Protein phosphorylation is a common post-translational modification in eukaryotic cells and has a wide range of functional effects. Here, we used mass spectrometry to search for phosphorylated residues in all the proteins of influenza A and B viruses – to the best of our knowledge, the first time such a comprehensive approach has been applied to a virus. We identified 36 novel phosphorylation sites, as well as confirming 3 previously-identified sites. N-terminal processing and ubiquitination of viral proteins was also detected. Phosphorylation was detected in the polymerase proteins , glycoproteins (HA and NA), nucleoprotein (NP), matrix protein (M1), ion channel (M2), non-structural protein (NS1) and nuclear export protein (NEP). Many of the phosphorylation sites detected were conserved between influenza virus genera, indicating the fundamental importance of phosphorylation for all influenza viruses. Their structural context indicates roles for phosphorylation in regulating viral entry and exit (HA and NA); nuclear localisation ; and protein multimerisation . Using reverse genetics we show that for NP of influenza A viruses phosphorylation sites in the N-terminal NLS are important for viral growth, whereas mutating sites in the C-terminus has little or no effect. Mutating phosphorylation sites in the oligomerisation domains of NP inhibits viral growth and in some cases transcription and replication of the viral RNA genome. However, constitutive phosphorylation of these sites is not optimal. Taken together, the conservation, structural context and functional significance of phosphorylation sites implies a key role for phosphorylation in influenza biology. By identifying phosphorylation sites throughout the proteomes of influenza A and B viruses we provide a framework for further study of phosphorylation events in the viral life cycle and suggest a range of potential antiviral targets. Eukaryotic cells regulate the function of many of their proteins through the reversible phosphorylation of serine, threonine or tyrosine residues. It is known that some influenza virus proteins are phosphorylated, but few sites of phosphorylation have been identified. We used mass spectrometry to identify 39 sites of phosphorylation, most of them novel, in proteins from influenza A viruses and an influenza B virus (a separate genus in the orthomyxovirus family) - to the best of our knowledge, this is the first time this has been attempted for all the proteins in a virus. By integrating sequence conservation data and structural information we were able to propose functions for most of these sites, providing a foundation for further studies, and we assessed experimentally the contribution of multiple phosphorylation sites in the influenza A virus nucleoprotein (NP) to viral growth and to transcription and replication of the genome. In addition, by identifying phosphorylation sites that are common to both influenza A and B viruses, we suggest that phosphorylation at these sites is a highly conserved aspect of influenza biology that may provide targets for antiviral therapy. Influenza viruses cause serious and widespread disease in humans and livestock. Influenza A viruses can infect a wide range of birds and mammals, including humans The proteins encoded by influenza viruses undergo a variety of post-translational modifications. In eukaryotic cells, phosphorylation of serine, threonine or, less frequently, tyrosine, is a common reversible protein modification that can have a wide range of effects on activity, stability, subcellular localisation and protein-protein interactions Here, we use liquid chromatography and tandem mass spectrometry (LC-MS/MS) to search for sites of phosphorylation in the proteomes of influenza A and B viruses. The same approach also allowed N-terminal processing to be identified, as well as a site of ubiquitination (in the influenza B virus M1 protein). To the best of our knowledge, this is the first time mass spectrometry has been used to simultaneously assess the phosphorylation of all proteins in a virus. In addition to three previously-identified sites, we report 22 novel sites of phosphorylation in influenza A viruses and14 novel sites of phosphorylation in influenza B viruses. Comparisons of experimental data and consensus sequences show that phosphorylation sites are conserved within and in some cases between genera. As the influenza A and B virus genera are estimated to have diverged several thousand years ago In the following sections we give an overview of the viruses and methodologies used in the study, give details of N-terminal processing of viral proteins, and then describe the location of phosphorylation sites in the matrix protein M1 and the ion channel M2, in the non-structural protein NS1 and the nuclear export protein NEP, in the viral glycoproteins, and in the polymerase and NP. By analysing the conservation and structural context of phosphorylation sites we propose that in influenza A and B viruses phosphorylation sites can affect viral entry and exit (HA and NA), regulate nuclear localisation  and affect protein multimerisation . Finally, we present experimental evidence that phosphorylation sites in influenza A virus NP contribute to viral growth in tissue culture and play a role at various points in the viral life cycle.For influenza A viruses, we focussed on the well-studied H1N1 laboratory strain A/WSN/33 (WSN), using virions purified from the growth media of infected MDBK cells . In addiIn addition to protein harvested from virions, proteins of the WSN virus were purified from lysates of human 293 T cells and from MDBK cells. In one approach tagged PB2 was used to purify material from infected cells ; all co-2 or IMAC resin, and the use of a more sensitive mass spectrometer, referred to in the text as ‘optimised methods’) greatly increased the number of phosphorylation sites detected. The phosphorylation sites found in influenza A and B viruses are summarised in Proteins were prepared for mass spectrometry by either excising them from polyacrylamide gels or by precipitation. Proteins were digested with trypsin to produce charged peptides, which were analysed by LC-MS/MS using the Central Proteomics Facilities Pipeline (CPFP) When analysing WSN and B/Brisbane/60/2008 virions, we pooled data from multiple experiments, providing between 43% and 94% coverage of each protein detected, with each tryptic peptide detected an average of 18 times . For WSNIn addition to phosphorylation it was possible to detect N-terminal acetylation and methionine excision, which are both common post-translational modifications in eukaryotic cells The matrix protein M1 of influenza A and B viruses is known to be phosphorylated at multiple sites, predominantly serines but also threonine For influenza A viruses, even without enrichment for phosphopeptides we detected phosphorylation in the N-terminus of WSN, though it was unclear from the mass spectrum whether this was at T9 or Y10. Using optimised conditions, we again detected phosphorylation at this position, with phosphorylation of Y10 giving the best match to the observed mass spectrum , S2. We For the influenza B virus we observed a similar pattern of M1 phosphorylation. Without using optimised conditions we detected phosphorylation in the N-terminus, most likely at S2 or T7, though its localisation to Y10 cannot be ruled out , S2. WitComparison of the sites of phosphorylation in M1 proteins of influenza A and B viruses indicates a number of common features . In aligThe N-terminal domain of influenza A virus M1 has been crystallised and has a flattened shape, with its opposing faces being positively and negatively charged T37 and T108 both form part of another surface of the N-terminal domain, in this case in loops that pass between the positively and negatively charged faces . T108 isThe C-terminal domain of influenza A virus M1 has not been resolved by X-ray crystallography, but a combination of modelling and experimental studies suggest that it consists of alpha helices connected by loops The M2 protein forms a tetrameric ion channel in the viral envelope and is subject to a number of post-translational modifications, including disulphide bond formation, palmitoylation, fatty acylation, and phosphorylation We clearly detected phosphorylation of either S64 or T65 in M2 of influenza A viruses even, in the case of the PR8-derived M2 of the CVVs, without using optimised conditions , S2. WhiThe M2 protein is translated from a spliced version of the mRNA encoding M1, with splicing taking place in codon nine of the M1 open reading frame. As the nine N-terminal residues common to M1 and M2 do not include a tryptic cleavage site, the N-termini of the two proteins could be clearly distinguished in this study. Despite being common to both proteins, residues S2/T5 and T9 are phosphorylated in M1 but not in M2. This is presumably a difference in phosphorylation, due to the different N-terminal structures of the two proteins. However, we cannot exclude the possibility that this difference is due to a chance failure to detect phosphorylated M2 peptides.The M2 proteins of influenza A, B and C viruses are all phosphoproteins Mutating the cytoplasmic loop serine of influenza A and C viruses reduces the ability of M2 dimers to assemble into tetramers The non-structural protein NS1 is known to be phosphorylated, predominantly on threonine A recently published report noted phosphorylation of S48 The nuclear export protein (NEP) of influenza A and B viruses is involved in the nuclear export of the viral genome in the form of ribonucleoprotein complexes (RNPs) The NEP phosphorylation site is adjacent to a previously identified nuclear export signal (NES) As with NLSs, phosphorylation in or near to NESs can promote or inhibit nuclear export through a range of mechanisms The haemagglutinin (HA) and neuraminidase (NA) proteins of influenza viruses are known to be subject to post-translational modification, notably glycosylation For HA of the influenza A virus WSN (H1 subtype), we detected phosphorylation of T358. After cleavage of HA0, T358 forms residue 15 of the fusion peptide of the HA2 fragment, which inserts into the endosomal membrane to allow viral fusion  [72], , anFor WSN virus without the use of optimised conditions, phosphorylation was readily detected at either S402 or S403 . We deteOptimised mass spectrometry conditions allowed us to detect additional sites of NP phosphorylation in the influenza A virus WSN. In virions, phosphorylation was detected for S9/Y10, S165 , S457 and T472/S473. In RNPs purified from infected cell lysates phosphorylation was detected at S9/Y10, S165 , Y296/S297, and S377/S378. All of these residues are highly conserved in influenza A viruses, with the exception of S377 , and S473 . Replacing the residue with glutamic acid, which is approximately similar in size and charge to phosphoserine, reduced the titre by approximately 20-fold at 30 h p.i., suggesting that although S402 phosphorylation is readily detected in the virion, constitutive phosphorylation of this residue is not optimal for viral growth. Although S403 is an alanine in the majority of influenza A virus strains, an S403A mutation caused a 10–100-fold growth defect at 30 h p.i., suggesting a specific requirement of WSN for serine at this position. A similar defect was seen when both S402 and S403 were mutated to alanines, removing the phosphorylation sites entirely (data not shown).To assess the roles of NP phosphorylation sites in transcription and replication  we performed RNP reconstitutions in 293 T cells . The majMass spectrometry allows the detection of specific sites of protein phosphorylation. By applying this technique to a selection of influenza A viruses and an influenza B virus we have identified 39 phosphorylation sites, 36 of them novel. Unmodified peptides were also detected, consistent with phosphorylation typically acting on a subset of the proteins present in the cell. Strikingly, we found that a number of phosphorylation sites were conserved between different strains and even genera, underlining the fundamental importance of phosphorylation in the life cycles of influenza viruses.Patterns of phosphorylation were similar between WSN grown in MDBK cells and reassortants of the similar PR8 virus grown in embryonated eggs and MDCK cells. This suggests that the similarities between these viruses are more important in determining phosphorylation patterns than the substantial differences between the hosts in which they were grown. From this it is reasonable to infer that the phosphorylation sites reported in this study will, in most cases, be similar to those found in other hosts, including in natural infections. Of the phosphorylation sites detected in PR8 reassortants only one was not also detected in WSN . As detection of phosphorylation by mass spectrometry is a stochastic process we consider this likely to reflect sampling variation rather than a difference in phosphorylation patterns. For the same reason, failure to detect phosphorylation at particular sites in this study , nuclear localisation , and multimerisation . In addition, a number of phosphorylations did not have an obvious function, and it is likely that some of these phosphorylations are non-essential, arising through interactions with cellular kinases that confer no fitness advantage to the virus The functions of a number of phosphorylation sites in NP of the influenza A virus WSN were tested experimentally, and were found to contribute to viral growth in cell culture and to RNP activity to varying extents. In arguing for a functional role for phosphorylation, studies of this sort are suggestive, though further studies will be required to address the possibility that the mutations may introduce unrelated structural changes, or that viral fitness and/or RNP activity may depend on the residues being present but unphosphorylated. However, by combining arguments from evolutionary conservation, structural context and experimental evidence, a convincing case can be made for the existence of multiple functional phosphorylation sites.Like most viral proteins NP is multi-functional Phosphorylation provides a promising target for antiviral chemotherapy, particularly as targeting cellular kinases reduces the capacity for viral escape mutations. Treatment of cells with protein kinase inhibitors interferes with multiple stages in the influenza virus life cycle, including nuclear import, transcription, protein synthesis, nuclear export and viral budding 2 in Modified Eagle Medium with Earle's salts  supplemented with 2 mM L-glutamine and 10% fetal calf serum (FCS).Madin-Darby Bovine Kidney epithelial (MDBK) cells, Madin-Darby Canine Kidney epithelial (MDCK) cells and 293 T human embryonic kidney cells were maintained at 37°C and 5% COInfluenza A/WSN/33 virus (WSN) Plaque assays were performed on MDBK cells using standard techniques. RNP reconstitutions were performed in 293 T cells using segment 6 (NA) vRNA as a template, and RNA accumulation measured by primer extension, PAGE, autoradiography and phosphorimaging, as described previously g/5 min/4°C, and placed on a rotating wheel for 1 h at 4°C in lysis buffer ). The soluble fraction was separated by centrifugation at 17 000 g/3 min/4°C, diluted 1∶5 with binding buffer ) and incubated overnight at 4°C with 200 µl of 50% suspension Strep-Tactin Superflow high capacity resin (IBA GmbH). The resin was washed four times with wash buffer , and proteins were eluted in 2 ml of elution buffer , 2 mM d-desthiobiotin) for 2 h on a rotating wheel at 4°C. Elution fractions were subsequently concentrated using Amicon Ultra-4 (3K MWCO) centrifugation devices.Affinity purifications of PB1-TAP using a Tandem Affinity Purification (TAP) tag 9–1010 plaque-forming units (PFU) of virus for WSN) at 2 days p.i. The medium was clarified by low-speed centrifugation , then layered onto a cushion of 30% sucrose in NTC  and pelleted by ultracentrifugation (112 000 g/90 min/4°C in an SW 28 rotor (Beckman Coulter)). Pellets were resuspended in NTC and spun through a 30–60% sucrose gradient in NTC (209 000 g/150 min/4°C in an SW 41 Ti rotor (Beckman Coulter)) to produce a visible band of virus which was drawn off with a needle and pelleted through NTC (154 000 g/60 min/4°C in an SW 41 Ti rotor) and resuspended in a small volume of NTC . A similar method was used to purify CVVs from infected eggs. Briefly, infected allantoic fluid was harvested, filtered and mixed with sodium azide. Virus was pelleted by ultracentrifugation, resuspended and spun on 10–40% sucrose gradients to produce a visible band of virus which was harvested and pelleted by ultracentrifugation. Samples of virus were taken to determine plaque titre; separated by SDS-PAGE and Coomassie or Silver stained according to standard techniques; or fixed with 2.5% glutaraldehyde, 2% paraformadehyde and 0.1% picric acid in 100 mM cacodylate buffer (pH 7.0), adsorbed onto formvar-coated grids, negative-stained with 2% aqueous uranyl acetate and examined by transmission electron microscopy using a FEI Tecnai 12 electron microscope.Viruses grown in cell culture were harvested from the growth media of four T175 flasks of infected cells ) to remove detergent and salts; the entire sample was then cut out of the gel with a clean scalpel. As an alternative method, some WSN samples were boiled in 1.25% SDS, precipitated in −20°C acetone, and resuspended in 8 M urea, 25 mM ammonium bicarbonate. Whole cell lysates and TAP-purified samples were separated by SDS-PAGE and stained with Coomassie; bands of the appropriate electrophoretic mobility were excised with a clean scalpel. Samples were then washed with 50 mM ammonium bicarbonate in 50% acetonitrile, reduced with 10 mM DTT and 55 mM iodoacetamide or chloroacetamide and digested with 0.5 µg trypsin (Promega) at 37°C for 16 h. Peptides were extracted with 0.1% formic acid in 50% acetonitrile, lyophilised in a SpeedVac (Thermo Savant) and desalted using an in-house manufactured C18 purification tip. Enrichment for phosphopeptides using TiOAll LC-MS/MS experiments were performed using either an Ultimate 3000 nano HPLC system  run in direct injection mode, coupled to an LTQ XL Orbitrap mass spectrometer or a Q Exactive mass spectrometer . Separation of peptides was performed by reverse-phase chromatography using a 15 cm (LTQ XL Orbitrap) or 25 cm (Q Exactive) by 75 µm inner diameter picotip analytical column , packed in house with Reprosil-Pur C18-AQ phase, 3 µm particle size , at a flow rate of 300 nl/min. Samples were typically resolved on a 120 min gradient. The LTQ XL Orbitrap mass spectrometer was operated in a “Top 5” and the Q Exactive in a “Top 10” data-dependent acquisition mode. Charge state +1 ions were rejected from selection and fragmentation and dynamic exclusion with 40 s was enabled.Bos taurus for MDBK cells, Canis lupus familiaris for MDCK cells, Gallus gallus for chicken eggs and Homo sapiens for 293 T cells) and with common contaminants and decoy sequences. For WSN the viral proteome was expanded to include experimentally confirmed and hypothetical proteins, as well as a translation of all six complete forward and reverse-sense reading frames from each viral segment. To identify peptides, CPFP uses iProphet Mass spectra were analysed using the Central Proteomics Facilities Pipeline (CPFP) http://platform.gisaid.org) or from the NCBI influenza virus resource (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html) and aligned with MAFFT Full-length influenza protein sequences were downloaded from GISAID  Candidate vaccine viruses (CVVs) were purified from embryonated chicken eggs or the culture medium of infected MDCK cells, separated by SDS-PAGE and silver stained. (B) 293 T cells were infected with A/WSN/33 PB2-Cstrep, and affinity purification was used to purify PB2-Cstrep and associated proteins from cell lysates. Proteins were separated by SDS-PAGE and silver stained. Key proteins are identified by electrophoretic mobility. (C) Affinity purification of PB1-TAP (with co-purifying PB2 and PA) and, separately, TAP-NP from transfected 293 T cells. Protein was separated by SDS-PAGE and stained with Coomassie Brilliant Blue; the indicated viral proteins were excised from the gel and submitted for LC-MS/MS.(TIF)Click here for additional data file.Figure S2Coverage of sequences. The full sequences of all proteins to which peptides were matched, with peptides assigned by CPFP shaded. Peptides with N-terminal acetylation (tylation  were sco(PDF)Click here for additional data file.Figure S3Representative spectra. Fragment ion spectra for N-terminal peptides and for phosphorylated peptides, annotated by Mascot or CPFP. The clearest spectrum showing each modification referred to in the text is presented (including overlapping peptides and unmodified peptides where available). Each spectrum was manually inspected.(PDF)Click here for additional data file.Table S1Protein sequence coverage.(DOC)Click here for additional data file.Table S2Summary of phosphopeptides.(DOC)Click here for additional data file.Table S3N-terminal modifications.(DOC)Click here for additional data file.Table S4Number of sequences used in alignments.(DOC)Click here for additional data file.


The control of the growth morphologies of ice crystals is a critical issue in fields as diverse as biomineralization, medicine, biology, civil or food engineering. Such control can be achieved through the ice-shaping properties of specific compounds. The development of synthetic ice-shaping compounds is inspired by the natural occurrence of such properties exhibited by antifreeze proteins. We reveal how a particular zirconium acetate complex is exhibiting ice-shaping properties very similar to that of antifreeze proteins, albeit being a radically different compound. We use these properties as a bioinspired approach to template unique faceted pores in cellular materials. These results suggest that ice-structuring properties are not exclusive to long organic molecules and should broaden the field of investigations and applications of such substances. Crystallization processes are the basis of the very existence of industries. In particular, the control of the growth kinetics and morphologies of ice crystals is far from being only a mere laboratory curiosity, and is of critical industrial importance in fields as diverse as biology, chemistry, medicine, frozen food, or civil engineering. In materials science, the control of the ice morphologies is of particular interest for the ice-templating processing route. Ice-templating is developed as a bioinspired, versatile processing route for porous materials and complex composites, yielding novel hierarchical structures with unprecedented convenience 5, 6).In ice-templating, ice crystals act both as a templating agent for the porosity and their growth as an autoassembly driving force. Ice crystals are removed by sublimation, yielding a complex porous scaffold where the porosity is a replica of the ice crystals. The characteristics and morphologies of the porous scaffolds are the results of complex interactions occurring between the growing crystals and the suspended particles during the solidification step. The number of parameters to be accounted for and their interdependence make any prediction and control extremely challenging (7-10) and the results unpredictable.One key objective of current investigations is to gain a robust and predictive control of the porous structure, independently of nature and characteristics of the particles being used. The easiest way to achieve this is to control directly the morphology of the solvent crystals at the crystallographic level. Additives can affect numerous parameters of the system, such as the viscosity, surface tension, pH, freezing temperature, interactions between the particles in suspension, and so forth, all of them having an influence over the crystal growth characteristics. Due to this complexity, the investigations related to the influence of additives have so far been exclusively empirical  complex, very similar to those of the so-called antifreeze proteins (AFPs). We discovered these properties by accident, while investigating the depletion-induced dispersive properties of ZRA in colloidal suspensions. ZRA is also able to alter the growth morphologies of ice crystals while maintaining their crystal structure. We use this effect to control the morphology of porous materials. ZRA represent a novel family of ice structuring compounds, previously unidentified, and could therefore broaden the field of investigations of such substances.Many living organisms and natural species are able to survive at relatively low temperatures in harsh environments. Their survival is ensured through a supercooling of their body fluids, by as much as 2.2°C, effectively preventing or delaying ice crystallization. These antifreeze effects are originating from the AFPs Most of ISPs substances nevertheless suffer from inherent common limitations: their low availability, extremely high price, and low convenience of use. Although some food-grade ISP can be made for a few dollars per gram, easily available, inexpensive and stable alternatives are still highly desirable. The vast majority of ISPs identified to date share some common characteristics, such as a long organic chain with amphiphatic structures, and a precise surface-surface complementarity with the exposed ice surface Ice crystals in a colloidal suspension grow in a dendritic manner, typically exhibiting a lamellar or cellular morphology with a dendritic surface . When ZRThe effect of ZRA is almost independent of the nature of the particles being used in the freezing suspension. Faceting is observed with both oxides  and covalent (silicon carbide) ceramics  or organ16-18). When added to water, ZRA is known to exhibit three different ionic complexes, depending on the pH 3A and Zr(OH)3A2-, A being the acetate group. The present results point towards the Zr(OH)3A2- complex (type 3), which is the dominant complex at pH>3. It is therefore radically different from the long organic compounds currently investigated or synthesized for their ice structuring properties.Very little is actually known about ZRA. It is mostly investigated for its stabilization properties of suspensions . Above the isoelectric point, ice has a negative surface charge. The Zr(OH)3A2- complex having also a negative charge, it is not likely that it will adsorb at a negative surface. Also of interest is the observation that this pH range, around the isoelectric point of ice, corresponds to the lowest surface charges of ice. Moving towards lower pH, the surface charge of ice is increasing, and the resulting electrostatic interactions between the ZRA complex and the ice surface might perturb the interactions controlling the growth kinetics and morphologies.The degradation of faceting at pH>4.3 might be explained by the change of surface charge of ice. Ice exhibits an isoelectric point, in the range 3-4.6  to decorate the crystals boundaries and reveal their morphologies. The in situ radiography reveals a rounded and isotropic morphology of the ice crystals in absence of ZRA . CrystalThe macroscopic growth kinetics (propagation of the freezing front) in presence of ceramic particles reveals no difference when ZRA is incorporated in the suspension . Its effThe incorporation of ZRA results in the presence of zirconium oxide in the ice-templated samples, which can therefore be used as a marker, revealing the location of ZRA in the frozen structures. The SEM observations  reveal aWe determined the crystallographic structure and orientation of the growing crystals in particles suspensions, using in situ X-Ray diffraction (XRD), on a setup equipped with a cooling stage. The suspension is cooled at 5°C/min, and during cooling, the (100) and (002) peaks of ice are followed in the 22-25° 2θ range . As soonAt lower growth velocities, for instance in the conditions where in situ freezing was achieved , the cryISPs molecules attach to equivalent planes around the crystals , and eveA non-colligative effect of the concentration of ZRA is observed. Such behavior is typical of ISPs too, where a threshold of concentration below which the AFP cannot inhibit ice growth exists. Increasing the concentration of ZRA or ISPs above the threshold does not affect the faceting effects or antifreeze mechanisms observed. Such behavior is believed to arise from the adsorption/inhibition mechanism of ISPs at the surface of ice crystals.ZRA shares some similarities with common ISPs. The moiety is similar to that of ISPs, with hydroxyl and acetate groups. Acetic acid is known to adsorb at the surface of ice and dimerise there 3A2- complex is controlling the incorporation of water molecules onto the surface of the ice crystals. The conformation of this ionic complex should exhibit enough affinities with the ice crystal lattice to adsorb at the surface. The incorporation of water molecules onto the growing surface effectively becomes the rate-limiting step, resulting in a faceted growth.The morphology of growing crystals is dependent of their growth rate-limiting step. The growth rate of rough crystals is controlled by diffusion of water molecules to the growing surfaces. Faceting of the crystals is observed in a diffusion free regime, where the rate-limiting step is the incorporation of molecules into the crystals 3A2- ionic complex (molecular weight: 327), a structure very different from the long organic molecules, which exhibit a typical molecular weight of 5 000-50 000 The ice-shaping mechanism is very similar to that of the usual ISPs and ISPs substitutes known to date. The most striking difference with currently used and developed compounds is their nature and size. Compounds that can bind and shape ice in biological systems have up to now all been macromolecules - proteins, glycoproteins or polysaccharides. Salts, including acetates, have been reported to enhance the effects of these macromolecules in binding to ice, but have not been reported, so far, to have an ice shaping activity on their own. The active antifreeze form of ZRA is the Zr(OH)The two most interesting aspects from a practical and industrial point of view are the stability and the price, ZRA being an inexpensive (approximately 0.1$/g) and easy to synthesize compound, in particular in comparison to AFPs, which typically sells for 5 000 to 10 000$/g. The inorganic nature might improve the stability, in comparison to the organic alternatives, which use is restricted to low and mild temperatures. Beyond ZRA, this suggests that we may now look in a new direction in the quest and investigations of ice structuring compounds, not being limited to long organic compounds.The first application of this novel ice-structuring compound is demonstrated here in the processing of porous materials by ice-templating. We are able to process bulk ceramic and polymer samples exhibiting a microhoneycomb structure, with a pore size of a few micrometers, continuous over more than a centimeter. To the best of our knowledge, such structures cannot be processed otherwise. Microhoneycombs of silicon have been processed by complex microtechnology and etching approaches, which are all materials-specific, and cannot be used to obtain structures of large dimensions (centimeters). Such structures should be of particular interest for applications as microreactors in chemical engineering, and the versatile nature of the process will allow selecting the right material, depending on the targeted application. Because the size of the cavities can be tailored, the fabrication of sound wave guides for a specific frequency sound absorption could also be achieved by this technique. Beyond porous materials, such substances can also be used to control biomineralization, and open new avenues in the autoassembly processes.The suspensions are prepared by mixing the ceramic powder or the polymer in aqueous suspension, the zirconium acetate and the binder  AIRVOL 205 sold by Air Products & Chemicals, Inc. or polyethylene glycol PEG6M sold by Merck) in distilled water. Experiments are carried out with either α-Alumina , yttria-stabilized zirconia , silicon carbide  or PTFE . Two zirconium acetate were tested, both in-house prepared (Saint-Gobain) and commercially available . The amount of zirconium acetate in the suspension corresponds to a concentration of zirconium provided by the zirconium acetate (g/L). This concentration is evaluated by measuring the mass of zirconia obtained after having subjected the compound introducing the zirconium acetate to stoving at 110°C for 16 hours, and then to baking in air (loss on ignition) at 1000°C for 2 hours. The mass of zirconium is obtained by multiplying the weighted mass of zirconia by the ratio of their molar masses, i.e. about 91/123. The concentration of zirconium provided by zirconium acetate is obtained by dividing this mass of zirconium, in grammes, by the volume in litres of the liquid phase of the suspension. Zirconium acetate is first mixed with distilled water; the binder is then added, preferably after having been dissolved in water, and the ceramic powder is finally added. The pH is measured between 30 minutes and 1 hour after the introduction of the last constituent. The addition of zirconium acetate may suffice to stabilize the pH of the slip within these ranges. If such is not the case, the pH may be adjusted by adding organic and/or inorganic acids or bases. The suspension is then ball-milled for 10 hours.Acta Materialia55, 1965 (2007) or S. Deville, E. Saiz, R. K. Nalla, A. P. Tomsia, Science311, 515 (2006).The suspension is poured into a PTFE mould and cooled from the bottom, using a liquid-nitrogen cooled copper rod. The cooling rates are adjusted through a thermocouple and a ring heater placed around the copper rod. Details of the experimental setup can be found in previous papers, such as S. Deville, E. Saiz, A. P. Tomsia, Once freezing is completed, the samples are freeze-dried for at least 48 hrs in a commercial freeze-dryer , to ensure a complete removal of the ice crystals.A binder removal step is performed with the following cycle: temperature rise at a rate of 600°C/h up to 500°C, steady stage of 1 hour at 500°C, temperature decrease to room temperature. Ceramic samples are densified by a high temperature sintering treatment. The sintering cycle of zirconia samples is the following: temperature rise at a rate of 600°C/h up to 1350°C, steady stage of 3 hours at 1350°C, temperature decrease at a rate of 600°C/h to room temperature. The sintering cycle of alumina samples is the following: temperature rise at a rate of 300°C/h up to 1350°C, steady stage of 3 hours at 1350°C, temperature decrease at a rate of 300°C/h to room temperature. The silicon carbide samples shown in the SOM are not sintered.http://imagej.nih.gov/ij/, 1997-2011). The mean pore size is determined by analysis of these images according to the erosion/expansion method described in Maire et al., J. Eur. Ceram. Soc., 27SEM observations are performed using either a TM1000 from Hitachi or a Nova NanoSEM 230 from FEI. The EPMA analysis is performed with a JEOL JXA8530F. For pore size measurements, the samples are infiltrated with an epoxy resin, and the cross-section perpendicular to the freezing direction mirror-polished before observation. The SEM micrographs were analyzed using the ImageJ software  and (002) peaks of ice are followed in the 22-25° 2θ range, with a Bruker LynxEye linear detector. As soon as the peaks are detected, meaning that the ice crystals reached the sample surface, a complete acquisition is performed in the 20-60° 2θ range. This procedure ensures that no ice-crystals formed from crystallization of the ambient humidity are observed.3. The energy is set to 20.5 keV. The distance between the sample and the detector is 20 mm. Because of the extremely high coherence of the X-ray beam on this beam line, absorption is not the only source of contrast in the obtained radiographs and phase contrast is also present, but in a small amount. A set of 1200 projections is taken within 180°. The detector was a CCD camera with 2048×2048 sensitive elements coupled with an X-ray-sensitive laser screen. Dynamics of solidification is followed by X-ray radiography, with an acquisition frequency of 3 Hz, which allows to precisely follow the interface evolution in two dimensions. The frozen structures after complete solidification are characterized in three dimensions afterwards using a low speed high-resolution tomography acquisition.The solutions are prepared by dissolving the zirconium acetate in distilled water, and adding potassium iodide  as a marker. The freezing and frozen solutions are scanned using a high-resolution X-ray tomograph located at the ESRF (beam line ID 19) in Grenoble (France). X-ray tomography is performed at a voxel size of (1.4 µm)Figure S1SEM micrographs of ice-templated silicon carbide with zirconium acetate (18 g/L of Zr), perpendicular to the solidification direction. Scale bar: 40 µm.(TIF)Click here for additional data file.Figure S2SEM micrographs of ice-templated PTFE with zirconium acetate (18 g/L of Zr), perpendicular to the solidification direction. Scale bar: 60 µm.(TIF)Click here for additional data file.


However, stimulation with the cAMP-elevating agent forskolin and the β-adrenergic receptor agonist CL 316,243 resulted in a PKA (protein kinase A)-dependent phosphorylation and 14-3-3 binding of SIK2. Phosphopeptide mapping of SIK2 revealed several sites phosphorylated in response to cAMP induction, including Ser358. Site-directed mutagenesis demonstrated that phosphorylation of Ser358, but not the previously reported PKA site Ser587, was required for 14-3-3 binding. Immunocytochemistry illustrated that the localization of exogenously expressed SIK2 in HEK (human embryonic kidney)-293 cells was exclusively cytosolic and remained unchanged after cAMP elevation. Fractionation of adipocytes, however, revealed a significant increase of wild-type, but not Ser358Ala, HA (haemagglutinin)–SIK2 in the cytosol and a concomitant decrease in a particulate fraction after CL 316,243 treatment. This supports a phosphorylation-dependent relocalization in adipocytes. We hypothesize that regulation of SIK2 by cAMP could play a role for the critical effects of this second messenger on lipid metabolism in adipocytes.SIK2  is a member of the AMPK (AMP-activated protein kinase) family of kinases and is highly expressed in adipocytes. We investigated the regulation of SIK2 in adipocytes in response to cellular stimuli with relevance for adipocyte function and/or AMPK signalling. None of the treatments, including insulin, cAMP inducers or AICAR (5-amino-4-imidazolecarboxamide riboside), affected SIK2 activity towards peptide or protein substrates  SIK  2, also called QIK (Qin-induced kinase), is an AMPK (AMP-activated protein kinase)-related kinase family member  with abu175 by LKB1, which is required for SIK2 catalytic activity, as demonstrated by the complete lack of activity in the Thr175Ala mutant, or in LKB1-deficient cells ATP was from PerkinElmer and phosphocellulose P81 paper was from Whatman. Hydromount was from National Diagnostics. AMARA peptide, HDAC5tide (where HDAC is histone deacetylase) and IRS1tide (where IRS1 is insulin receptor substrate1) were synthesized by Dr Grahame Bloomberg  and Sakamototide was synthesized by Pepceuticals. Adenoviral vectors for HA–SIK2 wild-type, Ser358Ala and Ser587Ala were produced by Vector BioLabs.3T3-L1 cells were from A.T.C.C. and DMEM (Dulbecco's modified Eagle's medium), FBS , dexamethasone, IBMX (isobutylmethylxanthine), insulin (differentiation of 3T3-L1 fibroblasts), phenformin, forskolin, ionomycin, CL 316,243, tetracycline, fish skin gelatine and HA (haemagglutinin)–agarose were all from Sigma. Flp-in™ T-REx™-293 cells, Flp-recombinase, blasticidin B, hygromycin, Hoechst nuclear stain, pre-cast Novex SDS/PAGE Bis-Tris gels and SDS sample buffer were all from Invitrogen. Penicillin/streptomycin was from VWR. Complete protease inhibitor cocktail was from Roche and AICAR was from Toronto Research Chemicals. H89 was from Biomol and insulin (for stimulation) was from Novo Nordisk. Protein G–Sepharose and glutathione–Sepharose were from GE Healthcare. [γ-563], anti-AMPK, anti-(AMPK phosphoThr172), anti-COX (cytochrome c oxidase) IV, anti-phosphoPKA substrate and anti-phosphoPKB substrate antibodies were all from Cell Signalling Technology. Anti-(PKB phosphoSer473) was from Invitrogen, anti-HA, anti-GAPDH  and anti-FLAG–HRP (horseradish peroxidase) antibodies were all from Sigma, anti-14-3-3 antibody was from Santa Cruz Biotechnology and anti-H3 antibody was from Millipore. Anti-(Na/K ATPase) antibody was from Novus Biologicals. The following antibodies were raised in rabbit and affinity-purified by Innovagen: anti-SIK2  used for immunoprecipitation and immunoblotting and anti-(SIK2 phosphoSer358)  used for immunoblotting. An antibody against phosphorylated T-loop SIK isoforms (residues 175–189 of human SIK1) was raised in sheep, and purified by the DSTT , University of Dundee, Dundee, U.K. The anti-BMH1/2 antibody used for 14-3-3 far-Western bloting was a gift from Professor Carol MacKintosh, University of Dundee, Dundee, U.K. Anti-AMPKα1 antibody for the immunoprecipitation kinase assay was provided by Professor D. Grahame Hardie, University of Dundee, Dundee, U.K. Secondary antibodies conjugated with HRP were from Invitrogen (anti-rabbit), Pierce (anti-sheep) and GE Healthcare (anti-mouse). Alexa Fluor™ 594 anti-mouse antibody was purchased from Invitrogen.The following primary antibodies were used for Western blotting: anti- was performed overnight in DMEM containing 5% FBS, 1% penicillin/streptomycin and 0.5% BSA at 37°C. Adipocytes were subsequently resuspended in KRH (Krebs–Ringer–Hepes), defined in ATP (300 c.p.m./pmol) and 0.5 μg of HDAC5 (1–550) protein for 20 min at 30°C. Following polyacrylamide gel electrophoresis and colloidal Coomassie Blue staining, the gels were fixed, dried and analysed in an FLA 3000 Fujifilm reader using Fuji imaging plates type BAS-III. One unit of activity was defined as that which catalysed the transfer of 1 nmol of 32P/min to the substrate.Cell lysates (5–50 μg) were incubated for 1 h on a shaking platform with 3 μg of anti-SIK2 or 1 μg of anti-AMPKα1 antibody conjugated to 5 μl of packed Protein G–Sepharose, alternatively anti-HA–agarose. The IPs (immunoprecipitates) were then further processed as described in . Phosphoisoforms , HDAC5tiproteins ,21, was proteins . The stoEscherichia coli-expressed GST–14-3-3ζ bound to glutathione–Sepharose (described below), were heated in SDS sample buffer, before loading on to a precast 4–12% Novex gradient gel and transferred on to a nitrocellulose membrane as described previously ATP-labelled peptides followed by analysis with Edman sequencing (results not shown). HDAC5tide and Sakamototide were selected as peptide substrates for SIK2 kinase activity measurements.The kinase domain of SIK isoforms and AMPK are well conserved and SIK has been shown to phosphorylate AMPK-targeted sequences in both intact cells and in cell-free assays. In order to identify more selective and efficient substrates for the measurement of SIK2 activity, we evaluated four peptides  derived from known AMPK- and SIK-targeted residues in ACC , CRTC2 (Sakamototide), HDAC5 (HDAC5tide) and IRS1 (IRS1tide). All substrates were tested using purified recombinant wild-type or kinase-inactive  GST–SIK2 expressed in HEK-293 cells A, as welubstrate C and 1D.2+ ionophore ionomycin and insulin /ATP ratio, the cAMP-elevating agent forskolin, the Ca insulin A. These onomycin B. PKB Sen of PKA A. None oDAC5tide A and SakDAC5tide C. ActiviDAC5tide A. Phosph 316,243 B. None oTo investigate the requirement of PKA for cAMP-induced phosphorylation of SIK2, we used the PKA inhibitor H89. As shown in 3-adrenergic receptor agonist CL 316,243 respectively  and Ser90 (HEK-293 cells). These phosphopeptides, however, displayed low abundancy and were therefore not studied further. The region containing potential T-loop phosphorylations, for example Thr175, was not covered in our analysis, probably owing to the large size of this tryptic peptide (65 amino acids). Colloidal Coomassie Blue staining, excision and mass fingerprinting of the relevant bands in the HA–IP revealed that SIK2 bound to all seven of the 14-3-3 isoforms   A and 4B.343, Ser358, Thr484 and Ser587) were subjected to site-directed mutagenesis analysis in HEK-293 cells, in order to investigate their role in the cAMP-induced phosphorylation and 14-3-3 binding of SIK2. Mutation of Thr484 and Ser587 to alanine did not affect the overall PKA phosphorylation or the 14-3-3 binding of HA–SIK2, as judged by the presence of endogenous 14-3-3 in the anti-HA–SIK2 IPs  phosphorylation was observed between the wild-type and Ser358Ala SIK2, or following forskolin stimulation.Selected sites that emerged from the phosphopeptide mapping  SIK1 was monitored as a control. Confocal imaging revealed that the subcellular localization of wild-type and Ser575Ala SIK1 was nuclear in non-treated cells  to obtain a cytosol and crude particulate fractions. The fractions were characterized using cytosolic (GAPDH), nucleic (H3), mitochondrial (COX IV) and plasma membrane (Na/K ATPase) markers. The particulate fraction contained nuclei, mitochondria and plasma membrane (P=0.0121). This coincided with a significant decrease in wild-type HA–SIK2 in the crude particulate fraction (P=0.0027), suggesting a relocalization of SIK2 upon cAMP stimulation. These data demonstrate a functional importance of Ser358 phosphorylation for the regulation of SIK2 localization in response to cAMP in adipocytes.A common function of 14-3-3 proteins is to trap its binding partners in the cytosol , and we membrane C, but no358, rather than Ser587, is the main site phosphorylated and responsible for the binding of SIK2 to 14-3-3 proteins. In contrast with SIK1, SIK3 and the Par-1/MARK family of AMPK-related kinases, which bind 14-3-3 in resting cells  in 3T3-L1 adipocytes  do not participate in SIK2 regulation in adipocytes.AMPK is known to be activated in response to elevation of intracellular [Caipocytes  and otheipocytes –35. Furt of SIK2 , may be gnalling –37. Alth358, binding to 14-3-3 and a subsequent movement away from potential substrates. We hypothesize that SIK2 in this manner could mediate effects of cAMP on adipocyte gene expression or participate in the acute regulation of lipid storage by cAMP, such as catecholamine-induced lipolysis.A key question that remains to be addressed is the biological function of SIK2 in adipocytes, and how it is affected by the regulation we describe. Recently, a total body SIK2 knockout mouse model was described, with no major effects on body mass . One cou


The highest levels of biliary metabolite and PAH in water were measured at the Odra River (the Bohumín site), which is located in relatively heavily industrialized and polluted region. Statistically significant positive correlation between biliary 1-OHP and sum of PAH in water was also obtained .This study investigated polycyclic aromatic hydrocarbons (PAH) pollution of selected rivers in the Czech Republic. Integrated evaluation was carried out using combination of chemical and biological monitoring, in which we measured content of 1-hydroxypyrene (1-OHP) in chub bile and priority PAH in water samples obtained by exposing the semipermeable membrane devices at each location. The concentrations of 1-OHP in bile samples and sum of priority PAH in water sampler ranged from 6.8 ng mg protein The monitoring of sum of these priority pollutants is often used for assessment of PAH occurrence in the ecosystem anthracene; it was detectable only at two sites.The results of concentration of sixteen priority PAH in water samples obtained by exposing the SPMD at sampling sites are summarized in In time of SPMD exposing, indicator fish were caught and bile samples were collected for determination of 1-OHP at all sites. The numbers of individuals, length, and weight of chubs for each location are given in −1), and the highest one at the Bohumín site, the Odra River (165.2 ng mg protein−1). The same trend was also recorded in the average values at the monitored locations. The highest mean value was observed at the Bohumín site—the Odra River (106.6 ± 30.2 ng mg protein−1), and it was significantly (P < 0.05) higher than those obtained at the Obříství site—the Labe River (6.8 ± 2.1 ng mg protein−1); the Schmilka site—the Labe River (12.1 ± 2.1 ng mg protein−1); the Topělec site—the Otava River (12.6 ± 1.3 ng mg protein−1); the Srbsko site—the Berounka River (15.4 ± 2.2 ng mg protein−1); the Zelčín site—the Vltava River (18.2 ± 1.0 ng mg protein−1); the Nespeky site, the Sázava River (18.8 ± 2.3 ng mg protein−1); the Pohansko site, the Dyje River (22.7 ± 3.7 ng mg protein−1); and the Bechyně site, the Lužnice River (23.0 ± 6.7 ng mg protein−1). No significant differences were found between the Bohumín site, the Odra River and the Lanžhot site, the Morava River (44.6 ± 7.3 ng mg protein−1) and the Židlochovice site, the Svratka River (72.8 ± 9.0 ng mg protein−1).The results of biliary 1-OHP are presented in P < 0.01) between biliary 1-OHP and sum of priority PAH was also obtained (rs = 0.806).Highly statistically significant positive correlation  formation of DNA adducts in liver and blood [Chemical monitoring such as measurement of pollutants content in the aquatic environment  and residue levels in the aquatic organisms is the most widely used method for assessment of aquatic contamination. Unfortunately, the presence of contaminants does not, by itself, indicate adverse effects on the organisms; it is appropriate to combine chemical monitoring with biological assessment. During biological assessment the responses of aquatic organisms to pollution stress can be evaluated by measurement of biochemical markers levels. Biomarker is defined as a change in a biological response which can be related to exposure to or toxic effects of environmental chemicals . Many exnd blood , 16. Resnd blood . In our nd blood .Leuciscus cephalus L.) [Oreochromis niloticus) [Anguilla anguilla) [Gadus morhua) [Pleuronectes vetulus) [Rutilus rutilus L.), [Salmo trutta m. fario L.) [Based on the previous studies , 11, 19,alus L.) , 19, nilloticus) , Europeanguilla) , Atlanti morhua) , Englishvetulus) , roach (ario L.) , 9.−1 to 165.2 ng mg protein−1. The mean content of 1-OHP varied significantly between several sites, with the highest mean concentration in bile from the Odra River at Bohumín site. This mean concentration was more than fifteen times higher than that at the Obříství site, the Labe River, where the lowest content of 1-OHP was detected. Results of biliary metabolite correspond with concentrations of priority PAH in water samples, because the highest PAH contamination was also observed at the Bohumín site, Odra River, and the lowest one at the Obříství site—the Labe River. The PAH levels at the Bohumín site—Odra River were more than thirty times higher than those at the Obříství site—the Labe River. The Odra River flows through a heavily industrialized  and urbanized region in the north of the Czech Republic. Several studies confirmed that the occurrence of various pollutants including PAH arises from the intensive anthropogenic activities in this region [−1) to 1,500 ng L−1. The maximum detected value is several times higher than we present in our study. The differences may be caused by different sampling methods, because in our study we used continuous sampling using SPMD, while in the study by Wolska and Namieśnik [In this study we detected 1-OHP in all bile samples, the individual results were very variable and ranged from 2.2 ng mg proteins region –23. Blahs region  monitores region  studied amieśnik  the convamieśnik . In receamieśnik , 24–26.P < 0.01, rs = 0.806) between biliary 1-OHP and PAH in water samples. Similar findings also document positive relationship between biliary metabolites and content of PAH in both water [Leuciscus cephalus L.) and bream (Abramis brama), the correlation coefficients were 0.81 and 0.76, respectively. They also confirmed relationship between 1-OHP and content of pyrene in sediment . This finding indirectly confirms the assumption of an appropriate relation between pyrene concentration and sum of priority PAH occurring in particular environmental compartment.The results from our study show strong positive and significant correlation (th water , 13 and th water . Hosnedlth water  documentIn conclusion, the results of the presented study indicate that the use of 1-OHP as biomarker in fish is useful tool for monitoring of PAH contamination in the aquatic ecosystem. Determination of this metabolite is also frequently used as a useful biochemical marker for assessment of human exposure to PAH, especially in smokers, drivers, or workers in aluminum plants, oil refineries, and coal-burning facilities. The analyses of 1-OHP in human are provided in urine samples , 29.


Wheat protein is responsible for various kinds of allergic disease. Wheat-dependent exercise-induced anaphylaxis (WDEIA) is one of the most important clinical phenotypes of adult wheat allergy. Relatively homogenous clinical and immunological presentations of this phenotype have already been described. More recently, some studies have shown that sensitization to hydrolyzed wheat protein (HWP) in cosmetics can induce allergy to HWP-containing food as well as contact allergy to HWP-containing cosmetics.We experienced treating five Japanese women with WDEIA with atypical presentation. They presented exercise-induced anaphylaxis after ingestion of normal wheat products, but also had episodes of skin and/or rhinoconjunctival contact allergy to hydrolyzed wheat protein (HWP)-containing facial soap. Furthermore, case histories and serological analyses of these patients indicated that the development of their WDEIA was induced by primary sensitization to HWP in the facial soap they used and accompanying sensitization to natural wheat protein.All of the patients had started to use the same facial soap product containing HWP 1 to 3 years prior to the onset of WDEIA. At first, they used the facial soap without any problem. When they continued to use the soap, they started to experience itchiness and urticaria of the eyelids or face after using the soap. Their symptoms of allergy to facial soap resembled prodromal symptoms that develop following the combination of wheat ingestion and exercise. A significantly higher IgE reactivity to HWP than to natural wheat protein was observed in these patients. Inhibition analyses of their sera showed that IgE reactivity to HWP was not inhibited by natural wheat extracts, whereas those to natural wheat extracts were fully inhibited by HWP, indicating that the primary sensitizer of these patients is HWP.Our report is important in that it indicates the possible role of HWP included in cosmetics in the induction of allergy to natural wheat products.


LCA did notinhibit the enzymatic kinase activity of EphA2 at 100 µM (LANCE method)confirming to target the Eph-ephrin protein-protein interaction. Finally, LCAinhibited cell rounding and retraction induced by EphA2 activation in PC3 cells.In conclusion, our findings identified a hit compound useful for the developmentof molecules targeting ephrin system. Moreover, as ephrin signalling is a keyplayer in the intestinal cell renewal, our work could provide an interestingstarting point for further investigations about the role of LCA in theintestinal homeostasis.Eph-ephrin system plays a central role in a large variety of human cancers. Infact, alterated expression and/or de-regulated function of Eph-ephrin systempromotes tumorigenesis and development of a more aggressive and metastatictumour phenotype. In particular EphA2 upregulation is correlated with tumourstage and progression and the expression of EphA2 in non-trasformed cellsinduces malignant transformation and confers tumorigenic potential. Based onthese evidences our aim was to identify small molecules able to modulateEphA2-ephrinA1 activity through an ELISA-based binding screening. We identifiedlithocholic acid (LCA) as a competitive and reversible ligand inhibitingEphA2-ephrinA1 interaction (Ki = 49 µM). Since eachephrin binds many Eph receptors, also LCA does not discriminate betweendifferent Eph-ephrin binding suggesting an interaction with a highly conservedregion of Eph receptor family. Structurally related bile acids neither inhibitedEph-ephrin binding nor affected Eph phosphorylation. Conversely, LCA inhibitedEphA2 phosphorylation induced by ephrinA1-Fc in PC3 and HT29 human prostate andcolon adenocarcinoma cell lines (IC The Eph receptor tyrosine kinases belong to the largest family of tyrosine kinasereceptors. To date 16 members, across many species, have been identified Eph-ephrin system has been extensively studied in embryogenesis where it plays acritical role in tissue boundaries formation and neuronal circuits development High levels of this receptor have been found in several cancer types including brain,lung, breast, ovarian, prostate, colorectal, and kidney malignancies. MoreoverephrinA1, the physiological EphA2 receptor ligand, is often downregulated when EphA2is up-regulated and vice versa For all these reasons EphA2 receptor represents a promising target in cancer therapyand different strategies are under evaluation by several research groups in order todevelop specific kinase inhibitors or ligands able to interfere with protein-proteininteraction.Although a first class of small molecule able to antagonize ephrin binding to theEphA4 and EphA2 receptors has been recently identified With this in mind, we performed an ELISA binding assay screening on an“in-house” chemical library . This apAll culture media and supplements were purchased from Lonza. Recombinant proteinsand antibodies were from R&D systems. Cells were purchased from ECACC.Leupeptin, aprotinin, NP40, MTT, tween20, BSA and salts for solutions were fromApplichem; bile acids, EDTA and sodium orthovanadate were from Sigma. Human IgGFc fragment was from Millipore (AG714).2 at37°C.PC3 human prostate adenocarcinoma cells were grown in Ham F12 supplemented with7% fetal bovine serum (FBS) and 1% antibiotic solution. HT-29human colon adenocarcinoma cells were maintained in EMEM supplemented with10% FBS, 1%NEAA, 1% sodium piruvate and 1%antibiotic solution. T47D human breast tumor cells were grown in RPMI 1640 with10% FBS and 1% antibiotic solution. All cell lines were grown in ahumidified atmosphere of 95% air, 5% CO2PO4, 1.15 g/lNa2HPO4, pH 7.4). The day after wells were washedthree times with washing buffer  andblocked with 300 µl of blocking solution (PBS +0.5% BSA) for 1hour at 37°C. Compounds were added to the wells at proper concentration in1% dimethyl sulfoxide (DMSO) and incubated at 37°C for 1 hour.Biotinylated ephrinA1-Fc (R&D BT602) was added at 37°C for 4 hours at 30ng/ml in displacement assays or in a range from 1 to 2000 ng/ml in saturationstudies. Wells were washed three times and incubated with 100 µl/wellStreptavidin-HRP (Sigma S5512) solution  for 20 minutes at room temperature, then washedagain for three times and incubated at room temperature with 0.1 mg/mltetra-methylbenzidine (Sigma T2885) reconstituted in stable peroxide buffer and 0.02%H2O2 (30% m/m in water), added immediatelybefore use. The reaction was stopped with 3N HCl 100 µl/well and theabsorbance was measured using an ELISA plate reader  at 450 nm.Our chemical collection  was stoc50 value was determined using one-site competition non-linearregression and Kd values of the curves with or without antagonists werecalculated using one-binding site non-linear regression analysis with Prismsoftware (GraphPad Software Inc.). The Ki was obtained using Schildplot The IC2, 100 µl per well. The plates werewashed three times with PBS and blocked for 1 hour with BSA 1%. Cellswere treated with proper concentration of substance or DMSO 0.5% for 30minutes in vials on a shaker. After that, plates were washed with PBS andincubated with 100 µl 5×105 cells/ml for 1 hour at37°C. Finally, plates were washed with PBS and adhering cells werequantified using MTT colorimetric assay.96 well ELISA high binding plates were incubated overnight at 4°C withephrinA1-Fc 1 µg/cm5cells/ml incomplete medium until they reached ∼40% confluence and serum starvedovernight. The day after cells were treated with compounds under study, vehicleor standard drug, stimulated with the proper agonist, rinsed with sterile PBSand solubilized in lysis buffer. The lysates were resuspended and rocked at4°C for 30 minutes and then centrifuged at 14000 xg for 5 minutes. Theprotein content of supernatant was measured with BCA protein assay kit (Thermoscientific), standardized to 200 µg/ml and transferred into a clean testtube ready to be used.Cells were seeded in 12-well plates at concentration of 10EphA2-, EphB4- and EGFR-phosphorylation were measured in cell lysates usingDuoSet®IC Sandwich ELISA  following manufacturer's protocol. Briefly, 96 well ELISAhigh binding plates (costar 2592) were incubated overnight at room temperaturewith 100 µl/well of the specific capture antibody diluted in sterile PBSto the proper working concentrations. The day after wells were washed andblocked for 1 hour at room temperature. After that, wells were washed and 100µl/well of lysates were added at room temperature for 2 hours; wells werewashed and incubated with Detection Antibody at room temperature for 2 hours.The phosphorylation was revealed utilizing a standard HRP format with acolorimetric reaction read at 450 nm.4 cells/well and preincubated for 5 min at 22°C inpresence of either HBSS or LCA. Cells were stimulated with 5 nM IGF1 or 100 nMinsulin for 10 minutes, lysed and a fluorescence acceptor  added for 2 hours. Afluorescence donor (streptavidin coupled-beads) coated with an antibody towardsIRKβ or IGFR1β was incubated for 2 hours and the signal was measured atλex = 680 nm and λem = 500 nmand 600 nm using a microplate reader .Stimulant or inhibitory effects of LCA towards IRKβ and IGFR1β activitywere tested using alpha Technology  in HepG2 orA431 cells, respectively. Cells were seeded in microplates at4×104 cells/well into 96-well plate, the following daygrowth media was exchanged with HBSS buffer +20 mM HEPES and cells wereallowed to equilibrate for 75 min with or without 100 µM LCA andstimulated with 0.1 nM VEGF. Impedance measurement was monitored for 10 minutes.VEGF receptor tyrosine kinase inhibitor II  was used as a standard reference.Stimulant or inhibitory activities of LCA towards VEGFR activity were tested inHUVE cells using cellular dielectric spectroscopy. Cells were seeded at5×10All results were expressed as a percent inhibition of the control response to 5nM IGF1, 100 nM insulin or 0.1 nM VEGF. These assays were performed at CEREP.Evaluation of LCA effects on the kinase activity of human EphA2 was performed bymeasuring the phosphorylation of the substrate Ulight-TK peptide (50 nM) usingthe LANCE detection method 5cells/ml and the day aftertreated with compounds or 0.5% DMSO for 2, 24, 48, or 72 hours. MTT wasadded at the final concentration of 1 mg/ml and incubated for 2 hours. Theresulting formazan crystals were washed with PBS 100 µl/well and thensolubilized with DMSO 200 µl/well. The absorbance was measured at 550 nmusing an ELISA plate reader and the results were expressed as the ratio betweenabsorbance of the cell treated with the compounds and untreated cells.Cell viability was evaluated using the MTT colorimetric assay. Cells were platedin 96-well plates at a density of 10PC3 cells were grown on 12-well culture plates and starved overnight in Ham-F12medium with 0.5% FBS. DMSO  orcompounds were incubated for 20 min, before stimulation with 0.5 µg/mlephrinA1-Fc or Fc for 30 minutes. During this time cells were observed andpictures were taken from the same field, before and after incubation, under amicroscope . Cell rounding was evaluated using ImageJ program.To identify compounds interfering with EphA2-ephrinA1 binding we immobilizedEphA2-Fc- ectodomain on proper ELISA plates and binding ofbiotinylated-ephrin-A1-Fc was detected using the colorimetric reaction developedby streptavidine-HRP and tetramethylbenzidine.Selectivity and specificity of the assay were tested using not-biotinylatedephrin-A1-Fc as a ligand of the EphA2-Fc receptor .As expeAll the compounds of the chemical collection were incubated for 1 hour at theconcentration of 200 µM and only lithocholic acid resulted tosignificantly reduce EphA2-ephrinA1 binding.50) we charted a displacement curveusing increasing concentration of LCA (in the range of 12.5–400 µM)towards biotinylated-ephrinA1-Fc at a concentration corresponding to itsKD. In these conditions we obtained a dose-dependent displacementand we calculated a pIC50 of 4.24±0.068  having a slope of0.8618. A slope between 0.8 and 1.2 is associated with a competitive binding.Finally, the pKi resulting from the intersection of the interpolated line withthe X-axis resulted to be equal to 4.31±0.03 (corresponding to a Ki of 49µM). We repeated displacement experiments incubating 200 µM LCA for1 hour and washing some wells before adding 50 ng/ml ephrinA1-Fc. Thedisplacement was detected only where the washing was not performed, suggestingthe reversibility of the LCA binding to EphA2-ephrinA1 system , deoxycholic (DCA) and chenodeoxycholic (CDCA). CA, DCA and CDCA did notdisplaced EphA2-ephrinA1 binding whereas LCA decreased it by68.2%±4.3. In order to calculate the inhibitory concentrationreducing binding of 50% , whereas incubation with LCAup to 100 µM did not modify enzymatic activity  which was recognized by an Europium-labeled anti-phospho antibodyand resulted in light emission . LCA 100 µM was completely inactive when tested towards thephosphorylation of EGF receptors induced by EGF both in PC3 and HT29 cells whereas Previous studies showed that PC3 cells express mainly EphA2 receptors and theiractivation lead to cell retraction and rounding In the present work we showed for the first time the interaction of lithocholic acid(LCA), a secondary bile acid, with Eph-ephrin system. We demonstrated that LCAcaused a reversible and competitive displacement of the biotinylated ligandephrinA1-Fc from the receptor EphA2-Fc in cell-free binding studies and we pointedout the antagonistic properties of LCA in phosphorylation studies in different celllines.Bile acids had been considered for long time only as detergent molecules necessaryfor lipid solubilization and absorption in the intestine during digestion. However,many studies have explored the hypothesis that bile acids also work as regulatorymolecules. A recent paper Consistently, our work suggests that LCA can act through the interaction withspecific receptors. In fact we provided evidence of a competitive antagonism towardsEph-ephrin binding. First of all we obtained the proper displacement of saturationcurves, the proper slope of the Schild plot and the reversibility of the binding. Insecond place LCA inhibited Eph-kinase phosphorylation induced by ephrinA1-Fc on PC3and HT29 cell lines but it did not affect enzymatic activity confirming to targetthe Eph-ephrin protein-protein interaction. Conversely, LCA was inactive againstother RTKs such as EGFR, VEGFR IRKβ, IGFR1β in cellular functional studiesdemonstrating that LCA interfered neither with kinase domain nor withprotein-protein interaction of these RTKs. Moreover, the Ki of LCA towardsEphA2-ephrinA1 interaction was six times lower than its critical micelleconcentration Since Eph–ephrin binding is highly promiscuous also LCA does not discriminateEph-receptor subclasses A and B. Therefore we can speculate an interaction with ahighly conserved region essential for both EphA and EphB receptor binding to theirphysiological ligands. Ephrin recognition by Eph receptors is mediated by ephrinG–H loop (key) that inserts into a hydrophobic Eph receptor channel shaped byD–E and J–K loops (lock). LCA interfered with Eph-ephrin protein-protein interaction with a higher affinitytowards ephrin A system than ephrin B system. Functional studies, carried out ondifferent cell lines, showed that LCA is an antagonist of Eph receptors, because itwas able to inhibit the phosphorylation of both EphA2 and EphB4 receptors whenstimulated with ephrinA1-Fc and ephrinB2-Fc, respectively. Notably, in accordance tobinding studies, LCA showed to have a higher efficacy in inhibiting EphA2phosphorylation than EphB4 phosphorylation.The inhibition of Eph-ephrin system could be very useful in the regulation of tumorprogression. In fact, several studies highlighted an important role forEphA2-ephrinA1 and EphB4-ephrinB2 interaction in tumor angiogenesis Moreover, the present work showed that LCA was able to inhibit cell rounding andretraction in PC3 cell line upon EphA2 stimulation, suggesting that this moleculecan actually antagonize the effects mediated by EphA2. The inhibition of EphA2 couldbe advantageous in cancer therapy whenever EphA2 activation mediates tumorprogression as previously demonstrated on mammary tumors and melanoma cells invivo. Consequently, LCA could play a role in the intestinal homeostasisand an alteration of its physiological amount could modify the expression and thesignaling of ephrin receptors and ligands. In this way the correct segregation,proliferation and differentiation mechanisms, underlying the tissue homeostasis,could be alterated. Therefore, our findings could be useful for further studiesaimed to explain the correlation between the concentration of fecal secondary bileacids (mainly DCA and LCA) and the colorectal cancer incidence, highlighted byseveral epidemiology studies In addition to the pharmacological features our discovery suggests intriguingpathophysiological implications. In fact, the expression levels of Eph-receptors andephrin-ligands have a critical role in the organizing cell renewal of the intestineTable S1List of the compounds used in the ELISA-based binding study.(PDF)Click here for additional data file.Figure S1EphrinA1-Fc competitively displaced biotinylated-ephrinA1-Fc binding toEphA2. The Calculated Ki was 102 ng/ml and the Hill slope was 1.19.(PDF)Click here for additional data file.Figure S2Cytotoxicity of bile acids on PC3, HT29 and T47D cells after 2 hours ofincubation with the indicated compounds. Data are means±SEM.(PDF)Click here for additional data file.


In allergic asthma, IgE increases airway remodelling but the mechanism is incompletely understood. Airway remodelling consists of two independent events increased cell numbers and enhanced extracellular matrix deposition, and the mechanism by which IgE up-regulates cell proliferation and extracellular matrix deposition by human airway smooth muscle cells in asthma is unclear.Characterise the role of the two IgE receptors and associated signalling cascades in airway smooth muscle cell remodelling.Primary human airway smooth muscle cells  were stimulated with human purified antibody-activated IgE. Proliferation was determined by direct cell counts. Total collagen deposition was determined by Sircol; collagen species deposition by ELISA. IgE receptors were silenced by siRNA and mitogen activated protein kinase (MAPK) signalling was blocked by chemical inhibitors.IgE dose-dependently increased extracellular matrix and collagen deposition by airway smooth muscle cells as well as their proliferation. Specifically in cells of asthma patients IgE increased the deposition of collagen-type-I, -III, –VII and fibronectin, but did not affect the deposition of collagens type-IV. IgE stimulated collagen type-I and type-VII deposition through IgE receptor-I and Erk1/2 MAPK. Proliferation and deposition of collagens type-III and fibronectin involved both IgE receptors as well as Erk1/2 and p38 MAPK. Pre-incubation (30 minutes) with Omalizumab prevented all remodelling effects completely. We observed no changes in gelatinase activity or their inhibitors.Our study provides the molecular biological mechanism by which IgE increases airway remodelling in asthma through increased airway smooth muscle cell proliferation and deposition of pro-inflammatory collagens and fibronectin. Blocking IgE action prevents several aspects of airway smooth muscle cell remodelling. Our findings may explain the recently described reduction of airway wall thickness in severe asthma patients treated with humanised anti-IgE antibodies. Increased IgE is a major pathology of allergic asthma which stimulates chronic inflammation and airway wall thickening leading to narrowing of the airway lumen Airway wall remodelling consists of several independent mechanisms including (i) sub-epithelial mesenchymal cell proliferation; (ii) increased extracellular matrix (ECM) deposition; and (iii) changes of the local ECM composition The thickening of the airway wall in asthma is largely caused by hypertrophy and hyperplasia of airway smooth muscle cells (ASMC) which express and respond to the Igε−RI and Igε−RII There is evidence that allergens and IgE can penetrate through the basement membrane towards tissue forming cells and activate them. In asthma, the function of the epithelium as a barrier is deranged and thus may allow allergens to get into direct contact with ASMC Therefore, we investigated the direct effect of purified human IgE on proliferation, ECM and collagen species deposition in ASMC derived from asthma and non-asthma patients.Bronchial biopsies were obtained from 16 patients  after written consent and approval by the local ethic committee of both Basel, Switzerland (Ref. Nr.: EK 05/06). None of the participants used any anti-asthma therapy for 1 week prior to the study. ASMC were isolated as described earlier Highly purified human IgE  was incubated overnight with an activating anti-IgE antibody before stimulating ASMC for 24, 48 or 72 hours. Purified human IgG treatment (Diatek) served as control. Prior to experiments confluent ASMC were serum starved for 24 hours. IgE activity was blocked by the humanized clinically used anti-IgE antibody Omalizumab .bis-(3-ethylbenzo thiazoline)-6-sulfonic acid was added for 20 minutes and stopped by 0.3N H2SO4. The absorbance (405/490 nm) was read with ELISA plate reader  The ECM composition was analysed by ELISA by a modified procedure published earlier 3H]-proline incorporation in confluent ASMC as described earlier The deposition of total ECM and collagens was determined by [MAPK Erk1/2 and p38 were inhibited with 0, 1, or 5 µM of either SB203580 (p38 MAPK) or PD98059  for 24 hours before being stimulated.To study the role of the two IgE receptors on IgE-induced ECM deposition, cells were pre-treated with 0, 1, or 5 µM of IgE receptor specific siRNAs (Santa Cruz Biotech) for 24 hours. After IgE stimulation, half the concentration of the siRNAs was added every 24 hours.Total RNA was extracted using a Qiagen kit following the instructions of the distributor. Real time PCR was performed for Col1A1, Col3A1, Col4A1, Col7A1 and fibronectin using the primers and conditions listed in 5/cm2 and serum deprived for 24 hours before stimulation. After 2 days, the cells were harvested and manually counted in a hematocytometer (improved Neugebaur) Cells were seeded at a density of 1×10The Null-hypothesis was that neither IgE nor Omalizumab affected ECM deposition and composition. Data analysis was performed with ANOVA (Bonferroni correction) and p<0.05 was regarded as significant.In The deposition of total ECM and of total collagens was dose dependently increased by IgE over 48 hours . IgG stiInvestigating the effect of IgE on different collagen types we observed a dose dependent significant increase of collagen type-I deposition by ASMC of asthma patients, while the effect on non-asthma patient cells was less strong . TGF-β1 Collagen type-III deposition was higher in cells from asthma patients compared to controls, which was further increased by TGF-β1 and IgE stimulation . IgG didIgE did not up-regulate collagen type-IV deposition, while TGF-β1 doubled its deposition, while IgG had no effect . CombineCollagen type-VII deposition was stimulated by TGF-β1; but for IgE only the two highest concentrations up-regulated its deposition by ASMC of asthma patients only, IgG had no effect . Pre-incBased on earlier studies with human serum containing high levels of IgE, we investigated its effect on fibronectin deposition. As shown in Finally, we observed that IgE dose-dependently stimulated the proliferation of ASMC, with a significantly stronger effect on ASMC of asthma patients at concentrations >1 µg/ml . FCS 5%. The rolAs shown in This study presents further evidence that IgE directly activates ASMC and thereby modifies the composition of the ECM during airway wall remodelling. Our data indicate that the IgE induces the deposition of the pro-inflammatory collagens type-I and fibronectin mainly independent of up-regulating transcription of the corresponding gens. This effect of IgE on ASMC is mainly mediated through Igε-RI and ERK1/2 MAPK activity. The same signalling pathway also controls IgE-induced ASMC proliferation. Despite the observation that Igε-RI is the major IgE receptor, Igε-RII and p38 MAPK also contribute to airway remodelling, however, to a much lower extend.3 indicates that airway remodelling is a much faster and more flexible pathology than expected. Airway remodelling in asthma was earlier linked to a disease specific pathological structure and function of the airway wall forming cells and ECM composition The hypothesis that IgE actively induces airway wall remodelling has recently being supported by two clinical studies performed in patients with severe asthma Collagen type-I increased ASMC proliferation, while it is not clear if it alters ASMC contractility The content of collagen type-III in airway wall was increased in childhood and adult asthma; but only in adults, the ratio of collagen type-III in the airways is correlated with severity rgens increased the deposition of collagen type-VII in the basement membrane which was linked to the loss of epithelial cell integrity In the context of airway inflammation, the role of collagen type-VII has not been studied well. In a rhesus monkey model inhaled house dust mite allein vivo deposition of fibronectin in asthmatic airway and found either no change or even a reduction The role of fibronectin in asthma is controversial. Two studies analysed the Our study indicates that the major effect of IgE on remodelling is mediated via Igε-RI and ERK1/2 MAPK and explains the increase of collagen type-I and –III deposition as well as of proliferation. In other cell types, including epithelial and mast cells, IgεR-I activated both Erk1/2 and p38 MAPK de novo synthesis and deposition of pro-inflammatory collagen types in the airways. If this observation could be confirmed in long term therapy with anti-IgE antibodies, it may explain the clinically documented improvement of lung function.Our data indicate that anti-IgE therapy may have a reductive effect on airway remodelling by reducing the


Retrospective case series.To identify the variation of C2 vertebral artery groove (VAG) based on the thin-slice computed tomography (CT) scan and choose an individual screw placement method to decrease risk of malposition.C2 pedicle screws can be successful anchors for a variety of cervical disorders. However, variations of VAG may cause malposition and breach when C2 transpedicle screw was inserted. Recognizing the variations of vertebrae artery groove (VAG) in C2 and choosing an individual screw placement method (transpedicle or translaminar) may be helpful for avoiding violation and decreasing the operation risk in upper cervical surgery.e  and parameter a . Subsequently, individual strategy was used to avoid the VAG violation.From January 2009 to December 2010, a total 45 patients with upper cervical disorders underwent 1–mm-thin-slice CT scans along the C2 pedicle direction to obtain the consecutive spectrum of C2 VAG were included in this study. The C2 VAG  was subgrouped based on parameter n = 74); translaminar screws were inserted in type II subgroup (n = 16). Postoperative CT scans showed that there were two pedicle screws violated into the artery groove, and no translaminar screw breached into the vertebrae canal. All the other screws were in right position. None of the 45 patients had severe complications such as spinal cord injury, dura tear, and infection.The variations of C2 VAG in these 45 patients include the following: type I 53 (58.9 %), type II 16 (17.8 %) type III 13 (14.4 %), and type IV 8 (8.9 %). Transpedicle screws of C2 were used in types I, III, and IV VAGs  with minimal complication for C2 screw fixation. Segmental screw fixation of the upper cervical spine using transpedicle screws at C1/C2 and/or laminar screws at C2 are becoming increasingly popular . HoweverFrom January 2009 to December 2010, there were total 45 consecutive patients with upper cervical disorders received preoperative thin-slice (1 mm thickness) CT scans along C2 pedicle direction to obtain the consecutive picture of C2 VAG  and parameter e . For more easy use of “safe zone” on the consecutive CT scan pictures, parameter e could be measured by the number of scan layer for the VAG apex just show, and parameter a could be measured by the diameters of the C2 pedicle on the VAG entrance slice of CT scan  was performed to assess the extent of decompression of the spinal cord. Dynamic X-rays and CT scans with reconstruction view were performed during follow-up visits at 3, 6, and 12 months after surgery to check the position of the implants and bone fusion. All the patients were followed up for 13–28 months (mean 14 ± 3 months). The JOA score was used to evaluated the spinal function and improvement 3 months after surgery.p < 0.05. Statistical analyses were performed using SPSS 13.0 for windows.A paired Student’s test was performed for pre- and postoperative result. The level of significance was set at Total 45 consecutive patients were included in this study, consisting of 25 men and 20 women with a mean age of 43 years (range from 15 to 63 years). 41 patients had segmental screw fixation of C1–C2 alone: 15 patients with os odontoideum associated with atlantoaxial dislocation, three with rheumatoid arthritis with evidence of instability or impaction, 23 with traumatic atlantoaxial dislocation. The other four patients underwent C2 screw placement as a part of an occipitocervical reconstruction: two patients with traumatic C1 lateral mass fracture and two had atlas tuberculosis.a and parameter e measured in the thin-slice CT scan spectrum of C2, the subgroup of C2 groove in these 45 patients includes the following: type I 53 (58.9 %), type II 16 (17.8 %) type III 13 (14.4 %), and type IV 8 (8.9 %). In the types I, III, and IV grooves, the pedicle size was confirmed to be large enough to accommodate a 3.5-mm screw; therefore, 73 transpedicle screws of C2 were used. The other 17 C2 VAG were recognized as type II subgroup because the groove shows a high ride shape, and the diameter of axis pedicle in the last layer is very small (lesser than 4.5 mm).The “safe zone” for screw implant is poor, while the thickness of lamina is enough for 3.5-mm screw placement; therefore, 17 translaminar C2 screws were inserted.According to the parameter p < 0.01 %), MRI showed that the mean cervicomedullary angle (CMA) changed from 136.3° ± 8.8° to 159.5° ± 9.22° (p < 0.01 %). All patients had different extents of improvement on the spinal function, and the average JOA improve from 9.5 ± 1.3 to 14.8 ± 1.6 (p < 0.01 %).The postoperative CT scan shows that there are two pedicle screws violated into the artery groove Figs. , 5, , a kind of CT scan spectrum of C2 VAG could be easily obtained. Compared with the single-slice image of C2 pedicle, the consecutive CT scan spectrum could provide us a kind of holography of the C2 VAG with more integrated and rich information. Through this C2 VAG CT spectrum, we could reconstruct a kind of model figure of C2 VAG easily just like the three-dimensional reconstruction CT image based on parameter a and e” in the coronal section of pedicle will provide a “safe zone” for screws placement. The larger the a/e value is, the safer for pedicle screw implantation. When the a/e lesser than 4.5/4.5 mm (type II subgroup), it is difficult and dangerous to place a 3.5 mm screw, as violation could happen easily.When inserting the screw through C2 pedicle, the room surrounded by the “a and e” is bigger than 4.5 × 4.5 mm, which could provide a relative safe room for pedicle placement. However, in the subgroup type II, the “safe zone” of “a and e” is lesser than 4.5 × 4.5 mm, which should be regard as contradiction for pedicle screw placement. Therefore, an alternative method of C2 translaminar screw should be recommended. The major advantage of C2 translaminar screw is the elimination of the potential risk of arterial injury by placing screws only within the posterior column [For the subgroup of types I, III, IV, the “safe zone” of “r column . In our r column –13. Yeom