Extensor tendon irritation is one of the most common complications following volar locking plate osteosynthesis (VLPO) for distal radius fractures. It is most likely caused by distal screws protruding the dorsal cortex. Shorter distal screws could avoid this, yet the influence of distal screw length on the primary stability in VLPO is unknown. The aim of this study was to compare 75 to 100 % distal screw lengths in VLPO.A biomechanical study was conducted on 11 paired fresh-frozen radii. HRpQCT scans were performed to assess bone mineral density (BMD) and bone mineral content (BMC). The specimens were randomized pair-wise into two groups: 100 % (group A) and 75 % (group B) unicortical distal screw lengths. A validated fracture model for extra-articular distal radius fractures (AO-23 A3) was used. Polyaxial volar locking plates were mounted, and distal screws was inserted using a drill guide block. For group A, the distal screw tips were intended to be flush or just short of the dorsal cortex. In group B, a target screw length of 75 % was calculated. The specimens were tested to failure using a displacement-controlled axial compression test. Primary biomechanical stability was assessed by stiffness, elastic limit, and maximum force as well as with residual tilt, which quantified plastic deformation.Nine specimens were tested successfully. BMD and BMC did not differ between the two groups. The mean distal screw length of group A was 21.7 ± 2.6 mm (range: 16 to 26 mm), for group B 16.9 ± 1.9 mm (range: 12 to 20 mm). Distal screws in group B were on average 5.6 ± 0.9 mm (range: 3 to 7 mm) shorter than measured. No significant differences were found for stiffness (706 ± 103 N/mm vs. 660 ± 124 N/mm), elastic limit (177 ± 25 N vs. 167 ± 36 N), maximum force (493 ± 139 N vs. 471 ± 149 N), or residual tilt (7.3° ± 0.7° vs. 7.1° ± 1.3°).The 75 % distal screw length in VLPO provides similar primary stability to 100 % unicortical screw length. This study, for the first time, provides the biomechanical basis to choose distal screws significantly shorter then measured.The online version of this article (doi:10.1186/s13018-015-0283-8) contains supplementary material, which is available to authorized users. Recent studies have reported complication rates following volar locking plate osteosynthesis (VLPO) for distal radius fractures of up to 18 % . Two of Campbell’s Operative Orthopaedics [Dorsal screw protrusion might be an avoidable complication, especially for extra-articular fractures (AO-23 A3), which are the most common ones , 8. The opaedics  recommenopaedics .Shorter distal screws are the most promising approach to avoid dorsal screw protrusion. Therefore, it is indispensable to investigate the effect of distal screw length on the primary stability of VLPO. Consequently, the aim of this study was to compare 75 to 100 % distal screw lengths in VLPO using human fresh-frozen radii and an established biomechanical fracture model for extra-articular distal radius fractures (AO-23 A3). The study’s null hypothesis was that unicortical 100 % distal screw lengths provide superior primary stability compared to 75 % distal screw lengths in VLPO.This biomechanical study was conducted on fresh-frozen human radii using a validated fracture model for extra-articular distal radius fractures (AO-23 A3). The local ethics committee approved the study (LMU #409-13). The outcome parameters of interest were stiffness, elastic limit, maximum force, and residual tilt of the distal fragment.Eleven paired fresh-frozen radii were obtained from the Centre of Anatomy and Cell Biology, Medical University of Vienna, Austria. Radii were randomized pair-wise, side alternating into a 100 % unicortical distal screw length group (group A) and a 75 % distal screw length group (group B). They were then cut to 14-cm length. High-resolution peripheral quantitative computer tomography scans  were performed. Radii presenting previous fractures, severe osteoarthritis, or bone lesions were excluded. Bone mineral density (BMD) and bone mineral content (BMC) were computed  to assesThe general preparation has been outlined in detail previously . In brieA drill guide block (Medartis A-2723 01/02) was mounted onto the distal plate, which assured uniform distal screw orientation. Following drilling, distal screw length was measured. Distal locking screw lengths were chosen according to the previously defined groups. For group A (100 %), the screw tips were intended to be flush or just short of the dorsal cortex Fig. . In grouFollowing distal screw insertion, a 10-mm dorsal wedge osteotomy simulating a dorsally unstable fracture was performed using an oscillating handsaw. The osteotomy location resembled the in vivo fracture location and was chosen based on previous studies , 13. CarEach specimen was then embedded using two custom-made aluminium jigs. The load axis was defined proximally by the medullary canal and distally slightly dorsoradial to the centre of the crista subdividing the fossa lunata and scaphoidea. The proximal 40 mm of the shaft and a shallow edge of the distal articular surface of the radii were embedded in polyurethane  Fig. .Fig. 2IlA proximal constrained setup was used Fig. . The embSpecimens were tested to failure using a displacement controlled axial compression test. Following preconditioning to exclude settling effects , the specimens were loaded at 1 mm/s until either a 20 % force drop or 3mm displacement was reached , 15. PhoR2 > 0.998). The elastic limit corresponded the last data point of the elastic range. Stiffness was defined as the slope of the regression line within the elastic range. Maximum force was defined as the force where the slope of the tangent line dropped below 95 % of the stiffness. In one case, the slope did not reach this threshold and the global maximum force was chosen. Residual tilt was determined using the motion tracking system to quantify the overall plastic deformation. It was defined as the angle between the initial and final testing position of the distal jig and assessed by rigid registration of the initial and final marker positions .The analysis of the biomechanical outcome parameters revealed no differences between the 75 and 100 % distal screw length group for any parameter Table . TherefoExtensor tendon irritation and attritional tendon ruptures are two of the most common complications following VLPO. Both can be caused by distal screws protruding the dorsal cortex –6. ShortThe authors are only aware of two studies, investigating the effect of distal screw length on the primary stability of VLPO, both with inherent limitations. Greenberg et al.  presenteIn general, the validity of a biomechanical study relies on the test setup used. We tried to apply a best-evidence setup based on previous experiments and literature , 13. PreThe results of our study are corroborated by comparison to literature. As outlined above, the biomechanical setups published for distal radius fractures vary significantly. This not only alters the biomechanical behaviour of the mode, which consequently leads to diverging results, but also hampers inter-study comparison. Still, similar maximum force values were reported in previous studies applying a comparable setup , 29. MorAlthough various biomechanical parameters associated to failure of the osteosynthesis have been assessed, the actual failure mode has not. Possible failure modes include screw-bone, screw-plate, or plate failure. These could be influenced by distal screw length. First, shorter screws reduce the screw-bone contact area, which might increase the local damage around the screws during loading and therefore influence total plastic deformation. In this study, residual tilt was chosen as a surrogate parameter to quantify total plastic deformation . Other sA further limitation might be the used axial loading protocol, as it does not account for all loading conditions during early rehabilitation. Although few authors conducted specific bending and torsion tests , most biThis biomechanical study was able to demonstrate that 75 % distal screw length can provide similar primary stability as unicortical 100 % distal screw length in VLPO. This study, for the first time, provides the biomechanical basis to choose distal screws significantly shorter then measured. Future clinical studies are required to validate this approach in vivo and investigate on the possible reduction of dorsal screw protrusion incidences and subsequent extensor tendon problems.


Drosophila or primary cilia in vertebrates, which is thought to be essential for its activation and function, but the underlying mechanisms remain poorly understood. Here we show that Hh stimulates the binding of Smo to a plasma membrane-associated kinase Gilgamesh (Gish)/CK1γ and that Gish fine-tunes Hh pathway activity by phosphorylating a Ser/Thr cluster (CL-II) in the juxtamembrane region of Smo carboxyl-terminal intracellular tail (C-tail). We find that CL-II phosphorylation is promoted by protein kinase A (PKA)-mediated phosphorylation of Smo C-tail and depends on cell surface localization of both Gish and Smo. Consistent with CL-II being critical for high-threshold Hh target gene expression, its phosphorylation appears to require higher levels of Hh or longer exposure to the same level of Hh than PKA-site phosphorylation on Smo. Furthermore, we find that vertebrate CK1γ is localized at the primary cilium to promote Smo phosphorylation and Sonic hedgehog (Shh) pathway activation. Our study reveals a conserved mechanism whereby Hh induces a change in Smo subcellular localization to promote its association with and activation by a plasma membrane localized kinase, and provides new insight into how Hh morphogen progressively activates Smo.Hedgehog (Hh) signaling controls embryonic development and adult tissue homeostasis through the G protein coupled receptor (GPCR)-family protein Smoothened (Smo). Upon stimulation, Smo accumulates on the cell surface in  A study of both fruit flies and vertebrate cells shows that the plasma membrane-associated kinase Gish/CK1γ promotes transduction of the Hedgehog signal by phosphorylating Smoothened. Drosophila to humans. Deregulation of Hh signal transduction contributes to a wide range of human disorders, including birth defects and cancer. The seven-transmembrane protein Smoothened (Smo) is an obligatory and conserved Hh signal transducer, but how Hh stimulates its activity remains unclear. Here we identify a plasma membrane associated kinase, Gilgamesh (Gish)/CK1γ, as a positive regulator of the Hh signaling activity. We find that Gish activates Hh signaling by phosphorylating a specific site in the Smo C-terminal intracellular tail. Phosphorylation of Smo by Gish is required for maximal activation of Smo and depends on membrane association of Gish and prior phosphorylation of Smo by protein kinase A (PKA). We also find that Hh stimulates the association of Smo with Gish after it travels to the plasma membrane, thus facilitating its phosphorylation by Gish. Finally, we provide evidence that CK1γ is found at the primary cilium in mammals and phosphorylates Smo to activate the Hh pathway. Our results uncover a conserved role of Gish/CK1γ in the regulation of Smo phosphorylation and provide new insight into the molecular underpinning of how Hh signal is transduced across the plasma membrane.The secreted glycoprotein Hedgehog (Hh) plays a conserved role in embryonic development and adult tissue homeostasis in species ranging from  Drosophila or primary cilia in vertebrates, where Smo adopts an open and active conformation to relay the Hh signal to the intracellular signaling components [Hedgehog (Hh) signaling plays an essential role in embryonic development and adult tissue homeostasis, and its deregulation has been implicated in congenital diseases and cancers –6. Hh exmponents –15.Drosophila, unphosphorylated or hypophosphorylated Smo adopts a closed, inactive conformation [Phosphorylation plays a critical role in the regulation of Smo conformation and subcellular localization . In Drosormation  and is uormation ,18. Uponormation –22, whicormation . In addiormation ,18. In aormation –26. Gprkormation . In mammormation . Cell suormation ,27; howeDrosophila and mammals [Drosophila Smo is phosphorylated at more than 26 Serine (Ser)/Threonine (Thr) residues, many of which have not been well characterized [Hh functions as a morphogen to specify different developmental outcomes in a concentration-dependent manner ,3. As su mammals ,19,24, scterized .gilgamesh (gish) in Drosophila [Drosophila eye [CK1γ, which is encoded by osophila , is a meosophila . Gish/CKosophila ,31. Gishhila eye , olfactohila eye , and plahila eye . Mammalshila eye , making -PKA12/SmoDN) with a wing-specific Gal4 drivers MS1096 (MS>SmoDN) [UAS-RNAi line (V106826) targeting Gish, the Drosophila homologue of mammalian CK1γ, enhanced the “fused wing” phenotype caused by MS>SmoDN , partially suppressed the “fused wing” phenotype caused by MS>SmoDN  ,24,25. WMS>SmoDN . Using iMS>SmoDN . Moreove fashion . On the MS>SmoDN .MS>SmoDN-induced wing phenotype, even though it was expressed at levels similar to the wild-type Gish  caused a similar "fused wing" phenotype, albeit more severe than that caused by MS>SmoDN , a Ser/Thr kinase acting downstream of Smo ,34,35. IMS>SmoDN . HoweverDN ED10639 (BL#9481), also enhanced the fused wing phenotype caused by MS>SmoDN  further enhanced the wing phenotype  induced at 48–72 h AEL survived to late third instar larval stages but did not affect the expression of Hh target genes ptc and engrailed (en) when localized in A-compartment cells near the A/P boundary  induced at 24–48 h AEL in the Minute background occupied large areas in late third instar wing discs  [1-3S/T, in which the underlined S/T is the CK1 site, whereas X represents any amino acid [601-700) but not the mutated CL-II site (GST-Smo601-700 CL-II SA) was phosphorylated by a recombinant CK1 in an in vitro kinase assay (PS)E(PT)NDI(PS)STW as an antigen  substituted (SRSSSFFKR) or deleted, respectively  but not the cytosolic variants (HA-GishCS and HA-GishΔC) could support CL-II phosphorylation in response to Hh  moiety fused to its C-terminus (Myc-Smo-Ub) . We confPrevious studies suggest that Hh stimulates Smo phosphorylation by PKA and CK1α/ε at three clusters of Ser/Thr residues in the middle region of Smo C-tail , and thaWe next determined whether PKA site phosphorylation and CL-II phosphorylation were induced by different levels of Hh. S2 cells stably expressing Myc-Smo were treated with Hh-conditioned medium containing different levels of Hh-N  for 4 h. Cell lysates were immunoprecipitated with anti-Myc antibody, followed by western blot analysis with either anti-SmoP687, which recognized the phosphorylated PKA site (S687) as well as two downstream CK1 sites [MS1096 Gal4 driver (MS>Smo-CFP) induced ectopic expression of dpp-lacZ, which is a low-threshold Hh target gene  or SmoSD123-CFP to generate SmoCL-IISA-CFP and SmoSDCL-IISA-CFP, respectively see . Consistget gene ; howeverpression . While Mh levels , SmoSDCLmutation . Hence, ng discs . By contng discs , consistC765 (C765>Smo) in wing discs carrying smo3 mutant clones. Our previous study revealed that the levels of Smo derived from C765>Smo were only slightly higher than that of endogenous Smo [C765>Smo-CFP completely rescued the expression of both ptc and en in smo3 mutant clones located near the A/P boundary  in the context of SmoSDGPSD, which contains phospho-mimetic mutations in the three PKA/CK1 phosphorylation clusters (SD123) and two Gprk2 sites (GPSD) , to geneboundary . In addiboundary . SmoSDCLpression , consistpression . On the SmoSD123 . Althougr levels , SmoSDalmoSDGPSD . We alsoOur previous studies revealed that phosphorylation of Smo C-tail induced a conformational change resulting in its dimerization/oligomerization, as indicated by an increased fluorescence resonance energy transfer (FRET) between C-terminally tagged CFP and yellow fluorescent protein (YFP) ,24. We fCS, and HA-GishΔC  but failed to bind Myc-SmoΔC570, which lacks the C-tail , suggestFinally, we determined whether Smo-Gish association is regulated by PKA-mediated phosphorylation of Smo. We found that mutating the PKA phosphorylation sites to Ala (SA123) attenuated Hh-stimulated Smo-Gish complex formation, whereas the phospho-mimetic mutations of PKA sites and adjacent CK1 sites (SD123) increased both the basal and Hh-stimulated Smo-Gish complex . The infGli-luciferase (Gli-luc) reporter assay, EYFP-CK1γ but not EYFP-CK1γ-ΔC stimulated Shh pathway activity -tagged Xenopus CK1γ (EYFP-CK1γ) was accumulated on the plasma membrane as well as on the primary cilium, whereas a cytosolic form of CK1γ, EYFP-CK1γ-ΔC, which lacks the C-terminal palmitoylation site , failed activity . By contactivity , suppresactivity . Our preactivity . On the rylation . We founrylation . By contrylation . Becauserylation , CK1γ-strylation , and Gliactivity , suggestDN [K73R and CK1γD164N) failed to block cilium-independent Smo phosphorylation induced by SAG ; howeved by SAG . These rDrosophila Smo and S1 site in mammalian Smo). We demonstrated that CL-II phosphorylation is required for the optimal Smo activation and Hh pathway activity. Interestingly, we found that Gish/CK1γ regulates Smo phosphorylation and Hh pathway activity depending on its plasma membrane/ciliary localization. We propose that cell surface/ciliary accumulation of Smo, which is facilitated by Hh stimulation, promotes its association, phosphorylation, and activation by Gish/CK1γ —for example, other members of CK1 family—may partially compensate for the loss of Gish, although these kinases do not phosphorylate CL-II as effectively as Gish.In this study, we generated an antibody (Smo4P) that recognized phosphorylated CL-II and demonstrated that CL-II phosphorylation was stimulated by Hh. Moreover, we found that CL-II phosphorylation was mediated by Gish in a manner depending on its membrane association. Both mutation . One posen in smo mutant clones located near the A/P boundary  prevented Hh-stimulated Smo cell surface accumulation, Smo-Gish association, and CL-II phosphorylation  prevented its ciliary localization, its association with mammalian Smo (mSmo), and its ability to phosphorylate mSmo and promote Shh pathway activity. Furthermore, depleting primary cilia by expressing a dominant negative for of Kif3b (DN-Kif3b) also affected CK1γ Smo interaction and phosphorylation by CK1γ. Taken together, these results suggest that only ciliary localized but not the cytosolic CK1γ is capable of phosphorylating Smo to promote Shh pathway activation. As Shh induces mSmo ciliary accumulation, we propose that ciliary accumulation of mSmo may facilitate its binding to and phosphorylation by CK1γ, which contributes to optimal Smo activation . BecauseMS1096 and C765 [UAS transgenes are: UAS-Smo-PKA12/SmoDN, UAS-Smo-PKA123/SmoSA123, and UAS-SmoSD123 [UAS-Smo-CFP, UAS-SmoSD123-CFP, UAS-SmoSDGPSD-CFP, and UAS-Smo-GPS1A2A-Fg/SmoGPSA [UAS-Myc-Gish (BL#41764), UAS-Myc-GishΔC (BL#41769), and UAS-Myc-GishKD (BL#41766); UAS-Flag-CK1α and UAS-Flag-CK1ε [UAS-GishRNAi . Xenopus CK1γ DNA constructs are: pCS-EYFP-CK1γ, pCS-EYFP-CK1γ -ΔC, pCS-CK1γD164N, and pCS-CK1γK73R [smo3 [gishKG03891/gishP (BL#13263), Gprk2Δ15 [Df(3R)ED10639 (BL#9481). gishΔ4 was generated by imprecise excision of the P-element from gishKG03891. GishCS, SmoCL-IISA , SmoCL-IISD , SmoSDCL-IISA, SmoSDCL-IISD, and SmoSDall were generated by PCR-based site-directed mutagenesis and confirmed by DNA sequencing. Mutant clones were generated by standard FRT/FLP mediated mitotic recombination or the MARCM system as previously described [gishΔ4 , gishΔ4 clones in Minute background  hs-CD2/FRT82B gishΔ4), gishP/P wings expressing SmoDN  hs-CD2/FRT82B gishKG03891), smo clones with or without expressing smo transgenes .All flies were raised on standard yeast and molasses-based food at 25°C. Gal4 drivers used in this study are and C765 ,24,48. USmoSD123 ; UAS-Smolag-CK1ε ; and UASK1γK73R . Mutant escribed ,49. MutaDrosophila S2 cells were cultured in Drosophila SFM (Invitrogen) with 10% fetal bovine serum, 100 U/ml of penicillin, and 100 mg/ml of streptomycin at 24°C. Transfections were carried out using the Calcium Phosphate Transfection Kit . Hh-conditioned medium treatment was carried out as described [3VO4, 1% NP-40, 10% Glycerol, and 0.5M EDTA (pH8.0). Cell lysates were incubated with protein A–Sepharose beads (Thermo scientific) for 1 h at 4 C° to eliminate non-specific binding proteins. After removal of the protein-A beads by centrifugation, the cleared lysates were incubated with Myc (HA or Flag) antibody for 2 h or overnight. The complexes were collected by incubation with protein A–Sepharose beads for 1 h at 4 C°, followed by centrifugation. The immunoprecipitates were then washed three times for 5 min each with lysis buffer and were fractionated by SDS–PAGE. FRET analysis was carried out as previously described [http://www.ab-mart.com) using the synthetic phosphopeptide NDLN(PS)(PS)E(PT)NDI(PS)STW-C as antigen. The reactive antibody was purified by absorption on a phosphopeptide affinity column and was further purified by subtraction on a column containing a non-phosphopeptide DLNSSETNDISSTW-C. The resulting antibody was initially characterized by western blot analysis using GST-Smo601-700 and GST-Smo601-700CL-IISA.escribed . Unless escribed ,51. For escribed . Antibodescribed , mouse a32p-ATP, and CK1δ (New England Biolabs) and incubated at 30°C for 1.5 h in reaction buffer. Phosphorylation of GST-fusion proteins was analyzed by autoradiography after SDS-PAGE. For ptc-luc reporter assays, Cl8 cells were transfected with 1 μg ptc-luc reporter construct and 50 ng RL-PolII renilla construct in 12 well plates together with 1 μg Smo constructs. After 48 h incubation, the reporter assays were performed using the Dual-Luciferase reporter assay system (Promega). Dual-Luciferase measurements were performed in triplicate using FLUOstar OPTIMA (BMG LABTECH). Gli-luc reporter assays were carried out as previously described [In vitro kinase assay was performed as previously described . Brieflyescribed . Double-escribed . For theS1 Data(XLSX)Click here for additional data file.S1 Figptc-Gal4 . dpp-lacZ expression marks the A-compartment cells near the A/P boundary. (C–J) Adult wings of the indicated genotypes. Gish RNAi enhanced the MS>SmoDN  Gish expression visualized by immunostaining with a Gish antibody in a control wing disc  and a wing disc expressing UAS-Gish-RNAi by (TIF)Click here for additional data file.S2 FiggishΔ4 clones generated by the MARCM system were immunostained for GFP (green), Gish , Ptc , Ci , and En . Clones were induced at 48–72 h AEL and marked by GFP expression. Gish expression was abolished in gishΔ4 clones (B–B"). Neither ptc nor en expression was affected in gishΔ4 clones . (G–H‴) A control wing disc  or wing disc carrying gishΔ4 clones generated in the Minute background at 24–48 h AEL were immunostained with Ci, Ptc, and Gish antibodies. ptc expression was not affected in gishΔ4 clones marked by the lack of Gish signal (arrows in H–H‴).(A–F") Wing discs carrying control or (TIF)Click here for additional data file.S3 Fig(A) S2 cells stably expressing Myc-Smo were treated with control or Gprk2 dsRNA and with or without Hh-conditioned medium, followed by immunoprecipitation and western blot analysis with the indicated antibodies. Western blot (middle panel) and RT-qPCR (right) experiments confirm Gprk2 knockdown efficiency. Western blots of coimmunoprecipitation experiments from lysates of S2 cells transfected with Myc-Smo and the indicated CK1 or Gprk2 constructs. Cells were grown in the presence or absence of Hh stimulation for 24 h and exposed to MG132 (50 μM) for 4 h before harvesting.(TIF)Click here for additional data file.S4 Fig(A) Western blot of coimmunoprecipitation experiments from lysates of S2 cells transfected with Myc-Smo or Myc-Smo-Ub together with mC*-YFP (a constitutively active form of PKA). Both Myc-Smo and Myc-Smo-Ub were phosphorylated by mC* at S687.(B) S2 cells were transfected with Myc-Smo or Myc-Smo-Ub either alone or together with mC*-YFP, followed by immunostaining to visualized cell surface Myc-Smo or Myc-Smo-Ub and mC*-CFP. Coexpression with the constitutively active form of PKA resulted in cell surface accumulation of both Myc-Smo and Myc-Smo-Ub.(TIF)Click here for additional data file.S5 Figdpp-lacZ and GFP expression in wing discs expressing the indicated Smo construct in the presence or absence Flag-Gish. Coexpression of Flag-Gish with Smo-CFP promoted its activity as indicated by more robust ectopic dpp-lacZ expression and enhanced overgrowth of the wing disc (B–B' compared with A–A'). Coexpression of Flag-Gish with SmoCL-IISA-CFP did not cause discernable change in the expression of dpp-lacZ or disc growth (D–D' compared with C–C'). (E–F‴) Wing discs carrying smo3 mutant clones expressing C765>Smo-CFP (E–E‴) or C765>SmoCL-IISA-CFP (F–F‴) were treated with 50 nM LMB for 2 h prior to immunostaining with GFP, Ci, and En antibodies. Both Smo-CFP and SmoCL-IISA-CFP promote Ci nuclear localization in smo mutant clones near the A/P boundary.(A–D') (TIF)Click here for additional data file.S6 Figsmo3 mutant clones expressing C765>SmoGPSA12 were immunostained to show the expression of GFP (green), which marks the smo mutant cells, Ci (red), and En  or Ptc . Expression of SmoGPS1A2A with C765 rescued ptc but not en expression in smo3 mutant clone near the A-P boundary (arrowheads). (C–D'') Wing discs expressing C765>SmoSD123-CFP (C–C") or C765>SmoSDGPSA-CFP (D–D") were immunostained to show the expression of GFP, Ptc, and En. SmoSDGPSA exhibited reduced activity compared with SmoSD123.(A–B‴) Wing discs carrying (TIF)Click here for additional data file.


Phosphorylation of Ser46 and Thr55 of p53-TAD increases its binding ability; however, the role of XPC-AF phosphorylation remains elusive. Here we describe a system for real-time and simultaneous monitoring of the phosphorylation and p62-PH affinity of p53-TAD and XPC-AF using nuclear magnetic resonance (NMR) spectroscopy. Unexpectedly, among seven reported kinases that presumably phosphorylate Ser46 and/or Thr55 of p53-TAD, only two specific and high-efficiency enzymes were identified: JNK2α2 for Ser46 and GRK5 for Thr55. During interaction with p62-PH, four different affinity complexes resulting from various phosphorylation states of p53-TAD by the kinases were identified. The kinetics of the site-specific phosphorylation reaction of p53-TAD and its affinity for p62-PH were monitored in real-time using the NMR system. Isothermic calorimetry showed that phosphorylation of Ser129 of XPC-AF increases binding to p62-PH. Although CK2 was predicted to phosphorylate Ser122, Ser129 and Ser140 from its sequence context, it specifically and efficiently phosphorylated only Ser129. Simultaneous monitoring of the phosphorylation and augmentation in p62-PH binding identified a key residue of p62-PH for contacting phosphorylated Ser129. In summary, we have established an NMR system for real-time and simultaneous monitoring of site-specific phosphorylation and enhancement of affinity between phosphorylation domains and their target. The system is also applicable to other posttranslational modifications.Posttranslational modifications have critical roles in diverse biological processes through interactions. Tumor-suppressor protein p53 and nucleotide excision repair factor XPC each contain an acidic region, termed the acidic transactivation domain (TAD) and acidic fragment (AF), respectively, that binds to the pleckstrin homology (PH) domain of the p62 subunit of the transcription factor TFIIH. Human p53-TAD contains seven serine and two threonine residues, all of which can be phosphorylated. Similarly, XPC-AF contains six serine and two threonine residues, of which Thr117, Ser122 and Ser129 have been reported as phosphorylation sites  The acidic transactivation domain  at the N-terminus is important for p53 transcription activity through its interaction with transcriptional co-activators, co-repressors and general transcription factors. p53-TAD consists of two homologous subdomains, TAD1 (residues 1–42) and TAD2 (residues 43–61). Both domains function as hubs, contacting multiple partners in intricate networks.The tumor-suppressor p53 has critical roles in DNA repair, cell cycle arrest, apoptosis, senescence, metabolism and autophagy in response to cellular stress.6 Human p53-TAD has nine phosphorylation sites: Ser6, Ser9, Ser15, Ser20, Ser33, Ser37, Ser46, Thr18, and Thr55, all of which have been implicated in activation of p53 upon stress.7 For example, phosphorylation of Ser46 activates p53 apoptotic activity,9 whereas phosphorylation of Thr55 is involved both in G1 cell cycle progression via the degradation of p53,10 and in nuclear export of p53.11p53 is subjected to diverse posttranslational modifications, which contribute significantly to its specific and malleable interactions. In particular, p53-TAD is subject to phosphorylation.15 In addition, phosphorylation of Thr18 decreases the interaction of p53 with its main negative regulator HDM2/MDM2,16 and phosphorylation of Ser46 and Thr55 increases its p53 affinity for the pleckstrin homology (PH) domain of the p62 subunit of the general transcription factor TFIIH.18 p62 is also a critical hub protein, functioning in both transcription and DNA repair.As demonstrated for p53 and many key hub proteins, phosphorylation and other posttranslational modifications are essential for modulating intermolecular interactions. For instance, single-site and multiple-site phosphorylation of Ser15, Thr18, Ser20, Ser33, Ser37, Ser46, and Thr55 in human p53-TAD increase the affinity of p53 for the TAZ1/CH1, KIX, TAZ2/CH3 and NCBD/IBiD domains of its co-activators, CBP and p300.21 XPC-AF contains six serine and two threonine residues, of which Thr117, Ser122 and Ser129 have been reported as phosphorylation sites in vivo,23 although their phosphorylation roles are unknown.Similar to p53-TAD, the acidic fragment  of the nucleotide excision repair factor XPC binds to p62-PH.24 to study the interaction of the p53-TAD, as well as XPC-AF, with p62-PH. First, we identified highly specific kinases for p53-TAD, namely c-Jun NH2-terminal kinase 2α2 (JNK2α2) for Ser46 and G protein-coupled receptor kinase 5 (GRK5) for Thr55, and verified the high efficiency of these kinases by analyzing the kinetics of the phosphorylation reactions. After demonstrating the existence of four different affinity complexes of p53-TAD and p62-PH arising from the different phosphorylation states of p53-TAD, we simultaneously monitored the site-specific phosphorylation and augmenting interaction of p53-TAD with p62-PH in real time. For XPC-AF, we first showed that Ser129 was specifically and efficiently phosphorylated by casein kinase II (CK2), which strengthened binding of XPC-AF to p62-PH. We then monitored the enhancement in complex formation mediated by Ser129 phosphorylation in real time by NMR, identifying a key residue of p62-PH for interaction with phosphorylated Ser129 of XPC-AF.A large number of studies have demonstrated that, in response to stress, p53 is stabilized and activated by altering its interactions with its binding partners via phosphorylation and other posttranslational modifications. However, few methods for monitoring its changing interactions in real time upon modification at an atomic level have been described thus far. To address this issue, here we have used real-time nuclear magnetic resonance (NMR) spectroscopy25 and protein kinase Cδ (PKCδ)26 for Ser46; extracellular signal-regulated kinase 2 (ERK2)27 and GRK528 for Thr55; and CK1, CK2 and JNK2α2, three kinases that are known to phosphorylate residues Ser20, Ser33, Ser37, Ser46, Thr18 and Thr55.14 We added each candidate kinase (2.5 μg) to 100 μl of 50 μM15N-labeled p53-TAD (residues 1–73) es 1–73)  and monies 1–73) .CDK5 and PKCδ did not phosphorylate any residues under the conditions used . We therM15N-labeled p53-TAD as a substrate, 1.0 μg of JNK2α2 achieved ~100% of phosphorylation at Ser46 within 1 h  We also quantitatively examined the effects of phosphorylation of p53-TAD2 on binding to p62-PH by using isothermal titration calorimetry (ITC) and NMR. Single phosphorylation of Ser46 or Thr55 increased the binding ~4-fold (Kd=6 μM) or ~5-fold (Kd=5 μM), respectively, and dual phosphorylation further enhanced the binding ~25-fold (Kd=1 μM).18 Here we asked whether the four complexes with different affinities could be distinguished on a 1H,15N heteronuclear single-quantum coherence (HSQC) spectrum during the phosphorylation reaction. To investigate this, we compared the spectra of 15N-labeled p62-PH complexed with unphosphorylated, Ser46-phosphorylated, Thr55-phosphorylated and Ser46, Thr55 double-phosphorylated p53-TAD2 peptides. Notably, many signals from interacting residues and nearby residues of p62-PH, such as E58 and T74, could be distinguished from each other in the four different affinity complexes , doubly phosphorylated on Ser46 and Thr55, and p62-PH of TFIIH using NMR and revealed that phosphorylated Ser46 and Thr55 make electrostatic contacts with K18/K19 and K54, respectively, of p62-PH .18   . Similar109–156)  and is l complex . Signal  complex .Kd) of 132 nM  that was unphosphorylated and phosphorylated at Ser129 by using ITC. The unphosphorylated XPC peptide bound to p62-PH with a dissociation constant  bound to its targets have been determined,34 a structure containing phosphorylated p53-TAD has only been solved for p53-TAD2 phosphorylated at Ser46 and Thr55 bound to p62-PH.18 Interestingly, in contrast to the amphipathic helix structures of unphosphorylated p53-TAD1 and p53-TAD2 observed in the other complexes, phosphorylated p53-TAD2 in this structure forms an extended string-like structure enabling it to make extensive and optimal contacts , two moderate-affinity complexes (p53-TAD singly phosphorylated at Ser46 or Thr55) and the high-affinity complex (p53-TAD doubly phosphorylated at Ser46 and Thr55)—could be distinguished from each other via the signals from p62-PH on a spectrum . Thus thspectrum . Quantitp53-TAD2 . E58 is of Ser46 .1H and 15N atoms of the protein backbone to monitor the altering intermolecular interaction, but other atoms will offer different types of structural information. For example, monitoring 13C atoms in a protein backbone can enable the observation of changes in the secondary structure of a protein of interest.In this way, real-time NMR monitoring is extremely useful in evaluating the impact of phosphorylation on protein–protein interactions, as well as enzymatic activity, specificity and kinetics. In the present study, we used 23 Thr117, Ser122 and Ser129 in XPC-AF are phosphorylated in vivo; to date, however, no studies on the role of these phosphorylated residues have been reported. Notably, the binding site for p62-PH identified in XPC-AF during the NMR chemical shift perturbation experiment contained a serine residue, Ser129 . Other posttranslational modifications such as acetylation and methylation perturb the local environment around the modified site,13C/15N-labeled human p53-TAD (residues 1–73) was prepared as previously described.18The 13C/15N-labeled human TFIIH p62-PH domain (residues 1–108) was prepared as previously described.47The 13C/15N-labeled human XPC-AF (residues 109–156) was expressed as a hexa-histidine-tagged product in a pET15b vector  in Escherichia coli Rosetta 2 (DE3) pLysS . The cells were grown at 37 °C in M9 minimal medium containing [15N]ammonium chloride and [13C]glucose. The products were expressed by induction with 1 mM isopropyl-β-d-thiogalactopyranoside. After 20–22 h of growth, the cells were harvested, and the cell pellet was suspended in buffer A (20 mM Tris–HCl (pH 8.0), 10% glycerol, 1m NaCl). The cells were lysed by sonication and centrifuged. The supernatant was loaded onto a Ni-nitrilotriacetic acid–agarose  column, equilibrated with buffer A. The sample was washed with buffer A containing 20 mM imidazole-HCl and eluted by 500 mM imidazole-HCl. Peak fractions were pooled, and the buffer was changed to thrombin cleavage buffer  using Amicon Ultra devices . The sample was digested with thrombin for 13–20 h at 25 °C to remove the histidine tag. After concentration using Amicon Ultra, the sample was applied to a Superdex30  column equilibrated with 20 mM potassium phosphate (pH 6.8) and 550 mM NaCl.The Unphosphorylated, Ser46- and Thr55-phosphorylated and Ser46, Thr55 double-phosphorylated peptides of human p53-TAD2 (residues 41–62), and unphosphorylated and Ser129-phosphorylated peptides of human XPC (residues 124–142) were purchased from Sigma Genosys Japan .M13C/15N-labeled human p53-TAD. For XPC, 2.5 μg of CK2 was added to 150 μl of 50 μM13C/15N-labeled human XPC-AF. Phosphorylation was conducted in 20 mM potassium phosphate (pH 6.8), 1 mM ATP, 10 mM MgCl2 and 10% D2O and monitored at 25 °C on a Bruker AVANCE III HD 600 MHz spectrometer  equipped with a triple-resonance TCI cryogenic probe. The 1H,15N HSQC spectra shown in 1H,15N HSQC spectrum shown in 48 and analyzed by NMRView.49CDK5, PKCδ, ERK2, GRK5 and JNK2α2 were purchased from Upstate Merck Millipore  and CK1 and CK2 were purchased from New England Biolabs . For p53, 2.5 μg of each kinase was added to 100 μl of 50 μet al.24 For p53, 1.0 μg of JNK2α2 and 2.5 μg of GRK5 were added to 150 μl of 25 μM13C/15N-labeled human p53-TAD alone and in complex with 13C/15N p62-PH in 20 mM potassium phosphate (pH 6.8), 1 mM ATP, 10 mM MgCl2, 5 mM deuterated dithiothreitol (DTT) and 10% D2O. For XPC, 2.5 μg of CK2 was added to 150 μl of 50 μM13C/15N-labeled XPC-AF alone and in complex with 13C/15N-labeled p62-PH in 20 mM potassium phosphate (pH 6.8), 1 mM ATP, 10 mM MgCl2, 5 mM deuterated DTT and 10% D2O. The phosphorylation of p53 TAD at Ser46 by JNK2α2 and at Thr55 by GRK5 was monitored by 1H-15N SOFAST-HMQC50 and 1H-15N fast HSQC,51 respectively. The phosphorylation of XPC-AF at Ser129 by CK2 was monitored by 1H-15N fast HSQC. Each experiment was repeated twice. Spectra were processed by NMRPipe and analyzed by NMRView. Build-up curves showing the time course of phosphorylation of p53-TAD at Ser46 by JNK2α2 were calculated by using data from Leu45, Ser46, Asp48, Ile50 and Gln52  in 20 mM potassium phosphate (pH 6.8), 1 mM ATP, 10 mM MgCl2, 5 mM deuterated DTT and 10% D2O. Unlabeled p62-PH was added to 15N-labeled XPC-AF at a molar ratio of 1:1.2 (XPC:p62) in 20 mM potassium phosphate (pH 6.8), 5 mM deuterated DTT and 10% D2O. 1H,15N HSQC spectra were taken before and after the addition of unlabeled samples at 32 °C on a Bruker AVANCE III HD 600 MHz spectrometer equipped with a triple-resonance TCI cryogenic probe. Backbone resonances of p62-PH and XPC-AF in their unbound and bound forms were assigned with a combination of CBCA(CO)NH, CBCANH, HNCO and HN(CA)CO.52 Spectra were processed by NMRPipe and analyzed by NMRView. Chemical shift change (Δδ) was calculated as Δδ={(Δδ1H)2+(Δδ15N/5)2}1/2.Unphosphorylated, Ser46- and Thr55-phosphorylated and Ser46, Thr55 double-phosphorylated peptides of human p53-TAD2 were added to Kd values of p62-PH and XPC peptide (residues 124–142) were measured by ITC using a VP-ITC calorimeter . Calorimetric titrations between 100 μM XPC in the syringe (25 × 20 μl injections) and 2 ml of 10 μM p62-PH in the cell were carried out at 20 °C in 20 mM potassium phosphate (pH 6.8). Each injection took place in 4 s with a preinjection delay of 210 s and a syringe stirring speed of 307 r.p.m. Data were analyzed using the Origin software package . Kd values were presented as means±s.d.The


N2-Src is a poorly understood neuronal splice variant of the ubiquitous C-Src tyrosine kinase, containing a 17 amino acid insert in its Src homology 3 (SH3) domain. To characterise the properties of N2-Src we directly compared its SH3 domain specificity and kinase activity with C- and N1-Src in vitro. N2- and N1-Src had a similar low affinity for the phosphorylation of substrates containing canonical C-Src SH3 ligands and synaptophysin, an established neuronal substrate for C-Src. N2-Src also had a higher basal kinase activity than N1- and C-Src in vitro and in cells, which could be explained by weakened intramolecular interactions. Therefore, N2-Src is a highly active kinase that is likely to phosphorylate alternative substrates to C-Src in the brain. Rabbit polyclonal anti-Src pY527 and anti-Src pY416 were from Cell Signaling Technologies . Mouse anti-β-actin was from Proteintech . Mouse anti-FLAG (clone M2), anti-mouse and anti-rabbit HRP-conjugated secondary antibodies and 2X SDS sample buffer were from Sigma . Unless stated otherwise, all other reagents were from Sigma.2.2Escherichia coli were prepared by inserting cDNA encoding His-tagged rat Srcs lacking the first 80 amino acids into pGEX4T-1  containing the human protein tyrosine phosphatase 1B (PTP1B) catalytic domain with a 3C protease site at its C-terminus . Plasmids for expressing recombinant active Δ80C-Src, N1- or N2-Src in terminus A. FLAG-tterminus  into pGEterminus  were ligterminus D. A plas2.3E. coli cultures at an O.D600 = 1 were induced with 0.5 mM IPTG and incubated at 18 °C overnight, shaking at 250 rpm. Following purification with glutathione resin (Genscript), the His-tagged kinases were cleaved from GST-PTP1B by incubation with PreScission 3C protease  at 4 °C overnight. The purified kinases were diluted 1:1 in kinase storage buffer , and stored at −80 °C. Approximately 0.5 mg pure kinase was obtained from a 1 l culture of E. coli.Recombinant His- and GST-fusion proteins were expressed and purified according to a previously described protocol with minor modifications for Src kinase expression 2.42, pH 7.5) and initiated by the addition of pre-warmed ATP  and incubated at 30 °C for the indicated times. Kinase and substrate concentrations were varied as indicated in the figure legends. Assays were terminated by transfer to ice and the immediate addition of 2X SDS sample buffer. Twenty percent of the reaction was separated on a 15% SDS–PAGE gel and transferred to PVDF membrane . Substrate phosphotyrosine content was determined by immunoblotting with mouse anti-phosphotyrosine (1:1000) and anti-mouse-HRP (1:5000). Immunoreactivity was visualised by incubation of immunoblots with enhanced chemiluminescence reagent (Millipore) and exposure to autoradiography film . To ensure equal protein loading, samples were stained with Coomassie gel stain to detect the substrates. Densitometric analysis of protein bands on immunoblots was performed with ImageJ Phosphorylation reactions were prepared in kinase reaction buffer . Where appropriate, statistical analyses of the data were performed with SigmaPlot software using one- or two-way ANOVA with post hoc pairwise comparisons by Tukey tests.To measure enzyme kinetics, the samples representing each substrate concentration phosphorylated by C-, N1- and N2-Src kinases were separated on a single gel. To normalise densitometry across multiple gels and produce curves across the full concentration range, a standard phosphorylated protein sample (8.3 μM YA phosphorylated by C-Src) was included on each gel. The highest value obtained by densitometry analysis of each curve was set to 1 and all other values were normalised accordingly. Due to the non-quantitative measurement of 2.54 cells were plated per well of a 24 well plate and transfected 24 h after plating at a ratio of 1 μg plasmid cDNA:2 μl EcoTransfect reagent according to the manufacturer’s instructions . Cells were lysed 48 h after transfection in SDS sample buffer and analysed by immunoblotting with anti-FLAG (1:1000), anti-Src-pY416 (1:1000), anti-Src-pY527 (1:1000), anti-phosphotyrosine (1:1000), anti-β-actin  and appropriate HRP-conjugated secondary antibodies (1:5000).B104 rat neuroblastoma cells were cultured in DMEM  supplemented with 10% foetal calf serum and 1% penicillin/streptomycin (Invitrogen). For the analysis of Src-FLAG expression and phosphorylation by immunoblotting, 3 × 1033.1E. coli is problematic, due to the toxicity of tyrosine phosphorylation in bacteria that do not support this form of post-translational modification To perform a direct comparison between the substrate specificities of C-, N1- and N2-Src, we developed an in vitro kinase assay using recombinant truncated Src kinases (lacking the first 80 amino acids) to phosphorylate peptide substrates fused to the C-terminus of GST. The expression of active Src kinases in The substrates comprised an ideal Src kinase substrate sequence  was determined by immunoblotting with an anti-phosphotyrosine antibody, which was quantified by densitometry  or after (Class II) the PxxP motif D. It wasM for YA D.Kms for the phosphorylation of YP1 and YP2 by N1-  is a key step in the activation of the Src kinase domain and it has been previously reported that N1-Src has a higher constitutive activity than C-Src We next determined the kinases’ phosphorylation status in cells, where they are subject to physiological regulation. In addition to detecting pY416, phosphorylation of Y527 was also analysed. Y527 phosphorylation at the C-terminus of Src by Csk promotes intramolecular association with the SH2 domain and is indicative of the inactive form of the kinase. Cell lysates from B104 rat neuroblastoma cells transfected with FLAG-tagged C-, N1- or N2-Src were analysed by immunoblotting with anti-pY416 and anti-pY527 antibodies. We observed no pY416 signal in untransfected cells and a weak signal in cells transfected with C-Src, however, pY527 immunoreactivity was readily detected for C-Src B. Therefbona fide N-Src substrates.To assess whether transfection of the apparently highly active N-Srcs resulted in increased cellular substrate phosphorylation, we probed the same lysates for total phosphotyrosine content. This revealed that the major phosphorylated proteins within the cells are the neuronal Srcs themselves B, presum3.4either of these interactions, akin to an ‘OR-gate’ switch, is sufficient to permit auto-phosphorylation on Y416 for full activity 248SKPQTQGLAKDA) was first delineated by deletions and mutations in the SH3 domains of Src and Hck that increased kinase activity Km for C-Src phosphorylation of YL was 5.2 ± 1.1 μM  and SH3 binding to the SH2:kinase linker. Evidence is accumulating to suggest that the disruption of ± 1.1 μM B, simila and YP2 D. This wectively B. This i3.5Having observed differential specificity of the N-Srcs for ideal SH3 ligands, we investigated the phosphorylation of an established in vivo C-Src substrate in brain, synaptophysin, a synaptic vesicle membrane tyrosine phosphoprotein , N2-Src has a high auto-phosphorylation activity, likely driven by weak intramolecular interactions. The N-Src SH3s have a weak affinity for peptide substrates containing Class I and Class II C-Src SH3 ligands and future work is required to establish the in vivo substrates of the N-Srcs and the structural basis of high affinity binding to the N-Src SH3 domains. Such studies could facilitate the development of isoform-specific inhibitors that can distinguish the kinases by disrupting SH3 ligand binding.Taken together, we have shown that in cells and in vitro


Trypanosoma brucei gambiense and T. b. rhodesiense. There is a need for simple, efficient, cost effective methods to identify new molecules with unique molecular mechanisms of action (MMOAs). The mechanistic features of a binding mode, such as competition with endogenous substrates and time-dependence can affect the observed inhibitory IC50, and differentiate molecules and their therapeutic usefulness. Simple screening methods to determine time-dependence and competition can be used to differentiate compounds with different MMOAs in order to identify new therapeutic opportunities.New therapeutics are needed for neglected tropical diseases including Human African trypanosomiasis (HAT), a progressive and fatal disease caused by the protozoan parasites T. brucei. Using this method, we identified tideglusib as a time-dependent inhibitor whose mechanism of action is time-dependent, ATP competitive upon initial binding, which transitions to ATP non-competitive with time. The enzyme activity was not recovered following 100-fold dilution of the buffer consistent with an irreversible mechanism of action. This is in contrast to the T. brucei GSK3β inhibitor GW8510, whose inhibition was competitive with ATP, not time-dependent at all measured time points and reversible in dilution experiments. The activity of tideglusib against T. brucei parasites was confirmed by inhibition of parasite proliferation (GI50 of 2.3 μM).In this work we report a four point screening methodology to evaluate the time-dependence and competition for inhibition of GSK3β protein kinase isolated from Altogether this work demonstrates a straightforward method for determining molecular mechanisms of action and its application for mechanistic differentiation of two potent TbGSK3β inhibitors. The four point MMOA method identified tideglusib as a mechanistically differentiated TbGSK3β inhibitor. Tideglusib was shown to inhibit parasite growth in this work, and has been reported to be well tolerated in one year of dosing in human clinical studies. Consequently, further supportive studies on the potential therapeutic usefulness of tideglusib for HAT are justified. T.brucei GSK3β inhibitors, and observed tideglusib to have a time-dependent, ATP-competitive mechanism that differentiated it from rapidly reversible inhibitors, such as GW8510. Adjusting the enzyme assays to account for these mechanisms showed that GW8510 and tideglusib had similar activities for TbGSK3β. However, this similarity did not translate to cellular activity, where GW-8510 was more active than tideglusib . These data suggest that factors other than TbGSK3β MMOA differentiate the effect of these molecules against T. brucei.Drug discovery for neglected tropical diseases must use efficient methods due to limited resources. One preferred drug discovery strategy is target-based drug discovery. In this strategy it is assumed that drug action begins with binding of a drug to its target. However, while binding is required, it is not sufficient to describe all the molecular interactions that translate binding to a therapeutically useful response. The contribution of aspects of the molecular mechanism of action (MMOA) such as time-dependence and substrate competition can influence concentration response relationships. To address this, a four point MMOA methodology was developed to evaluate time-dependence and substrate competition. We used this method to evaluate the MMOA for  New medicines are needed for neglected tropical diseases (NTDs). Drug discovery and development in all disease areas is inefficient due to high failure rates that increase the costs. The impact of the high failure rates on NTDs is very significant as a result of limited resources afforded to these diseases because of a lack of commercial markets for the medicines . There iTrypanosoma brucei. There are currently an estimated 30000 cases of HAT, with approximately 70 million people in 36 African countries at risk /([drug] + KI) and also known as the fractional occupancy). When inhibition of product formation is rapid and observed immediately following simultaneous addition of inhibitor and substrate, the inhibitor and substrate binding are in equilibrium. The contribution of a longer lived and more stable complex, (E’I), formed by conformational rearrangement of the initial complex (EI) can be detected experimentally following a preincubation of the enzyme and inhibitor prior to addition of substrate  binding with an enzyme (E+ I → EI) . Bindingubstrate . The preo is the enzyme rate (velocity), Vmax is the maximal velocity, [S] is the substrate concentration, Km is the Michaelis constant for the substrate, [I] is the inhibitor concentration and KI is the equilibrium dissociation constant of the inhibitor. The reaction velocity for a competitive inhibitor will change at different substrate concentrations and will depend on the ratios of [I]/KI and [S]/Km. This change can be theoretically calculated by the following equation derived from the equation for competitive inhibition,1 and S2 are the two substrate concentrations; v1 and v2 are the respective velocity of product formation. For the studies in this report we run the kinase assays with substrates at approximately 0.5 Km (10 μM for ATP) and 5x Km (100 μM for ATP). We chose 100 μM ATP because the ADP-Glo assay does not work well above 100 μM ATP and 10 μM ATP then gives us a better window. At inhibitor concentrations near KI (approximately the IC50 at low substrate concentration) then I/KI = 1 and (1 + I/KI) = 2. The theoretical shift for a competitive inhibitor in this system where the Km is 20 μM and ATP concentrations are 10 and 100 μM is 3.7. When there is no competition, no reduction of inhibition will be observed between the two ATP concentrations (non-competitive behavior).Competition occurs when the binding of two molecules (substrate and inhibitor) to the same protein is mutually exclusive (when one is bound the other is not). Competition of an inhibitor with substrate under equilibrium conditions will decrease the amount of inhibitor bound  and decrease the amount of inhibition. The higher the substrate concentration, the less inhibition. The loss of inhibitory activity at equilibrium will depend on the relative concentrations of the inhibitor and substrate as well as their respective equilibrium constants. The effect of a competitive inhibitor on the rate of an enzyme reaction is described by the following relationship.T. brucei was expressed in E. coli with a C-terminal His tag and purified to homogeneity as described in Materials and Methods. Conditions for the enzyme assay were optimized for linearity with time and enzyme concentration using GSM as a phosphoryl acceptor and measuring ADP production using Promega ADP-Glo reagents. The substrate versus rate plots showed saturable kinetics for the two substrates GSM and ATP, with Kms of 23 μM and 21 μM, respectively and Vmax of 31 and 34 s-1 in the two studies  and 22.6 nM (N = 5), respectively. The IC50 values for GW-8510 were 14.5 nM and 15.1 nM for preincubated reactions and non-preincubated reactions, respectively.ICion time . PreincuThe four point methodology was demonstrated for tideglusib and GW8510 . TidegluIn ATP competition studies the percent inhibition of activity by GW8510 was less at higher ATP concentrations (100 μM) than lower ATP (10 μM), 28% vs 67%, respectively irrespective of preincubation time . TidegluFurther evaluation of time dependence under more physiological conditions of no preincubation and higher ATP concentration was evaluated using enzyme progress curves. Progress curves measure the progress of the enzyme reaction with time . In thes50 of 5 nM following preincubation, with no evidence for covalent binding [Tideglusib and GW8510 were evaluated for irreversibility by measuring the TbGSK3β activity following a 100-fold dilution with reaction buffer. In these experiments tideglusib and GW8510 [100 nM] were preincubated with 50 nM TbGSK3β for 30 min at which time the reaction was diluted 100 fold into a solution containing 100 μM ATP and 80 μM GSM. The activity was then measured for up to two hours . The act binding . These i binding . The res50 in the HTS screens. A good starting concentration for the time-dependent studies is the IC25 (concentration at 25% inhibition), since time-dependent inhibition will increase with preincubation time. For the competition analysis, use of an inhibitor concentration equal to the IC50 at substrate Km  will allow observation of the decrease in inhibition due to higher substrate concentrations  was 2.3 μM , tideglusib (at 5 or 10 μM) markedly slowed trypanosome growth .In practice, this four point MMOA method can be applied at any stage in the drug discovery process. In early discovery, hits are identified in screens at one or a few concentrations and confirmed with IC50 measures in receptor competition assays with and without preincubation [et al have recently described a dual point competition association assay for assessing ligand-receptor binding kinetics. Their approach was found to be useful for ranking molecules [It is worth mentioning that this four point MMOA method is a simplification of more rigorous methods that are common practice in biochemistry and pharmacology labs. Typically studies to look for time-dependence and competition are accomplished by investigating complete dose-response curves whereby the results inform a detailed understanding of the molecular interaction. In preparation for these definitive studies, smaller experiments are used to establish conditions. We have found that the four point MMOA studies are suitable for screening purposes and can be sufficiently reliable to differentiate compounds. In previous work, we applied a similar strategy to differentiate kinetics for antagonists binding to CCR5 using ICcubation . Guo et olecules . The fouUsing the four point MMOA method, tideglusib was determined to be a time-dependent inhibitor of TbGSK3β. Its effectiveness increased with time until its inhibition activity was similar to GW8510, a well-documented potent TbGSK3β inhibitor Figs 3 a. Both tiT. brucei, and demonstrates the value of early MMOA determination to help understand target specific hypotheses. Many factors may contribute to the difference in activity including poor solubility of tideglusib that limits cellular activity and/or that the cellular activity of GW-8510 maybe due in part to a non-TbGSK3β mechanism. While further studies are needed to evaluate these issues, the understanding of their different TbGSK3β MMOAs will assist in designing and correctly interpreting the data.Adjusting the enzyme assays to account for these mechanisms showed that GW8510 and tideglusib had similar activity for TbGSK3β under more physiological conditions of preincubation and 100 μM ATP . HoweverT. brucei.The MMOA for tideglusib inhibition of TbGSK3β identified with the four point MMOA assay is consistent with that observed for inhibition of human GSK3β as reported by Dominquez and coworkers . Using aT. brucei GSK3 homologs (TbGSK3 short), is necessary for cell growth and viability and thus may serve as a potential drug target for the treatment of HAT [In humans, GSK3β is a regulatory kinase for over 40 different proteins in a variety of pathways, and has been implicated in a number of diseases including: Type II diabetes (Diabetes mellitus type 2), Alzheimer's Disease, inflammation, cancer, and bipolar disorder . Furthert of HAT . In a ret of HAT . Tideglut of HAT , 36. Blot of HAT .T.brucei GSK3β inhibitors and observed tideglusib to have a time-dependent mechanism which differentiated it from rapidly reversible inhibitors, such as GW8510. Tideglusib was shown to inhibit parasite growth in this work and has been reported to be well tolerated in one year of dosing in human clinical studies [In summary, we used the four point MMOA method to evaluate the MMOA for  studies –37. ConsS1 Fig(A). Tideglusib in 10 μM GSM. (B). Tideglusib in 80 μM GSM. All reactions preincubated or not preincubated with TbGSK3β for 30 min at room temperature at 0.06 μM. Experiments run with 10 μM ATP. Assays with 30 min preincubation were preincubated with inhibitor, TbGSK3β, GSM peptide, and buffer. ATP was mixed to initiate reaction. 0 minute preincubation contained inhibitor, GSM peptide, ATP, and buffer. TbGSK3β was mixed to initiate reaction. Reactions were run at room temperature for 5 minutes and stopped at 80°C. ADP formed was measured by Promega ADP-Glo kit. Black = 30 minute preincubation Grey = 0 minute preincubationTime dependent inhibition by preincubation of TbGSK3β with tideglusib in 10 μM and 80 μM GSM peptide. (TIF)Click here for additional data file.


Both hypoallergens were able to induce Met e 1-recognizing IgG antibodies in mice, specifically IgG2a antibodies, that strongly inhibited IgE from shrimp allergy subjects and Met e 1-sensitized mice from binding to Met e 1. These results indicate that the two designer hypoallergenic molecules MEM49 and MED171 exhibit desirable preclinical characteristics, including marked reduction in IgE reactivity and allergenicity, as well as ability to induce blocking IgG antibodies. This approach therefore offers promises for development of immunotherapeutic regimen for shrimp tropomyosin allergy.Designer proteins deprived of its IgE-binding reactivity are being sought as a regimen for allergen-specific immunotherapy. Although shrimp tropomyosin (Met e 1) has long been identified as the major shellfish allergen, no immunotherapy is currently available. In this study, we aim at identifying the Met e 1 IgE epitopes for construction of hypoallergens and to determine the IgE inhibitory capacity of the hypoallergens. IgE-binding epitopes were defined by three online computational models, ELISA and dot-blot using sera from shrimp allergy patients. Based on the epitope data, two hypoallergenic derivatives were constructed by site-directed mutagenesis (MEM49) and epitope deletion (MED171). Nine regions on Met e 1 were defined as the major IgE-binding epitopes. Both hypoallergens MEM49 and MED171 showed marked reduction in their  Food allergy is a type 1 hypersensitivity disorder that affects up to 10% of the general population Metapenaeus and Penaeus spp Metapenaeus ensis (Met e 1), which exhibits specific serological IgE reactivity with serum samples from shrimp allergy patients Penaeus aztecus (Pen a 1) At the molecular level, the muscle protein tropomyosin was identified as the major shrimp ingestion-related allergen in Although food avoidance and epinephrine injection are currently the first-line treatments in patients with anaphylaxis, allergen-specific immunotherapy (SIT) is the major strategy for clinical management of allergy as it has the capacity to modify the course of the disease. However, conventional modalities for SIT using native allergens are constrained due to the potential risk of allergic side-effects during treatment. In this context, hypoallergen with low/no IgE reactivity is desirable for SIT. Notably, the nature of allergenic epitopes and hypoallergens might greatly affect the SIT outcome such as the induction and generation of blocking antibodies, shifting of the Th1/Th2 paradigm and induction of peripheral tolerance by recruitment of regulatory T cells Metapenaeus tropomyosin Met e 1. The second objective of this study is to construct hypoallergenic derivatives of Met e 1 by introducing point mutations within the IgE-binding epitopes identified, or by deleting these epitopes. The IgE reactivity, allergenicity, immunogenicity and the inhibitory potential of the hypoallergen-induced antibodies towards IgE antibodies of subjects allergic to shrimp and Met e 1-sensitized mice To circumvent this issue, we have chosen a two-pronged approach in designing shrimp tropomyosin hypoallergens. In this study, the first objective is to define the major IgE-binding epitopes of Serum samples were obtained from 12 subjects (aged 3–17 years) with confirmed clinical history of allergic responses to shrimp and positive skin prick test . SpecifiA written consent was obtained from the parents of the children enrolled in the study . The use and storage of human sera were approved by the Joint Chinese University of Hong Kong - New Territories East Cluster Clinical Research Ethics Committee with a written informed consent (CREC Ref. No. CRE-2010-514). All animal protocols were approved by the Animal Experimentation Ethics Committee, The Chinese University of Hong Kong (ref No. 11/006/GRF and 463911), in accordance with the Department of Health (Hong Kong) guidelines in Care and Use of Animals. All experiments were performed under licenses granted from the Government of Hong Kong Special Administrative Region.There were three independent methods used to predict the immunodominant allergenic epitopes including 1) computational prediction of IgE binding epitopes, 2) ELISA against overlapping peptides that span the entire Met e 1 sequence, and 3) dot-immunoblotting of overlapping peptides against the entire Met e 1 sequence. 18 overlapping peptides spanning the full-length (274 amino-acids) Met e 1 were commercially synthesized (GenScript). Each peptide had 20 amino acids (except for peptide 18 that contains 19 amino acids) with five amino acids overlapping with the adjacent peptides at the N-terminus. Individual peptides were dissolved in distilled water, aliquoted and stored at −20°C until required.Three computational models from the Immune Epitope Database (IEDB) Analysis Resource were employed to predict the major linear IgE-binding epitopes of Met e 1, including Bepipred Antibody Epitope Prediction, Kolaskar & Tongaonkar Antigenicity model and Emini Surface Accessibility Prediction. Bepipred Antibody Epitope Prediction predicts the location of IgE-binding epitopes based on the hidden Markov model and propensity scale method 2SO4. Absorbance was measured at 450 nm using an ELISA plate reader (Bio-Rad). All absorbance values were background-corrected, in which the background absorbance was the OD value of Met e 1-coated wells incubated with secondary and tertiary antibodies only. All the above procedures were performed at room temperature. The plates were washed with PBS/0.5% Tween-20 (PBST) three times and PBS once between each step and all dilutions were made in 1% BSA/PBS.For peptide ELISA, 3 µg of each peptide were coated on 96-well plates  in 0.05 M carbonate buffer overnight. After blocking with 1% BSA/PBS for 1.5 h, the plates were incubated with individual serum samples (150 dilution) at room temperature for 2 h. Thereafter, the plates were incubated with biotinylated goat anti-human IgE (Vector) in 11000 dilution for 45 min followed by incubation with Avidin D, Peroxidase labeled antibody (Vector) in 11000 dilution for 30 min. The plates were then developed with TMB substrate reagent set (BD Biosciences) for 15 min and the reaction was terminated by 2 N HFor dot-immunoblotting, 3 µg of each peptide (3 µL) were spotted onto a 0.2 µm nitrocellulose membrane (Bio-Rad). The membrane was allowed to air-dry and thereafter fixed with 2.5% glutaraldehyde/PBS for 10 min Salmo salar , Epinephelus coioides , Siniperca chuatsi  and Thunnus thynnus   . All nin01050.1) . To cons01050.1) . This muEscherichia coli BL21 (DE3) (Invitrogen) by culturing in MagicMedia (Invitrogen) at 37°C overnight. His-tagged recombinant Met e 1 (rMet e 1), MEM49 and MED171 were purified using the HisPur Cobalt Spin Column (Thermo Scientific) according to the manufacturer’s instructions. Protein concentration was determined by BCA assay (Sigma Aldrich) while the purity was determined by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining.cDNA coding for the full length shrimp tropomyosin Met e 1 and the encoding sequences of MEM49 and MED171 were cloned into His-tag expression vector pET30(a)+ (Novagen) and expressed in 3–4 weeks old female Balb/c mice were acquired from the Laboratory Animal Services Centre, The Chinese University of Hong Kong. All animals were maintained on a shrimp-free diet and housed in pathogen-free conditions. To induce Met e 1 hypersensitivity in mice, sensitization was performed as described previously by intragastric administration of 0.1 mg of recombinant tropomyosin plus cholera toxin on days 0, 12, 19 and 26 and challenged on day 33 3. Blood was collected 4 h after the last injection for the determination of antibody levels.For immunization experiments, 5–6 weeks old female Balb/c mice were intraperitoneally immunized three times on days 0, 7 and 14 with 0.1 mg purified rMet e 1, MEM49 or MED171 adsorbed to 1 mg Al(OH)2SO4.To examine the IgE reactivity to rMet e 1, MEM49 or MED171, 96-well ELISA plates were coated with 5 µg/mL of either rMet e 1, MEM49 or MED171 in 0.05 M carbonate buffer overnight at 4°C, blocked with 1% BSA/PBS for 2 h and incubated with serum samples from shrimp allergy subjects or Met e 1-sensitized mice (110 dilution) overnight at 4°C. After washing, plates were incubated with biotinylated anti-human (Vector) or anti-mouse IgE antibodies (BD Pharmigen) and Avidin D, Peroxidase labeled antibody (Vector), each at 11000 dilution at room temperature for 1 h and 30 min, respectively. The plates were then developed with TMB substrate reagent set (BD Biosciences) for 15 min and the reaction was terminated by 2 N H2a antibodies raised in rMet e 1, MEM49 and MED171 immunized mice, sera in serial dilutions (1400 to 125600) were incubated in the rMet e 1, MEM49 or MED171 coated plates (5 µg/mL) for 2 h at room temperature. The plates were then incubated with goat anti-mouse IgG or anti-mouse IgG2a (Southern Biotech) in 12000 dilution for 45 min followed by incubation with Avidin D, Peroxidase labeled antibody (Vector) in 11000 dilution for 30 min. The plates were then developed with TMB substrate reagent set (BD Biosciences) for 5 min and the reaction was terminated by 2 N H2SO4.To determine the reactivity of IgG and IgGin vivo allergenicity of MEM49 and MED171. Backs of naïve Balb/c mice were shaved, followed by intradermal injection of Met e 1-specific IgE-containing sera  under isoflurane narcosis. Two hours later, mice were injected intravenously with a mixture of 100 µL of 0.5% Evan’s blue dye (Sigma Aldrich) and 0.1 mg rMet e 1, MEM49 or MED171. Thirty minutes after dye-rMet e 1 administration, mice were sacrificed by cervical dislocation and skins of their backs were immediately inverted for the measurement of blue region diameters.Passive cutaneous anaphylaxis was performed to determine the no inhibitor–ODinhibitor)/ODno inhibitor]×100% and expressed as percentage inhibition.Competitive inhibition ELISA was performed to evaluate the blocking capacity of hypoallergen-induced blocking antibodies. Briefly, rMet e 1 was used to coat 96-well plates (5 µg/mL) overnight at 4°C and blocked with 1% BSA/PBS for 2 h. Plates were then washed and blocked with 100 µL of 110 diluted sera from mice immunized MEM49, or MED171 overnight at 4°C. Thereafter, 100 µL of sera from shrimp allergy patients or Met e 1-sensitized mice at a predetermined dilution (110–120 dilution) were added and incubated at room temperature for 2 h. The wells were then washed and followed by the addition of biotinylated anti-human or anti-mouse IgE antibodies, HRP-Avidin D and developed as described above. The blocking ability of the induced IgG antibodies was determined using the equation [ followed by the Student-Newman-Keuls test using SigmaStat 3.1. The difference was considered statistically significant at p<0.05.By ELISA, sera from patients with shrimp allergy (n = 12) had significantly higher IgE reactivity against five peptides  when compared with other peptides (p<0.05) . None ofAllergenic regions on Met e 1 were also defined based on the intensity of peptide spots and the frequency of recognition in dot-immunoblotting . A peptiThree online immunoinformatics models were applied to define the IgE epitopes. . Seven e25–30, Met e 143–60, Met e 187–103, Met e 1146–154 Met e 1161–165, Met e 1191–211, Met e 1236–241, Met e 1247–255 and Met e 1269–281, respectively , which matches with our initial expectation.The second hypoallergen MED171 was designed by deleting all IgE-binding epitopes, which results in a smaller-sized truncated tropomyosin variant of only 171 amino acid residues. With the disruption of all epitopes and possibly its structural flexibility as in tropomyosin, IgE reactivity and allergenicity of MED171 should be more significantly abolished. From our data, both variant showed significant reduction in their + T cell epitopes mapped by Ravkov et al. 2a antibodies in mice immunized with MEM49 or MED171, but not with the wild type allergen Met e 1. The Th1-driven allergen-specific IgG2a antibody in mouse and IgG4 antibody in human induced during SIT are considered to be blocking antibodies and correlate well with clinical improvements 2a responses even a pro-Th2 adjuvant was used during immunization. Such production of specific IgG2a and absence of Met e 1-specific IgE might correspond to the Th1-driving potential of the two hypoallergens. Most importantly, these antibodies were able to significantly block IgE of both shrimp allergy subjects and Met e 1-sensitized mice from binding to Met e 1. Such inhibitory and Th1-inducing potential are beneficial and it is likely that a MEM49- or MED171-based vaccine will modulate shrimp tropomyosin-induced allergic responses.We noted that most of the human shrimp tropomyosin CD4in vivo and in vitro IgE reactivity and allergenicity of the two designer shrimp tropomyosin hypoallergens MEM49 and MED171 when compared to the wild type allergen Met e 1 and more importantly, robust IgG antibodies’ responses with inhibitory potential to Met e 1-specific IgE antibodies of shrimp allergy subjects and Met e 1-sensitized mice. Finally, this work signifies an important discovery that could potentiate the development of prophylactic and/or therapeutic therapies in shellfish allergy.To our knowledge, this is the first study providing experimental evidence of a shellfish allergen-specific IgG blocking antibodies induced by hypoallergens. Our results demonstrate significant decrease in the Figure S1Salmo salar , Epinephelus coioides (orange-spotted grouper), Siniperca chuatsi (Mandarin fish) and Thunnus thynns (Atlantic bluefin tuna). Amino acid deviations within each IgE-binding epitope (framed) were identified and subsequently mutated into the homologous sequence of fish tropomyosins (bold letters shaded in gray) for the construction of hypoallergen MEM49.Comparison of the tropomyosin sequences for the construction of hypoallergen MEM49. Tropomyosin sequence of Met e 1 was compared to that of four fish species (TIF)Click here for additional data file.Figure S2(A) Surface probability score of each amino acid residue of Met e 1 in Emini Surface Accessbility Prediction. (B) Antigenic propensity score of each amino acid residue of Met e 1 in Kolaskar & Tongaonkar Antigenicity. (C) Epitope score of each amino acid residue of Met e 1 in Bepipred Linear Epitope Prediction.Computational prediction of tropomyosin IgE-binding epitopes. (TIF)Click here for additional data file.Table S1Clinical characteristics and shrimp tropomyosin-specific IgE of the shrimp allergy patients included in this study. 12 patients 3–17 years old with documented history of shrimp allergy were recruited in this study for mapping the major IgE-binding epitopes of Met e 1 and characterizing the IgE reactivity of the hypoallergens.(DOCX)Click here for additional data file.


It has been suggested that SrRuO3 can be robust against some intrinsic/external perturbations but fragile against some others in terms of electro-transport and magnetism, and it is our motivation to explore such stability against the local site cation disorder. In this work, we prepare a set of SrRuO3-based samples with identical averaged A-site size but different A-site cation disorder (size mismatch) by Ca and Ba co-substitution of Sr. It is revealed that the electro-transport and magnetism of SrRuO3 demonstrate relatively high stability against this A-site cation disorder, characterized by the relatively invariable electrical and magnetic properties in comparison with those of SrRuO3 itself. A simple electro-transport network model is proposed to explain quantitatively the measured behaviors. The present work suggests that SrRuO3 as an itinerant electron ferromagnetic metal possesses relatively high robustness against local lattice distortion and cation occupation disorder.It is known that the electro-transport and magnetism of perovskite alkaline-earth ruthenate oxides are sensitive to the lattice distortion associated with the A-site cation size. Orthorhombic CaRuO In these cases, the competition between the correlation and electron kinetic energy is marginal, allowing more fascinating consequences in terms of quantum states and magneto-transport behaviors against intrinsic and extrinsic stimuli, even if these stimuli are weak83 with A = Sr, Ca, and Ba131415162RuO4 and A3Ru2O7etc also exhibit rich quantum phase transitions and exotic states including superconductivity18193 to outline the underlying physics.As the knowledge at textbook level, transition metal oxides with typical compact ABO3 perovskites, the ionic size mismatch between the A and B sites is the origin for lattice symmetry variation and structural distortions realized by oxygen octahedra rotation and/or tilting, which are often discussed within the framework of structural tolerance factor213-type mode16d-orbitals and O 2p-orbitals, the electronic structure and magnetism can be remarkably dependent of the A-site cation and its radius (RA). The perovskites ARuO3 manifest the physics of electron correlation within this framework too93 (SRO) and CaRuO3 (CRO) both exhibit the orthorhombic (o-) structure91415243 (BRO) may have poly-type structure depending on the synthesis details14262728271430For ABOPbnm (Pnma) space group and its lattice constants are a = 5.57108 Å, b = 5.53543 Å, and c = 7.8504 Å14Ru-O1-Ru) and 162.4° (ϕRu-O2-Ru) with the A-site size RA-XII ~1.4401 Å where subscript XII refers to the twelve-coordinate value for Sr2+ 1434353637For a consideration of structural consistence, we confine our discussion on the o-SRO and o-CRO as well as c-BRO in the present work. The lattice structures of these compounds are shown in 2+ site) and Ru-site chemical substitution by isovalent or heterovalent ions, which has been demonstrated to impose substantial influence on the electro-transport and magnetism. We first consider one extreme end: o-CRO. Different from SRO, the Ca2+ ionic radius is RA-XII ~1.339 Å, making more seriously distorted lattice and more bent Ru-O-Ru bond ϕ ~148° much smaller than that of o-SRO323940T but no antiferromagnetic (AFM) order is developed42ΘW ~ −68 K, suggesting an AFM background. However, o-CRO remains to be a metal although its electrical resistivity (ρ) dependence of T is much weaker than most metallic transition metal oxides32ρ is comparable in magnitude with that of o-SRO, while reported electrical resistivity data from various groups are scattered3245RA for ~7% regarding o-SRO and o-CRO is not a small quantity, leading to remarkable differences in lattice parameters and distortion between them, as summarized in 143 are robust or fragile against structural variations.Nevertheless, electro-transport and magnetism of SRO can be either fragile against some other structural and chemical perturbations. One example is the A-site 32ρ283281415163 with intrinsic or external perturbations.Similar case can be found if one considers the other extreme end: c-BRO. Given that the Ba2+ and Ba2+ have been carried out141719323353545556572+ by Ca2+ or Ba2+. We only present a short outline of the consequence of Ba-substitution at low level and Ca-substitution over a broad range. For Srx1−CaxRuO3 (SCRO), the Ca substitution makes the lattice seriously distorted, characterized by reduced ϕRu-O1-Ru and ϕRu-O2-Ru and stretched Ru-O bond lengths32χ at T > Tc begins to deviate the Curie-Weiss behaviors in accompanying with the reduced Tc9173233χ(T) behavior associated with the appearance of Griffiths (phase) state141732ρ-x dependence. For Srx1−BaxRuO3 (SBRO), the Ba substitution gives rise to distinctly different consequence14χ(T) above Tc. Therefore, one can discuss the physics within the conventional framework of correlated physics. Since Ba2+ has bigger ionic size and stronger ionic character than Sr2+ has, two physical ingredients are expectedW, which would enhance the ferromagnetism and improve the electron itinerancy. The other is the competition between the Ba-O bonding and Ru-O bonding which eventually elongate the Ru-O bond at higher x level, making narrow W, low Tc, and inactive electron itinerancy with increasing x. The latter ingredient seems to be dominant for c-BRO, explaining the low Tc and large ρ of c-BRO with respect to o-SRO9Besides a number of investigations on o-CRO and o-SRO in bulk and thin film forms16174950513 do depend largely on a number of structural and electronic factors, such as the A-O bond length, A-site ionic character (e.g. electronegativity), RuO6 oxygen octahedral rotation and tilting, and Ru-O bond length etc. Nevertheless, so far every trial by means of isovalent or heterovalent substitution has always caused more than one occurrence of these factors and thus a general scenario for predicting the electro-transport and magnetism of ARuO3 and other ruthenates is yet complicated181956603, these disorder-induced phenomena have not yet well understood in spite of some fragmental works available60To this stage, one understands that the electro-transport and magnetism of ARuO3 originates from several aspects. First, the Anderson localization as a well-known concept in condensed matters has been emphasized in correlated electron systems too623 manganites has been well investigated and the itinerant electron localization and spin-glass behavior with increasing disorder were identified5964656665688246071The main motivation to address the A-site cation disorder-induced phenomena in ARuO2+ and Ca2+ at Sr2+-site, keeping the average Sr2+-site size invariant but the local disordering is induced, which should increase with increasing substitution level. Different from earlier works on manganites65In this work, we proposed the idea of co-substitution of Ba3, the A-site ionic size disorder is usually measured by the mean square momentum or size disorder variance σ2 652+, Ca2+, and Ba2+. The chemical formula becomes Srx1−xRuO3 with the co-substitution level x. The A-site size variance is:For a perovskite ABOyi and Ri are the atomic fraction and ionic radii of i-type ions at A-site, and 〈…〉 stands for configuration averaging. In our work, condition 〈RA〉 = RSr at Sr2+ state is always maintained in order to exclude effect from variation of the average A-site size. Therefore, we must have the following relationships:where σ2 and x can be used as a measure of the A-site disorder.noting that both x are marked aside. It is seen that all the peaks for different x can be properly indexed by the standard o-SRO reflections without identifiable impurity phase. The relative intensity of each peak shows no obvious dependence on x, suggesting the invariable lattice structure with increasing x.Before we investigate the effect of A-site disorder on the electro-transport and magnetism, a characterization of the structural and chemical homogeneity of the as-prepared samples is necessary. First, the X-ray diffraction (XRD) θ–2θ spectra of a set of samples are presented in x. Second, gradual evolution of the peak profile details with increasing x can be detected, taking the reflections (202) and (040) shown in x = 0 become broadening with x and merged at x = 0.20, suggesting the local lattice distortion due to the A-site size mismatching. Similar behavior can be seen for reflections (204), (323), and (161), as shown in For a clearer clarification, several amplified reflections at 2θ ~22.65°, 46.30°, and 76.85° are presented in x = 0.10, 0.15, and 0.20 are presented in 2 are inserted for reference since it was found that sample x = 0.20 contains very weak signals from RuO2. The best fitted lattice parameters for the three samples are listed aside and the parameters for all the samples are listed in χ2 < 1.8) although fluctuations are inevitable. One may claim that the macroscopic lattice structure of o-SRO remains nearly invariable to the Ca/Ba co-substitutions. Nevertheless, the local lattice delicate distortion with increasing A-site disorder is detectable, characterized by the full-width at half maximum Δ(2θ) data for three 2θ angles as a function of x, as shown in x, marking the enhanced local lattice distortion from the A-site disorder.For a quantitative evaluation of the possible lattice distortion, we adopt the Rietveld refinement package to fit the measured XRD data and the data for three samples x = 0.15 is presented in x = 0.20.We also checked the cation distribution homogeneity in these samples by electron probe microanalysis to exclude possible chemical segregation. The SEM image of microstructure of sample ρ(T) data for several samples x are plotted in TR) in the metal-like ρ(T) curve14x = 0) exhibits a clear itinerant electron FM transition at Tc ~161 K, responsible for the anomaly at TR14M(T) curves are plotted in Tc, while the dM/dT data gives the maximal point at Tm, as shown in TR, Tc, Tm, α) together with M and ρ at several T are plotted in Now we turn to the electro-transport and magnetism in response to the increasing A-site disorder. The measured ρ(T) curve upward and the anomaly leftward simultaneously, leading to slight reduction of TR. However, such a shift is insignificant and the metal-like ρ(T) dependences remain little changed. Second and similarly, the M(T) curve also shifts downwards and leftwards with gradual reduction of Tc and Tm. Either, the M(T) dependences remain little change. Third, the linear ρ − T data above TR can be fitted by relation ρ = αT + b with monotonously increasing coefficient α(x), suggesting more scattering due to the local lattice distortion. Far below TR (T ≪ TR), the ρ − T data can be reasonably described by relation ρ − ρ0 ~ T 2  dependence. First, the M(T) curves under the ZFC and FC conditions with cooling and measuring fields of ~1.0 kOe are plotted in x = 0 and 0.10 respectively. We see a clear separation of the M(T) curves under the ZFC and FC modes with a small cusp right below Tc for all the samples including pure SRO. Although SRO is often seen as a ferromagnetic metal, such a feature of ZFC-FC separation in polycrystalline bulk SRO, thin film SRO, and even single crystal SRO, was reported in earlier works7576M(T) data do show ferromagnetic transition around Tc, but the clear separation in M(T) curve between the ZFC and FC modes is also a well-established feature. Certainly, such a feature is not necessarily related to spin-glass behavior in the strict sense, one may argue that this ferromagnetism may be sensitive to defects and local lattice distortion inevitable in the samples. Additional investigation is needed, e.g. by measuring the ac magnetic susceptibility and spin relaxation153274The effect of the A-site disorder on the ferromagnetism of o-SRO can be illustrated too from the M-H loops at T = 5K for several samples are plotted in Ms for sample x = 0 reaches ~1.2 μB/f.u., consistent with earlier reportsMs down to ~0.85 μB/f.u at x = 0.20. It is known that Ru ion in ARuO3 is in the Ru4+ state with electronic configuration d4. The low-spin (S = 1) state contributes an effective moment of ~2.8 μB above Tc, as predicted by the low-spin moment of 2[S(S + 1)]1/2 78Ms is only ~30% given x = 0.20, indicating that the magnetism of o-SRO is not as sensitive as expected to the A-site disorder. For the coercive field, it is revealed too that the A-site disorder enhances the coercive field up to 0.65 T at x = 0.20. Earlier work revealed that the ferromagnetic SrRuO3 thin films deposited on LaAlO3 substrates exhibit a coercive field of ~10 kOe, while the coercive field for single crystal SrRuO3 is 3 kOe38x1−CrxO3 (0 ≤ x ≤ 0.12) showing substitution-dependent coercive fieldOn the other hand, the Seff, and the dc magnetic susceptibility χ above Tc can be described by32While that the magnetism of SRO is rigidly ferromagnetic or spin-glass like remains to be an issue, one may investigate the stability of magnetism of o-SRO to the A-site disorder by evaluating the spin clustering tendency in the co-substituted samples. We assume the effective moment as χ−1(T) data for several samples are presented in T ρ(T2) data. The best fitting of the high-T χ−1 data using Tc and Seff, as marked in the plots. Several seminal characters can be highlighted. First, for all the samples, the χ − T data at T > Tc can be well described by Seff is roughly ~4.10 μB, independent of x and bigger than the effective moment of Ru4+ (~2.8 μB)78T paramagnetic phase favors a spin-cluster state instead of the single-spin ensemble. Second, an obvious deviation of χ−1 from the fitting as T tends close to Tc from the high-T side, is identified, suggesting a Griffiths phase-like feature, as extensively discussed for o-SRO and SCRO systems1417T ρ data do fit the T 2-dependence, as shown by the linear solid (red) line for each caseThe  Ru4+ ~2. μB78T paρ(T) dependence to see its consistence with measured data.As a complementary part to the above discussion, one may propose a simplified model to fit the electro-transport behavior quantitatively. Due to the random occupation of the A-site by Sr, Ca, and Ba, the simplest approach is to develop an electro-resistor network and evaluate the 4+-Ru4+ bonds in the lattice. Since the highest x is 0.20, one may assume that the Ca and Ba occupations in the lattice are well separated without the nearest-neighbored Ba-Ba, Ca-Ca, and Ba-Ca pairing. If one A-site is occupied by ion A′ (=Ca or Ba), it has twelve nearest-neighbor Ru4+-Ru4+ bonds whose resistivity is assigned as ρA′-A′ and twenty-four next nearest-neighbor Ru4+-Ru4+ bonds whose resistivity are assigned as ρSr-A′. The left are resistors with resistivity ρSr-Sr. It should be noted that any variation of the A-site ionic size would influence the surrounding nearest-neighboring ρA′-A′ and the next nearest-neighboring ρSr-A′, and the consideration of ρSr-A′ takes into account partially the itinerary feature of Ru4+ electrons. Here, the values of ρA′-A′ and ρSr-Sr at a given T are taken from the measured data of polycrystalline o-CRO, c-BRO, and o-SRO respectively. While the data for c-BRO are taken from ref. ρCa-Ca(T) curve is from our earlier work8232ρ(T) data are plotted in To proceed, one constructs a three-dimensional lattice as shown in ρA′-A′ and ρSr-Sr, one has to determine ρSr-A′ (A′ = Ca and Ba) as a function of T too. A physically sufficient argument on a derivation of ρSr-A′ is quite challenging since it can be related with the details of electronic structure associated with the lattice distortion which is unavailable to us. Given the fact that o-SRO accommodates the itinerant electron transport, ρSr-A′ can be approximately treated as the geometric mean of ρSr-A′ and ρA′-A′, i.e. ρSr-A′ = (ρSr-Sr × ρA′-A′)1/2 83ρ(T) for the three-dimensional cubic resistor networks of different substitution level x are evaluated based a sufficient configuration averaging. As examples, the measured (mea.) and evaluated (sim.) data are presented in x = 0.02, 0.07, and 0.20, respectively. A good consistence of the measured (mea.) and evaluated (sim.) data over the whole T-range has been demonstrated, illustrating on the other hand the implication of the resistor network model as a quantitative description of the electro-transport behaviors.Besides x = 0.20 does not change the qualitative behaviors of o-SRO. Qualitatively, this stability has two-fold origin. On one hand, the Ru4+ 4d-orbitals are more extended than 3d-ones, making the p-d hybridization between Ru4+ and O2− much less sensitive to the variation of Ru-O-Ru bond lengths and angles142+ benefits to the bandwidth broadening and thus electrical conductivity. A low-level Ba-substitution (<0.15) benefits to the electro-transport which counteracts to the A-site disorder induced damage to the electrical conductivity14So far, our data from various aspects seem to confirm the high stability of electro-transport behaviors and magnetism against the A-site disorder. The variance up to Besides, it is known that the A-site size disorder in manganites may drive the electronic phase separation653 would offer rich quantum states with distinctly different electrical and magnetic properties due to the competitive electron correlation (U) and bandwidth (W). This argument applies particularly to CaRuO3 which is believed to be on the verge of magnetic ordering. Nevertheless, the present work suggests the high stability of o-SRO from the electro-transport and magnetism against the A-site disorder. This high stability can also be a good annotation to the good electrical conductivity of o-SRO as electrode material which is robust against imperfection, defects, and other perturbations245260Finally, as a general remark, it is well-accepted that ARuOx = 0.20 has been revealed, it is demonstrated by a series of electro-transport and magnetism characterizations that the itinerant electron ferromagnetism of o-SRO can be maintained, exhibiting the high stability against the sufficient A-site disorder. The possible origin for this stability is discussed, which is the consequence of delicate balance between two local lattice distortion induced effects. One is the Ca-substitution induced AFM background and the other is the Ba-substitution induced bandwidth broadening.In conclusion, we have conducted systematic experiments on the impact of A-site cation disorder (size variance) on the electro-transport and magnetism of polycrystalline o-SRO upon the Ca/Ba co-substitution of Sr. While spatially random Ca/Ba occupation up to x1−(Ca0.6296Ba0.3704)xRuO3 (SCBRO) samples which exhibit different size variance dependent of x. It is noted that the Ca/Ba-substitution level x is equivalent to the A-site size variance σ2. For a rough estimation, the size variance corresponding to x = 0.20 reaches up to σ2 ~0.0034 Å2, which is on the similar order of magnitude as the size variance of manganites specifically proposed for the A-site size disorder investigations, e.g. σ2 ~0.005 Å2 for Sm0.55(Ca0.6Ba0.4)0.45MnO3.We prepared a series of Srx up to 0.20 were synthesized using the conventional solid state sintering. The highly purified powders of oxides and carbonates were mixed in stoichiometric ratios, ground, and then fired at 1000 °C for 20 h in air. To reduce the effect of trapped space charges near the grain boundaries of polycrystalline samples, the sample volume density should be close to 100%. The resultant powders were reground and pelletized under 1000 psi pressure to disks of 2 cm in diameter, and then the pellets were sintered at 1300 °C for 24 h in air in prior to natural cooling down to room temperature.The SCBRO polycrystalline samples with Kα radiation at room temperature. The refinement of high resolution XRD data was performed using the standard Rietveld method. The chemical composition and its spatial homogeneity down to nanoscale were checked using electron probe microanalysis installed with scanning electron microscopy  which was used to observe the microstructure. The transmission electron microscopy (JEOL 2100F microscope) was also used to characterize the microstructure.The samples crystallinity and structure were checked by X-ray diffraction (XRD) with Cu M) as a function of T and magnetic field H was measured using Quantum Design superconducting quantum interference device magnetometer (SQUID) in zero-field cooled (ZFC) and field-cooling (FC) modes respectively. The cooling field and measuring field were both 1.0 kOe unless stated elsewhere. The electro-transport and specific heat were characterized using physical properties measurement systems (PPMS) from the Cryogenic Co. Ltd and from the Quantum Design Inc. The electrical resistivity ρ as a function of T and H was obtained.The samples’ magnetization .