Mitosis requires extensive rearrangement of cellular architecture and of subcellular structures so that replicated chromosomes can bind correctly to spindle microtubules and segregate towards opposite poles. This process originates two new daughter nuclei with equal genetic content and relies on highly-dynamic and tightly regulated phosphorylation of numerous cell cycle proteins. A burst in protein phosphorylation orchestrated by several conserved kinases occurs as cells go into and progress through mitosis. The opposing dephosphorylation events are catalyzed by a small set of protein phosphatases, whose importance for the accuracy of mitosis is becoming increasingly appreciated. This review will focus on the established and emerging roles of mitotic phosphatases, describe their structural and biochemical properties, and discuss recent advances in understanding the regulation of phosphatase activity and function. Protein phosphorylation is the most profuse post-translational modification in eukaryotes. It plays a critical role in controlling protein function, activity, and molecular interactions. The phosphorylation state of a given protein is established by the balance of activities between protein kinases and protein phosphatases. Kinases catalyze the transfer of a phosphate group to tyrosine (Tyr), threonine (Thr), or serine (Ser) residues, while protein phosphatases are responsible for the counteracting hydrolytic removal of the phosphate moiety. Phosphoproteomic analyses revealed that protein phosphorylation occurs primarily at Ser and Thr residues , with Tyr phosphorylation accounting for only up to 2% of the total phosphoproteome ,5. BasedSerine/threonine phosphatases are distributed through three broad families in accordance to their primary structure and catalytic mechanism: the phosphoprotein phosphatase (PPP), the protein phosphatase metal-dependent (PPM), and the haloacid dehydrogenases (HAD) families . The mosA PP1 holoenzyme contains one of four isoforms of the PP1 catalytic subunit—PP1α, PP1β, PP1γ1, or PP1γ2  and one or two of the numerous regulatory subunits ,19,20,21(0–1)[V/I]x[F/W] and is present in nearly 70% of identified PIPs [More than 200 regulatory subunits of PP1 have been described. These are generically designated as PP1-interacting proteins (PIPs) and its diversity provides the cell with a wide array of different PP1 holoenzymes to meet specificity and regulatory requirements ,28. PIPsied PIPs ,34. Struied PIPs . Additioied PIPs ,28. The ied PIPs ,37,38. DCrystallography studies show that binding to PIPs does not impose a discernible conformational change on the PP1 catalytic domain. Instead, most PIPs limit PP1 activity towards specific substrates through a combination of functional domains involved in PP1-anchoring, PP1-inhibition, substrate-recruitment, and/or subcellular-targeting ,27,28. TThe PP2A catalytic subunit has two isoforms (PP2ACα and PP2ACβ) that share 97% of their sequence identity with each other ,49. LikeThe PP2A scaffold subunit contains 15 tandem helical HEAT  repeats assembled into an elongated, horseshoe-shaped structure  Figure A. PP2A with no discernible differences in their substrate binding pockets . These sAlthough recurrently underrepresented in global phosphoproteomes, tyrosine phosphorylation plays a critical role in numerous signal transduction pathways that underlie a broad spectrum of fundamental physiological processes. Protein tyrosine phosphatases (PTPs) are defined by the active-site signature motif HCX5R, in which the cysteine residue acts as a nucleophile essential for catalysis ,71. ProtThe human genome encodes three CDC25 phosphatases: CDC25A, CDC25B, and CDC25C ,88,89. TThe unidirectional transitions between the phases of the cell cycle is governed by reversible phosphorylation and proteolysis. The highest occupancy of phosphorylation sites is observed in mitosis. Over 32,000 protein phosphorylation/dephosphorylation events occur as cells go into, progress through, and exit from mitosis ,108,109.Activation of CDK1-Cyclin B has long been established as the trigger that drives the transition into mitosis. The activity of CDK1 throughout the cell cycle is regulated by the quantity and subcellular localization of Cyclin B and by the phosphorylation status of CDK1 itself. Expression of Cyclin B increases during the G2 phase of the cell cycle, thus allowing formation of the CDK1-Cyclin B complex before mitotic entry . The actIn addition to CDK1-Cyclin B, multiple other mechanisms contribute to regulate CDC25 phosphatases B. These Mitotic kinases have also been shown to operate as active regulators of CDC25 phosphatases B. CentroData from Xenopus oocytes revealed that prompt CDC25C activation further requires the activity of PP1 phosphatase B. DuringAlong with increased CDK1-Cyclin B activity, the inhibition of PP2A-B55 phosphatase is essential for mitotic compliance A. B55-tyMitotic entry is accompanied by the condensation of DNA into chromosomes, each comprised of two sister-chromatids held together from DNA replication until anaphase onset by proteinaceous ring-like cohesin complexes ,180 FigA. These Centromeres are protected against WAP1-dependent cohesin removal by the activity of SGO1-associated PP2A-B56 ,214,215.Drosophila and Caenorhabditis elegans embryos, as well as in human cultured cells. The mechanism by which PP4 promotes the maturation of centrosomes remains unknown, but the phosphatase appears to be necessary for proper centrosomal localization of PLK1 [Accurate segregation of chromosomes relies on the formation of a bipolar spindle structure . This re of PLK1  and acti of PLK1 . Convers of PLK1 .C. elegans, PP4 directly dephosphorylates the Katanin catalytic subunit homologue MEI-1 to promote microtubule-severing activity in meiosis [Regulation of microtubule dynamics at the centrosomes and spindle poles relies in part on the activity of the microtubule-severing Katanin complex ,235, who meiosis ,238.Xenopus egg extracts [C. elegans embryos, the regulatory subunit, PR72/RSA-1, targets PP2A to centrosomes through a direct interaction with the PCM-associated RSA-2 protein. Centrosomal PP2A-PR72/RSA-1 promotes microtubule outgrowth and spindle stability by controlling KLP-7 and the spindle assembly factor, TPX2/TPXL-1. The PP2A-PR72/RSA-1 complex restricts the levels of KLP7 through an unknown mechanism and directly mediates the accumulation of TPX2/TPXL-1 at centrosomes, thus increasing the rate of microtubules’ nucleation as well as the length of centrosomal microtubules [Plus-end growth of microtubules emanating from centrosomes relies on the activity of the microtubule-polymerase ch-TOG/XMAP215/MSPS , whose mextracts . The C-textracts . The numextracts ,241. In otubules . No obviotubules ,244, wheotubules ,246,247.otubules ,249. Thiotubules ,251,252.otubules ,254,255.otubules . In addiotubules ,258,259.otubules ,261. Theotubules ,262. Altotubules . Followiotubules . These potubules ,265. Thiotubules ,274,275.Setting two independent microtubule-organizing centers at opposite poles of a mitotic cell requires the separation of duplicated centrosomes, which typically occurs before nuclear envelope breakdown in organisms that undergo an open mitosis . CentrosC. elegans, PP6 was also shown to positively regulate pulling forces during anaphase by promoting the cortical localization of LGN and NuMA homologues [Phosphatases not only control the assembly of the mitotic spindle, but also its orientation. The position that the spindle assumes in mitosis defines the axis of cell division and plays a key role in cell fate determination in tissues. Depletion of PP4 from progenitor cells of the mouse neocortex causes premature differentiation into neurons and severe defects in cortical layering and brain cytoarchitecture . Biochemmologues ,321. HowTo ensure faithful genome partitioning, sister kinetochores must become attached to microtubules emanating from opposite spindle poles. This attachment configuration is termed amphitelic or bioriented and enables the dividing cell to segregate sister chromatids towards opposite sides during anaphase and in this way distribute one copy of each chromosome per daughter nucleus. Amphitelic attachments assume an end-on arrangement with the plus-end of microtubules embedded within the kinetochore. This is believed to provide sufficient stability to sustain the force released by the depolymerisation of kinetochore-associated microtubules during anaphase . HoweverA central player in the formation of stable end-on attachments is the NDC80 complex at the outer-kinetochore, which directly binds microtubules through the CH domain and the N-terminal tail of NDC80/HEC1 ,333,334.Because BUBR1 associates with KNL1 in the absence of microtubules, the binding of PP2A-B56γ to BUBR1 places the phosphatase in the close proximity of NDC80 to efficiently counteract Aurora B destabilizing activity and enable initial end-on microtubule interactions ,375,381 C. elegans, timely association of the SKA complex with bioriented kinetochores is facilitated by dephosphorylation of the NDC80 N-terminal tail [The PP1γ-KNL1 complex further suppresses kinetochore Aurora B activity, hence enhancing the stability of amphitelic attachments ,386. Disnal tail . Whethernal tail . Althougnal tail . The accnal tail . Kinetocnal tail ,395,404.nal tail ,406 and nal tail . Pools onal tail ,408. Impnal tail . As chronal tail .Several other PIPs have been shown to act as important kinetochore-targeting factors of PP1, but their precise impact on microtubule attachment has been difficult to discern. For instance, PP1 bound to MYPT1 was shown to antagonize the phosphorylated T-loop of PLK1, which consequently renders the kinase inactive ,410. ConTo ensure that each daughter nucleus receives one copy of the genome, chromosome segregation can only occur after all sister-kinetochore have established stable amphitelic attachments. Timely control of anaphase onset is achieved through the spindle assembly checkpoint (SAC), a conserved signaling pathway primarily instated by unattached/tensionless kinetochores that inhibits the ubiquitin-dependent proteolysis of Securin and Cyclin B. Degradation of Securin triggers the cleavage of centromeric cohesion by the protease Separase to enable sister chromatid separation, whereas reduced levels of Cyclin B lead to inactivation of CDK1, which consequently reverts the cell into an interphase state. According to the prevailing mechanistic model of SAC signaling, unattached/tensionless kinetochores catalyze the assembly of a diffusible protein tetramer known as mitotic checkpoint complex (MCC) that binds to the E3 ubiquitin ligase anaphase promoting complex/cyclosome (APC/C) and thereby prevents it from targeting Securin and Cyclin B for degradation ,431,432.Monopolar spindle 1 associates with unattached kinetochores and phosphorylates KNL1 on its MELT repeats, creating docking sites for the hierarchical recruitment of additional SAC proteins necessary for MCC assembly ,445,446 Drosophila cells was shown to dephosphorylate the T-loop of MPS1 to ensure the inactivation of residual MPS1 molecules remaining at kinetochores and thereby efficiently avert the phosphorylation of SAC substrates during metaphase [Once all kinetochores attach to microtubules and become stably bioriented, the SAC must be silenced to permit APC/C activation and initiate anaphase. This primarily entails mechanisms that prevent and revert the phosphorylations catalyzed by MPS1. The region of the NDC80 CH domain that interacts with MPS1 partially overlaps with the microtubule binding interface , which setaphase  (Figure etaphase , but theetaphase ,465.Suppressing MPS1 activity at bioriented kinetochores is not sufficient to promptly silence the SAC, as the phosphorylations carried out to instate the checkpoint still need to be reversed. This task has been almost consensually attributed to PP1 ,479,480.C. elegans [Interestingly, PP2A-B56γ bound to BUBR1 might also directly contribute to dephosphorylate phospho-MELT motifs  and was  elegans .Once the SAC is switched off and APC/C-CDC20-dependent degradation of Securin and Cyclin B occurs, the cell becomes irreversibly committed to exit mitosis. Resumption of an interphase state requires remodeling of the spindle architecture, reassembly of the functional nuclear envelope, and decondensation of chromatin . This exDrosophila cells suggest that PP2A-B55 might also target Lamin and NUP107 to direct their assembly into the nuclear envelope during mitotic exit [Reformation of the nuclear envelope and associated nuclear pore complexes around chromatin occurs at the end of mitosis to establish functional interphase nuclei ,507,508.tic exit . Furthertic exit  (Figure C. elegans was recently shown to require the contribution of PP1c/GSP-2 bound to ELYS/MEL-28 [Several findings indicate that PP1 is equally important for reassembly of functional nuclear envelopes. Binding to AKAP149 localizes PP1 on the assembling nuclear envelope, where it dephosphorylates Lamin B to promote its polymerization and accelerates nuclear lamina assembly ,525,526 S/MEL-28 . HoweverAs the nuclear envelope reassembles, the segregated chromatin decondenses to re-establish an interphase structure compatible with DNA replication and transcription . This reThe accuracy of mitosis requires tight spatiotemporal control of phosphatase activities. Although PP1 does contribute to mitotic exit progression, the bulk of CDK1-mediated phosphorylations are removed by PP2A when in complex with B55 . Thus, PImportantly, active PP2A-B55 seems to follow a timely defined dephosphorylation program so that events in anaphase, telophase, and cytokinesis are carefully coordinated and regulated to ensure a successful cell division ,164. It The mechanism controlling PP1-mediated inactivation of GWL remains, however, somewhat contentious. It has been proposed that PP1 activity is globally repressed in mitosis by a CDK1 inhibitory phosphorylation on its C-terminus ,558,559 The activity of PP2A-B56 at kinetochores is modulated to avert deleterious dephosphorylation during early mitosis, when correction of erroneous attachments is often required. This is accomplished by BOD1, a small protein that shares sequence similarity with ENSA and Arpp19, and that specifically binds to PP2A-B56 when phosphorylated by CDK1 . BOD1-meIn the past decades, we have witnessed the emergence of protein phosphatases as critical regulators of mitosis. In recent years, the list of identified substrates and regulators has increased considerably, as has our understanding of phosphatases’ roles in mitotic progression.Structural and biochemical determinants of phosphatase holoenzyme assembly and specificity have been unveiled. Research has made important advances in the functional characterization of protein phosphatases and has generated critical molecular knowledge to better understand their spatiotemporal control. Importantly, these achievements have provided new and renovated perspectives on the regulation of mitotic events, whose accurate execution is absolutely critical for genomic stability.Nonetheless, enduring unanswered questions deserve further investigation. It remains relevant to dissect the regulatory network underlying the activity of CDC25 isoforms. The field will also benefit from a comprehensive identification of the precise substrates that are targeted by specific Ser/Thr phosphatase holoenzymes and of the upstream signaling inputs controlling the subcellular localization and activity of the different holoenzymes. These have been hampered by the inability to routinely tackle specific interactions of phosphatases with substrates or regulators. Further structural insights into SLiM-mediated interactions might reveal critical mechanisms to enable the disruption of specific phosphatase complexes in a way that is compatible with systematic studies. Furthermore, the development of specific inhibitors targeting phosphatase-SLiM contacts should also fuel their potential use as therapeutic strategies in disease. This might represent a valuable alternative, or complement, to protein kinase inhibitors that are often associated with on-target resistance.


Obesity is associated with higher postmenopausal breast cancer incidence. Visfatin level alteration is one of the mechanisms by which obesity promotes cancer. Ligand-independent activation of estrogen receptor alpha (ERα) is also associated with carcinogenesis. The activity of ERα is modulated through phosphorylation on multiple sites by a number of protein kinases. Here we investigated the effect of visfatin as a novel adipocytokine on the phosphorylation and activity of ERα in MCF-7 breast cancer cells. We showed that exogenous administration of visfatin significantly increased the phosphorylation of ERα at serine 118 (Ser118) and 167 (Ser167) residues. Visfatin-induced Ser118 phosphorylation was diminished after treatment of cells with U0126 (MEK1/2 inhibitor). Furthermore, our results showed that visfatin-induced Ser167 phosphorylation is mediated through both MAPK and PI3K/Akt signaling pathways. Inhibition of the enzymatic activity of visfatin by FK866 had no effect on phosphorylation of ERα. We also showed that visfatin enhanced the estrogen response element (ERE)-dependent activity of ER in the presence of 17-β estradiol (E2). Additional study on T47D cells showed that visfatin also increased Ser118 and Ser167 phosphorylation of ERα and enhanced ERE-dependent activity in the presence of E2 in these cells. Breast cancer (BC) is the most prevalent malignancy in women worldwide with nearly 234,190 new cases in the United States in 2015 . EstrogSeveral studies showed that obesity is associated with increased risk of postmenopausal BC. Current factors contributing to the development of BC in obese women include increased levels of sex steroid hormones such as estrogen, insulin resistance, increased levels of insulin-like growth factors (IGFs), oxidative stress, adipocytokines such as adiponectin, leptin and visfatin , penicillin/streptomycin and trypsin were purchased from Gibco . Recombinant visfatin was obtained from Peprotech . Epidermal growth factor (EGF), 17-β estradiol (E2), FK866, LY294002, and U0126 were purchased from Sigma-Aldrich . Phenol red-free RPMI 1640, Opti-MEM I reduced serum medium and lipofectamine 2000 transfection reagent were purchased from Invitrogen Corporation . Horseradish peroxidase-conjugated goat anti-rabbit IgG and polyclonal antibodies against human ERα, pERα (Ser118) and pERα (Ser167) were purchased from Santa Cruz Biotechnology . Cignal ERE reporter assay kit and dual-luciferase reporter assay kit were purchased from Qiagen  and Promega , respectively.2 atmosphere. For ERα protein expression and phosphorylation studies, cells were seeded at the density of 3.5 × 105 cells per well in 6-well culture plates with 2 ml phenol red-free RPMI 1640 containing 5 % dextran-coated charcoal-stripped FBS (cFBS) in order to eliminate the false positive effects of phenol red  FBS and 1 % penicillin/streptomycin at 37 °C under a 5 % COFor analysis of ERα protein expression and phosphorylation, MCF-7 cells were cultured and treated as described previously. After washing with ice-cold phosphate-buffered saline (PBS), cells were lysed with RIPA buffer  containing protease and phosphatase inhibitors. The cell lysate was then incubated on ice for 30 min followed by centrifugation at 13,000 g for 15 min at 4 °C. The supernatant was transferred into a clean microtube and stored at -70 °C until used. Protein concentration was determined by Bicinchoninic acid assay.Each sample was mixed with SDS-PAGE loading buffer and heated at 100 °C for 5 min. Then equal amounts of protein (50 μg) were loaded onto 8 % (v/v) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred to polyvinylidene difluoride membrane after separation. Nonspecific binding of the membrane was blocked with 5 % BSA in TBST  under agitation for 2 h at room temperature (RT). Then the membrane was probed with primary antibody against the protein of interest diluted in blocking buffer overnight at 4 °C. After washing with TBST, the membrane was incubated with the secondary antibody conjugated with horseradish peroxidase (1:5000 diluted in blocking buffer) for 1 h at RT. Immunoreactive bands were visualized with the ECL detection kit  according to the manufacturer's instructions using a Chemiluminescence Imaging System . Protein levels were quantitated by densitometry using ImageJ software and the expression level of each protein was normalized to corresponding β-actin expression level.4 cells per well in 96-well culture plates with 150 μL phenol red-free RPMI 1640 containing 5 % cFBS. After 24 h, cells were transfected with ERE reporter construct (according to the manufacturer's guidelines) using 1 μL lipofectamine 2000 in 100 µl Opti-MEM I reduced serum medium. After 6 h of transfection, cells were shifted into the fresh phenol red-free medium containing 0.5 % cFBS. After 24 h of transfection, cells were treated with 0, 12.5, 25, 50 and 100 ng/ml of visfatin with or without 1 nM E2 to test for their effects on ERE-dependent transcriptional activation by measuring luciferase activity. After 12 h of treatment, cells were washed with 150 μl PBS at RT and then lysed with 20 μl 1X passive lysis buffer. The luciferase activity was measured using dual-luciferase reporter assay kit according to the manufacturer's instructions. The light produced by luciferase enzyme was determined by LUMO Microplate Luminometer as relative light unit (RLU) and corrected with Renilla internal control. Three independent experiments were performed, and the ERE-dependent transcriptional activity was quantified as fold induction compared to that obtained from control vehicle-treated cells.Cignal ERE reporter assay kit (Qiagen) was used to investigate the effect of visfatin on ERE-dependent transcriptional activity of ER. MCF-7 cells were seeded at the density of 1×10The T47D cell line was grown and seeded in the same conditions described for MCF-7 cell line. To evaluate the effect of visfatin on ERα phosphorylation, T47D cells were treated with 0 and 50 ng/ml of visfatin for 12 h. The cell lysates were obtained using RIPA buffer and protein levels of ERα and its phosphorylated forms (Ser118 and Ser167) were determined by western blot analysis. To investigate the effect of visfatin on ERE-dependent transcriptional activity, T47D cells were transfected with ERE reporter construct as described for MCF-7 cell line. After 24 h of transfection, cells were treated with 0 and 50 ng/ml of visfatin with or without 1 nM E2 for 12 h. Finally, the luciferase activity was measured and corrected with Renilla internal control.(p) <0.05 was considered as a statistically significant difference.All statistical analyses were conducted by GraphPad Prism 6.0 . Data are the mean ± SE acquired for at least three independent experiments. One-way ANOVA and Dunnett's multiple comparison test were used to compare means in different groups, and a probability value In this study, the effect of different concentrations of visfatin  on ERα protein expression and phosphorylation (Ser118 and Ser167) was investigated in MCF-7 cell line. As shown in Figure 1AERα can be phosphorylated at Ser118 in response to estradiol binding or activation of protein kinase signaling pathways such as MAPK, RET, and GSK3, which suggests that this phosphorylation plays an important role in ERα function. As described in the previous section, visfatin phosphorylated the ERα at Ser118 in MCF-7 cell line. Visfatin-induced phosphorylation was investigated in the presence of 50 ng/ml of visfatin together with FK866 (NAMPT inhibitor), LY294002 (PI3K/AKT inhibitor) or U0126 (MEK1/2 inhibitor) separately. As illustrated in Figure 2On the other hand, treatment with U0126 completely abrogated the phosphorylation of ERα at Ser118. These results indicated that MAPK mediates visfatin-induced phosphorylation of Ser118 residue of ERα.Ser167 is a major site of phosphorylation in response to activation of the MAPK pathway . It canIn order to examine whether visfatin is able to modulate ERE-dependent transcriptional activity, MCF-7 cells were transiently transfected with the ERE-luciferase reporter construct as previously explained. Figure 4As shown in Figure 5An association between obesity and BC has been revealed by different studies, although the mechanism underlying this relationship remains to be fully understood. Altered adipocytokine secretion in obese women plays an important role in BC progression by influencing cell proliferation, invasive growth, apoptosis, and angiogenesis. It has also been reported that ligand-independent activation of ERα can promote BC in obese women  especiaIt has been reported that estradiol and growth factors such as EGF and IGF-1 stimulate the phosphorylation of Ser118 residue of ERα . MAPK, Ser167 is another important site of ERα phosphorylation that influences ER activity and has been investigated extensively. It has been revealed that Ser167 is phosphorylated by AKT, p90RSK, mTOR/p70S6K, ERK1/2 MAPK and casein kinase II . Our stVisfatin is the secretory form of NAMPT, the main enzyme responsible for NAD production. Therefore, it is suggested that some of the functions of visfatin may be mediated by NAD production. However, FK866 which is the inhibitor of the enzymatic activity of NAMPT had no effect on visfatin-induced ERα phosphorylation. This finding shows that enzymatic activity of visfatin is not involved in ERα phosphorylation and it is a direct effect of visfatin on signaling pathways induced by tyrosine kinase receptors. So far, no specific receptor has been identified for visfatin. Classic mechanism of ER function occurs after interaction with E2, through receptor dimerization and binding to its specific response element known as estrogen response elements (EREs) . ER phoMitra Nourbakhsh and Shahnaz Khaghani  contributed equally as corresponding authors.This work was financially supported by Tehran University of Medical Sciences [grant number 93-02-30-25163].The authors declare that they have no conflict of interest.


The essential splicing factor Cwc24 contains a zinc-finger (ZF) domain required for its function in splicing. Cwc24 binds over the 5′ splice site after the spliceosome is activated, and its binding prior to Prp2-mediated spliceosome remodeling is important for proper interactions of U5 and U6 with the 5′ splice site sequence and selection of the 5′ splice site. Here, we show that Cwc24 transiently interacts with the 5′ splice site in formation of the functional RNA catalytic core during spliceosome remodeling, and the ZF-motif is required for specific interaction of Cwc24 with the 5′ splice site. Deletion of the ZF domain or mutation of the conserved ZF residues greatly weakened the association of Cwc24 with the spliceosome, and lowered the affinity and specificity of its interaction with the 5′ splice site, resulting in atypical interactions of U5, U6 and Prp8 with the 5′ splice site, and aberrant cleavage at the 5′ splice site. Our results reveal a crucial role of the Cwc24 ZF-motif for defining 5′ splice site selection in the first splicing step. The spliceosome catalyzes the removal of intervening sequences from precursor mRNA in a two-step process. It consists of five small nuclear RNAs (snRNAs), U1, U2, U4, U5 and U6, and a range of protein factors . The snRNineTeen Complex) is then added to the spliceosome, which is required to stabilize the interactions of U5 and U6 with the pre-mRNA during formation of the active spliceosome , YSCC038 (MATa prc1 prb1 pep4 leu2 trp1 GAL1-CWC24::URA3) and EGY48 (MATa trp1 ura3 his3 LEU::pLexop6-LEU2).The following yeast strains were used: BJ2168  was obtained from GE Healthcare Inc. Proteinase K was purchased from MD Bio Inc., and SuperScript III was purchased from Invitrogen.Anti-Ntc20, anti-Prp8, anti-Prp2, anti-Spp2 and anti-Cwc24 are polyclonal antibodies produced by immunizing rabbits with corresponding recombinant proteins expressed in et al.  glycerol) and distributed onto a 10-cm culture dish. The dish was placed on ice under a UV254 nm lamp at a distance of ∼10 cm in a UV Stratalinker 1800 (Stratagene Inc.), and irradiated at an energy level of 0.125 J/cm2. After deproteinization, RNA was precipitated and resuspended in 78 μl of H2O and then mixed with 10 μl of E. coli tRNA (10 mg/ml), 10 μl of Buffer S , 1 μl of RNasin (40 U/μl) and 1 μl of 5′-biotinylated oligonucleotide U5-Cbio or U6-Abio at 300 μM. The mixture was boiled for 2 min, slow cooled to room temperature and precipitated with a 50 μl bed volume of streptavidin-Sepharose by incubation at 4°C for 1 h. The precipitate was heated for 2 min to release the selected RNAs, which were fractionated on a 5% polyacrylamide (29:1), 8 M urea gel . Individual crosslinked products were excised from gels and eluted. Following precipitation, the amount of RNA was estimated by counting in a Scintillation counter . For primer extension, 0.055–0.15 fmole of crosslinked RNA, 2 × 105 cpm or 50 fmole of 5′-32P-labeled oligonucleotide Pre-III and 10 nmole each of four dNTPs was mixed in a 13 μl annealing mixture, heated to 65°C for 5 min, and incubated on ice for at least 1 min, and then mixed with a 7 μl prewarmed reaction mixture to give a final concentration of 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 5 mM dithiothreitol (DTT), 40 U of RNasin and 200 U of SuperScript III reverse transcriptase. The mixture was then incubated at 50°C for 1 h. Extension products were analyzed by electrophoresis on 10% polyacrylamide (19:1)-8 M urea gels (Thermo Scientific Owl S3S Aluminum-Backed Sequencer).UV-crosslinking of U5 and U6 to pre-mRNA was performed according to Chan et al. ,21. Each254 nm at 0.8 J/cm2 , and then collected into a microcentrifuge tube. The sample was then added to a denaturation mix to give a final concentration of 1% SDS, 1% Triton X-100 and 0.1 M dithiothreitol (DTT). The mixture was boiled for 2 min and allowed to cool to room temperature in a water bath. The mixture was diluted 10-fold with a buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 1 mM EDTA, 0.05% NP-40 and 0.2 mg/ml tRNA, and subjected to immunoprecipitation. The precipitates were washed with the same buffer without tRNA and then digested with proteinase K, followed by phenol–chloroform extraction and ethanol precipitation. For primer extension analysis, RNA was isolated from 0.4 to 1 ml of UV-irradiated splicing reaction mixtures. Each RNA sample was mixed with 2 × 105 cpm or 50 fmole of 5′-32P-labeled oligo Pre-II or Pre-V, denatured by heating at 65°C for 5 min, and then quickly chilled on ice. Primer extension reactions were carried out with SuperScript III reverse transcriptase at 50°C for 1 h. Reaction products were analyzed on 10% polyacrylamide (19:1)-8 M urea gels (Thermo Scientific Owl S3S Aluminum-Backed Sequencer).For RNA–protein crosslinking, each 25 μl of the splicing reaction mixture with 2 nM pre-mRNA was spread onto a 10-cm culture dish placed on ice, irradiated with UVE. coli lysates that expressed recombinant His-tagged SUMO-Brr2C for 1 h at 4°C. Escherichia coli lysates from 0.1 OD600 cells and 10 pmole of recombinant Cwc24 proteins were used for each reaction. Beads were washed four times with 1 ml of wash buffer . Washed beads were resuspended in 1 × SDS-PAGE loading buffer and boiled for 5 min prior to fractionation on SDS-PAGE.Recombinant V5-tagged Cwc24 mutant proteins bound to 10 μl of protein A-Sepharose beads conjugated with 3.5 μl anti-V5 antibody were incubated with To map Brr2 domains interacting with Cwc24, we fused Brr2 deletion mutants to the LexA-DNA binding domain in plasmid pEG202, and Cwc24 was fused to the Gal4-activation domain in plasmid pACT2. To map Cwc24 domains interacting with Brr2C, we fused Brr2C to the LexA-DNA binding domain in plasmid pEG202, and Cwc24 deletion mutants were fused to the Gal4-activation domain in plasmid pACT2. Each pair of plasmids was transformed into yeast strain EGY48 together with the reporter plasmid pSH18-34, and expression of β-galactosidase was assessed to determine protein–protein interactions.We previously reported that deletion of any region of Cwc24 that includes the ZF domain impairs splicing . Here, wCwc24 was previously shown to interact with the Ski2-like RNA helicase Brr2 ,39. The 254 nm. Following denaturation, Cwc24-pre-mRNA crosslinking was analyzed by immunoprecipitation with anti-V5 antibody  were found to associate with the spliceosome when levels were increased to micromolar concentrations Figure , but theWe have previously shown that depletion of Cwc24 by a genetic or antibody approach does not completely abrogate splicing activities in extracts, but does result in aberrant cleavage at the 5′ splice site . Since ZACT1 pre-mRNA  or mutated (C144A), the association of Cwc24 with the spliceosome is greatly destabilized with only very low levels of spliceosomes being co-precipitated with Cwc24 even when the mutant proteins are present at concentrations more than 100-fold greater than for wild-type protein. Our crosslinking analyses revealed that the Cwc24 ZF mutants were capable of interacting with the pre-mRNA. At a concentration of 35 nM, the C144A mutant could crosslink to the pre-mRNA with an efficiency only slightly less than that of wild-type protein, albeit with lower specificity. In agreement with this result, electrophoretic mobility shift assays (EMSA) revealed that Cwc24 binds RNA nonspecifically, and the C144A mutation did not significantly alter its RNA binding capability (data not shown). Whereas wild-type Cwc24 crosslinked primarily to G1 and U2 of the 5′ splice site, crosslinking of the C144A mutant protein was relatively weak at these positions and was extended further upstream to the -2 position on the 5′ exon, indicating that an intact ZF structure is important for specific interaction of Cwc24 with the 5′ splice site. Cryo-EM structures of both human and yeast Bice site ,47. Neveice site ,49. Cwc2analysis . It is pOur Cwc24 ZF mutants showed a dominant-negative property in splicing reactions when provided at concentrations of 350 nM or more. These increasing protein concentrations resulted in enhanced association of the mutant proteins with the spliceosome. Therefore, although the mutant proteins do not tightly bind to the spliceosome, they interfere with spliceosomal binding of wild-type Cwc24 protein. Furthermore, they fail to promote the catalytic reaction upon binding to the spliceosome, with the residual splicing activities also resulting in aberrant cleavage at the 5′ splice site. These results suggest that an intact ZF is important for both stable association of Cwc24 with the spliceosome, and for its subsequent function in orchestrating the catalytic center of the spliceosome for branch formation.Our two-hybrid and pull-down assays show that Cwc24 interacts with the C-terminal domain of Brr2. Brr2C has been shown to interact with several spliceosomal components, particularly for proteins required for the catalytic and disassembly steps (including DExD/H-box proteins Prp2 and Prp16), and may serve as a platform for recruiting splicing factors to the spliceosome ,40,50. Wact spliceosome  1-127 of Cwc24, as in the C1 mutant, completely abolished its ability to interact with Brr2C, whereas the N-terminal segment of aa 1-137 is sufficient for the interaction. Although showing no growth defect, the C1 mutant is partially impaired for its function in the splicing reaction, requiring a >10-fold protein concentration to acquire comparable splicing activity as wild-type . The afficeosome , indicat51 residue of U6 to positions −1 and +2 of the 5′ splice site was also only seen in the absence of Cwc24 (Previously, we showed that U5 and U6 interaction with the 5′ splice site on activated spliceosomes was aberrant in the absence of Cwc24 . U5 was of Cwc24 . These ract complex reveals that the ZF-domain is positioned between the 5′ splice junction and the α-finger of Prp8, thus preventing Prp8 from contacting the 5′ splice site (A model for how Cwc24 may mediate formation of the RNA catalytic core through Prp2-mediated spliceosome remodeling is presented in Figure ice site  (Supplemice site . Bindingice site . MoreoveDrosophila Cwc24 ortholog mdlc has been shown to be required for the maintenance of neuronal differentiation. Knockdown of mdlc gene results in reduced levels and aberrant splicing of the pros transcript, indicating that Mdlc not only has a function in promoting splicing efficiency, but is also involved in splice-specific regulation (Saccharomyces cerevisiae, it may be more loosely required in other organisms where splicing regulation is ubiquitous. The level of the protein may impact splicing of various introns to different extents in fly and human, resulting in differential splicing patterns.The gulation . In lighgkz733_Supplemental_FileClick here for additional data file.


To the EditorThe only therapy that is able to modulate the cause of IgE‐mediated allergy and to attain a long‐term effect is allergen‐specific immunotherapy (AIT). In conventional subcutaneous AIT, the vaccine consists of an extract from an allergen source that contains major and minor allergens as well as non‐allergenic proteins. To reduce IgE‐mediated side‐effects caused by the injection of intact allergens, chemically modified extracts with less IgE‐binding activity, named allergoids, have been used for AIT since the 1980s. Most allergoids are obtained by treating allergen extracts with form‐ or glutaraldehyde, which react with free amino groups and form covalent inter‐ and intramolecular bonds, resulting in the formation of high‐molecular‐weight aggregates. Allergoids were intended to increase the efficacy of AIT and to reduce the frequency of injections, as the reduced IgE‐reactivity makes it possible to apply higher vaccine doses. Clinical efficacy of allergoids has been shown and reviewed in the pastIn spite of their reduced IgE‐binding and allergenicity, allergoids have been shown to be immunogenic and to induce allergen‐specific IgG, especially IgG1 and IgG4.®, Allergopharma, Reinbeck, Germany; for characteristics see+ basophils, even though the vaccinations were interrupted after the 3rd maintenance dose before the grass pollen season according to the treatment schedule.Sera and whole blood samples of ten GP‐allergic patients  undergoing subcutaneous AIT with a vaccine containing a formaldehyde‐modified GP‐extract .The FAB inhibition with sera from patients who had undergone AIT with unmodified allergens reached a mean of 40% after 4 months and increased significantly to 86% after 13 months of AIT Figure B. MoreovThus, preseasonal AIT with formaldehyde‐modified GP‐allergens induced IgG1‐ and IgG4‐antibodies specific for GP‐extract as well as for defined major grass allergens in our patient group, indicating—in line with previous studiesMost of the data reported on blocking antibodies induced by AIT derive from studies using vaccines with unmodified allergens. We employed two different biological assays to evaluate the IgE‐blocking activity in sera of allergoid‐treated patients: one involved the inhibition of complex formation between IgE and allergen (FAB) and the other the capacity to inhibit IgE cross‐linking (BAT) Figure D,E. Bothet alNeither the individual concentration of allergen‐specific IgG antibodies nor the decrease in serum levels during the interruption of vaccination correlated with the individual blocking capacity. For instance, in spite of the general reduction in GP‐specific IgG, IgG1 and IgG4 after 12 months, the IgE‐blocking activity had further increased, suggesting that not total IgG levels are relevant, but rather other properties, such as epitope specificity or affinity of the IgG antibodies. In line, it has been shown for AIT with unmodified allergens that the IgE‐blocking activity of post‐AIT sera does not correlate with the levels allergen‐specific IgG4 but rather with IgE‐blocking capacity.In contrast to the treatment with allergoids, the individual data of the BAT‐inhibition assay were more coherent in patients treated with unmodified allergens. We conclude that in a vaccine containing native allergens, most IgE‐epitopes are present and accessible, so that a greater variety of antibodies can be induced which may lead to a more uniform total IgG response and high total blocking capacity. Inversely, the lower blocking capacity observed in sera of patients with allergoid‐treatment is most probably due to the modification of the tertiary structure of the allergens. Studying immune responses to AIT with unmodified birch pollen, we previously found that the majority of IgG4 antibodies recognize similar epitopes on the major birch pollen allergen (Bet v 1) as IgE antibodies of the same patient.In summary, this study demonstrates for the first time that antibodies induced by AIT with allergoids possess functional IgE‐blocking capacity in vitro. As the ex vivo data on basophil activation induced by GP‐extract revealed a marked reduction during vaccination in these patients Figure B and theFH has retrieved fees for organizing education and for attending symposia from ALK, Hørsholm, Denmark and Allergopharma, Reinbeck, Germany. SM has received reimbursement for attending a scientific symposium from Allergopharma. The remaining authors have no conflict of interest to declare. Click here for additional data file. Click here for additional data file. Click here for additional data file.


A similar inhibitory action of CSE was also observed in human platelets. CSE inhibited arachidonic acid–induced TXA2production in murine platelets with an IC50value of 7.32 ± 2.00%. Accordingly, the inhibitory effect of CSE on collagen-induced aggregation was significantly blunted in platelets lacking the TXA2receptor compared with the inhibitory effect in control platelets. In contrast, the antiplatelet effects of CSE in platelets lacking each inhibitory prostanoid receptor, prostaglandin (PG) I2receptor and PGE2receptor subtypes EP2and EP4, were not significantly different from the effects in respective control platelets. Among the enzymes responsible for TXA2production in platelets, the activity of cyclooxygenase (COX)-1 was inhibited by CSE with an IC50value of 1.07 ± 0.15% in an uncompetitive manner. In contrast, the activity of TX synthase was enhanced by CSE. The results indicate that CSE inhibits COX-1 activity and thereby decreases TXA2production in platelets, leading to inhibition of platelet aggregation.The results of studies that were performed to determine whether cigarette smoking affects platelet function have been controversial, and the effects of nicotine- and tar-free cigarette smoke extract (CSE) on platelet function remain to be determined. The aim of this study was to determine the effect of CSE on platelet aggregation and to clarify the mechanism by which CSE affects platelet function. CSE inhibited murine platelet aggregation induced by 9,11-dideoxy-9α,11α-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U-46619), a thromboxane (TX) A Cigarette smoking has been recognized as a risk factor for various diseases including cancers, cardiovascular diseases, and respiratory diseases.Nicotine is the representative bioactive agent contained in cigarette smoke, and the effect of nicotine on platelets has been investigated in some studies. Nicotine potentiated thrombin- or adenosine diphosphate (ADP)-induced platelet aggregation.2production and intracellular concentration of cyclic adenosine monophosphate (cAMP) in platelets. Finally, we examined whether CSE affects activities of cyclooxygenase (COX) and TX synthase, enzymes responsible for TXA2production.In this study, we attempted to determine the effect of nicotine- and tar-free cigarette smoke extract (CSE) on platelets and to clarify the mechanism by which CSE affects platelet function. We first examined the effect of CSE on platelet aggregation. Then we investigated whether CSE can change thromboxane (TX) AU-46619 and arachidonic acid were purchased from Cayman Chemical . Collagen and ADP were purchased from Nycomed Pharma  and Sigma-Aldrich , respectively. Cigarettes were purchased from Japan Tobacco Inc. , and the cigarette brand used in this study was Hi-Lite, in which one cigarette contains 1.4 mg nicotine and 17 mg tar. Cambridge glass fiber filters were purchased from Heinrich Borgwaldt GmbH . 3-Isobutyl-1-methylxanthine (IBMX) was purchased from Sigma-Aldrich.2receptor subtype EP2(EP2–/–mice) or EP4(EP4–/–mice), PGI2receptor IP (IP–/–mice), or TXA2receptor TP (TP–/–mice) have been reported.4–/–mice, have a genetic background of C57BL/6 mice. EP4–/–mice have a mixed genetic background of C57BL/6 and 129/Ola mice.4–/–mice, F2-WT mice having a similar genetic background were used as controls. All the experiments, which were approved by the Asahikawa Medical University Committee on Animal Research, were performed on 11- to 23-week-old male mice.Generation and maintenance of mice lacking the prostaglandin (PG) ENicotine- and tar-free CSE was prepared according to a previously reported method2, and 5 mM KCl, pH 7.4). Diluted blood was then centrifuged at 90 gfor 5 minutes, and PRP was obtained by collecting the upper phase. Platelet-poor plasma (PPP) was prepared by further centrifuging the remaining lower phase at 1,500 gfor 10 minutes. In platelet aggregation studies, the number of platelets in PRP was adjusted to 3 × 105platelets/μL with PPP, and the final concentration of trisodium citrate was adjusted to 0.38%. To prepare washed platelets, a one-tenth volume of 77 mM EDTA (pH 7.4) was added to PRP and the mixture was centrifuged at 900 gfor 15 minutes. The platelet pellet was washed once with a washing buffer  and centrifuged at 900 gfor 15 minutes. Finally, the platelet pellet was suspended in modified Tyrode's buffer  and the number of platelets was adjusted to 106platelets/μL. For human platelet preparation, informed consent was obtained from healthy volunteers who had not taken any medications and cigarettes for at least the past 14 days. Blood was drawn by venepuncture and was then centrifuged at 140 gfor 10 minutes. PRP was obtained by collecting the upper phase and PPP was prepared by further centrifuging the remaining lower phase at 1,500 gfor 15 minutes. The number of platelets in PRP was adjusted to 3 × 105platelets/μL with PPP, and the final concentration of trisodium citrate was adjusted to 0.38%. The experiments using human platelets were approved by the Asahikawa Medical University Research Ethics Committee.Platelet-rich plasma (PRP) and washed platelets of mice were prepared as reported previously–/–mice, the concentration of collagen was increased (2.5–3.5 μg/mL) to induce a degree of aggregation similar to that in PRP prepared from WT mice. For human platelets, U-46619 and collagen were added at concentrations to induce aggregation of 45 to 50% (0.5–0.8 μM) and 45 to 55% (0.5–1.0 μg/mL), respectively.Platelet aggregation was examined by a previously reported method using an aggregometer 2. After further incubation for 5 minutes at 37°C, the reaction was terminated by the addition of ice-cold 1 N HCl (25 μL), and the platelet suspension was centrifuged at 20,400 gfor 10 minutes at 4°C. The supernatant was neutralized with a one-fifth volume of 1 M Tris (pH 10.4), and the content of TXB2was measured by using a TXB2enzyme immunoassay Kit .Washed platelets (100 μL) were preincubated in the absence of calcium and fibrinogen for 5 minutes at 37°C, and then CSE was added 5 minutes before the addition of 1 μM arachidonic acid or 10 nM PGHgfor 10 minutes at 4°C. The content of cAMP in the supernatant was determined by using a radioimmunoassay kit  after trichloroacetic acid had been extracted three times with water-saturated diethyl ether.Washed platelets (100 μL) were preincubated in the presence of 1 mM IBMX, an inhibitor of phosphodiesterase, for 10 minutes at 37°C, and then CSE was added. After further incubation for 10 minutes at 37°C, the reaction was terminated by the addition of 30% trichloroacetic acid (25 μL), and the platelets were disrupted by sonication. The solution was centrifuged at 20,400 m) and the maximum velocity (Vmax) of COX-1 within the Michaelis–Menten kinetics.COX-1 and COX-2 activities were measured using a COX Fluorescent Inhibitor Screening Assay Kit . Briefly, CSE was added to a solution containing ovine COX-1 or human COX-2, and the mixture was incubated for 5 minutes at room temperature. To initiate the reaction, arachidonic acid was added to the mixture, and then the sample was further incubated at room temperature. After 2-minute incubation, the fluorescence was measured with an excitation wavelength of 535 nm and an emission wavelength of 590 nm using a microplate reader . To determine the manner of the inhibitory action of CSE on COX-1, we examined the effect of CSE (3%) on the Michaelis constant . At a concentration of 3%, CSE alone did not induce platelet aggregation or platelet shape change , left. ± 0.14% . CSE al± 0.19% . On the± 0.19% . CSE alctively ,F.2is known as a potent stimulator of platelets, and it is produced when platelets are activated. Therefore, it works as a central regulator of platelet activation in a positive feedback manner. To determine whether CSE affects TXA2production in platelets, we examined the effect of CSE on arachidonic acid-induced TXA2production. For assessment of the degree of TXA2production, we measured the content of TXB2, a stable TXA2metabolite. To exclude TXA2production associated with platelet aggregation, we performed the experiment in a buffer without calcium and fibrinogen, which enable aggregation. Under this condition, CSE inhibited TXA2production in a concentration-dependent manner with an IC50value of 7.32 ± 2.00% . Since TP-mediated signaling plays a role in collagen-induced platelet aggregation, the concentration of collagen was increased to induce a degree of aggregation in TP–/–platelets similar to that in WT platelets. In TP–/–platelets, the inhibitory effect of CSE on collagen-induced aggregation was attenuated significantly compared with that in WT platelets; the IC50values were 3.51 ± 0.13% and 0.92 ± 0.15%, respectively , EP4(EP4–/–platelets), or IP (IP–/–platelets). U-46619 induced similar degrees of aggregation in these platelets as well as WT platelets and platelets prepared from F2-WT mice (F2-WT platelets). In both EP2–/–and IP–/–platelets, the inhibitory effects of CSE on U-46619-induced aggregation were not significantly different from the inhibitory effect in WT platelets. The IC50values were 1.35 ± 0.14%, 1.46 ± 0.16%, and 1.14 ± 0.10%, respectively (4–/–platelets, the inhibitory effect of CSE was also not significantly different from that in F2-WT platelets. The IC50values were 1.31 ± 0.15% and 1.42 ± 0.18%, respectively (It is well known that PGIctively . In EPctively . These 2, and EP4, we examined whether CSE affects the intraplatelet concentration of cAMP, a second messenger of the inhibitory prostanoid receptors. CSE had no significant effect on the cAMP concentration in washed platelets prepared from WT mice (To confirm that CSE does not affect the signaling of the inhibitory prostanoid receptors, IP, EPWT mice , indica2synthesis was suppressed by CSE (50values of 1.07 ± 0.15% and 0.80 ± 0.12% (2synthesis in platelets. It has been shown that COX-2 was expressed in only a few platelets prepared from healthy donors.mof COX-1 from 3.37 ± 0.40 to 1.21 ± 0.41 μM and also decreased Vmaxfrom 139.1 ± 6.8 to 44.6 ± 4.6 arbitrary fluorescence units (Since TXA by CSE , we nex± 0.12% . To dete units , indica2to TXA2. We finally examined whether CSE affects the activity of TX synthase. To assess TX synthase activity, we added PGH2to the platelet suspension and examined the increase in the content of the stable TXA2metabolite TXB2in the medium. In contrast to the effect on COXs, CSE enhanced TXA2synthase activity in a concentration-dependent manner with an EC50value of 1.80 ± 0.57% (TX synthase works downstream of COX, converting PGH± 0.57% .2production is reduced,Previous studies have shown that cigarette smoking is a major risk factor for cardiovascular diseases.We first examined the effect of nicotine- and tar-free CSE on platelet aggregation. CSE potently inhibited murine platelet aggregation induced by U-46619 or collagen –C. Th2production and that the inhibitory effect of CSE on collagen-induced aggregation was suppressed significantly in TP–/–platelets compared with the inhibitory effect in WT platelets (2production. In EP2–/–, EP4–/–, or IP–/–platelets, the inhibitory effect of CSE on U-46619-induced aggregation was not significantly different from that in respective control platelets (sexpressed in platelets, including inhibitory prostanoid receptors, do not participate in the inhibitory effect of CSE on platelet aggregation.We next intended to clarify the mechanism by which CSE inhibits platelet aggregation. We showed that CSE inhibited arachidonic acid–induced TXAatelets . These atelets ,B. Fuatelets . These 2production in platelets. CSE inhibited COX-1 activity in a concentration-dependent manner, and the pattern of inhibition was uncompetitive (2to PGH2, the direct precursor of TXA2. Our results indicated that CSE binds to the COX-1–PGG2complex and stabilizes it, resulting in an uncompetitive inhibition of PGH2production. In contrast to the inhibitory action on COX-1 activity, CSE enhanced TX synthase activity in a concentration-dependent manner (2production in platelets. Like the action of CSE, it has been shown that catechol inhibited arachidonic acid–induced platelet aggregation and that the inhibition of COX activity and TXA2production by catechol played a role in its antiplatelet effect.COX-1 is an enzyme that is responsible for TXAetitive . In thi manner . These Notably, CSE also inhibited the activity of COX-2 , a well2production.This is the first report showing that CSE, the gas phase constituents of cigarette smoke, inhibits platelet aggregation and that its antiplatelet effect is derived from the inhibition of platelet COX-1 activity and resultant reduction in TXA


The dynamic response analysis of horizontal and inclined layered sites using large-scale shaking table tests in various directions, including the dip direction, strike, vertical direction, slope direction and direction perpendicular to the interface of layered sites is conducted in this study. The Fourier spectrum and response spectrum characteristics in the horizontal site are first investigated in this study, and the dynamic responses of the inclined layered sites are then studied and compared to the corresponding responses of the horizontal layered site. The influence of dip angle on the response spectrum is also studied. Construction is usually built on layered sites, including horizontal layered sites and inclined layered sites. A remarkable amount of researches regarding the dynamic response of horizontal layered sites have been carried out, and the results have been widely accepted 2]3]4][3][4]2][[4][3][4][3][4]2]6]. Neve. Neve6].As an approach to reveal the dynamic response characteristics directly, the large shaking table test has been widely employed by researchers to study the dynamic responses of slopes and earthquake-induced landslides 12]13][[13][12][[[13][12]The shaking table test was carried out at the Nuclear Power Institute of China (NPIC). The shaking table device has six degrees of freedom, the shaking table size is 6 × 6 m, the carrying capacity is 600 KN, the maximum lateral displacement is ±150 mm, the maximum vertical displacement is ±100 mm, the maximum acceleration under a full loading condition is 1.0 g horizontally and 0.8 g vertically, and the loading frequency range is 0.1–80 Hz.π theorem, the similitude ratio of this shaking table test is 10 for length, 1 for density, 1 for acceleration, 3.16 for time, 0.316 for frequency and 10 for elasticity modulus:f=V:f=Vf off of the horizontal layered site is 29.88 Hz based on Eqs (The resonance frequency d on Eqs  and 2)..f of theIn the vertical direction, all frequency components hardly change in wave propagation, and the amplification effect of the frequency components is slight, as shown in As seen in It can be concluded from the above analysis that in the horizontal site, the ratio of the Fourier spectrum in the gravel soil layer is larger than that in the soft rock layer in the horizontal direction, as illustrated in It is worth noting that in the gravel soil layer, the amplification coefficients decrease with increasing PGA, however, in the soft rock layer and hard rock layer, the higher amplifications are generally obtained for PGA = 0.20 g, whereas the lower ones are obtained for 0.50 g. This implies that the gravel soil layer begins to show nonlinear characteristics when PGA = 0.10 g, whereas the soft rock layer and hard rock layer begin to show nonlinear characteristics when PGA = 0.20 g. The reason for nonlinear response is that when the geotechnical material shear strain increases and shear modulus decreases with increasing PGA, the hysteresis curve will be fuller, and the dissipated shaking energy by the geotechnical material will increase, thus weakening the dynamic response of the site, therefore, the amplification coefficients decrease. The nonlinear response of the surface layer is also observed by Huang et al. in Taiwan  and BereThe acceleration response spectra of a single-degree-of-freedom (SDOF) system with a 5% damping ratio under the Kobe earthquake record with amplitudes of 0.10 g and 0.50 g were calculated as shown in β of the response spectra under the 0.50 g earthquake wave is close to that under the 0.10 g earthquake wave. Because of the significant amplification effect of the gravel soil layer, as shown in β of the response spectra of the gravel soil layer is larger than those of the soft rock layer and hard rock layer. In the short period , the sequence of the response spectrum amplification factor β is gravel layer > hard rock layer > soft rock layer. Whereas in the long period (T > 0.3 s), the sequence of the spectral response amplification factor is gravel layer > soft rock layer > hard rock layer. The result indicates that on the one hand, the buildings constructed on the gravel layer will suffer a stronger dynamic response than the soft rock layer and hard rock layer. On the other hand, with regard to the buildings constructed on the soft rock layer or hard rock layer, when the building period T ≤ 0.3 s, the buildings constructed on the hard rock layer will suffer a stronger dynamic response than those on the soft rock layer, whereas the building constructed on the soft rock layer will suffer a stronger dynamic response than those on the hard rock layer when the building period T > 0.3 s.A remarkable amount of research on the dynamic response of horizontal layered sites has been undertaken, and relevant research results have been widely applied to regional seismic safety evaluation and seismic design 2]4][30[4][302][[30[4][30The influence of dip angle on the Fourier spectrum and response spectrum are discussed in this study. The data employed to calculate the Fourier spectrum and response spectrum in this study were collected from the 5# accelerometer at the layered horizontal site, 21# at the 7.5° inclined layered site, 4# at the 10° inclined layered site and 11# at the 12.5° inclined layered site.To reveal the influence of dip angle on the frequency components, the Fourier spectra of the sites with different dip angles were calculated. The Fourier spectra in the dip direction, strike and vertical direction are shown in In the dip direction, as seen in In the strike, as the ratio of the Fourier spectrum is close to 1, the influence of dip angle on the 0–23 Hz frequency components can be ignored. In the frequency band of 23–40 Hz, the ratio of the Fourier spectrum of the 10° inclined layered site increases with increasing frequency, however, the ratios of the Fourier spectra of the 7.5° and 12.5° inclined layered sites decrease, and the reduction of the 7.5° inclined layered sites is slightly larger than that of the 12.5° inclined layered site. The above analysis shows that the dip angle has almost no influence on frequency components within 0–23 Hz. In the frequency band of 23–40 Hz, the frequency components of the 10° inclined layered site were amplified, whereas the frequency components in the 7.5° and 12.5° inclined layered sites were weakened, the reduction in the 12.5° inclined layered site is more serious than that in the 7.5° inclined layered site. The influence of dip angle on the Fourier spectrum conforms to the influence of dip angle on acceleration response in the strike, as shown in It is observed from Figs The ratio of the response spectrum was defined as the ratio of the response spectrum amplitude of gravel soil in the inclined layered site to that of gravel soil in the horizontal layered site. The ratio of the response spectrum represents the amplification effect on the response spectrum of the dip angle. All response spectra were calculated using the data collected from the top accelerators in the gravel soil layer, i.e., 5# accelerator for the horizontal layered site, 21# for the 7.5° inclined site, 4# for the 10° inclined site and 11# for the 12.5° inclined site. The ratio of the response spectrum of the X, Y, and Z directions under the 0.4 g El Centro earthquake record was calculated, as shown in T ≤ 0.1 s). Conversely, when T > 0.1 s, the ratio of the response spectrum decreases to less than 1 rapidly, especially in the dip direction, the ratio of the response spectrum then keeps a low value, which indicates that when T > 0.1 s, the response spectrum amplitude of the 12.5° inclined layered site is relatively lower than that of the horizontal layered site in the dip direction and strike. In the vertical direction, it can be seen that the ratio of the response spectrum remains approximately 1 when the dip angle is 7.5° and 10°. It indicates that in the vertical direction, the influence of the dip angle on the response spectrum in the 7.5° and 10° sites is not obvious. However, with regard to the 12.5° inclined layered site, the response spectrum will be obviously amplified when T is approximately 0.8 s, and the ratio of the response spectrum reaches its peak value of 2.86 at T = 0.68 s.In the dip direction, strike and vertical directions, when the dip angle is 7.5° or 10°, the ratio of the response spectrum is near 1. When the dip angle is 12.5°, in the dip direction and strike, the amplification effect of the response spectrum is obvious in the short period  has little influence on the dynamic response of constructions with a period of 1.00–4.00 s.It is worth noting that slope failure is the co-action result of acceleration both in the slope direction and the direction perpendicular to the interface. In practice, cases exist in which the slopes slide along the slope face with a small dip angle. For example, the landslide involving sliding failure along the slope face with a dip angle of 3–10° in the northeast PanZhihua airport in southwest China . This seθ > 12.5°) requires further study, and the research results in this paper require further practical verification and complement.The differences and similarities of spectral characteristics between inclined layered sites and horizontal layered sites were compared in this paper using large scale shaking table tests, and the corresponding results were quantitatively analysed. The largest dip angle of the inclined layered sites in this model test was just 12.5°, so the model site should be defined as a small dip angle inclined layered site. Because some obtained results in this paper cannot be explained according to the current state of knowledge, whether the test result is suitable for larger dip angle inclined layered sites Click here for additional data file.S2 Data(ZIP)Click here for additional data file.S3 Data(ZIP)Click here for additional data file.


A high-grade soft tissue sarcoma was diagnosed microscopically and immunohistochemistry revealed positive expression of VEGFr, PDGFr, SCF, and EGFR. Repeat surgical resections and targeted treatment with toceranib resulted in a stable remission for nearly two years.This case report documents the clinical and pathological findings in a dog that rapidly developed a high-grade sarcoma at the site of multiple vaccinations and follows the response to surgery and adjunct treatment with toceranib. An 11-year-old female spayed Labrador Retriever presented with dorsocervical subcutaneous masses at the injection site three weeks after receiving DA Injection-site sarcomas commonly occur in cats but are rare in other species , 2. MostAn eleven-year-old female spayed Labrador Retriever was presented for evaluation of multinodular subcutaneous masses in the dorsal cervical area .2PP-Lepto, Rabies, and Bordetella, had been injected into this same area during a wellness examination three weeks earlier. The dog's previous vaccination history included routine boosters at standard intervals administered at variable sites since puppyhood. Fine needle aspiration cytology of the masses revealed a mesenchymal spindle cell proliferation with a high level of atypia and minimal numbers of inflammatory cells. A wide surgical excision of the entire site with 3 cm margins was performed the following week. The excised tissue contained a regionally extensive, expansile, and infiltrative spindle cell neoplasm surrounded by a pseudocapsule and mild to moderate chronic inflammation with multifocal lymphonodular aggregates. The center of the neoplasm had undergone extensive necrosis, and the myxomatous matrix was admixed with grey-brown globular material. The neoplastic cells were fusiform to spindloid, formed interlacing bundles, and had moderate amounts of darkly eosinophilic cytoplasm. The nuclei were ovoid and had a stippled chromatin pattern with numerous, variably sized prominent nucleoli. There was marked anisokaryosis and anisocytosis. Binucleated and multinucleated cells as well as karyomegaly were multifocally observed. The mitotic count was 20 in 10 high powered fields , and there were occasional bizarre mitotic figures  , Plateleβ, was hypothesized. Toceranib was initially administered orally at a dose of 2.1 mg/kg and then increased to 2.8 mg/kg on a Monday-Wednesday-Friday schedule. No adverse effects were noted at these doses and no hypertension or proteinuria was detected. Repeat CBC and serum biochemistry profiles were normal throughout the course of therapy and thoracic radiographs did not show any evidence of metastatic disease. A small cluster of subcutaneous nodules was identified at the excision site fifty weeks after the first surgery and a third complete excision with 3 cm margins was performed. Histology confirmed a recurrence of the grade 3 soft tissue sarcoma and the toceranib was continued. At the time of submission of this manuscript, the dog remains in remission ninety-three weeks after initial diagnosis.Based on the expression of VEGFr and PDGFr, a response to targeted tyrosine kinase inhibitor therapy with toceranib, a small molecule inhibitor of VEGFr2 and PDGFrInjection-site sarcomas have been rarely reported in dogs. In a pathological study, soft tissue sarcomas in fifteen dogs that developed at sites where previous vaccinations may have been given were compared with canine soft tissue sarcomas that developed at nonvaccination sites, as well as with injection-site sarcomas from cats. A histologic pattern consistent with that of feline injection-site sarcomas  was repoThe association of feline soft tissue sarcomas with rabies and feline leukemia virus vaccinations was made over twenty years ago , and subSarcomas in cats have been reported to develop as early as 4 months and up to several years after vaccination, although in many instances the prepatent period is unknown . Feline Toceranib phosphate is an oral, small molecule split receptor tyrosine kinase (RTK) inhibitor that is licensed for use in the US and Europe at the maximally tolerated label dose of 3.25 mg/kg for high-grade mast cell tumors in dogs. Toceranib possesses antiangiogenesis and antitumor activity due to its potent inhibition of targets, including VEGFr, PDGFr, and KIT . BiologiIn the dog in this report, the expression of VEGFr, PDGFr, EGFr, and SCF was demonstrated by immunohistochemistry. Toceranib, since it targets VGEFr and PDGFr, was selected with the intent of reducing the potential for local regrowth and distant metastasis . The dosIn conclusion, the association of vaccinations with the occurrence of a high-grade soft tissue sarcoma several weeks after the injections in the dog in this report and the morphologic phenotype of the reported sarcoma is most consistent with a diagnosis of an injection-site sarcoma. This association does not constitute definitive proof that the vaccinations caused the sarcoma. Expression of tyrosine kinase receptors was investigated by immunohistochemistry, and a targeted therapy was elected based on VEGFr and PDGFr expression. Aggressive surgical resection of the soft tissue sarcoma and treatment with toceranib appeared to be useful in maintaining a sustained remission.


Solanum tuberosum leaves get frost killed at −3 °C and are unable to frost harden. The basic nature of frost injury to potato leaves is not clear. By employment of infrared differential thermal analysis (IDTA) in combination with viability assessment, we aimed to clarify the mechanistic relationship between ice formation and frost injury. During controlled freezing of potato leaves two distinct freezing events were detected by IDTA. During the first freezing event, the ice wave propagated via the xylem and spread out within 60 s throughout the whole leaf. When leaves were rewarmed after this freezing event, they did not show any frost injury symptoms. We suggest that this non-lethal first ice wave is restricted to the extracellular space. When leaves remained exposed after this exotherm, a second freezing event with a diffuse freezing pattern without a distinct starting point was recorded. When thawed after this second freezing event, leaves always showed frost damage suggesting intracellular freezing. The freezing behavior of potato leaves and its relation to frost damage corroborates that control of ice nucleation is a key for frost protection.Infrared thermography has been widely used to study freezing processes in freezing resistant plants but hardly in freezing susceptible species.  Unfortu problem . S. tubet harden . Under ft harden ,26 and prden [−1 . The basrden [−1 . In an eo −10 °C . HoweverS. tuberosum leaves tolerate ice or get immediately killed upon ice formation. This should allow addressing the question of the basic nature of frost injury in S. tuberosum leaves. The results may have consequences for potential future means allowing frost protection in this important crop plant.We aimed to study the freezing process in potato leaves by IDTA in combination with a viability assessment in order to find out whether 22.1Solanum tuberosum (var. Agria Bio). During the summer season of 2018 (May till August) potato plants were cultivated in a field plot (2 m2) of the Botanical Garden of the University of Innsbruck . The field plot contained natural soil (sandy silt) with modest compost fertilization. Plants were regularly watered. Sampling of leaves was started right after the maximum canopy height had been reached. Only young but fully expanded leaves from the canopy surface were used for the experiments. Leaves were selected randomly and always detached in the morning before experiments were performed. The leaf petiole was immediately submersed and cut under water before the transport to the laboratory, which lasted less than 3 min. The leaves remained well watered till the onset of the experiments.Freezing processes were studied in leaves of Air temperatures were recorded by a TidBit data logger . Monthly mean/minimum temperatures were in May 17.2/8.3 °C, in June 18.7/9.6 °C, in July 20.5/11.4 °C and in August 20.9/6.1 °C.2.2S. tuberosum leaves was conducted inside the cooling compartment of temperature-controlled freezers as described previously in detail [−1 to target freezing temperatures. As the temperature of ice nucleation also has effects on freezing injury in potato leaves [Pseudomonas syringae van Hall 1902) which occurred at a mean temperature of −2.3 °C ± 0.4 SE. In all freezing experiments leaf temperatures were monitored via copper constantan thermocouples  that were connected to a data logger .Controlled exposure to subzero temperatures of n detail . As cooln detail , in all o leaves , ice nuc2.3S. tuberosum were measured by the established method of differential thermal analysis . During2.4S. tuberosum, infrared differential thermal analysis  was  was S. t2.5To check whether leaves were ice susceptible or ice tolerant, leaves were exposed during the freezing treatment to subzero temperatures at −2.5 °C ± 0.2 SE for 2 h and were either ice inoculated or kept in the supercooled state. Experiments were conducted in a freezer with an acrylic glass lid, which allowed for the monitoring of the leaves throughout the whole freezing procedure from the outside. When potato leaves freeze, they typically slightly bend. In the artificially ice inoculated leaves (N = 6), ice nucleation was triggered via the cut surface of the leaf petiole using the following experimental setting: The leaf petiole was wrapped in moist cotton wool that was additionally soaked with a suspension of INA bacteria. The leaves that were intended to stay supercooled received no such treatment (N = 14).2.6Right after the low temperature treatment, frost injured leaves were highly fragile and got limp. Slight blackish discolorations appeared within hours. Leaves were kept in plastic bags on wet paper towels at room temperature for full development of frost damage. Final leaf viability was visually assessed 4 d after the freezing treatment. Intense blackish discoloration of the leaf blade indicated frost damage.3S. tuberosum were subjected to controlled freezing experiments down to −3.0 °C. They were either inoculated with ice or not. In half of the leaves ice nucleation was triggered, the other half remained supercooled. While the ice nucleated leaves showed tissue infiltrations and were limp, the supercooled leaves seemed visually unaltered after the low temperature treatment. 4 d afterwards, frost injuries manifested as blackish discolorations in leaves that had been ice nucleated  the leaves were rewarmed at moderate rates to non-freezing temperatures. Despite ice formation, the leaves did not show any symptoms of frost damage 4 d after the freezing treatment  was observed when ice was initiated at about −4 °C as compared to similar leaf tissue frozen to the same freezing temperature but with ice, which was initiated at about −1 ° C [Strikingly, when leaves were rewarmed after the first freezing exotherm, they did not show any symptoms of frost injury. This suggests that the first freezing event is not lethal. There is some indication in the literature that potato leaves might tolerate freezing . In a tht −1 ° C . This maS. tuberosum leaves tolerate ice, even though only for a short time period (10–78 min). IDTA reveals that ice initially propagates via the vascular tissue, but at the same time ice captures the whole leaf blade. By IDTA it is not possible to localize where exactly ice forms and propagates in the mesophyll during this initial freezing event. The observed freezing pattern could be extra-tissue freezing [Our findings support that freezing  where icWhen leaves remained exposed after the first freezing exotherm under the experimental conditions a second freezing event could be detected. This freezing process had a diffuse pattern and occurred all over the leaf blade. No distinct starting point of this freezing could be depicted. After this second freezing event when leaves had thawed, they were water soaked and limp, which indicated frost damage. Given that during the first freezing event extra-tissue water has frozen, during this second freezing event remaining supercooled water inside the living cells must have frozen. This suggests an intracellular freezing process. Preliminary cryo-microscopic results give evidence that mesophyll cells of potato do not show a freezing cytorrhysis that is commonly the result of freeze dehydration but freeze intracellularly during the second freezing event . The timS. tuberosum is unable to frost harden and gets frost killed around −3 °C [S. tuberosum examined could survive below −2.5 °C [nd −3 °C . None of −2.5 °C . Our res −2.5 °C ,26 being −2.5 °C  must be http://www.mdpi.com/2076-3417/9/5/819/s1. Video S1: First exothermic event: IDTA sequence obtained on a leaf of S. tuberosum during a controlled freezing treatment at a cooling rate of 3 K·h−1. Whitening shows the spreading of ice after ice nucleation in the leaf petiole over the entire leaf blade. This first exotherm lasted approximately 1 min (playback speed 4x), Video S2: Second exothermic event: IDTA sequence obtained on a leaf of S. tuberosum during a controlled freezing treatment at a cooling rate of 3 K·h−1. The second freezing event is diffuse without a distinct starting point (playback speed 4x). The video depicts only a part of the second freezing event.The following are available online at Supplementary materials


Nature, Eunyoung Park et al. reported structures of autoinhibited and active BRAF–MEK1–14-3-3 complexes. This finding reveals the detailed mechanism underlying RAF regulation, which could facilitate the development of novel therapeutic strategies to overcome RAF-related cancer.1In a recent paper published in 2 Previous studies have partially demonstrated the molecular mechanisms of the Raf/MEK/ERK cycle.2 Initially, GTP-bound RAS recruits Raf to the plasma membrane, which drives RAF to release inhibitory 14-3-3 protein from the N-terminus and form a dimer, thereby switching from an autoinhibited state to an activated state. Then, the activated Raf dimers enable MEK recruitment and phosphorylation, thereby transmitting the signal down to ERK. Finally, ERK signaling implements a negative feedback loop in which ERK phosphorylates several inhibitory sites in distinct regions of activated RAF, resulting in a release from the activated RAS and the disruption of RAF dimers. Some studies have revealed that BRAF and MEK are pre-associated in the quiescent state. In addition to its role in physiological processes, aberrant RAF activation in the key pathway contributes to causing cancer; thus, RAF kinase has been considered a target for anticancer treatment. Several Raf kinase inhibitors have been developed for clinical use and have achieved remarkable clinical outcomes. Nevertheless, most patients relapse within a year of treatment due to acquired resistance. Some RAF inhibitors unexpectedly promote RAF dimerization to induce the upregulation of ERK signaling, while some break the autoinhibitory status by disrupting the interactions of BRAF kinase with its N-terminal region.3 In the past decades, structures of isolated domains or fragments of the kinases targeting this pathway have been characterized to elucidate the mechanism of RAF/MEK/ERK6; however, the lack of a deeper structural characterization with intact structural information of RAF regulation has hindered the development of novel RAF/MEK/ERK kinase inhibitors.The RAF/MEK/ERK signal transduction pathway regulates a wide set of cellular events, including cell proliferation, differentiation, and survival, via phosphorylation cascades in all eukaryotic cells.1 The mammalian homologs of RAF kinases  have three conserved regions called CR1, CR2, and CR3. CR1 at the N-terminal site contains the RBD (RAS binding domain) and the CRD (cysteine-rich domain) domain, the CR2 region consists of a binding site for the 14-3-3 protein, and CR3 at the C-terminal site has another binding site for the 14-3-3 protein, a phosphorylated motif and a serine/threonine kinase domain. The cryo-EM structure of an autoinhibited BRAF/MEK1AA/14-3-3 complex  reveals the inhibitory mechanisms of the 14-3-3 dimer (PDB:6NYB). In an autoinhibited state, the active site of the BRAF kinase domain faces away from the 14-3-3 domain but faces MEK1 with extensive contact between the C-lobes of both kinases. ATP-γS is bound in the BRAF active site cleft, while ADP seems to be in a MEK active site. A 2.6 Å crystal structure of GDC-0623 and the ATP analog AMP-PNP-bound BRAF/MEK1AA complex reveals high similarity with the cryo-EM structure (PDB:6PP9). A helix-like turn, called an inhibitory turn, stacks with hydrophobic residues in the glycine-rich loop and the β3 strand and the c-helix stabilize the inactive conformation. Rotation of the N-lobe results in a less open active site compared with other reported non-nucleotide bound BRAF structures, which implies the importance of ATP binding in the autoinhibited states. Two 14-3-3 proteins form a cradle-like space for the direct binding of the N-terminal CRD domain of BRAF, which blocks interface residues H510, D565, and Y566 for BRAF dimer formation and obstructs the CRD domain for membrane recruitment of BRAF and RAS-driven activation. The CRD domain is located in the center of the complex and exhibits β strands with two zinc ions stabilized at coordination sites. Two loops of the CRD domain (residues 239-245 and 253-260), which mediate the association of the domain with the membrane, extensively contact the highly conserved region of 14-3-3. As previously reported, R239 and T241 of BRAF are the key residues that bind 14-3-3. The pS365 and pS729 regulatory segments occupy the recognition groove on a separate side of the 14-3-3 dimer and interact with the kinase C-lobe and C-terminus of BRAF, respectively. The structure of the autoinhibited BRAF/MEK1AA/14-3-3 complex gives direct evidence that MEK also participates in stabilizing the inactive state of BRAF.To understand the normal regulation of RAFs, Park et al. recently reported the cryo-EM complex structures of full-length BRAF with MEK1 and 14-3-3 proteins in both the autoinhibited and active status.S365A/MEK1AA/14-3-3 complex reveals a symmetrical back-to-back dimeric BRAF with MEK1 bound to each BRAF, while the 14-3-3 dimer rearranges to bridge the S729-phosphorylated tails of the two BRAFs in the active state (PDB: 6Q0J). BRAF S729 is stoichiometrically phosphorylated, but T599 and S602 exhibit negligible phosphorylation, although it is believed that autophosphorylation of T599 and S602 is critical for BRAF activation,7 which is consistent with the BRAF/14-3-3 complex. Interestingly, BRAFS365A/MEK1AA/14-3-3 with only one MEK bound to the BRAF dimer was observed under the same preparation conditions, which reveals an asymmetric position regarding the kinase dimer. The asymmetric structure indicates that 14-3-3 can regulate the affinity for MEK and control its release.Furthermore, the BRAFThe structures of the BRAF–MEK1–14-3-3 complexes reveal an inactive and active conformation of BRAF, thereby providing mechanistic insights into RAF regulation. In the autoinhibitory state, the CRD and its membrane binding surface are mainly buried by the interaction with the 14-3-3 dimer and other fragments of RAF, whereas the RBD is exposed, allowing the activated RAS to recruit the stationary complex to the membrane. Upon RAS binding to RBD, BRAF kinase is released from 14-3-3 and forms an open monomer. Finally, the 14-3-3 domain rearranges to bridge the pS729 site of two RAF open monomers, thus promoting the formation of active RAF dimers (Fig.


To investigate the primary clinical value of atlantoaxial pedicle screw placement assisted by a modified 3D-printed navigation template.We retrospectively analyzed the cases of 17 patients treated from June 2015 to September 2016 with atlantoaxial pedicle screw placement assisted by a modified 3D-printed navigation template. All procedures were performed prior to surgery, including thin-slice CT scanning, medical image sampling and computerized 3D modeling of the atlantoaxial joint, optimal pedicle screw trajectory determination, and anatomical trait acquisition for the atlantoaxial pedicle, spinous process of the axis, vertebral lamina and posterior lateral mass, and design of a reverse template. During surgery, a navigation template was tightly attached to the atlantoaxial joint to assist in pedicle screw placement. Surgeons subsequently used an electric drill to remove the template through a guide channel and then placed the atlantoaxial pedicle screw. Observed indexes included the VAS score, JOA improvement rate, surgery duration, and blood loss.Surgery was successful in all 17 patients, with an average operation duration of 106±25 min and an average blood loss of 220±125 ml. Three days postoperatively, the VAS score decreased from 6.42±2.21 to 3.15±1.26. Six months postoperatively, the score decreased to 2.05±1.56. The postoperative JOA score increased significantly from 7.68±2.51 to 11.65±2.72 3 d after surgery and to 13.65±2.57 after 6 months. Sixty-eight pedicle screws were inserted successfully, with 34 in the atlas and 34 in the axis. According to the Kawaguchi standard, 66 screws were in grade 0 (97.06%), and 2 were in grade 1 (2.94%). The pre- and postoperative transverse and sagittal screw angles showed no significant differences.Atlantoaxial pedicle screw placement assisted by a modified 3D-printed navigation template is worth recommending due to the improved accuracy in screw placement, improved patient safety and beneficial clinical effects. Posterior pedicle screw fixation of the atlantoaxial joint is a widely recognized treatment option by spinal surgeons due to its advantages of long-term stability, high fusion rates, and good position correction From June 2015 to September 2016, 17 patients with atlantoaxial dislocation or deformity were treated in our department and subsequently followed for 6-8 months. There were 11 males and 6 females ranging in age from 25 to 56 years with a mean age of 43.3 years. Of the 17 patients, 13 were treated for atlantoaxial dislocation, and 4 were treated for atlantoaxial deformity. All patients suffered from neck and shoulder pain, including 5 patients with accompanying cervical cord compression. Imaging examination showed that all 17 patients suffered from cervical instability or dislocation. Fifteen of the patients could fully recover before the surgery, while the other 2 could only generally recover. After surgery, there was no obvious pressure on the anterior aspect of the spinal cord. This study was approved by the ethics committee.We used a LightSpeed 16-slice spiral CT machine  for the imaging examinations. The scanning range included three segments from the first cervical vertebra to the third cervical vertebra with a 1.25-mm slice thickness, 5.0-mm pitch, 9.37-mm/s table speed, 120-kV voltage, and 200-mA current. Scanned images were saved in DICOM format, and the DICOM files were imported into the 3D image producing and editing software Mimics 17.0  to construct 3D models of the target cervical area. To avoid damage in the trajectory to the pedicle cortex, preliminary simulation of pedicle screw placement was conducted with computer-aided design software Creo2.0  using a cylindrical simulated screw with a diameter of 3.5 mm to observe the positional relationship between the trajectory and pedicle in the transverse, sagittal and coronal views of the 3D image. The axis of the cylinder 3.5 mm in diameter was used to build a screw placement guide tube with an inner diameter of 2.1 mm and a vertebral lamina with a length of 15 mm. The template location surface was formed by removing the interfering parts of the template and 3D model after designation of the screw hole position. A cylindrical range pole 3.5 mm in diameter was rebuilt based on the preset axis line of the screw hole. The range pole was moved approximately 10 mm toward the screw hole, and the average thickness of the template was set at approximately 6 mm. The template file was imported into a Formlab 3D printer  to print the cervical pedicle screw placement template. Ethylene oxide was subsequently used to disinfect the template, which was later sealed for future use.In vitro testing demonstrated the accuracy of the modified 3D-printed navigation template. After feasibility of the treatment was assured, the navigation template was disinfected with ethylene oxide before use on patients. All patients maintained an atlantoaxial reduction position with a skull traction weight of 3-6 kg. After receiving general anesthesia, patients in the prone position were incised in the middle of neck to expose the posterior atlantoaxial region with complete dissection of the muscles and ligaments attached to the lamina and spinous process. The disinfected modified 3D-printed navigation template was later attached to the lateral mass, lamina, and spinous process of the corresponding vertebra. Next, after close observation, the surgeons drilled into the vertebral bone cortex to a depth of 10 mm with an electric drill and a 2.0-mm-diameter bit, and assistants subsequently attached the template. Parallel with the direction of the guide posts, the electric drill was used in the guide channels to investigate optimum screw placement and to observe the post’s direction and movement. Probes were used after drilling to detect whether the internal walls of the screw channels were intact. Threads and pedicle screws with a diameter of 3.5 mm were placed once the intact screw channels were confirmed. After screw placement and lateral observation using a C-arm X-ray machine, the screw position was confirmed, and the surgeons kept patients in the optimum reduction position after adjusting their heads and necks. Bone grafting was completed after screw-rod fixation. Finally, drainage tubes were inserted, and the incision was sutured outward.A preoperative simulation surgery was conducted using the 3D-printed template in this study. Kirschner wire was drilled through the guide channel of the modified 3D-printed navigation template and the pedicle to ensure that the wire was completely in the pedicle and would not break the pedicle cortex. After surgery, patients were kept under strict bed rest with the cervical spine held stationary and were turned over axially as needed. The drainage flow of the operative site was observed, and the drainage tube was removed if the drainage flow was less than 50 ml/24 h without cerebrospinal fluid leakage. Before surgery, the patients had taken an antibiotic for 24-48 h, a dehydrant for 3-5 d, and a gastric acid suppression agent for 3-5 d. Adrenalineand was added for 3-5 d if there was impaired spinal cord function before surgery. Patients wore a cervical support device to perform bedside exercises 3 d after surgery.Regular atlantoaxial CT scanning was used to observe the position of the pedicle screw. The accuracy of screw placement was assessed with the Kawaguchi standard by observing the positional relationship between the screw and pedicle, as follows: Level 0: the screw is completely in the pedicle; Level 1: the screw is out of the cortex of the pedicle by less than 2 mm, and no screw-related complications occurred, including nerve or vertebral artery injury; Level 2: the screw is out of the cortex of the pedicle by more than 2 mm, and no screw-related complications occurred, including nerve or vertebral artery injury; Level 3: Screw-related complications occurred, including nerve or vertebral artery injury. Levels 0 and 1 were considered to represent accurate screw placement.Using deviation analysis in Mimics software, the transverse and sagittal angles of the pre- and postoperative trajectories were measured and compared The VAS score for cervical and shoulder pain was recorded 1 d before, 3 d after and six months after surgery. In this scoring system (with a full score of 10), 0 represents no pain, and 10 represents insufferable pain. The JOA score was used (with a full score of 17) to evaluate the patient’s cervical nerve function; in this system, the higher the score, the better the nerve function.In vitro testing was performed to compare the accuracy of screw placement with the modified 3D-printed navigation template with that of the traditional freehand, C-arm-guided and navigation-guided techniques. The results showed that the accuracy of the 3D navigation template was 0.992, which was significantly higher than that of the freehand (0.962), C-arm-guided (0.954) and navigation-guided (0.941) techniques.The surgeries on all 17 patients were successful, with a mean procedure time of 106±25 min and a mean operative blood loss of 220±125 ml. Sixty-eight pedicle screws were inserted successfully, including 34 screws in the atlas and 34 in the axis. After surgery, there were no nerve or vertebral artery injuries or complications, including incision infections or cerebrospinal fluid leaks. During the follow-up period, there were no indications of screw loosening or breakage. According to the Kawaguchi standard, 97.06% of the 68 successfully placed pedicle screws were in grade 0 (66/68), and 2.94% were in grade 1 (2/68). The success rate was 100% . Mimics The results showed that there were no significant differences in the transverse or sagittal angle preoperatively or postoperatively (mean P>0.05, The postoperative VAS score significantly decreased from 6.42±2.21 to 3.15±1.26 3 d after surgery and to 2.05±1.56 after 6 months. The postoperative JOA score significantly increased from 7.68±2.51 to 11.65±2.72 3 d after surgery and to 13.65±2.57 after 6 months. No spinal nerve injuries occurred .Posterior atlantoaxial pedicle screw fixation has become the “gold standard” for atlantoaxial fixation. Due to the complex structure of the atlantoaxial area, traditional surgical methods pose great risks to vascular and nervous tissue. Therefore, improving the accuracy of atlantoaxial pedicle screw placement has become an urgent need for spinal surgeons. The use of various computer-aided methods can improve screw placement accuracy and safety, thereby reducing the risk of spinal cord and vascular injury Compared with traditional techniques, the modified 3D-printed navigation template has channels on both sides, as well as internally shifted guide posts with a length of 30 mm and diameter of 3 mm. The modified 3D-printed navigation template has many advantages over traditional techniques in atlantoaxial pedicle screw placement, the most notable of which are great improvements in accuracy and safety. The modified 3D-printed navigation template also exhibited the following advantages: ① reduced placement deviation, more accurate screw placement and intraoperative identification of template shaking and displacement via observation of the post position; ② screw placement deviation avoidance via the use of an electric drill parallel with the channel using the guide posts as a reference; ③ improved intraoperative template stability and reduced intraoperative template shaking and displacement via assistants holding the additional long posts acting as guides at opposite sides to repair the navigation template position; and ④ reduced required intraoperative space via limiting the space expansion of internally shifted guide posts on both sides, leading to less tissue traction and dissection. Therefore, screw placement deviation can be reduced if the template is fully attached to the posterior vertebral parts, as ensured by post-oriented full dissection of the posterior soft tissues.in vitro test demonstrated the accuracy of the modified 3D-printed navigation template. Once the feasibility of the treatment was assured, the modified 3D-printed template was disinfected and sterilized before use in patients. Pedicle screw placement assisted by the modified 3D-printed navigation template was conducted in 17 patients. The mean surgery duration for treating atlantoaxial dislocation and deformity was 106±25 min and 186±125 min, respectively; the mean operative blood loss was 220±125 ml and 312±207 ml, respectively. These results demonstrate the advantages of shorter operation times and decreased blood loss compared to traditional surgery, which is in agreement with the results of the study conducted by Hu et al. A preoperative simulation surgery was conducted using the 3D-printed template in this study. Kirschner wire was drilled through the guide channel of the modified 3D-printed navigation template and the pedicle. Kirschner wire was inserted completely into the pedicle, and the cortex of the pedicle was not broken. The in vitro on the model to ensure that the screw trajectory did not deviate from the posterior pedicle before clinical application. Additionally, probes were used for investigating the walls of the screw placement channels, and screw placement was evaluated to define screw positions for safe surgery. ⑤ Placement deviation may be generated if the navigation template is not closely attached to the bones posterior to the vertebral lamina; therefore, it is of great importance to allow the template to closely attach to the bones by the complete removal of the corresponding spinous process, lamina, lateral mass and dorsal soft tissues. Furthermore, pedicle screw fixation is not suggested for an atlantoaxial pedicle with a diameter less than 3.5 mm.The modified 3D-printed navigation template also has limitations similar to those of traditional methods. ① With UV-curable resin being the main material, the 3D-printed navigation template was fragile after production, resulting in cuttings forming at the base of the template. Therefore, more attention should be focused on reducing screw placement deviation by enabling an electric drill to run parallel with the guide channels. In this research, the diameter of the guide channels, 2.1 mm, matched the drill bit diameter. Additionally, the paraffin oil used in the channels for lubrication intraoperatively reduced obstruction resistance and avoided template wear caused by the friction of the drill against the channels. ② High-temperature disinfection and other operations can lead to template deformation; therefore, ethylene oxide is recommended for both disinfection and sterilization. ③ More complex preoperative designs should be considered. ④ Deviations may occur in template production; however, we conducted preoperative experiments In conclusion, this study demonstrates that it is safe and feasible to perform atlantoaxial pedicle screw placement with high accuracy and no neurovascular injuries using a modified 3D-printed navigation template. These results indicate that use of the modified 3D-printed navigation template is an acceptable new method for the localization and orientation of atlantoaxial pedicle screw placement, which is of great clinical value. However, due to the small number of samples in this study, further clinical research with larger samples and multiple centers is warranted.Each author has made an important scientific contribution to the study and has assisted with the drafting or revising of the manuscript.