The tumor suppressor Smad4/DPC4 is an essential transcription factor in the TGF-β pathway and is frequently mutated or deleted in prostate, colorectal, and pancreatic carcinomas. We recently discovered that Smad4 activity and stability are regulated by the FGF/EGF and Wnt signaling pathways through a series of MAPK and GSK3 phosphorylation sites located in its linker region. In the present study, we report that loss-of-function associated with 2 point mutations commonly found in colorectal and pancreatic cancers results from enhanced Smad4 phosphorylation by GSK3, generating a phosphodegron that leads to subsequent β-TrCP–mediated polyubiquitination and proteasomal degradation. Using chemical GSK3 inhibitors, we show that Smad4 point mutant proteins can be stabilized and TGF-β signaling restored in cancer cells harboring such mutations. Bone Morphogenetic Protein(BMP)Cyclin-dependent kinase(CDK)Caco-2cell lineDeleted in prancreatic carcinoma 4(DPC4)Glycogen Synthase Kinase 3(GSK3)Mitogen-Activated Protein Kinase(MAPK)pancreaticcancerprostatecancerbeta-Transducing Repeat-containing protein(beta-TrCP)Wnt-induced STabilization Of Proteins(Wnt/STOP).1 The TGF-β pathway exerts potent antitumor effects and its misregulation often results in cancer progression.2 In the canonical TGF-β signaling pathway, activated TGF-β receptors phosphorylate C-terminal serine residues of the transcription factors Smad1/5/8 for bone morphogenetic proteins (BMPs) or Smad2/3 for the TGF-β/activin branch of the pathway.3 The transcription factor Smad4, also called deleted in pancreatic carcinoma 4 (DPC4), functions as a co-Smad that binds to receptor-phosphorylated Smads (R-Smads) and is an essential downstream determinant in TGF-β signaling that was, until recently, considered a constitutive component of the pathway.3The transforming growth factor-β (TGF-β) family of cytokines constitutes the largest group of growth factors in humans and regulates many aspects of cell behavior such as differentiation, migration, and apoptosis.4 Smad4 appears to be the main barrier to tumor progression and is frequently mutated, with devastating effects, in prostate and pancreatic carcinomas.5 Mutations in Smad4/DPC4 have been identified in approximately 50% of pancreatic adenocarcinomas,6,7 suggesting a pivotal role during pancreatic cancer progression. Tumors lacking functional Smad4 are more proliferative, invasive, and angiogenic, and consequently more likely to form metastases.8 Among patients undergoing surgical removal of pancreatic adenocarcinoma, survival was found to be significantly longer for patients whose tumors express Smad4 protein.8Although receptor-phosphorylated Smads or TGF-β receptors can be mutated or deleted in some cancers,9 Skp1-Cullin1-F-box (SCF) E3 ligases recognize specific protein substrates through their variable F-box proteins.10 β-transducin repeat-containing protein (β-TrCP), a member of the Fbw subfamily of F-box proteins, is known to recognize various phosphorylated substrates, including β-catenin,11 IkappaB,11,12 nuclear factor (NF)-kappaB B2,13 NF-kappaB p105,14 Snail,15 and TAZ.16,17 Interestingly, several of these proteins are substrates for glycogen synthase kinase-3 (GSK3), a Wnt- and phosphoinositide 3-kinase (PI3K)-regulated kinase that recognizes pre-phosphorylated substrates. For phosphorylation of a Serine/Threonine (Ser/Thr) residue, GSK3 prefers a phospho-Ser/Thr at the n + 4 position, which is known as the priming phosphorylation site.18The ubiquitin proteasome pathway regulates degradation of most proteins in mammalian cells, and E3 ubiquitin protein ligases (E3-ligases) determine the specificity and timing of the ubiquitination process for given substrates.19 We found that fibroblast growth factor (FGF)/epidermal growth factor (EGF) stimulation leads to Smad4 phosphorylation by Erk of the canonical MAPK site (PXTP) located at Threonine 277. Phosphorylation of this site recruits transcriptional co-activators, most likely p300, allowing Smad4 to reach peak transcriptional activity.19,20 However, MAPK also primes Smad4 for 3 subsequent GSK3-mediated phosphorylations that cause transcriptional inhibition and generate a phosphodegron that is used as a docking site by β-TrCP, leading to protein polyubiquitination and proteasomal degradation.19 Wnt signaling inhibits GSK3 activity22,23 and therefore potentiates Smad4 activity, particularly at low concentrations of TGF-β.19 Thus, the linker domain of Smad4 contains a growth factor-regulated transcription activation domain that integrates Wnt, FGF/EGF, and TGF-β signaling. This convergence of signaling pathways via Smad4 plays an important role during early Xenopus development, where it regulates cell competence to mesoderm induction on the dorsal side of the embryo.19We recently reported that Smad4 activity and stability are directly regulated through GSK3 phosphorylations in the linker region of Smad4 that are primed by the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (Erk).24,25 Our finding that β-TrCP binding to Smad4 is regulated by GSK3 phosphorylations raised the question of whether this increased binding between β-TrCP and Smad4 resulted from an increase in Smad4 GSK3 phosphorylations. In the present study, we report that Smad4 proteins harboring 2 point mutations commonly found in human pancreatic and colorectal cancers (Pro130Ser and Asn351His) have enhanced phosphorylation by GSK3 and are highly unstable. GSK3 inhibitors restored Smad4 stability and TGF-β signaling in smad4−/− mammary tumor cells expressing Smad4 mutant proteins. In addition, in the colon carcinoma Caco-2 cell line, which endogenously expresses the Smad4N351H mutation and is refractory to TGF-β treatment, TGF-β signaling can be restored by simply treating cells with the GSK3 inhibitor lithium chloride (LiCl). In conclusion, the present findings reveal that some mutations found in human cancers do not alter Smad4 function but rather inhibit TGF-β signaling by enhancing GSK3 phosphorylation of Smad4, which leads to its subsequent degradation. These mutant proteins have retained their ability to transduce the TGF-β signal, provided that their instability is reversed by inhibiting GSK3 activity.Interestingly, Wan et al. reported that point mutations commonly found in colorectal and pancreatic carcinomas enhance Smad4 degradation by increasing β-TrCP–mediated Smad4 polyubiquitination.19 Others had observed that point mutations commonly found in colorectal and pancreatic cancers enhance the interaction between Smad4 and β-TrCP.25 Our discovery that the binding of Smad4 to β-TrCP requires GSK3 phosphorylation suggested that Smad4 containing point mutations may have an increased susceptibility to GSK3 phosphorylation. This, in turn, would generate a phosphodegron recognized by β-TrCP, triggering polyubiquitination and degradation in the proteasome , had been reported to be rapidly degraded as a consequence of increased binding to β-TrCP.25 On the other hand, binding between Smad4 Ile383Lys (Smad4m383) and β-TrCP was not found to be significantly enhanced,25 and was used as a control. To test the role of the newly discovered GSK3 linker phosphorylation sites in the degradation of Smad4 mutants, we generated these point mutations with either intact linker GSK3 phosphorylation sites or mutated  GSK3 phosphorylation sites   .25,26 To determine the level of GSK3 phosphorylation of Smad4 mutant proteins, the western blot was also immunostained with an affinity-purified phospho-specific antibody raised against the 2 first Smad4 linker GSK3 phosphorylation sites  we found that all 3 mutants were less stable than the Smad4 wild-type (wt) protein   . We had Importantly, treatment of cells with the GSK3 inhibitor LiCl restored the stability of Smad4m130 and Smad4m351 to levels similar to those of Smad4wt . Taken t21 Cells were transfected with the TGF-β-specific reporter CAGA12-luciferase,28 and dual luciferase reporter gene assay experiments were carried out .31 Caco-2 cells were transfected with the TGF-β–specific reporter CAGA12-luciferase , incubated with or without LiCl, and treated for 8 h with different amounts of TGF-β1 ligand  increase Smad4 phosphorylation by the GSK3 kinase, thus triggering its degradation  that includes Thr277.19,20 In the absence of Wnt, Thr277 phosphorylation primes 3 GSK3 phosphorylations that generate a phosphodegron recognized by β-TrCP, inhibiting transcription and triggering Smad4 degradation .35–37 GSK3 has many other substrates in addition to β-catenin.36,38,39 The Smad4 GSK3 sites first came to our attention in the course of a bioinformatics screen for proteins containing 3 or more GSK3 phosphorylation sites in a row.19,36 Many other proteins have been found to be regulated by GSK3 sequestration in membrane organelles after Wnt treatment, in a process that has been designated as Wnt-regulated STabilization Of Proteins, or Wnt/STOP and, as Wnt signaling is maximal during the G2/M phase of the cell cycle, is considered to be essential for protein stabilization in preparation for cell division.39 Thus, it is conceivable that the biochemical changes caused by Smad4 point mutations might also apply to other proteins.Canonical Wnt stabilizes β-catenin by inhibiting its phosphorylation by GSK3.19 These mutations inactivate Smad4 by enhancing GSK3-mediated phosphorylations that generate a phosphodegron recognized by the E3 ubiquitin ligase β-TrCP, resulting in loss-of-function via protein degradation.19,25 It is surprising that mutations in the MH1 and MH2 domains  would similarly affect Smad4 phosphorylations at the distant linker region  were cultured in DMEM:Ham's-F12 (1:1 vol:vol). Cells were transfected with DNA constructs using BioT (Bioland) 24 h after plating. For all treatments with growth factors, cells were deprived of serum for 18 h and then treated with the indicated concentration of TGF-β1, FGF2 (50 ng/mL), or EGF (10 ng/mL) (R&D Systems). The Erk pathway inhibitor U0126 was used at a concentration of 40 μM. For GSK3 inhibition, cells were treated for the indicated time with the GSK3 inhibitor LiCl used at 30 mM or BIO (Sigma) used at 5 μM.HEK293 cells  were cultured in DMEM supplemented with 10% fetal bovine serum (GIBCO) at 37°C in 5% COGSK318 used at 1:25,000. Secondary antibodies used were IRDye 800CW donkey anti-rabbit IgG  and IRDye 680RD donkey anti-mouse IgG .The following antibodies were used in this study for the detection of overexpressed proteins: anti-Smad4 monoclonal , anti-GAPDH , and anti-pSmad4For western blot analyses, cells were cultured in 6-well plates, treated as indicated for 4 h, and then lysed in radioimmunoprecipitation assay buffer  supplemented with protease inhibitors (Roche #04693132001) and phosphatase inhibitors . Western blotting was performed using standard protocols. Odyssey™ Blocking Buffer (LI-COR) diluted in PBS (1:1 ratio) was used to block nitrocellulose membranes for 1 h at room temperature. All antibodies were diluted in PBS:Odyssey™ Blocking Buffer supplemented with 0.1% Tween 20. Blots were incubated with primary antibodies overnight at 4°C. Membranes were then washed extensively with Tris-buffered saline Tween 20 (TBST) and incubated with secondary antibodies for 1 h at room temperature. Images were acquired using an Odyssey 9120 infrared imaging system (LI-COR).18 PCR-based site-directed mutagenesis  was employed to generate all Smad4 mutants used in this study. Primers used were as follows: Smad4m130 (GTCTGTGTGAATTCATATCACTACG); Smad4m351 (GTTACTGTTCATGGATACGTG); Smad4m383 (GGTTGCACAAAGGCAAAGGT GTG); Smad4-GM (CATAACAGCGTACCACCTGGGCTGGAAGTAGGGCTGCACC ATAC). Mutations were confirmed by sequencing. HA-tagged human β-TrCP ΔF-box  and luciferase assays were performed with the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions using a Glomax Luminometer (Promega).For luciferase reporter gene assays, HEK293 or MDA-MB-468 cells were transiently transfected in 6-well plates at 80% confluency. The following day, cells were trypsinized and plated on 24-well plates. Cells were allowed to attach to the plastic surface, preincubated with LiCl (30 mM) for 2 h, and treated with the indicated concentrations of TGF-β1 (R&D Systems) for an additional 8 h. Growth factors were added at cell confluency of 60% or less; this is important because high confluency inhibits TGF-β signaling.P > 0.05; *, P ≤ 0.05); **, P ≤ 0.01).Results are given as the mean ± standard error of the mean (SEM). Statistical analyses were performed with Excel (Microsoft Corp.) applying the 2-tailed t test. Differences of means were considered significant at a significance level of 0.05 (n.s, not significant;


Plasmodium infection in Anopheles gambiae has been proposed to result from naturally occurring polymorphisms that alter the strength of endogenous innate defenses. Despite the fact that some of these mutations are known to introduce non-synonymous substitutions in coding sequences, these mutations have largely been used to rationalize knockdown of associated target proteins to query the effects on parasite development in the mosquito host. Here, we assay the effects of engineered mutations on an immune signaling protein target that is known to control parasite sporogonic development. By this proof-of-principle work, we have established that naturally occurring mutations can be queried for their effects on mosquito protein function and on parasite development and that this important signaling pathway can be genetically manipulated to enhance mosquito resistance.Susceptibility to A. gambiae MAPK kinase MEK to alter key residues in the N-terminal docking site (D-site), thus interfering with its ability to interact with the downstream kinase target ERK. ERK phosphorylation levels in vitro and in vivo were evaluated to confirm the effects of MEK D-site mutations. In addition, overexpression of various MEK D-site alleles was used to assess P. berghei infection in A. gambiae.We introduced SNPs into the A. gambiae. Furthermore, as expected, inhibition of MEK-ERK signaling due to D-site mutations resulted in reduction in P. berghei development relative to infection in the presence of overexpressed catalytically active MEK.The MEK D-site contains conserved lysine residues predicted to mediate protein-protein interaction with ERK. As anticipated, each of the D-site mutations  suppressed ERK phosphorylation and this inhibition was significant when both mutations were present. Tissue-targeted overexpression of alleles encoding MEK D-site polymorphisms resulted in reduced ERK phosphorylation in the midgut of A. gambiae, as in model organisms and humans, depends on the integrity of conserved key residues within the MEK D-site. Disruption of signal transmission via engineered SNPs provides a purposeful proof-of-principle model for the study of naturally occurring mutations that may be associated with mosquito resistance to parasite infection as well as an alternative genetic basis for manipulation of this important immune signaling pathway.MEK-ERK signaling in  Plasmodium, transmitted by female anopheline mosquitoes to humans during blood feeding. Prevention and treatment of the disease requires extensive efforts and coordination among the general public, health care organizations, and government agencies. Despite increased global efforts, malaria remains a top-ranked vector-borne disease and major global health concern, affecting over half of the world’s population every year  in the pDREAM 2.1 vector (Genscript) (wild type MEK or wtMEK) was used to generate five additional plasmids encoding MEK mRNA with various combinations of SNPs: pMEK1, pMEK2, pMEK3, pMEK4 and pMEK5 . The following conditions were used for plasmid replication: 15–17 cycles of denaturation at 95°C for 30 sec, primer annealing for 1 min at 55°C, followed by extension at 68°C for 1 min per 1 kb amplified. The products were treated with endonuclease DpnI (New England BioLabs) for digestion of the parental DNA template and purification of the selected mutation-encoding synthesized DNA. The nicked synthesized plasmid DNAs with the desired mutations were transformed into E. coli TOP10 chemically competent cells (Invitrogen). Eight to ten transformed colonies for every desired mutation were screened for plasmid DNA using the Qiagen Miniprep Kit and the manufacturer’s instructions (Qiagen). Among those, four to five plasmids were sequenced for confirmation of the introduced functional nucleotide changes .To introduce SNPs into the MEK-encoding plasmids were transfected into A. gambiae Sua5B cells using Effectene Reagent (Qiagen) and the manufacturer’s recommended protocol. In brief, 1×106 Sua5B cells in 2 mL medium were plated in 6-well tissue culture plates overnight at 28°C. At 24 h after plating, cells were transfected with 0.6 μg of plasmid DNA and incubated at 28°C. At 36 h post-transfection, medium was removed and cells were washed with ice-cold phosphate buffered saline (PBS) in preparation for immunoblotting.A. gambiae was described previously [A. gambiae carboxypeptidase promoter was engineered into the plasmid [The plasmid for transgene overexpression in adult female eviously . To ensu plasmid ,29. The A. gambiae were allowed to feed for 30 min on artificial blood meals at 16–24 h prior to MEK-encoding plasmid inoculation. For our studies, a mixture of 0.5 μg/μl MEK-encoding plasmid DNA, the in vivo transfection reagent jetPEITM (Polyplus-transfection Inc.) and glucose at a final concentration of 5% [For each experiment, at least twenty laboratory reared 3–5 d old female on of 5%  was injeA. gambiae Sua5B cells, cells were lysed in 200 μl cell lysis buffer . Cellular debris was removed by centrifugation at 14,000 × g for 10 min at 4°C. The resulting supernatants were mixed with Laemmli sample buffer  and the proteins were denatured at 95°C for 4 min prior to electrophoresis.To harvest proteins from Mosquito midguts were dissected into PBS and mixed to release blood, if any, by pipetting up and down. The midguts were washed in a filter column fitted with a fine mesh with a mixture of protease and phosphatase inhibitor cocktails (Sigma) in ice-cold PBS until all of the blood was removed. Fresh PBS mixture was added to loosen the midgut tissue from the filter, transferred to a fresh tube, and then centrifuged and prepared for electrophoresis as for cell culture lysate above.in vitro experiments. Levels of pERK in the midgut for each group of A. gambiae were normalized to GAPDH levels and then to pERK levels in control mosquitoes transformed with wtMEK plasmid construct for in vivo experiments.Proteins were separated on 10% SDS-PAGE polyacrylamide gels at 135 V for 1 h, 50 min. Proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories) for 1 h, 15 min at 7 V. Coomassie blue staining of the polyacrylamide gel was used to visually assess consistency of protein loading. Membranes were blocked in nonfat dry milk (5% w/v) in 1X Tris-buffered saline  containing 0.1% Tween (TBS-T) for 1 h at room temperature, and then reacted overnight in primary antibody at 4°C. The membrane was washed 3 times, 5 min each with 1X TBS-T followed by incubation with appropriate secondary antibody 4°C overnight. The membrane was washed again 3 times, 5 min each with 1X TBS-T and then incubated in SuperSignal West Dura Extended Duration Substrate (Pierce). The Kodak Image Station 4000MM Pro Imaging System  was used to capture the image of the membrane and Quantity One (Bio-Rad Laboratories) software was used for densitometry analysis of the antibody-bound proteins. Levels of phosphorylated ERK (pERK) in Sua5B cells for each treatment were normalized to total ERK levels for protein loading and then normalized to pERK levels in the control cells transfected with wtMEK plasmid construct for Primary antibodies and dilutions included anti-FLAG-M2  , anti-GAPDH  , anti-diphosphorylated ERK (pERK)   and anti-ERK1/2   . Anti-rabbit IgG-peroxidase   and anti-mouse IgG-peroxidase   were used as secondary antibodies for immunoblotting.free (Invitrogen). Quantitative RT-PCR was performed on an ABI Prism 7300 Sequence Detection System (Applied Biosystems). Primers and Taqman probes (Applied Biosystems) were designed to distinguish over-expressed alleles from endogenous A. gambiae MEK mRNA: MEK-RT forward, 5′CCGAGCAACATTCTTGTAAATAGCAGTGG3′; MEK-RT reverse, 5′AAGCGCTCGGGCGACATATAAC3′; S7 forward, 3′GAAGGCCTTCCAGAAGGTACAGA3′; S7 reverse 5′CATCGGTTTGGGCAGAATG3′; wtMEK probe, 6FAM-GATTCAATGGCCAATTCTTTTGTAGG-MGBNFQ; MEK2/5 probe, 6FAM-GATGAAATGGCCAATGATTTTGTAGG-MGBNFQ; and S7 probe, VIC-AGAAGTTCTCCGGCAAGCACGTCGT-6-carboxytetramethylrhodamine. Amplification conditions were defined as reverse transcription at 48°C for 30 min, AmpliTaq Gold activation at 95°C for 10 min, and then 40 cycles of denaturation at 95°C for 15 sec and annealing/extension at 60°C for 1 min.Total RNA was isolated from dissected individual midguts and carcasses  using TRIzol reagent (Invitrogen) and genomic DNA was removed using TURBO DNA-P. berghei for transmission to A. gambiae. When parasitemia reached 5-10% of peripheral red blood cells , mice were anesthetized and exposed to mosquitoes for feeding. Thirty 3–5 d old F0 female mosquitoes transformed for midgut-specific overexpression of wtMEK, pMEK2 or pMEK5 were aspirated into individual cartons. Non-transformed A. gambiae females in a fourth carton served as an additional control. Mosquitoes were allowed to rest for 24 h and starved 2–4 h prior to blood feeding on anesthetized P. berghei-infected mice for 30 min. All non-blood fed females were removed from the containers using a mechanical aspirator while the remainder were maintained at 19°C and 80% humidity. At 12 d post-blood feeding, mosquito midguts were dissected in PBS and stained with mercurochrome for direct counting of P. berghei oocysts.Female CD1 mice were infected with P. berghei were approved and in accord with regulatory guidelines and standards set by the Biological Safety Administrative Advisory Committee of the University of California, Davis. Experiments involving the use of animals were reviewed and deemed to be in accord with all relevant institutional policies and federal guidelines by the UC Davis Institutional Animal Care and Use Committee .Protocols involving the culture and handling of in vitro were analyzed by ANOVA (α = 0.05) for overall significance and by Bonferroni’s test for pairwise comparisons of means. Differences in exogenous MEK allele expression in vivo were analyzed by ANOVA (α = 0.05) and by Bonferroni’s test for pairwise comparisons of means. A Pearson’s r test was performed to assess the relationship between docking site mutations and ERK phosphorylation levels in midgut tissue of transformed F0 females (α = 0.05). Significant differences in control (non-transformed mosquitoes) mean oocyst counts among replicates were determined by one-way ANOVA (α = 0.05); no differences were detected, so replicates were combined for analyses. Significant differences in oocyst counts from combined replicates were detected by unpaired t-tests (α = 0.05). All calculations were performed using GraphPad Prism version 5.02 for Windows .Differences in levels of ERK phosphorylation in Sua5B cells A. gambiae MEK  or with pMEK2 (S243E/S247D) had 50-70% higher levels of ERK phosphorylation relative to cells transfected with wtMEK plasmid, which encoded the unaltered MEK allele  [A. gambiae cells in the absence of treatment [The substitution mutations in control) ,31. Howecontrol) ,31, wherA. gambiae MEK D-site should interact directly with two conserved aspartic acids in the CD domain of ERK  were collected for rearing.Consumption of blood initiate vitellogenesis in the female mosquito during which the fat body produces yolk protein precursors that are absorbed by the developing oocytes ,33. Penget al.  exploiteMEK alleles, 4 d old F0 adult female offspring of plasmid-injected A. gambiae were allowed to feed on blood for 30 min. Age-matched F0 offspring from mosquitoes from the same cohorts that were not injected with plasmid and, hence, not transformed were fed alongside transformed F0 females as controls to assess specificity of transcript detection. At 2 h post-feeding, midguts from six female A. gambiae in each group were dissected into PBS. TaqMan qRT-PCR with probes specific to exogenous variant MEK alleles revealed no detectable signals from midguts or carcasses of non-transformed F0 females; hence, relative levels of endogenous MEK mRNA in non-transformed females (NT) were used for comparison to variant MEK allele expression in transformed F0 females  than endogenous MEK mRNA levels in non-transformed mosquitoes . We reasoned that a mean reduction of approximately 23% in phosphorylated ERK levels for 4 of 5 replicates, although marginally not significant, could be biologically significant given our previous observations that incomplete inhibition of TGF-β1-induced midgut ERK phosphorylation by the MEK inhibitor PD98059 [P. falciparum growth in A. stephensi.Based on midgut-biased variant s Figure , we exams Figure A. As sho PD98059  could reP. berghei-infected mice for 30 min . Oocyst counts in mosquitoes overexpressing wtMEK were not significantly different from non-transformed (NT) mosquitoes (P = 0.11). The introduction of D-site mutations (pMEK5) reduced oocyst development (28.0 ± 5.8 oocysts) relative to mosquitoes transformed with pMEK2 (P = 0.07) to levels that were not different from mosquitoes overexpressing wtMEK (P = 0.11) and non-transformed mosquitoes (P = 0.48). These results confirm our previous observations that MEK-ERK signaling regulates parasite development and indicate that this regulation is dependent on MEK docking functionality.To determine whether overexpression of catalytically active MEK with D-site mutations would result in an infection phenotype similar to that induced by PD98059 inhibition of MEK , we allon Figure . As expeet al. [A. gambiae cells in vitro and in vivo. Further, these mutations can alter the success of malaria parasite infection in transformed F0 mosquitoes. Specifically, K3M and K6M mutations in the D-site , fibrinogen-related protein 1 (FREP1) and fibrinogen-related protein 30 (FBN30). RNAi-mediated silencing of FREP1 resulted in a significant decrease in infection prevalence while FBN30 transcript ablation increased infection intensity two-fold relative to controls [Plasmodium development, mutations such as these have thus far been used only to identify target genes for which to query the effects of RNAi-mediated silencing on infection phenotype. However, many immune gene products require protein-protein interaction to form complexes or post-translational modification to mediate specific cellular functions. As such, the work presented here indicates that functional SNP studies can be extended to determine the mechanisms by which coding sequence mutations specifically impact infection phenotype.Naturally occurring SNPs within  and ILP3 as well and TOLL6. Indeed,and TOLL6, raisingL6[et al. identificontrols . While tA. gambiae. In particular, we have demonstrated that engineered mutations in A. gambiae MEK recapitulate the effects of small molecule inhibition of MEK-ERK signaling in mosquito cells and on parasite infection [A. gambiae that are resistant to malaria parasite development and transmission.We have established proof-of-principle for the functional analysis of SNPs on protein function and infection susceptibility in nfection ,21. In aThe authors declare that they have no competing interests.MEK plasmids. AAB carried out the experiments in vitro and KC carried out the experiments in vivo. SL and LS conducted statistical analyses. SL and LS prepared the manuscript with AAB. All authors read and approved the final version of the manuscript.AAB and SL conceived and designed the experiments of the study. MC and ACB performed mutagenesis to generate the SNP-encoding


Selecting sites is an important part of a clinical trial. Selection of poor sites can lead to reduced recruitment and lower quality of data. Currently there are no tools available to assess either suitability of a potential site, or factors that could be addressed to improve suitability.In order to develop a ‘site selection tool’ to improve site selection, we surveyed Trial Managers and Chief Investigators to ask about their current practice for choosing sites in their trial. The aim was to identify ‘key performance factors’.A short online survey was distributed to all members of the UK Trial Managers Network and NIHR Chief Investigators. The questions asked about: use of any method for site selection; if and how potential sites are assessed for suitability; the definition of a successful site; and if and how site performance is assessed. Free text responses were assessed to identify themes, and grouped into ‘key performance factors’ which could be used to assess the suitability of a site for inclusion into a trial.The online survey was distributed to 597 members of the UK Trial Manager Network (UKTMN) and 159 NIHR Chief Investigators. Responses were received from 126 UKTMN members (21%) and 74 (47%) Chief Investigators. Results will be presented and discussed, including key performance factors and the themes identified.This survey presents an overview of current practice for choosing sites in UK non-commercial trials. Results will contribute to developing a ‘tool’ to improve site selection.


Multiple phosphorylation sites are identified in human TTP, but it is difficult to assign major vs. minor phosphorylation sites. This study aimed to generate additional information on TTP phosphorylation using phosphopeptide mapping and mass spectrometry (MS). Wild-type and site-directed mutant TTP proteins were expressed in transfected human cells followed by in vivo radiolabeling with [32P]-orthophosphate. Histidine-tagged TTP proteins were purified with Ni-NTA affinity beads and digested with trypsin and lysyl endopeptidase. The digested peptides were separated by C18 column with high performance liquid chromatography. Wild-type and all mutant TTP proteins were localized in the cytosol, phosphorylated extensively in vivo and capable of binding to ARE-containing RNA probes. Mutant TTP with S90 and S93 mutations resulted in the disappearance of a major phosphopeptide peak. Mutant TTP with an S197 mutation resulted in another major phosphopeptide peak being eluted earlier than the wild-type. Additional mutations at S186, S296 and T271 exhibited little effect on phosphopeptide profiles. MS analysis identified the peptide that was missing in the S90 and S93 mutant protein as LGPELSPSPTSPTATSTTPSR (corresponding to amino acid residues 83–103 of human TTP). MS also identified a major phosphopeptide associated with the first zinc-finger region. These analyses suggest that the tryptic peptide containing S90 and S93 is a major phosphopeptide in human TTP.Tristetraprolin/zinc finger protein 36 (TTP/ZFP36) binds and destabilizes some pro-inflammatory cytokine mRNAs. TTP-deficient mice develop a profound inflammatory syndrome due to excessive production of pro-inflammatory cytokines. TTP expression is induced by various factors including insulin and extracts from cinnamon and green tea. TTP is highly phosphorylated  Similar tandem C8C5C3H zinc finger sequences have been found in many species, ranging from human through yeasts and plants Tristetraprolin (TTP) is the prototypic member of a small family of tandem Cin vivoTTP protein expression is induced in various cell types by a number of factors including insulin in vivo labeling, site-directed mutagenesis, mass spectrometry and computational analysis. One major problem is that the major phosphorylation sites identified by mass spectrometry are not necessarily in agreement among different laboratories 52, S178 and S220 of mTTP  are the major phosphorylation sites in mTTP, but the human equivalent S60 is not identified as a major site in hTTP 105 and S316 of mTTP are phosphorylated in intact cells 113 or S323 of hTTP are not confirmed in transfected human cells Several approaches have been used to identify TTP phosphorylation sites including 90 and S93 in hTTP resulted in the disappearance of a major phosphopeptide peak in the HPLC chromatogram. The missing phosphopeptide identified by MS contained both S90 and S93, suggesting that the tryptic peptide is a major phosphopeptide in hTTP from transfected human cells.In this study, we extended our investigation on the identification of potential phosphorylation sites by phosphopeptide mapping, site-directed mutagenesis and mass spectrometry. Our results demonstrated that mutations at both SHEK293 cells were transfected with pHis-hTTP plasmids encoding wild-type and mutant proteins with serine and threonine to alanine mutation(s) in hTTP. Immunoblotting showed that all His-hTTP proteins were expressed in the transfected cells . ImmunosWild-type and a number of mutant His-hTTP proteins were purified by Ni-NTA beads and eluted with an imidazole solution. The purified proteins were separated by SDS-PAGE and stained with Coomassie brilliant blue (CBB) and silver reagents for observing the electrophoretic mobility of the proteins. CBB staining is less sensitive so that minor size differences between TTP protein bands could be seen on the gel. Silver staining is more sensitive, which could easily obscure the neighboring TTP bands, resulting in a smear or fat band instead of distinctive bands. CBB staining showed that the wild-type and mutant hTTP proteins with 1–4 mutations exhibited multiple distinctive bands on protein gels . Silver in vivo labeling studies showed that TTP was highly phosphorylated 32P]-orthophosphate following transfection with the wild-type plasmid pHis-hTTP. The wild-type protein was purified from the 10,000g supernatant by Ni-NTA affinity beads. SDS-PAGE followed by autoradiography showed that hTTP was essentially the only phosphoprotein purified by this procedure     clearly changed the elution pattern by increasing the retention time of the phosphopeptides  , S , or S  were sim96) . Two phosphopeptide peaks in fractions 37–43 disappeared in hTTP with these two mutations in addition to those mutations at S T271   or ST271 . The two protein .SEDLAAPGHPPVLR   .SCRR   . Mutatio8C5C3H zinc-finger region, and contained three potential phosphorylation sites at T106, T111 and S113 in hTTP. Five potential phosphorylation sites are located in the highly conserved zinc finger domains of hTTP: T106, T111 and S113 in the first zinc-finger region, and Y158 and S160 in the second zinc-finger region  exhibited little effect on the phosphopeptide mapping profiles. S186 in mTTP was shown to be one of the major sites phosphorylated by MK2 in vivo and in vitro52 and S178178 increases the relative ratio of TTP protein in the cytoplasm 52 to A52 in mTTP weakly reduces the assembly of TTP-14-3-3 protein complex, whereas mutation of S178 to A178 and of S52/178 to A52/178 substantially reduces the association of mTTP with 14-3-3 protein complex It is interesting to note that mutation at S90 and S93 sites in hTTP were minor phosphorylation sites because fewer unique phosphopeptides containing both sites were observed by MudPIT 66, S88, T92, S169, S186, S197, S218, S228, S276 and S296 were proposed as major sites in hTTP from transfected human cells for a number of reasons 66, S88, T92, S169 and S186 in hTTP observed by MudPIT were confirmed by LC/MS/MS, MALDI/MS/MS or protein sequencing; 2) S197, S218 and S228 strongly affected the electrophoretic mobility of hTTP; 3) More than four copies of phosphopeptides containing S197, S228, S276 and S296 were identified from triple digested hTTP by MudPIT; 4) [32P]-labeling studies showed that the truncated hTTP peptides containing S218 and S228 were highly phosphorylated in intact cells; 5) the MALDI MS analysis of the in-gel tryptic digest of hTTP showed ions corresponding in mass to tryptic peptide T20–21/T21–22 (aa 195–242/196–243) plus the addition of one, two, and three phosphate groups; 6) all of these sites were conserved in TTP from various mammalian species; and 7) S58, S176, S178, S220, T250 and S264 of mTTP were phosphorylated in vivo and/or in vitro66, S184, S186, S228, T257 and T271 of hTTP. More studies are needed to address the question of relevant contributions of various phosphorylation sites towards the total TTP phosphorylation status in intact cells.It is still difficult to assign the relative contributions of individual phosphorylation sites in TTP. The fact that S90A and S93A mutations in hTTP caused the disappearance of a major phosphopeptide suggests that mass spectrometry alone has limitations on assigning major vs. minor phosphorylation sites. In previous MS analysis, it was suggested that both SE. coli exhibits approximately 2-fold greater affinity for the TNF mRNA ARE than the protein purified from transfected human HEK293 cells It is thought that phosphorylation of TTP in cells can decrease its mRNA binding activity. For example, TTP expressed in human HEK293 cells and then dephosphorylated by CIAP was able to bind more tightly to an GM-CSF mRNA ARE probe than native, phosphorylated TTP The plasmids (pHis-hTTP) coded for recombinant His-hTTP proteins containing six histidine residues at the N-terminus of the full-length hTTP (GenBank accession no. NP_003398) 2 in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen) supplemented with 10% (v/v) fetal calf serum (FCS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 2 mM L-glutamine. The cells in 10-cm plates were transfected with 0.5 µg of pHis-hTTP and 4.5 µg of pBS+ carrier plasmids.HEK293 cells were transiently transfected with pHis-hTTP plasmids using the calcium phosphate precipitation method 2 for 3 h. The medium was then aspirated off. DMEM  with [32P]-orthophosphate (0.1 mCi/mL) was added to each dish 2 for 90 min.The transfected HEK293 cells were washed after being cultured overnight and incubated in fresh medium under the same conditions for 24 h. The old medium was removed from culture dish followed by washing with no-phosphate DMEM. To the dish was added no-phosphate DMEM plus 1% FCS (approximately 15 µM phosphate in the medium) and incubated at 37°C with 5% CO32P]-orthophosphate-medium was removed after in vivo radiolabeling. The cells were washed with PBS and lysed directly in the plate at 4°C for 1 h with 0.6 mL His-tag purification buffer  plus 10 mM imidazole. The lysate was transferred into microcentrifuge tubes and centrifuged at 10,000g for 10 min. Protein concentrations were determined with the Bio-Rad Dye assay kit (Bio-Rad Laboratories) and BSA as standards The UTP  and T7 RNA polymerase with the Promega’s RiboProbe In Vitro Transcription System . The binding reaction mixtures were incubated for 30 min at room temperature before digestion with 100 units of RNase T1  for 15 min at 30°C. The TTP−probe complexes and free probes were separated by 6% native PAGE and detected by autoradiography on X-ray film.TNF mRNA ARE binding activity was evaluated with the RNA gel mobility shift assay (GMSA) according to a previous procedure


Drosophila circadian clock, undergoes daily changes in phosphorylation. However, the physiological role of dCLK phosphorylation is not clear. Using a Drosophila tissue culture system, we identified multiple phosphorylation sites on dCLK. Expression of a mutated version of dCLK where all the mapped phospho-sites were switched to alanine (dCLK-15A) rescues the arrythmicity of Clkout flies, yet with an approximately 1.5 hr shorter period. The dCLK-15A protein attains substantially higher levels in flies compared to the control situation, and also appears to have enhanced transcriptional activity, consistent with the observed higher peak values and amplitudes in the mRNA rhythms of several core clock genes. Surprisingly, the clock-controlled daily activity rhythm in dCLK-15A expressing flies does not synchronize properly to daily temperature cycles, although there is no defect in aligning to light/dark cycles. Our findings suggest a novel role for clock protein phosphorylation in governing the relative strengths of entraining modalities by adjusting the dynamics of circadian gene expression.Transcriptional/translational feedback loops drive daily cycles of expression in clock genes and clock-controlled genes, which ultimately underlie many of the overt circadian rhythms manifested by organisms. Moreover, phosphorylation of clock proteins plays crucial roles in the temporal regulation of clock protein activity, stability and subcellular localization. dCLOCK (dCLK), the master transcription factor driving cyclical gene expression and the rate-limiting component in the  Drosophila, dCLOCK is a master circadian transcription factor that drives cyclical gene expression and is likely the rate-limiting component in the transcriptional/translational feedback loops that underlie the timekeeping mechanism. dCLOCK undergoes temporal changes in phosphorylation throughout a day, which is also observed for mammalian CLOCK. However, the role of CLOCK phosphorylation at the organismal level is still unclear. Using mass-spectrometry, we identified more than a dozen phosphorylation sites on dCLOCK. Blocking global phosphorylation of dCLOCK by mutating phospho-acceptor sites to alanine increases its abundance and transcriptional activity, leading to higher peak values and amplitudes in the mRNA rhythms of core clock genes, which likely explains the accelerated clock speed. Surprisingly, the clock-controlled daily activity rhythm fails to maintain synchrony with daily temperature cycles, although there is no observable defect in aligning to light/dark cycles. Our findings suggest a novel role for clock protein phosphorylation in governing the effective strengths of entraining modalities by adjusting clock amplitude.Circadian clocks are synchronized to local time by daily cycles in light-dark and temperature. Although light is generally thought to be the most dominant entraining cue in nature, daily cycles in temperature are sufficient to synchronize clocks in a large range of organisms. In  A large variety of life forms manifest circadian (≅24 hr) rhythms in behavior and physiology that are driven by endogenous cellular clocks or pacemakers Drosophila is the basic-helix-loop-helix (bHLH) PAS domain containing transcription factor termed dCLOCK  period (per) and timeless (tim), in addition to other clock genes and ccgs. Subsequently, the PER and TIM proteins interact in the cytoplasm and after a time-delay translocate to the nucleus where they function with other factors to inhibit the transcriptional activity of dCLK-CYC. Eventually, the levels of PER and TIM decline in the nucleus, facilitating another round of dCLK-CYC-mediated transcription. In a “secondary” stabilizing TTFL, the dCLK-CYC heterodimer induces the expression of PAR domain protein 1ε (pdp1ε) and vrille (vri), whose protein products  in turn activate and repress the expression of dClk, respectively, leading to daily cycles in dClk mRNA levels mammalian CLOCK; mCLK) and BMAL1 (homolog of CYC) that governs rhythmic expression of the negative regulators Per1-3, in addition to other clock genes and ccgs The rate-limiting component of the main TTFL in Although TTFLs constitute a major molecular framework for the oscillatory behavior of cellular clocks, posttranslational modifications of clock proteins are central to maintain proper timekeeping functions by regulating clock protein stability, sub-cellular localization and activity in vivoin vivodCLK also undergoes circadian changes in phosphorylation state, but in a manner different from that of PER Drosophila S2 cell culture system in combination with mass spectrometry to map phosphorylation sites on dCLK. Our results indicate that dCLK is highly phosphorylated (at least 14 phospho-sites). In S2 cells, mutated versions of dCLK where all the mapped Ser/Thr sites were switched to Ala (herein referred to as dCLK-15A) manifested increased E box dependent transcriptional activity without affecting interactions with other core clock partners such as CYC and PER. In flies, dCLK-15A protein is exclusively hypo-phosphorylated suggesting that we identified, at the very least, a major portion of the total phosphorylation sites found on dCLK in flies. Expression of dCLK-15A rescues the arrhythmicity of Clkout flies yet with an approximately 1.5 hr shorter period. Consistent with a role in regulating protein stability, the levels of dCLK15A are substantially higher compared to the control situation, which along with increases in transcriptional activity likely explains the faster pace of the clock. The daily peak levels in per/tim mRNA and protein reached higher values in dCLK-15A expressing flies, further supporting the notion that dCLK levels are normally rate-limiting in the clock mechanism. Surprisingly, the clock-controlled daily activity rhythm in dCLK-15A mutant flies fail to maintain synchrony with daily temperature cycles, although there is no defect in aligning to light/dark cycles. Together, our findings indicate that in animal systems, the post-translational modification of a master circadian transcription factor plays a critical role in setting the pace of the clock and regulating circadian entrainment.In this study, we used a simplified Drosophila S2 cells. This simplified cell culture system was successfully used to identify physiologically relevant phosphorylation sites on Drosophila PER dClk-V5 under the inducible metallothionein promoter (pMT). Total cell lysates were subjected to immunoprecipitation with anti-V5 antibody, followed by multi-protease digestion, titansphere nanocolumn phosphopeptide enrichment, and tandem mass spectrometry, as previously described As an initial attempt to better understand the role(s) of dCLK phosphorylation we sought to map phosphorylation sites using recombinant protein production in cultured In preliminary studies we individually mutated each of the identified phosphorylated Ser residues to Ala residues but did not see major effects on dCLK electrophoretic mobility, except for the S859A mutant version of dCLK, which manifested slightly faster electrophoretic mobility. . The tradCLK-16A interacts with either CYC or PER proteins to a similar extent as that observed for the wild-type version (dCLK-WT), demonstrating that dCLK-16A is not grossly misfolded . In addiDrosophila circadian timing system, we first evaluated the ability of a novel wildtype dClk transgene to rescue behavioral rhythms in the arrhythmic Clkout genetic background . Clkout is a newly described arrhythmic null mutant that does not produce dCLK protein . The transgene was constructed with a 13.9 kb genomic fragment that contains the dClk gene, which we modified by introducing a V5 epitope tag at the C-terminus of the dClk open reading frame for enhanced protein surveillance. Flies were exposed to standard entraining conditions of 12 hr light∶12 hr dark cycles  at 25°C, followed by several days in constant dark conditions (DD) to measure free-running behavioral periods. In the behavioral analysis, p{dClk-WT};Clkout flies manifested robust locomotor activity rhythms with normal ∼24 hr periods . In sharp contrast to flies harboring the control version of dClk, the two independent lines of p{dClk-15A};Clkout flies manifested generally weaker behavioral rhythms that are approximately 1.5 hr shorter compared to their wild-type counterparts . The circadian period was shortened to approximately 23 hr . Intriguingly, the levels of dCLK-15A were substantially higher compared to dCLK-WT throughout a daily cycle. Quantification of immunoblots indicated that the average daily levels of dCLK-15A are about 2.5 times more than those of dCLK-WT . In sharp contrast, during the beginning of the temperature entrainment regime although p{dClk-15A};Clkout flies also manifest two activity peaks, they occur much earlier at around the middle of the warm- and cryo-phases  circadian rhythms in a wide variety or organisms o-phases .dClk-15A};Clkout flies, the timing of the major activity peak occurring during the mid-warm phase appeared to progressively advance on subsequent days in TC  despite the fact that LL normally abolishes circadian rhythms dClk-15A};Clkout flies to maintain a more stable 24-hr phase relationship with the temperature cycle  and later  days of exposure to TC. We chose tim mRNA levels as a surrogate marker for clock dynamics because it normally has a robust high amplitude rhythm  of phosphorylation were switched to alanine, and examined circadian behavior in a Clkout genetic background. Our results indicate that global phosphorylation of dCLK is an important aspect of setting clock speed by regulating the daily levels and/or activity of dCLK. This is consistent with earlier work suggesting dCLK is the rate-limiting component in the central transcriptional/translational feedback loop (TTFL) in the Drosophila clock mechanism, and that increasing the levels of dCLK lead to shorter behavioral periods Phosphorylation of clock proteins plays diverse roles in circadian oscillatory mechanisms by regulating numerous aspects of clock protein metabolism/activity, including time-of-day dependent changes in stability, transcriptional activity and subcellular localization S2 cells . To examIn this study, we show that dCLK undergoes multi-site phosphorylation. Among the phospho-sites identified, seven serine residues are situated immediately N-terminal to a proline, indicating a major role for the CMGC group of kinases. Indeed, studies using cultured S2 cells suggested that dCLK is a potential target of several distinct CMGC kinases in vivo role of phosphorylation on dCLK degradation. If we did miss mapping some sites on hyper-phosphorylated dCLK that are critical for regulating stability it is possible that these sites can still be phosphorylated on dCLK-15A expressed in S2 cells but not in flies. For example, hyper-phosphorylation of dCLK might depend on prior phosphorylation at one or more of the 15 phospho-sites we identified, and this dependency might be more strict in flies compared to the S2 cell over-expression system. Hierarchical phosphorylation has been demonstrated for other clock proteins, such as Drosophila PER and mammalian CLK Elimination of phosphorylation sites from dCLK (dCLK-15A) leads to significant increases in the overall daily levels of dCLK in flies, which is well correlated with previous reports in S2 cells showing that hyper-phosphorylated dCLK is sensitive to degradation dper and tim mRNAs in p{dClk-15A};Clkout flies are higher compared to the control situation  bout of activity in LD is earlier compared to control flies  also showed progressively earlier evening activity bouts in thermal cycles  and long (29 hr) endogenous rhythms, respectively perS and perL flies only entrain to TC in LL dClk-15A};Clkout flies the amplitude of the PER abundance cycle is increased reaching higher peak values . All final constructs were verified by DNA sequencing.The pMT-dClk-V5 were established for dCLK purification. dClk expression was induced by adding 500 µM CuSO4 to the medium and cells were harvested 24 hr post-induction. 200 ml of culture (3×106 cells/ml) was used and harvested cells were lysed using modified-RIPA buffer  with the addition of a protease inhibitor cocktail (Roche) containing 1 mM EDTA, 25 mM NaF, and 1 mM Na2VO3. To extracts, anti-V5 antibody (Invitrogen) was added and incubated overnight with gentle rotation at 4°C followed by the addition of Dynabeads Protein A (Invitrogen) with a further overnight incubation. Beads were collected using DynalMPC. dCLK was eluted with modified Laemmli buffer  supplemented with 50 mM reducing agent TCEP  at 65°C for 20 min. Alkylation was performed by adding 0.5M IAA (iodoacetamide) for 20 min at room temperature in the dark. The eluate was resolved using 8% SDS-PAGE, and all the detectable dCLK bands of differing electrophoretic mobility excised (which under the conditions used was mainly the ‘intermediate’ phosphorylated band), subjected to protease digestion and analyzed by mass spectrometry. Mass spectrometry was performed as described in Schlosser et al. 2005. Data analysis was performed as described previously Hygromycin-resistant stable Schneider 2 (S2) cell lines expressing pMT-HA-Luciferase (luc) reporter assay was performed as described previously dClk-V5 and pMT-dClk-16A-V5 along with 10 ng of perEluc, 30 ng of pAct-β-gal-V5/His as indicated. dPER mediated repression of dCLK dependent transactivation was measured by transfecting 0–20 ng of pAct-dper together with 2 ng of pMT-dClk-V5 or pMT-dClk-16A-V5. One day after transfection, dClk expression was induced with 500 µM CuSO4 , and after another day cells were washed in phosphate buffered saline (PBS), followed by lysis in 300 µl of Reporter Lysis Buffer (Promega). Aliquots of cell extracts were assayed for β-galactosidase and luciferase activities using the Luciferase Assay System and protocols supplied by the manufacturer (Promega).S2 cells were obtained from Invitrogen and transfected using effectene following the manufacturer's protocol (Qiagen). Clkout flies were generated in one of our laboratories (P.E.H.) as follows: 5.2 kb deletion of dClk exon 1 and upstream sequences was generated by FLP-mediated recombination between FRT sites in the pBac Clk[f06808] and pBac Clk [f03095] dClk sequences. The remaining pBac insert was excised via pBac transposase induced transposition resulting in white-eyed flies harboring the deletion 5′ CGGAATATTGGACAACAAACAG 3′) and downstream of the f06808 insertion site (5′CAGCAGTGGAATCTTAATACAG 3′), and sequenced to confirm the endpoints of the deletion. This new dClk deletion allele was named Clkout.dClk-containing P[acman] transgene was generated using recombineering-mediated gap repair ATGTGGCGCGCCGCCCCAAAAATCCATAAATGCT) and clkLA-r (GTGTTGGATCCAGGGGTGTTATAGAGAGGGACA) for the left arm and clkRA-f (GTGTGGATCCGCAGAGTGAAACCTGTGCAA) and clkRA-r (ATATATGTGCGGCCGCTCCCGGTTATGAGTTTTTCG) for the right arm via PCR, and cloned as AscI-BamHI and BamHI-NotI fragments into AscI and NotI digested attB-P[acman]-ApR vector (modified to remove the SphI site) to form attB-P[acman]ClkLARA. Recombination-competent SW102 cells harboring BAC clone RP98 5K6 , which contains the dClk genomic region, were transformed with the attB-P[acman]ClkLARA vector (linearized with BamHI). Recombinants containing 15.5 kb of genomic sequence beginning ∼8 kb upstream of the dClk translation start and ending ∼2.5 kb downstream of the dClk stop codon were verified by PCR and sequencing and termed attB-P[acman]-Clk. To introduce a V5 epitope tag at the C-terminus of the dClk open reading frame (ORF), a 3′ genomic fragment of dClk (from 351 bp upstream to 1580 bp downstream of the translation stop) was cloned into pGEM-T vector . Sequences encoding V5 were introduced in-frame immediately upstream of the dClk stop codon using the Quickchange site directed mutagenesis kit  to create pGEM-T-dClk3′V5. The 3′ dClk genomic fragment in attB-P[acman]- dClk was swapped with the 3′ fragment in pGEM-T-dClk3′V5 using SphI and NotI to form attB-P[acman]- dClkV5. This transgene was inserted into the VK00018 attP site on chromosome 2 via PhiC31-mediated transgenesis To generate transgenic flies that produce wild-type dCLK tagged with V5 at the C-terminus, dClk wherein the codons for the 15 identified phospho-serine were switched to those for alanine was generated in multiple stages as follows: A genomic dClk sub-fragment from NheI to SphI site was isolated from P[acman]-dClk-V5 and subcloned into pSP72 vector where the multi-cloning sites were mutagenized to introduce a NheI site, and named this plasmid as pSP72-dClk(NheI/SphI). Next, we obtained a dClk sub-fragment spanning from the NcoI to SphI sites by restriction digestion of pSP72-dClk(NheI/SphI), subcloned the released fragment into pSP72 where the multi-cloning sites were mutagenized to introduce a NcoI site, and named this plasmid as pSP72-dClk(NcoI/SphI). We performed serial site directed mutagenesis with pSP72-dClk(NcoI/SphI) and finally made pSP72-dClk(NCoI/SphI)-S11A wherein codons for the serine residues at amino acids 209, 210, 211, 444, 450, 487, 504, 611, 645, 859, 902 on dCLK were all switched to those for alanine residues [(GenBank accession number NP_001014576)]. We purified the dClk(NCoI/SphI)-S11A insert by restriction enzyme digestion of pSP72-dClk(NCoI/SphI)-S11A and replaced the wild-type dClk(NcoI/SphI) insert, generating pSP72-dClk(NheI/SphI)-S11A. Next, a more 3′ genomic dClk sub-fragment from the SphI to NotI sites was subcloned into pSP72 vector where the multi-cloning sites were mutagenized to include NotI and NheI sites, and named this plasmid as pSP72-dClk(SphI/NotI). We performed serial site directed mutagenesis with pSP72-dClk(SphI/NotI) and made pSP72-dClk(SphI/NotI)-S4A wherein codons for the serine residues at amino acids 924, 934, 938, 1018 were switched to those for alanine. Finally, the genomic dClk(SphI/NotI)-S4A fragment was ligated with pSP72-dClk(NheI/SphI)-S11A generating pSP72-dClk(NheI/NotI)-S15A, and then dClk(NheI/NotI)-S15A fragment was switched with wild-type dClk(NheI/NotI) fragment in pacman-dClk-V5 plasmid yielding P[acman]-dClk-15A-V5. Transgenic flies were generated by BestGene Inc. . P[acman]-dClk-15A-V5 transformation vector was injected into flies carrying the VK00018 attP docking site on the second chromosome for site-specific integration dClk-15A-V5 transgene in a wild-type background were obtained and then crossed into a Clkout genetic background to yield dClk-15A-V5;Clkout.Transformation vector containing a genomic chi-square periodogram analysis and pooled to obtain a group average for each independent transgenic line or genotype. Power is a quantification of the relative strength of the rhythm during DD. Individual flies with a power ≥10 and a ‘width’ value of 2 or more (denotes number of peaks in 30-min increments above the periodogram 95% confidence line) were considered rhythmic. Actogram represents the locomotor activity data throughout the experimental period. Vertical bars in the actogram represent absolute activity levels for each 30 min intervals averaged for each given genotypes of flies. The strength of this measurement can be manipulated by using the function called hash density, which represent the number of times fly need to make beam crossing to be registered as one vertical bar. The hash density of the actogram was varied for better comparison depending on the activity levels of given genotypes of flies.The locomotor activities of individual flies were measured as previously described using the Drosophila Activity Monitoring system from Trikinetics . Young adult flies were used for the analysis and exposed to 4 days of 12 h light followed by 12 h dark [where zeitgeber time 0 (ZT0) is defined as the time when the light phase begins] at 25°C and subsequently kept in constant dark conditions (DD) for 7 days. Temperature entrainment  was performed in constant dark condition and in some cases, in the presence of constant light (>2000lux). Temperature cycles were 12 h of 24°C (cryo phase) followed by 12 h of 29°C  (where ZT0 is defined as the time when the cryo phase begins) for 4 days and subsequently kept at 24°C for 7 days. The locomotor activity data for each individual fly was analyzed using the FaasX software , which was generously provided by F. Rouyer . Periods were calculated for each individual fly using Protein extracts from S2 cells were prepared as previously described 2, and 0.1 mg/ml bovine serum albumin), divided into two equal aliquots. One aliquot of bead was treated with 200 units of λ protein phosphatase (NEB) and no addition was made to the other aliquot. Both aliquots were incubated for 30 min at 30°C with occasional shaking, and immune complexes analyzed by immunoblotting.For immunoprecipitation, cell extracts from S2 cells were prepared and 3 µl of anti-HA (12CA5) or anti-V5 antibody was added depending on the target protein sought, and incubated for overnight at 4°C with gentle rotation. The next day, 20 µl of Gammabind-sephase bead  was added with a further incubation of 3 hr at 4°C. The immune complexes were eluted with 1X SDS-PAGE sample buffer. For λ- phosphatase treatment, the purified immune complexes were resuspended in λ protein phosphatase buffer . 1 µg of total RNA was reverse transcribed with oligo-dT primer using Prime Script reverse transcriptase (TAKARA) and real-time PCR was performed in Corbett Rotor Gene 6000 (Corbett Life Science) using Quantitect SYBR Green PCR kit (Qiagen). Primer sequences used here are as follows; Figure S1dClk-V5. Mutated sites are indicated on the top. Expression of dCLK was induced 24 hr after transfection by adding 500 µM CuSO4 to the medium. Cells were harvested 24 hr after induction, and protein extracts were subjected to western blot analysis. dCLK was visualized with anti-V5 Ab. Please note that the decrease in the levels of dCLK S859A (A) was not reproducible as shown in (B). (C) Shown are the average values for relative E box dependent luciferase activity in the presence of 2 ng of wild-type (WT) or serine to alanine mutated version of pMT-HA-dClk-V5. dCLK-S875A was included as randomly chosen serine to alanine mutant.Analysis of CLK phosphorylation site mutants for electrophoretic mobility and transcriptional activity in S2 cells  S2 cells were transiently transfected with 500 ng of wild-type (WT) or serine to alanine mutated version of pMT-HA-(TIF)Click here for additional data file.Figure S2dClk (WT) or pMT-HA-dClk-15A (15A) singly (A) or in combination with 600 ng of pMT-dbt-V5 (B). Expression of dCLK and DBT was induced 24 hr after transfection by adding 500 µM CuSO4 to the medium. 24 hrs post induction, 10 µg/ml of cycloheximide (CHX) was treated to inhibit translation. Cells were harvested at the indicated time points and protein extracts were subjected to immunoblotting. dCLK was visualized with anti-HA (3F10) antibody. Shown are the representative blots for each analysis and relative levels of dCLK proteins were determined by measuring band intensities of immunoblot using image J software.Stability of dCLK-WT and dCLK-16A protein in S2 cells.  S2 cells were transiently transfected with 300 ng of pMT-HA-(TIF)Click here for additional data file.Figure S3dClk (WT) or pMT-HA-dClk-15A (15A). Expression of dCLK was induced 24 hr after transfection by adding 500 µM CuSO4 to the medium. Cells were harvested 24 hr after induction and protein extracts were first subjected to immunoprecipitation using anti-HA (12CA5) antibody and immune complexes were incubated in the absence (−) or presence (+) of λ phosphatase followed by immunoblotting. (B) Shown are the average values from three independent experiments for relative E box dependent luciferase activity in the absence (BL) or presence of pMT-dClk-V5 (WT) or pMT-dClk-15A (15A).dCLK-15A manifests similar alterations as dCLK-16A compared to dCLK-WT in terms of stability and transcriptional activity in S2 cells. (A) S2 cells were transiently transfected with 500 ng of pMT-HA-(TIF)Click here for additional data file.Figure S4dClk-WT};Clkout and p{dClk-15A};Clkout flies in light/dark cycles. Adult flies of the indicated genotype  were entrained with 12 hr∶12 hr light∶dark cycles for 4 days followed by 8 days in DD. Black and white bar on top of each actogram indicates when lights were off and on, respectively. Red arrowhead indicates when DD starts. The vertical black bars on each row of the actogram depict the activity of the fly . To better visualize rhythmic behavior, each day's worth of activity recordings was double plotted. HD, hash density of the actogram.(A–D) Representative daily locomotor activity patterns of p{(TIF)Click here for additional data file.Figure S5dClk-WT};Clkout and p{dClk-15A};Clkout flies in temperature cycles in the absence of light. Adult male files for a given genotype  were entrained in 12 hr∶12 hr temperature cycles of 24°C∶29°C for 9 days and maintained at 24°C for 7 days in the absence light. The vertical black bars on each line of the actogram depict fly activity . Each day's worth of activity recordings was double plotted to better visualize rhythmic behavior. Red horizontal bars and blue horizontal bars below each panel indicated thermo- or cryo-phases, respectively. The results clearly indicate that the offset in evening activity occurs progressively earlier in p{dClk-15A};Clkout flies even during TC. HD, hash density of the actogram.(A–D) Representative daily locomotor activities of p{(TIF)Click here for additional data file.Figure S6dClk-WT};Clkout and p{dClk-15A};Clkout flies in temperature cycles after exposure to constant light. Adult male files for a given genotype  were exposed to constant light for 6 days and then entrained in 12 hr∶12 hr temperature cycles of 24°C∶29°C for 7 days in the absence light. The red arrowhead indicates when the lights were turned off. The vertical black bars on each line of the actogram depict fly activity . Each day's worth of activity recordings was double plotted to better visualize rhythmic behavior. Flies became arrhythmic shortly after exposure to constant light. The results clearly indicate that the offset in evening activity occurs progressively earlier in p{dClk-15A};Clkout flies during TC. HD, hash density of the actogram.(A–C) Representative daily locomotor activities of p{(TIF)Click here for additional data file.Figure S7dClk-WT};Clkout and p{dClk-WT};+/+ flies. Adult flies of a given genotype (indicated at the top of panels) were collected at the indicated time in LD (ZT) and protein extracts analyzed by immunoblotting using the anti-dCLK antibody (gp208). Note that the levels of dCLK are higher in p{dClk-WT};+/+ flies.dCLK protein levels and phosphorylation in p{(TIF)Click here for additional data file.


It significantly decreased CYP3A4 activity in a dose- but not time-dependent manner. Results from Lineweaver–Burk plots showed that GA could inhibit CYP3A4 activity competitively, with a Ki value of 1.57 μM in HLMs. Moreover, CYP2C9 and CYP2C19 could also be inhibited significantly by GA with IC50 of 42.89 and 40.26 μM in HLMs, respectively. Other CYP450 isoforms were not markedly affected by GA. The inhibition was also confirmed by an in vivo study of mice. In addition, it was observed that mRNA expressions of the Cyps2c and 3a family decreased significantly in the livers of mice treated with GA. In conclusion, this study indicates that GA may exert herb-drug interactions by competitively inhibiting CYP3A4.Glycyrrhetinic acid (GA) has been used clinically in the treatment of patients with chronic hepatitis. This study evaluated the effect of GA on the activity of five P450(CYP450) cytochrome enzymes: CYP2A6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4, in human liver microsomes (HLMs) and recombinant cDNA-expressed enzyme systems using a HPLC-MS/MS CYP-specific probe substrate assay. With midazolam as the probe substrate, GA greatly decreased CYP3A4 activity with IC Gancao (licorice root) is a crude drug of Glycyrrhiza uralensis Fisch (licorice), G. glabra L. var. glandulifera Waldst. and Kit, is believed to harmonize formulations to carry the formula to the 12 regular meridians [Natural compounds are used in the treatment of refractory diseases more and more frequently because of their efficacy and low toxicity. The traditional Chinese medicine eridians ,4 antitueridians ,6, anti-eridians ,8, and meridians ,10. It ieridians ,12, antieridians , antialleridians , and anteridians ,16.in vitro studies in rat or human liver microsomes have demonstrated that CYP3A and CYP2C9 enzymes participate in the 22α- and 24-hydroxylation of GA, respectively [in vivo studies validating the herbal-drug interaction.Recently, several ectively ,18. Moreectively ,18. InduIn the present study, we evaluated the mechanism of inhibition mode of GA through cytochrome P450 isoforms (CYP450) and which confirmed an inhibitory effect in mice. The study provides evidence that a herbal-drug interaction may occur, depending on the drug or class of drugs administered, resulting from CYP450 inhibition.2, NADP+, glucose 6-phosphate, glucose 6-phosphate dehydrogenase, and potassium phosphate (monobic and dibaic) were chromatographic-grade chemicals purchased from Sigma Chemicals . Acetonitrile and methanol were of analytical grade and purchased from Sigma Chemicals . PrimeScript® RT reagent Kit With gDNA Eraser and SYBR® Premix DimerEraser™ were purchased from Takara Biotechnology . Glycyrrhetinic acid (GA) was purchased from National Institute for the Control of Pharmaceutical and Biological Products .The pooled human liver microsomes (HLMs) and human recombinant enzyme used in the incubation studies were purchased from BD Gentest Co. . Coumarin, 7-hydroxycoumarin, tolbutamide, 4-hydroxytolbutamide, (S)-mephenytoin, 4′-hydroxymephenytoin, metoprolol, α-hydroxymetoprolol, midazolam maleate, 1-hydroxymidazolam, propranolol, quinidine hydrochloride, tranylcypromine, fluconazole, ticlopidine, quinidine, ketoconazole, furafylline, and diethyldithiocarbamate were purchased from Sigma Chemicals . Formic acid, ethylic acid, ammonium formate, MgCln = 5 for each dose) for 15 days. After the end of study, mice were killed rapidly, decapitated to collect livers frozen in liquid nitrogen, and stored in −80 °C for further assays. The study was approved by the ethical committee of the Zhengzhou University (No. SYXK-2012-0001) and complied with national laws relating to the conduct of animal experiments.Eight-week old male C57BL/6 mice were obtained and maintained at the Animal Experimental Center of Zhengzhou University according to the Institutional Animal Care Guidelines in a light-dark (12 h–12 h), temperature (23 °C ± 2 °C) and humidity (50% ± 8%) controlled laboratory environment. Sterilizing water and standard diet were provided for the animals, and standard iron cages were used for six mice per cage. GA  was administered to mice  detection system. The mRNA gene expression levels were normalized to β-actin gene expression.Total RNA was extracted from frozen livers using TRIzol reagent. The purity and concentration of the RNA were calculated by measuring its absorbance at 260 and 280 nm. A PrimeScript® BCA Protein Assay Kit .Microsomes were prepared from each mouse liver according to the method of Zhao and colleagues . ProteinA previous validated method was followed for incubation of liver microsome, NADPH regeneration, sample preparation, and HPLC-MS/MS analysis .Compound assays were performed in triplicate using individual drug-probe substrates specific for each HLM-CYP or recombinant enzyme with HPLC-MS/MS detection. The concentration range of the inhibitors was set from 0.01 μM to 100 μM (five points).To investigate the inhibitory mode of GA, HLMs were pre-incubated with GA in potassium phosphate buffer (pH 7.4) for 0, 10, or 30 min in the presence of NADPH. Midazolam was then added and incubated for 30 min at 37 °C. Midazolam was used as a probe substrate at 5, 10, or 20 μM.To confirm the selective inhibition of CYP3A4 enzyme by GA, 10 pmol of human recombinant cDNA-expressed CYP3A4 was incubated with GA at 0.01 to 100 μM, with the NADPH system and midazolam as a selective CYP3A4 substrate for 30 min at 37 °C.p < 0.05 was considered statistically significant. The 50% inhibitory concentration (IC50) values were calculated by nonlinear regression using GraphPad Prism 5.0 . Lineweaver–Burk plots were obtained from SigmaPlot software .A value of 50 value of 8.195 μM, and a moderate inhibition of CYP2C9 and CYP2C19 activity was observed with IC50 values of 42.89 and 40.26 μM, respectively. However, GA caused no significant inhibition of CYP2A6, CYP2D6, or CYP2E1 activity (IC50 > 100 μM). The inhibitory effects of GA on five typical reactions catalysed by five human CYP enzymes were examined in HLMs. GA showed potent inhibition of CYP3A4 activity in HLM with an ICTo investigate the mode by which GA inhibits CYP3A4-catalyzed midazolam 1′-hydroxylation, CYP3A4 inhibitory activity was determined with or without pre-incubating microsomal mixtures for 0, 10, or 30 min at 37 °C in HLMs. GA strongly and dose-dependently inhibited CYP3A4-catalyzed midazolam 1′-hydroxylation in HLMs, but not time-dependently . To inve50 value of 7.498 μM , while the lower dose of GA only showed a slight decreasing tendency (p > 0.05). Moreover, GA showed no statistically significant effect on the expression of Cyp2a4 or Cyp2d22 (p > 0.05).The mRNA expression levels of genes encoding Cyps are shown in This study is the first to investigate the enzyme kinetics of the inhibitory effects of GA on CYP450 enzymes activities in HLMs, as well as in mice. GA significantly inhibits CYP3A4, CYP2C9, and CYP2C19 enzyme activity in HLMs and in mice. Moreover, GA significantly decreased the mRNA expression of Cyp 2c and 3a family members in mice.CYP3A4 is responsible for the metabolism of a variety of drugs and endogenous compounds in humans. CYP3A4 constitutes up to 60% of liver CYP isoforms. Over 50% of marketed drugs are metabolized by CYP3A4 ,22. At pGancao, has been widely used in the treatment of inflammatory diseases, especially liver disease. Complications may occur when patients are treated with drugs concomitantly with GA. Serious or even fatal adverse drug reactions (ADRs) may occur owing to alteration of CYP activity by drugs concomitantly with GA [GA, one of the bioactive compounds from  with GA . Herbal-et al. [in vitro evidence was provided. A study of Liu et al. [in vivo evidence of its inhibition of CYPs. In this study, GA showed potent competitive inhibition of CYP3A4 activity. No significant inhibition of CYP2A6, CYP2D6, or CYP2E1 activity was noted.There have been several studies about the metabolism of GA ,18, indiu et al.  evaluateGA could induce the activation of PAI 1 expression and NaDPH oxidase through direct binding to mineralocorticoid receptors . The CYPin vitro and in vivo evidence of potential drug interactions involving GA. Further clinical trials are needed to determine whether GA exerts adverse effects when co-administered with other drugs. Whether it exerts a favourable influence on other medical treatments also deserves further investigation.The results provide in vivo study in which the mRNA expression of Cyp 2c and 3a family members in mice was decreased significantly by GA. The result indicated that GA might cause herbal–drug interactions when co-administrated with substrates of CYP3A4, CYP2C9, and CYP2C19.This study demonstrated an inhibitory effect of GA on CYP3A4-catalyzed midazolam 1′-hydroxylation in that acts HLMs in a competitive manner. Moreover, GA may also inhibit CYP2C9 and CYP2C19 activity. This inhibition was also validated by an


Growth modulation with tension band plates (TBPs) and cannulated screws is the current mainstay of treatment for pediatric lower extremity angular deformities. Solid screws have been used as an alternative to cannulated screws to decrease the risk of screw failure, particularly in obese children. The downside of solid screws is the decrease in precision of placement. This study describes a surgical technique to insert solid TBP screws accurately.TBP insertion starts with the same conventional steps by inserting the guidewires into the epiphysis and metaphysis, straddling the physis. After fluoroscopic confirmation of the position of the guidewires, the cannulated drill bit is used to broach the cortex to a depth of 5 mm in the bone. A standard 4.5-mm cannulated screw from the TBP set is used to tap the screw tract over the guidewires for approximately three-quarters of the planned screw length. After removing the guidewires, the solid screws are then inserted in each hole to follow the tapped tracts.This technique was used in five patients including four with Blount disease and one with bilateral genu varum.It is recommended to use solid screws with TBPs in patients with a high body mass index to avoid screw fracture. Our technique describes using a cannulated screw as a tap to create a tract to ease accurate insertion of the solid screws, and prevent the solid screw from deviating outside the desired path. Lower extremity angular deformities such as genu varum or valgum are common in children. Treatment options include osteotomy  or growtTBPs use cannulated screws which provide ease and accuracy of screw insertion over a guidewire , 5, 8. AWe consider using solid screws for obese patients, as is commonly seen in adolescent Blount disease. The downside of solid crews is that they cannot be placed with the same high degree of precision as cannulated screws. The senior author developed a technique to insert solid screws into TBPs, while still maintaining the accuracy of the cannulated system.The surgical technique for inserting a TBP is similar to the standard technique described by Stevens . The imaThe epiphyseal and metaphyseal guidewires are then inserted through the eccentric holes by using the supplied drill guide, under fluoroscopic control for accurate placement straddling the growth plate. Two pilot holes are then drilled using a 3.2-mm cannulated step drill bit to a depth of 5 mm. The drill bit should not be drilled deeper than 5 mm, as this removes bone, weakens screw purchase and deceases pull-out strength.A 4.5-mm cannulated screw is then used as a tapping tool over the guidewire to tap at least three-quarters of the desired screw length. The guidewire is then removed to insert a 4.5-solid screw that follows the path created by the tapping Figs. , 2. ContSolid screws have been used with TBPs since 2008 in five patients, including four with Blount disease and one with bilateral genu varum. The five patients  had a mean age at surgery of 12.1 years (range 7.2−14.6 years). Figure The rationale behind temporary growth modulation for lower limb angular deformity is to allow for gradual correction while the physis is still growing and avoid the need for invasive osteotomy. Excellent results have been reported with the use of TBPs; however, fractures of the cannulated screws have been reported, mainly in obese patients with Blount disease , 9–11.Burghardt et al. proposed two modifications in order to avoid hardware failure. The first is to apply two TBPs side by side and the second is to use solid screws rather than cannulated screws . Solid sThe disadvantage of solid screws is that they are harder to accurately insert than cannulated screws. Here, we describe our technique to insert a solid screw into a TBP with ease and accuracy similar to cannulated screws. The surgical technique is consistent with that described by the manufacturer who recommends pre-drilling only 5 mm into the cortex, i.e., not all the way through the bone. The rationale for this is to increase the screw purchase within the bone, especially the metaphyseal cancellous bone. For this reason we do not drill the entire screw length when a solid screw is used. Instead, we use a cannulated screw to tap the screw trajectory over the guidewire. The ‘screw’ tap pushes the bone aside and impacts it, making it more dense. In comparison, drilling with a cannulated 3.2-mm drill bit over the entire length would remove bone and result in decreased screw purchase. The initial pass with a cannulated screw creates a clean channel for the solid screw to follow and helps avoid deviation of the solid screw. The solid screw must be inserted carefully without applying extra force or pushing on the screwdriver to avoid deviating from the pre-tapped tract close to the physis. A tied suture around the screw head can be used to prevent screw displacement in the soft tissue especially in obese patients as previously described in submuscular plating techniques .The fate of the extra cannulated screw which is used as a tap may depend on where you practice. In the USA, it is common hospital practice to discard any screw that is inserted into the body, even if it is immediately removed. This is due to concern that the screw may be weakened by insertion, e.g., if it is rubbing against the side of a plate during insertion. This practice is common in the USA during open reduction internal fixation of long bone fractures; a screw that is inserted and found to be too long or too short, is commonly discarded and replaced. However, in other parts of the world, this practice is less common, and a screw that is inserted briefly and removed can be recirculated into the tray to be used for another case. A third option is to select one long cannulated screw to be used as the ‘tap screw’ and keep it separate from the implant screws along with the instrument part of the tray, to be used over and over again, in a similar way as a tap is repeatedly used. Finally creating a special instrument (tap) supplied by the company is a good concept that could be considered by the instrument manufacturers.There is some controversy about the optimum position for screws in guided growth. At least one study argues for initial parallel placement . HoweverIn this study we describe a surgical technique to use a cannulated screw as a tap to clarify the desired screw trajectory prior to solid screw insertion. We believe the use of a tapping screw is better than drilling the full trajectory with a 3.2-mm drill bit, to increase the screw purchase with the cancellous bone.