and fT172A may trigger the trans-autophosphorylation activity of 5M variant. Interestingly, in comparison with 5M, the mixture of 5M and fT172A did not exhibit increased substrate phosphorylation activity for MBP . Moreover, the phosphorylation of MBP in the mixture of WT and fT172A significantly reduced compared to that in the mixture of WT , further confirming that phosphorylation of Thr172 is necessary for kinase activity of Stk1. A possible explanation is that the phosphorylated fT172A proteins compete against WT proteins for binding the substrate. Without phosphorylation of Thr172, phosphorylated fT172A is unable to regain activity. Together, these results suggest that autophosphorylation of Thr172 in the GT/S motif is mediated by a cis mechanism, while other activation loop serines/threonines are autophosphorylated by a trans mechanism.To test this hypothesis, we created a new Stk1-KD variant 5M (S159A/T161A/S162A/T164A/T166A) by replacing all the activation loop serines/threonines with Ala except Thr172. His-tag-free T172A (fT172A) was prepared by cleaving the His-tag of T172A  pLysS cells with pET28a constructs carrying coding sequences of different Stk1-KD variants. E. coli BL21 (DE3) pLysS cells transformed with pET28a vector served as a negative control. The expression of Stk1-KD variants were induced by IPTG, and total protein lysates were prepared by sonication. Despite the different expression levels of Stk1-KD variants, T172A, T172D, and 5M variants did not show autophosphorylation activities in the cellular content . To further investigate the impacts of other activation loop serines/theronines on the autophosphorylation activity of Stk1-KD, we directly evaluated the autophosphorylation levels of purified Stk1-KD variants. As expected, T172A, T172D, and 5M variants completely lost autophosphorylation abilities . While Ala substitution of Thr161 did not affect the in vivo autophsophorylation activity of Stk1-KD, the other kinase variants S159A, S162A, T164A, and T166A showed reduced autophosphorylation abilities in the cellular content . Collectively, Thr172 plays an essential role in the autophosphorylation activity of Stk1 in vivo.The importance of activation loop serines/threonines S. aureus  from the activation loop were successfully identified , indicative of a fully phosphorylated activation loop within Stk1 in the pathogen. Interestingly, these six phosphorylation sites could be identified after in vitro autophosphorylation of Stk1-KD , suggesting all of activation loop serines/threonines are required for the efficient catalytic activity of Stk1. This also implies that differential phosphorylation of activation loop modulates the kinase activity of Stk1. Interestingly, Ala or Asp substitution of Thr172 in GT/S motif results in non-functional kinases , indicating that phosphorylation of Thr172 is essential for activation and kinase activity of Stk1. Indeed, the necessity of phosphorylation of corresponding serine or threonine for kinase activity has also been reported with M. tuberculosis Mtb-PknA and B. anthracis Ba-Stk1 . Further evidence comes from the trans-autophosphorylation assays. fT172A and T172D did not show autophosphorylation activity, or trans-autophosphorylation activity to each other. However, WT Stk1-KD could autophosphorylate, and efficiently trans-autophosphorylate fT172A and phosphorylate MBP . To a less extent, similar results were also observed for 5M where Thr172 is intact. These results suggest that autophosphorylation of Thr172 in the GT/S motif is mediated by a cis mechanism, whereas other activation loop serines/threonines are trans autophosphorylated. Indeed, similar mechanism was also observed for Thr180 of Mtb-PknA [28].Subsequent homology modeling of Stk1-KD revealed that Thr172 is the closest residue to the γ-phosphate group of ATP compared to other activation loop serines/threonines. These imply that Stk1 adopts a Figures D). Unexpectedly, although WT was able to more efficiently trans-auotophosphorylate fT172A, phosphorylated fT172A significantly decreased the phosphorylation level of MBP . These results suggest that in the absence of phosphorylated Thr172, phosphorylation of other activation loop residues has no impact on the kinase activity of Stk1. This further supports that phosphorylation of Thr172 is the first step to activate Stk1. The importance of Thr172 was further confirmed by evaluating the autophosphorylation activity of Stk1-KD variants in E. coli cells, where T172A or T172D did not show autophosphorylation ability in the cellular content , indicating the essentiality of Thr172 for Stk1 activity both in vitro and in vivo.Ala replacement of serines/threonines in the GT/S motif of homologous Ser/Thr kinases led to inactive variants, which hinders evaluating the effect of phosphorylation of other activation loop residues on kinase activity in the absence of phosphorylation of GT/S motif. For Stk1, the trans-autophosphorylation of fT172A by 5M or WT may provide some clues. 5M could trans-auotophosphorylate fT172A, which did not increase phosphorylation for substrate MBP . In the inactive state, the unphosphorylated activation loop is folded into the catalytic cleft mimicking a substrate, which prevents the binding of substrates. Under unknown external stimulation, the binding of ATP would trigger the cis autophosphorylation of Thr172 to change the conformation of activation loop, leading to self-activation of Stk1. Subsequent interaction with another Stk1 molecule would induce the trans autophosphorylation of other activation loop residues for optimal kinase activity. Mutation of Thr172 leads to non-functional kinase, regardless of the phosphorylation status of other activation loop residues. The cognate phosphatase Stp1 dephosphorylates Stk1, regenerating the inactive, unphosphorylated kinase.Based on our current study, we propose an activation model for Stk1 (S. aureus. Given the conservation of the GT/S motif among the RD family of eSTKs, our findings also provide more insights into the regulation of activity of eSTKs. Thr172 and the stepwise activation of Stk1 may play key roles in virulence and pathogenesis of S. aureus, which awaits further studies. Kinases are well-known targets with similar structures, which may hamper the development of specific inhibitors. However, considering the high sequence variability and differential phosphorylation in activation loop of bacterial STKs, this study is also of practical importance for rational design of selective and effective Stk1 inhibitors for combating drug-resistant S. aureus including MRSA.In summary, our findings established that the phosphorylation of Thr172 of the GT/S motif in the activation loop is necessary for self-activation and kinase activity of Stk1 from WZ, XC, and ZL designed the research. WZ, XC, and SL performed the research. WZ and XC analyzed the data. WZ wrote the manuscript. ZL reviewed and modified the manuscript. All the authors revised and approved the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Drosophila larva overexpressing PAR1 . In 293T culture, MB decreased MARK4-mediated Tau phosphorylation in a dose dependent manner. Further studies revealed a two-fold mechanism by MB including down-regulation of MARK4 protein level through ubiquitin-proteasome pathway and inhibition of MARK4 kinase activity in vitro. This study highlights the importance of MARK4 as a viable target for Tauopathy and provides fresh insight into the complex mechanism used by MB to treat AD.Alzheimer’s disease (AD) is a neurodegenerative disease characterized by genotypic and phenotypic heterogeneity. Critical components of the two AD pathological pathways, Aβ-amyloidosis and Tauopathy, have been considered as therapeutic targets. Among them, much effort is focused on aberrant Tau phosphorylation and targeting Tau-phosphorylating kinases. Methylene blue (MB), a phenothiazine dye that crosses the blood-brain barrier, has been shown to hit multiple molecular targets involved in AD and have beneficial effects in clinical studies. Here we present evidence that microtubule affinity-regulating kinase (MARK4) is a novel target of MB. MB partially rescued the synaptic toxicity in  Drosophila, overexpression of PAR1, the fly homolog of MARK kinase, leads to elevated tau phosphorylation at S262 and S356 sites and increased toxicity. More importantly, the initial phosphorylation of Tau by PAR1 at S262/S356 sites is prerequisite for the subsequent multisite phosphorylation of Tau by other kinases, such as GSK-3β and CDK5Drosophila model revealed that kinases and phosphatases are the major factors to affect Tau toxicityAlzheimer’s disease (AD) is the most common neurodegenerative disease and causes progressive loss of memory and other cognitive functions. The pathological hallmarks of AD include amyloid plaque and neurofibrillary tangles (NFT). Tau, a highly soluble microtubule-binding protein, has been determined to be the major component of NFT, where it is hyperphosphorylated and becomes insoluble and filamentous13Methylene blue (MB) is a dye that has been widely used in a range of different fields, including uses as a medicine in treating various bacterial and viral infections and cancer9101213Drosophila model as well as in mammalian cell cultures. A demethylated analog of MB, Azure C (AC) was also tested. In a screening study, both AC and MB showed activity in regulating Tau stability through heat shock protein Hsp70Here we studied the effect of MB on MARK4 mediated Tau phosphorylation in a in vivo, we used Drosophila NMJ as the model system. Previously, it has been shown that PAR1/MARK is predominantly localized at NMJ and overexpression (OE) of PAR1 leads to decreased synapse formation and synaptic transmission2021in vivo.To determine whether AC, MB and OS  can affeTo extend our observation to mammalian system, we tested whether MB has inhibitory effect on MARK4 mediated Tau phosphorylation in mammalian cell cultures. 293T cells were transiently transfected with plasmids encoding human 4R2N Tau and HA tagged MARK4 (HA-MARK4-WT). A kinase dead form of MARK4 (HA-MARK4-KD) was included as a control. As shown in It is worth noting that we also detected high molecular weight forms of Tau (HMW Tau) when the cells were treated with high concentrations of MB and AC. The HMW Tau was seen as protein bands/smear larger than 75 kDa and appeared in both pTau and total Tau blots . One posSince endogenous MARK4 activity was detected, we repeated the experiment under more physiologically relevant condition without overexpression of HA-MARK4. Similar results were observed as shown in To evaluate the selectivity of kinase inhibition by these compounds, we also measured Tau phosphorylation by other endogenous kinases at additional sites including Serine 202, 214 and 396. GSK-3β and CDK5 among other kinases have been shown to be involved in Tau phosphorylation at these sitesDrosophila model, HA-MARK4-WT protein level was down regulated by MB in a dose dependent manner  E3 ubiquitin ligase complex19We provide evidence that down-regulation of MARK4 protein level by MB is specific and at least in part through degradation by the ubiquitin-proteasome system . Lee et in vitro kinase assay results indicate that MB directly inhibits Tau phosphorylation by MARK4 at Ser262 . Mammalian expression constructs of Tau, HA-MARK4 WT and KD are generous gifts of Dr. Bingwei Lu (Stanford University). (R)-MG132 was purchased from Cayman Chemical Azure C was purchased from Fluka/Sigma , methylene blue was purchased from EMD Chemicals  and olsalazine sodium was purchased from AK Scientific . Expression plasmid for the human full-length four-repeat Tau in The UAS-PAR1-Myc was described before37Third instar larvae were selected, dissected in PBS, fixed in 4% formaldehyde (Ted Pella) in PBS for about 15 minutes and washed ×3 in 0.1% Triton X-100 in PBS. FITC-conjugated anti-HRP (Jackson ImmunoResearch Laboratories) was used at 1:150. For Dlg staining, monoclonal mouse anti-Dlg (1:500)2 with Dulbecco’s modification of Eagle’s medium (DMEM) (Gibco/Life Technologies) with 10% fetal bovine serum (FBS) (Gibco/Life Technologies), 100 units/ml penicillin and 100 μg/ml streptomycin (Gibco/Life Technologies). For transfection, cells were plated in poly-D-lysine coated 12-well plates (BD) at 2.2 × 105 cells/well. After 40 h incubation, each well were transfected with 1 μg of plasmid DNA encoding human 4R2N Tau and/or HA tagged human MARK4 (WT or KD) using lipofectamine 2000 (Life Technologies) according to manufacturer’s instruction. 24 h after transfection, cells were treated with AC, MB or OS at indicated final concentrations for 3 h at 37 °C. When MG132 was used to inhibit proteasomes, cells were pretreated with 500 nM MG132 for 1 h at 37 °C followed by drug treatment in the presence of MG132.293T cells were cultured at 37 °C in 5% CO3VO4, 10 mM NaF, 60 mM β-glycerolphosphate, 10% glycerol, protease inhibitor cocktail), and then centrifuged at 13,000 g for 20 minutes at 4 °C. Supernatants were boiled in SDS sample buffer. For 293T cultures, cells were washed in ice cold 1 × PBS and lysed in CHAPS lysis buffer . After two free-thaw cycles, cell lysates were centrifuged at 13,000 rpm 4 °C for 10 min. Supernatant was used for BCA protein assay, immunoprecipitation or WB. Western blot was performed using the following primary antibodies: rabbit anti-PAR1 antibody21For fly larvae, dissected body-wall muscles were homogenized in lysis buffer . Recombinant protein was purified using Ni-NTA agarose (Qiagen).Purification of recombinant 4R2N hTau was carried out as described previously412) was mixed with 150 ng of GST-MARK4 (Sigma) and 150 ng of lab-made recombinant 4R2N hTau followed by addition of AC, MB or OS at the indicated concentrations. ATP was added last to a final concentration of 200 μM. The total volume was brought up to 15 μl with protease free H2O. The reaction was incubated at 30 °C for 30 min and then terminated by adding 4 × LDS buffer with 2-mercaptoethanol. After incubation at 70 °C for 10 min, the samples were analyzed by Western blot.For a typical reaction, 5 μl of 3 × kinase buffer  was performed for statistical analysis.How to cite this article: Sun, W. et al. Attenuation of synaptic toxicity and MARK4/PAR1-mediated Tau phosphorylation by methylene blue for Alzheimer's disease treatment. Sci. Rep.6, 34784; doi: 10.1038/srep34784 (2016).


Pedicle screw fixation in the upper cervical spine is a difficult and high-risk procedure. The screw is difficult to place rapidly and accurately, and can lead to serious injury of spinal cord or vertebral artery. The aim of this study was to design an individualized 3D printing navigation template for pedicle screw fixation in the upper cervical spine.Using CT thin slices data, we employed computer software to design the navigation template for pedicle screw fixation in the upper cervical spine (atlas and axis). The upper cervical spine models and navigation templates were produced by 3D printer with equal proportion, two sets for each case. In one set (Test group), pedicle screws fixation were guided by the navigation template; in the second set (Control group), the screws were fixed under fluoroscopy. According to the degree of pedicle cortex perforation and whether the screw needed to be refitted, the fixation effects were divided into 3 types: Type I, screw is fully located within the vertebral pedicle; Type II, degree of pedicle cortex perforation is <1 mm, but with good internal fixation stability and no need to renovate; Type III, degree of pedicle cortex perforation is >1 mm or with the poor internal fixation stability and in need of renovation. Type I and Type II were acceptable placements; Type III placements were unacceptable.A total of 19 upper cervical spine and 19 navigation templates were printed, and 37 pedicle screws were fixed in each group. Type I screw-placements in the test group totaled 32; Type II totaled 3; and Type III totaled 2; with an acceptable rate of 94.60%. Type I screw placements in the control group totaled 23; Type II totaled 3; and Type III totaled 11, with an acceptable rate of 70.27%. The acceptability rate in test group was higher than the rate in control group. The operation time and fluoroscopic frequency for each screw were decreased, compared with control group.The individualized 3D printing navigation template for pedicle screw fixation is easy and safe, with a high success rate in the upper cervical spine surgery. The difficult of posterior cervical spinal surgery is the focal point of this study. Nowadays, the primary system of internal screw placement in posterior cervical surgery is the screw-rod system, which iThe cervical pedicle screw internal fixation system is firmer and more stable than the lateral mass screws system. The study shows that the pull-out strength is twice that of the lateral mass screw ; thus, iHowever, the traditional upper cervical pedicle screw fixation is difficult, inefficient, and carries a high risk of spinal cord and vertebral artery injury , 7. EvenWhether using the traditional free-hand technique or CT and digital computer navigation technique, the purpose is to reduce the risk, improve the success rate, and guarantee effective pedicle screw fixation . In receThe study was approved by the Ethics Committee of The First Affiliated Hospital of Bengbu Medical College. All the participants provided their written informed consent to participate in this study. At our imaging center, we collected continuous tomography data of cervical spine 64-slice spiral CT from 13 patients with upper cervical spine diseases. The scanning parameters were: voltage, 120 kV; electric current: 150 mA; layer thickness, 0.5 mm; screw pitch, 0.5 mm; matrix, 512×512. Original DICOM images were disposed with Mimics15.0. The cervical spine was segmented and extracted, and then models with STL format after 3D reconstruction were exported. An equal proportion of cervical spine was printed with the 3D printer, idea-Print F100, and PLA printing material. Print parameters were set to a single-story height of 0.18 mm, model wall thickness of 1.00 mm, filling rate of 15.0%, and filling speed of 100 mm/s.Mimics. The optimal placement channel and entrance point were obtained through minor adjustments to ensure that the screws were located within long axis of the vertebral pedicle and to ensure that no walls pierced. A straight line was introduced in the virtual screw center, then saved and exported it in Initial Graphics Exchange Specification (IGS) format  format . The 3D ) format .The 3D reconstruction models of upper cervical vertebra were opened by NX 8.5 software. The spinous process, the transverse process and the vertebral arch lamina in upper cervical spine were extracted. The reverse templates consistent with the anatomical form of above osseous tissues were designed. The IGS format line was exported into NX8.5 as a reference and established 3D coordinate system. A concentric cylinder centered on the format line was designed in the 3D coordinate system. The internal diameter was 4 mm, and the external diameter was 8 mm. The virtual cervical screws in vertebral pedicle were adjusted to the best position. The length of screw was measured with a measuring tool in NX 8.5 software, and this provided reference for the depth of pedicle screw fixation. After subtracting from the vertebral body, the reverse template was summated with the cylinder, and finally formed the navigation templates STL files .The navigation template STL files were opened by idea-Print F100, which has its own processing software, ideaMaker. The navigation template of upper cervical spine was printed using the following print parameters: single slice height, 0.1 mm; model wall thickness, 2.0 mm; filling rate, 25%; filling speed, 100 mm/s. The base and external supports were also printed .A 3D printer then produces two sets per case of equal proportion. In the test group, pedicle screw fixation was guided by navigation template; in the control group, the screws were fixed under C-arm X-ray fluoroscopy. Both the operations were operated by the same chief physician of spinal surgery from our hospital.The cervical spine models were performed in a 64-slice spiral CT scanning after pedicle screw fixation. Accuracy of the screw fixation was evaluated with Mimics15.0 software. The data was measured at all the levels of each screw. The final data was based on the lowest level of screw fixation, and the average results were calculated from measurements taken three times. The fixation effects were divided into 3 types , 14, acct-test (inspection level: α = 0.05)The data of the test group and control group were analyzed with SPSS 17.0 software. We tested statistical differences of screw placement accuracy with chi-square test. Statistical differences on quantitative data of the screw placement was compared by paired Reconstructed data of this study were derived from 13 patients with upper cervical disease, and consisted of 6 males and 7 females, aged 25years to 57 years. Detailed information was shown in According to the reconstructed data, 13 pairs of upper cervical spine models, with two of each case printed; 19 navigation templates were printed, including 6 bilateral atlas navigation templates, 12 bilateral axis navigation templates and a right axis navigation template were printed. Atlas deformation in 6 cases with basilar invagination was serious and failed to produce a navigation template. Bilateral atlas and left axis in a case with in intraspinal tumor were severely damaged and failed to produce a navigation template.2 = 7.703, P = 0.021).A total of 74 cervical pedicle screws were placed in 2 groups. Of the 37 screws in test group, 32 were type I (86.49%), 3 were type II (8.11%), and 2 were type III (5.40%). Of the 37 screws in control group, 23 were type I (62.16%), 3 were type II (8.11%), and 2 were type III (29.73%). The acceptable rates of screw fixation in the test group and control group were 94.60% and 70.27%, respectively, while the unacceptable rates were 5.40% and 29.73%, respectively. The acceptable rates for the test group was significantly higher than that of the control group Click here for additional data file.