Our general approach yields an experimental descriptor for the intrinsic activity and the active-site utilization, aiding in the catalyst development process and enabling a previously unachieved level of understanding of reactivity trends owing to a deconvolution of site density and intrinsic activity.Carbon materials doped with transition metal and nitrogen are highly active, non-precious metal catalysts for the electrochemical conversion of molecular oxygen in fuel cells, metal air batteries, and electrolytic processes. However, accurate measurement of their intrinsic turn-over frequency and active-site density based on metal centres in bulk and surface has remained difficult to date, which has hampered a more rational catalyst design. Here we report a successful quantification of bulk and surface-based active-site density and associated turn-over frequency values of mono- and bimetallic Fe/N-doped carbons using a combination of chemisorption, desorption and  Iron and nitrogen doped carbon materials are widely studied electrocatalysts, however measurement of features such as intrinsic turn-over frequency and active site utilization has proved difficult. Here, the authors use a combination of chemisorption and spectroscopy techniques to determine these properties. Accordingly, current key goals in non-precious metal ORR catalysis comprise molecular insight in the catalytically active site15592459The electrochemical reduction of oxygen  in acid and alkaline environments is a tremendously important reaction for a variety of emerging electrochemical energy technologies and device components such as cathodes of metal air batteries234782510112A recent report by Zelenay and co-workersOur incomplete understanding of the role of the metal ion in the active-site structure originates in large parts from the lack of a suitable technique to probe the chemical composition, density and reactivity of the active sites in these types of catalysts. Molecular probes, such as cyanide and carbon monoxide, have been used in a number of unsuccessful efforts to poison active catalytic sites in rotating disk electrode (RDE) measurements262757Fe Mössbauer spectroscopy, we derive a measure for the active-site utilization of the (Fe-based) NPMCs, quantifying the limits of further activity enhancement. Moreover, the direct comparison of TOFs of mono- and bimetallic catalysts uncovers previously unexplored synergistic effects between two dissimilar metal ions in the enhancement of the ORR activity in alkaline.In the present work, we use PANI-derived Me–N–C catalyst materials as model systems to demonstrate the benefit of a combined active-site analysis by Mössbauer spectroscopy and CO pulse chemisorption/desorption. Unlike earlier studies, low-temperature CO pulse chemisorption and temperature-programmed desorption (TPD) are successfully applied as chemical interrogation tools to obtain quantitative insights into the density of active adsorption sites of the catalysts. To the best of our knowledge, this is the first time that pulse chemisorption and TPD are successfully applied to quantify surface adsorption sites of Me–N–C materials. Our analysis correlates the active-site density with the apparent catalytic reactivity in acid and alkaline media, yielding surface site-based metal-specific catalytic turn-over frequencies (TOFs). In combination with 2 to H2O, while their Tafel Plots . The results are summarized in To assess the stability of the catalysts, we performed a severe accelerated stress test (AST) by cycling the potential between 0.5 V2–H2) and the relative change in activity in dependence on the cycle number are shown in et al.et al. up to 10,000 cyclesThe fuel cell polarization curves, cycling stability plots O–H2 and t57Fe Mössbauer spectroscopy1721To obtain atomic-scale insight into catalytically active Fe centres, the two Fe-containing catalysts were investigated using 4 site (D1) and a ferrous intermediate-spin FeN4 site (D2). In previous reports, the D1-related site was identified as ORR active site in Fe–N–C catalysts1116172021IIIN4)–O2 site (D3)4 site 203+). Induced by the reduced particle size, these particles appear X-ray amorphous. It should be noted that the Mössbauer parameters of our doublet D4 are close to values previously found for a NFeN2+2…NH+/C site20−1 for δIso and 0.4 mm s−1 for ΔEQ). In this case, the higher isomer shift is caused by the additional coordination of our FeN4 site by two weak ligands in comparison with the fivefold-coordinated site NFeN2+2 in accordance with literature. Assuming that our D4 doublet should be assigned to that particular FeN2+2…NH+/C site, a significantly larger mass-related activity for the Fe–N–C catalyst versus the bimetallic catalyst would be expected, as only the Fe–N–C catalyst contains this site that is reported as catalytically highly active one. In addition, this catalytic site is ‘famous' for its fast deactivation, whereas all of our catalysts are rather stable in PEM-FC and RDE. Therefore, we assume that our D4 site is indeed different from that site.The first two doublets are typical for Fe–N–C catalysts16172021iso, and the quadrupole splittings, ΔEQ, for the different iron sites being similar in all catalysts, we conclude that there is no evidence for an alloy-like electronic interaction between iron and manganese. On the other hand, the data evidenced that the two catalysts contained substantially smaller fractions of inorganic Fe species (here only oxidized iron particles) compared with PANI-based catalysts reported earlier53S4, iron nitride and iron carbide. We attribute the absence of these phases in our catalysts to the smaller amount of ammonium peroxidisulphate (APS) employed in the synthesis . Consequently, other iron sulphide modifications were formed that were removed more effectively during AL. This effect was previously also found for other Me–N–C catalysts33With the isomer shifts, δmax).From the relative absorption area of each iron site and the absolute iron content in the final catalyst, one can estimate the relative abundance of each type of iron site. These values are given in x–peak is missing.X-ray photoelectron spectroscopy (XPS) was used to probe the near-surface nitrogen content of the Me–N–C catalysts. It confirmed the presence of a high fraction of nitrogen as required for the chemical coordination of the metal sites. 3,2 edge of both Mn-containing samples are compared. The catalysts reveal similar fine structures of two main contributions A1, A2 that appear at 639 eV, 640.5 eV and 649 eV, 651 eV, respectively, for the L3 and L2 edges. In addition, there are slight contributions B at 642.5 eV for L3 (653 eV: L2-edge). The fine structure resembles that of MnPc/Au measured under an incident beam angle of 30°, which is close to our experimental conditions. These energies describe the charge transfer from Mn 2p3/2 (L3) and 2p1/2 (L2) states into hybridized Mn d-statesx environment.X-ray absorption near edge spectroscopy (XANES) measurements were performed to characterize the Mn oxidation state in the two manganese-containing catalysts. In To quantify the density of active adsorption sites at the surface of the catalysts, CO pulse chemisorption experiments at 193 K followed by TPD of CO were carried out –N–C–3HT–2AL shows two separate desorption peaks. The slight down- and up-shift observed in the individual desorption peak temperatures of Mn and Fe sites in this bimetallic catalyst, respectively, seem to indicate some differences in their detailed CO desorption kinetics compared with the monometallic catalysts. Furthermore, comparing the experimental TPD peak integrals with the actual metal ratio of Fe:Mn=3:1 . Hence, it appears that the bimetallic nature has no impact on the ORR characteristics in acid, while we have seen that it substantially improves the catalyst stability during ASTs in acidic electrolyte ≈10 electrons per site per s)21120482 diffusion.Comparing our values with those previously reported, we achieve one of the highest TOF values for NPMC. The only TOF value that exceeds ours is related to a binary active site composed of a high-spin NFeNcatalystnCO (in nmol mg−1) equals the molar concentration of active surface sites contributing to the ORR process. By multiplying the measured values with Avogardo's constant NA, we get the mass-based surface site density MSD (2).Beside the TOF, the density of active sites is another important parameter for the optimization of catalysts. If each catalytic active site is blocked by one CO molecule, the molar amount of adsorbed CO per mg2 or CO molecules. Indeed, it was shown previously for Fe–N–C catalysts that fractions of D1-related sites are ‘hidden' in the bulk of the material and consequently do not contribute to the ORR activity164 sites provides a measure of the maximum number of possible ORR active sites referred to as the maximum mass-based site density MSDmax, given the perfect accessibility to all active FeN4 sites. Based on previous reported correlations between ORR activity and our D1 motif, we exclusively ascribe the ORR activity to the D1-related FeN4 species1617max can be calculated from Mössbauer spectroscopy by We would now like to compare our CO chemisorption derived mass-based site densities, MSD, with analogous mass-based site densities, derived from Mössbauer spectroscopy. However, we would like to point out the difference in the distinct probing characteristics of both methods: Mössbauer spectroscopy is a bulk method probing the total density of specific Fe sites, typically deviating from the number of Fe sites actually accessible for adsorption of OD1 [wt%] denotes the iron concentration of the D1 motif . For reasons of comparison, we have included two columns in max) either considering all FeN4 sites or only the D1-related FeN4 sites.We would like to point out that the exclusive assignment of ORR activity to D1 was based on the following requirements that we demand for an ORR active site: (1) a significant contribution of the site to the ORR based on structure activity correlation and (2) the correlation should be of general nature and hold for differently prepared catalysts rather than just a single preparation route. A contribution of D2 can be excluded due to previous results111617To characterize the catalyst performance and their possible enhancement through optimizing the catalyst surface area and mass-based site density, we introduce the ratioCO/MBS is plotted for the Fe-containing catalysts in CO/MBS are very similar for each two catalysts after 2HT–1AL and 3HT–2AL, which supports the notion of ΦCO/MBS representing an experimental meaningful descriptor for physically possible additional activity enhancements. To illustrate this, we point to the green dashed line in CO/MBS=1, which represents the optimal utilization of Fe sites in Me–N–C catalysts. In this case, all active Fe sites in a catalyst would be accessible by O2 (and CO) and contribute to the ORR process. Similarly, ΦCO/MBS offers projections of the maximum achievable catalyst current density of a specific family of catalysts, by dividing the experimentally observed mass activity by ΦCO/MBS. This is important as such projections allow a rapid and objective assessment and comparison of the theoretical promise of a specific preparation route or of specific precursors.to represent an active-site utilization factor quantifying what ratio of the total number of catalytically active sites in a catalyst is actually accessible for the ORR. This utilization factor Φmax. Due to the AL that was involved not only inactive but to the same degree active sites were removed. Hence, the utilization of active sites should have been improved by another approach to enhance the ORR activity. In contrast, assuming by similarity of these three metal combinations similar ΦCO/MBS factors for the Mn–N–C catalysts the change in the utilization factor going from 2HT–1AL to 3HT–2AL equals the increase in ORR activity .For the materials used here, our analysis uncovers that the mass activities could be further enhanced by a factor of 1.5x (for Fe–N–C–3HT–2AL) and 1.9x –N–C–3HT–2AL) provided a suitable preparation. This enhancement could be realized by optimizing the catalyst dispersion such that all active sites are located on accessible pore surfaces. For a better evaluation of the utilization factor, Mössbauer spectroscopy was also applied to the two iron-containing catalysts with 2HT–1AL. Their TOFs, mass-based site densities and the utilization factors are given in et al., was related to a balance of active-site destruction and utilizing active sites that were previously hidden in the bulkBeside the importance of the utilization factor for the catalyst activity (and the number of sites contributing), we believe that the utilization factor will be an important descriptor for the catalyst stability in the general concept of heterogeneous catalysis. For example, the ‘stable' performance of the PANI–Fe–C catalyst in a potentiostatic test at 0.6 V, as described by Ferrandon CO/MBS that serves as a quantitative descriptor for the promise and potential of a specific chosen preparation technique with respect to its activity and stability. As such, ΦCO/MBS enables an accelerated screening of the potential of different catalyst preparation techniques and, hence, is of general importance for future advances in the development of Fe-containing non-precious ORR catalysts.In conclusion, our combined analysis of non-precious ORR catalysts using CO adsorption and Mössbauer spectroscopy offers a previously unachieved quantification of active-site density and intrinsic TOF activity descriptors. The two experimentally accessible site density values yielded an active-site utilization factor Φ3 was added into 2 ml of pure aniline (Sigma-Aldrich) dissolved in 250 ml 0.5 M HCl. This solution was kept stirring for 1 h at 4 °C. Thereafter, 5 g APS ((NH4)2S2O8), as the oxidant, was added drop wise to inform the in situ polymerization of aniline. Finally, Ketjenblack EC-300 J (0.4 g), which was treated first in 0.5 M HCl , then refluxed in concentrated nitric acid  and subsequently washed with H2O was mixed with FeCl3/PANI under constant stirring. The mixture was kept under continuous stirring at room temperature for 48 h. The desired product was then refluxed and vacuum dried to obtain the precursor powder. To achieve the final catalyst, 3HT steps and 2ALs were performed , whereas each preparation was finalized by a HT step. Hence the Fe–N–C–3HT–2AL had the following sequence of preparation steps: HT1−AL1−HT2−AL2−HT3.Synthesis of Me–N–C catalysts involved a multistep preparation that is schemed in  compare . The fir2 was used as the metal precursor for Mn-based catalysts. For bimetallic catalyst, a molar ratio of Mn:Fe=1:1 was chosen as the optimal metal ratio. The nomenclature is similar to the iron-based catalysts.MnCl2 at 900 °C for 1+3 h .The metal-free reference catalyst was prepared similarly, but without any metal chloride during the oxidative polymerization. In this case, the starting precursors went through a HT in N2O, 150 μl isopropanol and 50 μl Nafion (5 wt%). This mixture was ultrasonicated with an US-finger for 15 min while the ink was placed in an ice bath. From this homogeneous ink, 10 μl were dropped on a glassy carbon disc with a diameter of 5 mm yielding a catalyst loading of 0.8 mg cm−2 for the NPMC. The Nafion to catalyst ratio was 1:4. Cyclic voltammetry was first performed at 10 mV s−1 in N2-saturated electrolyte at 1,500 r.p.m. Then, the solution was saturated with oxygen and linear sweep voltammetry was recorded with 10 mV s−1 at 1,500 r.p.m. Catalyst mass activity (Im) in units of mA mgcatalyst−1 was calculated according to Im=Ikin/Lcatalyst, where Lcatalyst is the geometric catalyst loading  and Ikin is the geometric kinetic current density, which was estimated according to: Ikin=I0.8 × Ilim/(Ilim−I0.8), using the geometric current density at 0.8 V versus RHE, I0.8, and the diffusion limited current density, Ilim. Stability tests were performed up to 9,000 potential cycles in the potential range between 0.5 and 1.3 V versus RHE in N2-saturated electrolyte with the scan rate of 50 mV s−1 on stationary electrode. All activity and stability tests were performed at room temperature in 0.1 M HClO4 (pH 1) and 0.1 M KOH (pH 13). All the results have been compared with a reference Pt/C 20 wt% (E-TEK) catalyst with a geometric Pt loading of 10 μgPt.The catalyst ink was prepared by mixing 15.7 mg of catalyst powder with 800 μl H−2 for NPMC and 0.2 mgPt cm−2 as homogeneous catalyst films were formed. The Nafion content in the dry catalyst layer was maintained at ca. 50 wt%. At the anode, Pt 46 wt%  was used. The same spray-painting method was used to form the anode layer. The cathode and anode were hot pressed onto two sides of one piece of a Nafion NRE 212 membrane. The geometric membrane electrode assembly (MEA) area was 10.0 cm2. Fuel cell testing was carried out in a single cell. Pure hydrogen and oxygen, humidified at 80 °C were supplied to the anode and cathode (relative humidity 100%). Noteworthy, the same absolute pressure of 1 bars was applied to both electrodes. Fuel cell polarization plots were recorded using a fuel cell test station . Recorded voltage was corrected for the resistance of the Nafion membrane as well as the recorded current was corrected for hydrogen cross over. The MEA voltage cycling tests were carried out by flow of nitrogen to the cathode in the voltage range of 0.6–1.0 V.The Fe–N–C–3HT–2AL and the –N–C–3HT–2AL catalysts were tested in the fuel cell cathode to evaluate their activity and durability under selected polymer electrolyte fuel cell conditions. The catalyst ink was prepared by ultrasonically mixing the catalyst powder with Nafion suspension  for 30 min. The as-prepared ink was applied to the gas diffusion layer by successive spray-painting until the cathode catalyst loading reached ca. 2 mg cm−1 as carrier gas. For each catalyst, an amount of 100–150 mg was mounted between quartz wool in a quartz reactor assembling in a furnace . The Mössbauer relevant γ-radiation was provided by a 50 mCi Co/Rh source. Calibration of the velocity scale was made with respect to an α-Fe foil. Spectra were fitted with the programme Recoil assuming lorentzian line shape of all absorption sites.Measurements were made for Fe–N–C and –N–C catalysts after 3HT–2AL and 2HT–1AL. About 75–100 mg of each sample were filled into an aluminium sample holder with a diameter of 1.5 cm and a height of 3 mm. The sample holder is closed on both sides by Tesa tape. Hence, in the path of the γ-ray 10–30 μgα X-rays as the excitation source. Peak positions were internally referenced to the C1s peak at 284.6 eV. Fitting of the N1s fine scans were made with the UNIFIT software with 30% Lorentzian line form in the fitting process. In addition, the surface-near elemental composition was determined.X-ray-induced photoelectron spectroscopy was carried out on an AXIS ULTRA DLD (Shimadzu) with monochromated Al KInitial and final metal contents were determined by optical emission spectroscopy using inductive-coupled plasma as ionization source, which was performed by Varian 715-ES. The samples were pre-treated by microwave at 180 °C to digest all moieties of the catalyst including carbon support. In a typical inductive-coupled plasma as ionization source sample preparation, ∼5 mg of the dry sample powder was weighted in a quartz tube and dissolved in a mixture of concentrated acids including: 2 ml sulphuric (98 wt%), 2 ml nitric (69 wt%) and 6 ml chloric (37 wt%) acids. Sample digesting was performed by Microwave (CEM SP-D Discover) at 120 °C for 10 min.How to cite this article: Sahraie, N. R. et al. Quantifying the density and utilization of active sites in non-precious metal oxygen electroreduction catalysts. Nat. Commun. 6:8618 doi: 10.1038/ncomms9618 (2015).Supplementary Figures 1-7, Supplementary Tables 1-7 and Supplementary References


This is an overview of the current knowledge on the plethora of molecular mechanisms tuning the activity of the ATP-consuming Na,K-ATPase to the cellular metabolic activity. Recent findings suggest that oxygen-derived free radicals and H2O2, NO, and oxidized glutathione are the signaling messengers that make the Na,K-ATPase “oxygen-sensitive.” This very ancient signaling pathway targeting thiols of all three subunits of the Na,K-ATPase as well as redox-sensitive kinases sustains the enzyme activity at the “optimal” level avoiding terminal ATP depletion and maintaining the transmembrane ion gradients in cells of anoxia-tolerant species. We acknowledge the complexity of the underlying processes as we characterize the sources of reactive oxygen and nitrogen species production in hypoxic cells, and identify their targets, the reactive thiol groups which, upon modification, impact the enzyme activity. Structured accordingly, this review presents a summary on (i) the sources of free radical production in hypoxic cells, (ii) localization of regulatory thiols within the Na,K-ATPase and the role reversible thiol modifications play in responses of the enzyme to a variety of stimuli  (iii) redox-sensitive regulatory phosphorylation, and (iv) the role of fine modulation of the Na,K-ATPase function in survival success under hypoxic conditions. The co-authors attempted to cover all the contradictions and standing hypotheses in the field and propose the possible future developments in this dynamic area of research, the importance of which is hard to overestimate. Better understanding of the processes underlying successful adaptation strategies will make it possible to harness them and use for treatment of patients with stroke and myocardial infarction, sleep apnoea and high altitude pulmonary oedema, and those undergoing surgical interventions associated with the interruption of blood perfusion.Control over the Na,K-ATPase function plays a central role in adaptation of the organisms to hypoxic and anoxic conditions. As the enzyme itself does not possess O One member of the P-type ATPases family, Na,K-ATPase, transforms the energy of phosphate bonds within the ATP to the energy of transmembrane Na/K gradient that is used to support the excitability of neurons and myocytes, control of intracellular Ca2+ levels and pH, intake of amino acids and fuel, and for sensing and signaling , that existed more than 3 billion years ago on our planet  system. Many other metabolic reactions in the cell also use oxygen as a necessary component. Thus, cells have developed systems that can sense fluctuations in oxygen concentration and perform different adaptations, and there are several links between hypoxia sensing and redox reactions that we briefly explore here.Oxygen is a key component of the cell energy production machinery driving Cambrian explosion ~500 Mio years ago. It plays a role of the final acceptor of the mitochondrial electron transport chain (ETC), which is coupled to ATP production by the H2; its half-life is as short as 2 × 105 M−1s−1 , and an oxidized form, water (H2O). Hydrogen peroxide half-life is quite greater than that of its predecessor, 103–104 M−1s−1 , a reaction that can be catalyzed by the oxidation of Fe2+ into Fe3+ in the so-called Fenton reaction  to form peroxynitrite (ONOO−), with a rate comparable to that of diffusion, 4–6 × 109 M−1s−1  by successive one-electron reductions Figure . Superox2, hypoxia would lower the production of ROS due to the law of mass action, i.e., as there is a lower oxygen concentration, one would expect to observe a decrease in ROS production. However, cell systems are often more complicated than the basic chemical systems in a test tube with multiple players and reactions involved in production and scavenging of ROS and RNS. Apart of thermodynamic and kinetic restrains, compartmentalization within the cell and species/cell type-specific settings affect the capacity of electron donors to react with the oxygen molecules and time course of these reactions. Indeed, there has been a long-standing controversy in the field, as there are a number of observations reporting lower ROS production in hypoxia   includes a fifth complex, complex V or ATP synthase that transforms the energy of proton gradient to phosphorylation of ADP to ATP  consists of four protein complexes within the mitochondrial inner membrane. Complexes I and II oxidize electron carriers NADH and FADH+ and transfers electrons to a series of Fe-S clusters which, in turn, reduce ubiquinone into ubiquinol  .Evidence obtained using transgenic animal models suggest different mitochondrial complexes of the mtETC are involved in generation of superoxide anion participating in redox signaling. Knocking down Rieske iron-sulfur protein (RISP) within the complex III indicates the key role of electron transport by this complex for HIF-1α stabilization under hypoxic conditions , has been suggested to require mitochondrial ETC Therefore, mitochondria-derived ROS produced in the first minutes of hypoxia  and/or their detoxification rate by the so-called antioxidant systems Sies, . Under tAs reviewed below terminal irreversible oxidation of the enzyme has two more consequences. It makes the regulatory thiols inaccessible for reversible thiol modifications and therefore renders the enzyme insensitive to redox changes and alterations in oxygen levels  whenever the latter has been formed.This is a fast reaction may compete with NO binding to the heme of soluble guanylate cyclase sGC heme  multiple targets get S-nitrosylated under hypoxic conditions. This NO synthase has high affinity to OS-glutathionylation , the formation of a mixed disulfide between a protein cysteine and that of glutathione. This thiol modification is produced in a number of chemical reactions  that does not require any catalysis, and several mechanisms requiring prior oxidative modification of the thiol residues .2 as an electron acceptor (reactions 8–9), oxidation of thiols to sulphenic acid (reaction 10–11) or to nitrosothiol (reactions 12–13)Reactions priming thiols to S-glutathionylation include prior formation of thiyl radicals with OS-glutathionylation of thiols in hypoxic cells is triggered by a local shift in GSSG/GSH ratio toward more GSSG H:NAD(P), GSH:GSSG, NO availability; Mieyal et al., Not all thiols are equally accessible for S-glutathionylation. Specific conditions that have to be fulfilled for the thiol to become glutathionylated are discussed below.The likelihood for a given thiol to undergo S-glutathionylation is defined by several factors: (i) dissociation constant pKa of a thiol (ii) microenvironment (amino acid composition in vicinity to the cysteine residue), (iii) accessibility of the group and steric restrictions, and (iv) redox potential of a thiol Nagy, . NucleopSteric restriction and localization of cysteine residue within the protein sequence are yet other parameters that should be taken into consideration when predicting S-glutathionylation sites  cysteine residues Figure . FifteenRegulatory S-glutathionylation Cysteine residues that were found S-glutathionylated within the α1 subunit isolated from duck salt gland were classified as targets for regulatory or basal S-glutathionylation -phosphine, TCEP) could not completely remove glutathione bound to the α subunit's thiols  and FXYD7 (brain-derived) Geering, . Phospho2S levels in a number of cells including chemoreceptory glomus cells  .Regulatory phosphorylation is known to control the activity, sensitivity to the cardiotonic steroids, and availability of the Na,K-ATPase on the plasma membrane. Changes in phosphorylation state of the enzyme were reported in response to hypoxic challenge and for a long time believed to be the only mechanism involved in “channel arrest” response of the Na,K-ATPase in hibernating animals  and novel isoforms PKC δ, ε, η, θ, and μ may be activated diacylglycerol (DAG), phorbol esthers and phosphatidylserine and are sensitive to the changes in Cae Figure .2+- and DAG-insensitive  by 35–50%, along activation of the novel isoforms (δ and ε)  making this isozyme PKC-insensitive. Phosphorylation efficiency may also vary depending on the PKC isoform. Conventional isoforms are more efficient in phosphorylation of the α1 isoform of the pump isolated from rat retinal cells than the novel isoforms δ and ε  at the N-terminus of the protein that forms the actuator domain. Ser18 that is considered as a major phosphorylation site is available for phosphorylation in the E2 conformation . All gasotransmitters are capable of activation of sGC that in turn triggers activation of cGMP-dependent protein kinase PKC (PKG)  is a redox-sensitive enzyme containing regulatory thiols  within the cytosolic M8-M9 loop. This loop may interact with the M10, C-terminus and the third NaIn the purified Na,K-ATPase protein preparation this target is accessible for phosphorylation in the presence of detergent  and initiate several signaling cascades  or systemic reduction in oxygen availability was shown to trigger release of cardiotonic steroids (bufadienolides or endogenous ouabain) into the circulation in rodents and humans  inhibition by thiol modifications (ii) modulation of the enzyme function/availability at the membrane due to the changes in phosphorylation, and (iii) interaction with nanomolar doses of endogenous cardiotonic steroids.2 availability. Based on the current knowledge the following scheme of responses may be suggested  . These conditions most likely favor S-glutathionylation of β subunit that is associated with activation of superoxide production by NOXes  in the presence of < 50 nM ouabain is not caused by its degradation but rather by a drop in protein synthesis (Zhang et al., 2+ levels and free radical production as well as activation of several kinases. Molecular mechanisms linking these processes to the regulation of HIF1α translation remain unclear.Recent data revealed the existence of intimate link between the activity of the Na,K-ATPase and the gene expression in hypoxic cells and tissues. Two factors that were recently suggested to impact the gene expression under hypoxic conditions include the shift in transmembrane Na/K gradients (Koltsova et al., AB is the author the general schematics of review, she contributed to all the sections and assembling of the review. AM, PH were contributing to the sections Introduction. Oxygen, and Oxygen Sensing from Evolutionary and Modern Perspectives, Versatility of oxygen sensing. Multiple Signals-Multiple Targets—Multiple Responses—Multiple Outcomes, and Oxygen and Redox-Sensitivity of the Na,K-ATPase. IP contribution were the sections Oxygen and Redox-Sensitivity of the Na,K-ATPase, Coordinated Regulation of Redox-Sensitive Protein Networks by Reversible Thiol, Oxygen-Sensitive Phosphorylation, and Regulation of the Na,K-ATPase Activity by Cardiotonic Steroids. All co-authors discussed the topics and writing the review. All of them agree with the final text.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In-plane frost growth on chilled hydrophobic surfaces is an inter-droplet phenomenon, where frozen droplets harvest water from neighboring supercooled liquid droplets to grow ice bridges that propagate across the surface in a chain reaction. To date, no surface has been able to passively prevent the in-plane growth of ice bridges across the population of supercooled condensate. Here, we demonstrate that when the separation between adjacent nucleation sites for supercooled condensate is properly controlled with chemical micropatterns prior to freezing, inter-droplet ice bridging can be slowed and even halted entirely. Since the edge-to-edge separation between adjacent supercooled droplets decreases with growth time, deliberately triggering an early freezing event to minimize the size of nascent condensation was also necessary. These findings reveal that inter-droplet frost growth can be passively suppressed by designing surfaces to spatially control nucleation sites and by temporally controlling the onset of freezing events. Frost inevitably forms on any subfreezing surface whose temperature is beneath the dew point. Water vapor can bypass the liquid phase and transform directly into ice, known as deposition or desublimation. However, in many cases the water vapor first condenses into supercooled liquid, which later freezes into ice. This indirect method of frost formation is known as condensation frosting and is more common than direct deposition in many cases, particularly at low supersaturation degrees and/or on hydrophobic surfacesUntil very recently, it was widely assumed that the primary mechanism of condensation frosting for surfaces promoting dropwise condensation was the heterogeneous nucleation of ice at the solid-liquid interface of each individual supercooled droplet2346789678101113143491617192122Curiously, it was observed that even when supercooled condensate was continually removed from a superhydrophobic surface via coalescence-induced jumping before heterogeneous nucleation occurred, frost still grew over the surface2125262124282930Stenocara desert beetle333435363739404243454750Inspired by the Here, we demonstrate that chemical patterns can be used to tune the spatial distribution of supercooled condensation and subsequently control the geometry and speed of inter-droplet frost growth. The success and rate of inter-droplet frost growth was found to be dependent upon two primary factors: the extent of spacing between hydrophilic regions where liquid nucleation occurred and the time allowed for condensation growth prior to the initial freezing event. For the first time, inter-droplet ice bridging could be completely halted by utilizing sufficiently sparse hydrophilic patterns and by quickly triggering a freezing event near the patterned condensation.Arrays of hydrophilic features were patterned against a hydrophobic backdrop on a silicon wafer using photolithography  is the saturation vapor pressure corresponding to the temperature of the condensing surface. Here, S = 1.2 for Ts = 5 °C and S = 3.5 for Ts = −10 °C. Note that the growth of patterned condensate on the triangle arrays was nearly identical to that on the circle arrays , there is a hyperbolic pressure distribution, ∇2P = 0, extending radially from the droplet’s saturated interface . The critical distance from the perimeter of the droplet to where nucleation can first occur on the hydrophobic surface is δIN = 22.5 μm for −10 °C .The dry zone on the hydrophobic surface can be modeled as a competition between the system’s in-plane and out-of-plane vapor pressure gradients. These pressure gradients could facilitate a dry zone in two different ways: by inhibiting the nucleation or by inhibiting the growth of liquid condensate. Let’s consider both in turn, beginning with an inhibited-nucleation dry zone of critical length Even when the vapor pressure is sufficient for nucleation to occur on the hydrophobic surface (at -10° C), it is possible that the nucleated droplet will simply evaporate under certain conditions. Nucleating embryos are typically ~1 nm in sizer is the radius of curvature of the droplet, R is the universal gas constant, T ≈ Ts and υ are the temperature and molar volume of the water droplet, and Ps and Pa are the saturated and actual vapor pressures about the droplet. Consider an isolated r = 10 nm hemi-spherical droplet that has recently nucleated on the hydrophobic surface and is situated near a wetted hydrophilic feature on either side. The vapor pressure about the droplet is Pa = 325 Pa at Ts = −10 °C, such that P∞ > Pa > Ps. Thus, even when nucleation is possible, the region will remain microscopically dry if the in-plane gradient driving evaporation exceeds the out-of-plane gradient facilitating condensation. The out-of-plane mass flow rate of vapor into the droplet scales as ζ is the concentration boundary layer thickness, while the in-plane (evaporation) mass flow rate gives δ is the edge-to-edge distance from the droplet to a wetted hydrophilic feature. The critical length scale of the inhibited-growth dry zone, δIG, can be estimated by balancing the evaporation and condensation rates, where For diffusive transport, the concentration boundary layer scales as:D is the diffusion coefficient of water vapor, β and ν are the volumetric thermal expansion coefficient and kinematic viscosity of air, and Ls ≈ 1 cm is the characteristic length of the condensing surfaceζ ≈ 0.8 mm and δIG ≈ 45 μm for −10 °C.where δIG > δIN for the conditions used in the present experiments at –10 °C, such that nanometric condensate is able to nucleate within the majority of the dry zone, but subsequently evaporates. For Ts = –10 °C, the predicted length of the inhibited-growth dry zone (δIG ≈ 45 μm) agrees with the experimental trend (~10 μm).Therefore D>) and surface coverage The average diameter  of D> was only measured for t ≥ 30 s to ensure that the substrate temperature had finished cooling to its steady-state temperature.where isolated droplet growth follows S = 1.2, α = 0.3873 for C (α ≈ 1/3 most likely due to the onset of some early (but not self-similar) coalescence events. No coalescence events occurred in the recorded time range (600 s) for C8P, and the resulting α = 0.3369 is in perfect agreement with the α ≈ 1/3 rule for isolated growth. Interestingly, the isolated growth of condensate followed α = 0.3029 for C4P and α = 0.191 for C2P, the latter of which is significantly lower than the α ≈ 1/3 rule for homogeneous surfaces. Compared to the control surface (C), the nucleation density was observed to be about 3 times larger for C4P and 13 times larger for C2P  or diminish (by increasing the boundary layer) the growth rate of condensation, depending upon the extent of supersaturation. However, even with the enhanced growth rates, it should be noted that the patterned surfaces exhibited smaller values of <D> compared to the control surface because the nucleation events were synchronized to minimize the variance in droplet volumes. At later times, where coalescence events were occurring at a steady-state surface coverage, 0.6 < α < 0.75. While this is lower than the theoretical trend of α ≈ 1, experimental measurements often hew closer to α ≈ 0.75; this discrepancy could be attributed to the presence of non-condensable gas, an over-simplified assumption of constant vapor flux, or the smallest droplets not being visible under the microscope objectiveD> actually decreased due to the coalesced droplets re-exposing many of the hydrophilic features to promote fresh nucleation events. This is in sharp contrast to condensation on the uniformly hydrophobic regions . This initial plateau value of While the concept of <analyzed . It was ρl is the density of liquid water and r ~ 10 μm is the radius of the bulge protruding from the stripe of water. The Reynolds number of the air then scales as ρg and μg are the density and viscosity of the air/vapor surrounding the water), which might be sufficient to induce a vapor flow toward the neighboring stripes.While the exact location along a stripe where a droplet bulges out seems be random for the first stripe where overflow occurs, a fascinating chain reaction follows where adjacent stripes proceed to bulge out droplets at the exact same location . In otheD> and Q*(t), is given by:To date, very little is known regarding the ability of patterned substrates to spatially manage condensate over a period of time. In addition to measuring <Nd(t) is the number of condensed droplets and Nf is the number of hydrophilic features within the same surface area. For a low supersaturation (S = 1.2), initially Q* ≈ 1 for C2P and C4P, followed by Q* < 1 upon the onset of coalescence  and the reduced quality of C8P was more exaggerated (up to Q* ≈ 1.44 compared to Q* ≈ 1.14 for S = 1.2).where lescence . An advaS*~L/D, predicts with high accuracy whether a frozen droplet is able to grow a frost bridge to a neighboring supercooled liquid droplet of initial diameter D and distance L from the frozen dropletS* > 1, the liquid droplet being harvested will completely evaporate before the ice bridge accumulates enough mass to connect, resulting in the stoppage of localized in-plane frost growthS* requires either tedious manual measurements of D and L for every dropletFinally, when spatially controlling condensation for the express purpose of tuning inter-droplet frost growth, the most relevant metric is the separation between the droplets relative to the size of the droplets. Previously, we have demonstrated that a dimensionless droplet separation coefficient, Q*(t) ≈ 1. Note that L is signified by (b − (<D(t)/2)) because the longest possible ice bridge is when the liquid droplet has almost entirely evaporated, which adds the radius of the droplet to the total length of the connecting bridge. It can be observed in D and L are approximately equal for all droplets at a given time for the C2P and C4P patterns , with the minor exception of droplets at the edge of the pattern which have fewer hydrophilic neighbors and are therefore slightly larger. By plotting S*(t) vs. t, it was observed that P and S both played significant roles regarding the length of time where S*(t) > 1 (S*(t) > 1 was extended by over an order of magnitude for S = 1.2 compared to S = 3.5, and by several times for C4P compared to C2P at the same supersaturation. These findings have important ramifications for the subsequent growth of frost over supercooled patterns of condensation.which applies to every droplet within the pattern, provided that *(t) > 1 . Indeed,Ts = −10 °C for chemical patterns of pitch P = 2 or P = 4, such that the quality of spatial control for the supercooled condensate was Q* ≈ 1. When the atmospheric conditions were kept identical to the previous experiments characterizing condensation (T∞ = 21 °C and H = 40 %), inter-droplet frost growth could not be measured repeatably due to an unexpected second mechanism of frost growth. In addition to the expected ice bridges that were grown locally from frozen droplets to neighboring liquid droplets, a surprising long-range freezing mechanism was consistently observed where a piece of ice suddenly appeared on a region of the surface far away from any pre-existing sources of ice (60kSi ≈ 150 W/m · K) and ejects ice far away from the mother droplet, would appear to be fundamentally different from the recently reported “frost halo” effect, which was exclusive to a highly insulating surface (kPMMA ≈ 0.19 W/m · K) and involves the localized evaporation and re-freezing of water vapor around the mother dropletThe growth of frost was characterized at s of ice . Once thT∞ = 24.0 ± 0.9 °C, H = 26 ± 2%, S = 2.7), the ice shrapnel effect never occurred. It is likely (but not certain) that the decrease in humidity was directly responsible for the disappearance of the ice shrapnel, as this is in agreement with a previous report that shattering did not occur for droplets freezing at sufficiently cold (and therefore less humid) air temperaturesFor the present study, it was found that the rate of frost growth was inconsistent when the effects of localized inter-droplet ice bridging were combined with the non-local ice shrapnel effect . InteresS*) exhibits a strong dependence upon both the pitch of the chemical pattern and the time allowed for condensation to grow , triangles (T2P/T4P), and stripes running parallel (HS2P/HS4P) or perpendicular (VS2P/VS4P) to the frozen water film . In eachf S* cf. . The valtime cf. . FurtherTs = −10 °C, the frost was always able to grow across the surface. This is because as the surface is cooled to Ts = −10 °C, S* > 1 is only true for roughly 20 s of condensation growth for P = 2 patterns and 60 s for P = 4, including the 30 s required for the cooling transient  plotted in While successful in preventing the ice from bridging to neighboring condensate, a single dry zone does have two long-term limitations. Firstly, far from the dry zone, the supercooled condensation is still free to grow, and will eventually freeze and grow ice bridges across the surface. This was evident in 0.1 μm/s , two ordbridging . FurtherδIG, can be theoretically estimated using the same flux balance approach used for calculating the condensation dry zones . The pressure gradients are driven by P∞ > Ps > Ps,i, where Ps and Ps,i are the saturation pressures of water and ice, respectively. The perimeter of the dry zone corresponds to the point where the net mass flux condensing into a liquid droplet from the ambient air is equal to the net mass flux evaporating from the droplet towards the ice. The condensed droplets bordering the dry zone can be approximated as an equivalent film of water of height hh ~ r, r being the mean droplet radius , which would likely increase the critical pitch of the hydrophilic patterns to maintain spatial control and magnify the effects of the pressure gradient between water and ice responsible for the dry zones. Therefore, the highly supersaturated conditions used here are actually somewhat extreme; it should be easier to pattern condensation and/or prevent inter-droplet ice bridging in a more natural environment. It should also be emphasized that silane monolayers, while ideal for fundamental studies, are prone to degradation under long-term condensation or frosting conditions. More durable hydrophobic coatings, such as grafted polymersThe present study characterized condensation and frost formation with the ambient environment at room temperature and fixed humidity, in contrast to natural systems where the air and surface temperatures are similar and the humidity can fluctuate widely. A recent study utilized an environmental chamber to chill the ambient air and widely vary the humidity, and confirmed that inter-droplet ice-bridging remains the primary driver of in-plane frost growth over this wider parameter spaceIn the present work, we triggered an early freezing event to locally dry out condensate and create a single dry zone on the chilled surface. While stable for several minutes at the very least, the long-term durability of the dry zone is doubtful due to the continued growth of water and ice far away from the dry zone. We suggest that a possible solution to this problem is triggering multiple freezing events simultaneously at an early time scale, for example creating an array of thin stripes of ice, such that multiple dry zones are created that overlap to permanently dry all of the liquid water away from the regions between the ice arrays. In other words, rather than patterning the supercooled condensation and triggering an isolated freezing event, it would now appear to be advantageous to pattern the freezing water itself!In conclusion, the spatial control of liquid condensation on chemical patterns, as well as the formation of a dry zone between ice and supercooled water, are both driven by a fascinating competition between in-plane and out-of-plane gradients in vapor pressure. Smooth chemical micropatterns can spatially control both the nucleation and growth of condensation even at very large supersaturations, provided that the distance between hydrophilic features is not too large. Compared to a uniform hydrophobic surface, condensation on a chemically patterned surface exhibited smaller average diameters and more homogeneous droplet volumes and inter-droplet separations. Therefore chemically patterned surfaces serve to maximize the spacing between condensate, such that the in-plane growth of frost via inter-droplet ice bridging can be delayed or even completely halted. Since the separation between droplets decreases with increasing duration of condensation, it was demonstrated that intentionally triggering an early freezing event on the surface is also crucial for halting ice bridging. Indeed, extremely early freezing events were shown to halt inter-droplet frost growth even on the non-patterned regions of the surface. Therefore, the inter-droplet growth of frost across a surface can be minimized or even stopped by spatially and/or temporally maximizing the separation between supercooled condensate upon the onset of freezing.2SO4:H2O2 at 25 °C for 10 min), rinsed extensively in deionized water, and dried with nitrogen. Clean wafers were functionalized with trichlorosilane (Sigma-Aldrich) through vapor deposition in a covered glass petri dish heated to 80 °C for 1 hr. Parylene was then deposited onto the silanized wafers using 800 mg of 2,2-paracyclophane precursor (Sigma) loaded into a parylene coater  run under standard operating conditions. Next, MP−P20 adhesion promoter (MicroChem) and S1818 photoresist (Shipley) were respectively spin-coated onto the wafer, both at 3,000 rpm for 45 s. After a 60 s bake at 115 °C, the wafer was exposed to ultraviolet light at 10 mW/cm2 at 365 nm for 5 s (Quintel Mask Alignment System) and developed in CD −26 for 2 min, rinsed with deionized water, and dried with nitrogen gas.Silicon wafers  were first cleaned with Piranha solution (3:1 Hμm thick) and the remaining photoresist (1.75 μm thick) while preserving the parylene that was underneath the photoresist. Immediately before use, the wafer was etched in oxygen plasma for 1–3 min to ensure that the exposed silicon oxide regions were hydrophilic, followed by a dry peel-off of the parylene film using tape and tweezersThe wafer was then etched with oxygen plasma in an inductively coupled plasma ion etching system  for 30 min. The etch rate under these conditions was approximately 62 nm/min, such that 30 min was sufficient to etch through the exposed parylene  was centered and focused under a 5× objective lens, such that the 2P, 4P and 8P arrays were all visible within the microscope’s field-of-view.A freshly prepared silicon sample was bonded to a Peltier stage (Deben MK3 Coolstage) with thermal paste and placed under a top-down optical microscope (Nikon Eclipse LV150). The ambient temperature was Once the field-of-view was set, the sample was immediately heated to 40 °C to dry the surface. The wafer was then cooled back down to 10 °C, followed by further cooling to either 5 °C or −10 °C to observe the resulting condensation with a digital camera (Nikon DS-5M) attached to the microscope. The transient of the Peltier stage was 5 ± 1 s to cool from 10 °C to 5 °C and 30 ± 1 s to cool from 10 °C to −10 °C. The forming condensation was imaged for 10 min, and the surface was then heated back to 40 °C to dry out the surface for the next trial. Three trials were obtained for both condensation temperatures and a new chip was used for each pattern type to minimize surface contamination.P = 2, 4, and 8 respectively. For arrays of stripes, there were 20, 39, and 77 stripes in the field of view for P = 2, 4, and 8 respectively. Each stack was cropped to analyze each array  in isolation. Each cropped stack was digitally sharpened and then converted to a binary mask. For the 4P and 8P arrays, the ‘Fill Holes’ process was performed to fill in holes in the droplets caused by reflected light, followed by the ‘Watershed’ process to ensure that droplets that were close to coalescing were not counted as a single droplet. The ‘Analyze Particles’ procedure was then performed to measure droplet diameters and surface coverage. For the 2P array, the droplets were too small for the ‘Fill Holes’ process to work accurately, so the following alternate approach was taken for the image processing. After sharpening the image and converting to binary, the ‘Close’ process was used to fill in the holes of the droplets, followed by ‘Erode’, ‘Watershed’, ‘Dilate’, and ‘Watershed’, respectively to separate nearby droplets from each other.A stack of images from each condensation video were analyzed in ImageJ, with a time interval of 3.0 s between each frame. For arrays of circles or triangles, there were 140, 507, and 1,925 hydrophilic features within the field of view for T∞ = 21.3 ± 0.3 °C and H = 40 ± 1%), the desired pattern was centered and focused under the microscope by condensing water at 5 °C, followed by drying at 40 °C and holding steady at 10 °C. The field-of-view under the microscope included a large rectangular hydrophilic region that bordered the hydrophilic arrays on one edge of the wafer. A 2.5 μL droplet of deionized water was deposited onto this rectangular hydrophilic pad, followed by cooling down to −10 °C to induce the growth of supercooled condensation on the hydrophilic array. The array in the field-of-view included 2P, 4P, and 8P features that were either circles, triangles, stripes running parallel to the deposited water droplet, or stripes running perpendicular to the deposited water droplet. To trigger inter-droplet ice bridging across the patterned condensation, a small piece of ice was gently touched against the side of the wafer containing the deposited water film to freeze it. To vary the rate of frost growth, the water pad was frozen at different times , where time zero corresponds to the instant where the surface finishes cooling to the set point temperature of −10 °C. The resulting frost growth was characterized by dividing the square root of the total area of each array by the time required for it to frost over. It should be noted that in addition to inter-droplet ice bridging, the frost growth for these experiments was also affected by ice shrapnel, where freezing droplets sprayed small particles of ice over the surface that induced non-local ice bridging events in addition to those caused by the initial freezing event.Under the same atmospheric conditions as with the condensation experiments  after reaching the steady-state temperature for all 2P and 4P shapes, with three trials performed for each parameter. The resulting frost growth was imaged to measure the averaged velocity of frost growth, in an identical manner to the first set of experiments. Finally, to capture a stable dry zone .


The C2 pedicle screw is more biomechanically stable and provides patients with increased postoperative range of motion in comparison to other methods of C2 fixation. However, as a result of the proximity of the C2 pedicle to the transverse foramen, there is a considerable risk of intraoperative morbidity due to vertebral artery injury laterally or vertebral canal breach medially. Other than the use of cadavers for the demonstration and practice of C2 pedicle screw placement, there are currently few other readily available teaching aids for the training of residents and fellows. Herein, we describe a simple and cost effective modality for the demonstration, evaluation, and practice of C2 pedicle screw placement in a laboratory setting. Atlantoaxial instability may occur as a result of trauma, tumors, congenital malformations, or inflammatory conditions of the upper cervical spine -2. FixatPosterior C1-C2 transarticular screws and C1-lateral mass screws with C2 pars screws are some of the more recently developed methods of C1-C2 fixation. The C2 pedicle screw was first described by Goel, et al. in the 1980s as part of a plate and screw construct that was utilized for posterior C1-2 fixation , 7-8. ThThe choice of any of these methods of fixation over another are influenced by anatomic constraints or variations, surgeon’s preference and/or experience , 9 as weThe C2 pedicle screw technique has numerous advantages over other methods of C2 fixation such as the C1-2 transarticular screw technique, the C2 lateral mass screw as well as the posterior wiring techniques. These advantages include increased range of motion postoperatively as well as stronger biomechanical stability. Based on a biomechanical study by Lehman, et al., C2 pedicle screws generated a greater insertional torque and pull-out strength in comparison to lateral mass, pars, and laminar screws. C2 pedicle screw placement is technically demanding and the precise and exact three-dimensional understanding of the anatomy of the region and vertebral artery is mandatory . Apart fHerein, we describe an easily applicable teaching aid that utilizes dried human C2 vertebrae to demonstrate the procedure while highlighting the avoidance of complications.The C2 pedicle is the portion of the C2 vertebra anterior to the pars, connecting the dorsal elements with the vertebral body. The entry point for the C2 pedicle screw is usually 5 mm rostral and 1 mm medial to the inferomedial aspect of the inferior articulating surface of C2. The screw is placed with 15-25 degrees of medial angulation with the thick medial wall of the C2 pedicle helping to redirect the screw if necessary, to prevent medial wall breakout and entry into the spinal canal. Utilizing dry vertebrae, the superior articulating surface of C2 is cut off at its lateral point of attachment with a high speed drilling disc , exposing the C2 pedicle and the entirety of the C2 transverse foramen as shown in Figure This provides the trainee with a better appreciation for the feel of the pedicle screw as it traverses cortical and cancellous bone, while having clear visual feedback of the position of the pedicle screw relative to surrounding structures in real time. The cut off superior articulating surface of C2 can be hinged off the odontoid process with a small metal spring as shown in Figure This allows for practice placement of the C2 pedicle screw by the trainee based on the landmarks and trajectory described above, as shown in Figure The placement of the screw can then once again be evaluated by lifting the hinged superior articular process and assessing the proximity of the pedicle screw to surrounding structures such as the vertebral artery, which is delineated with a red plastic tube in Figure This can be repeated as often as necessary until the trainee is comfortable with the technique.This cheap and simple teaching aid provides a much needed avenue for preparing trainees for real-life C2 pedicle screw placement while avoiding the associated risks of intraoperative training or the usually prohibitive cost of providing cadavers for training residents and fellows. It can also easily be applied to other technically challenging spinal instrumentation procedures such as C1 lateral mass and thoracic pedicle screw placement.


Dear Editor,Autophagy is a lysosome-based degradation pathway, characterized by formation of a double-membrane vesicle named the autophagosome. During autophagy, various cargos are engulfed and delivered to lysosomes (or the vacuole in yeast) for degradation. It is an evolutionarily conserved pathway and plays important roles in various physiological and pathological conditions such as development, immunity, cancer and neuronal degeneration  proteins. Phosphorylation plays an important role in the regulation of autophagy  search algorithm. Each search result had no close second ranking protein contaminant. The corresponding MS/MS spectra for each of the tentatively assigned phosphorylation sites were manually verified and annotated for the sequence-informative b and y fragment ions. We also manually verified the intensity of each phospho-peptide and compared the phosphorylation ratio between cells in nutrient-rich medium and starvation medium. Through this process, we identified that many Atg proteins, including Atg1, Atg3, Atg4, Atg6, Atg15, Atg16, Atg18, Atg27, Atg29 and Atg31, are phosphorylated. More than 60 sites were identified on these proteins purified from both fed and starved cells . We also generated some mutants in which combinations of different sites are mutated. We found that most single phosphorylation site mutations have no effect on autophagy, except a few sites on Atg1 which have been reported previously


Kynurenine aminotransferase II (KAT-II) is a 47 kDa pyridoxal phosphate (PLP)-dependent enzyme, active as a homodimer, which catalyses the transamination of the amino acids kynurenine (KYN) and 3-hydroxykynurenine (3-HK) in the tryptophan pathway, and is responsible for producing metabolites that lead to kynurenic acid (KYNA), which is implicated in several neurological diseases such as schizophrenia. In order to fully describe the role of KAT-II in the pathobiology of schizophrenia and other brain disorders, the crystal structure of full-length PLP-form hKAT-II was determined at 1.83 Å resolution, the highest available. The electron density of the active site reveals an aldimine linkage between PLP and Lys263, as well as the active site residues, which characterize the fold-type I PLP-dependent enzymes. N-methyl-d-aspartate (NMDA) receptor occurs by binding at the glycine binding site [Kynurenic acid (KYNA), with several specific biological activities, is known to antagonize all ionotropic glutamate receptors. The antagonisation of the ing site  and at ting site ,3, and aing site . Therefoing site . In addiing site ,7. More ing site . Furthering site , suggesting site ,11, the ing site ,13. KYNAing site ,15 whichRegardless of the evidence for the participation of KYNA in neuroprotection, the potential for cognitive damage in the central nervous system (CNS) which results from the involvement of glutamatergic and cholinergic neurotransmission needs appropriate consideration , especial-kynurenine (l-KYN), the main intermediate of the kynurenine pathway into KYNA, an endogenous neuroactive agent in CNS [It is believed that in the CNS of mammals kynurenine aminotransferase (KAT) isozymes are responsible for the synthesis of KYNA . Both KAt in CNS , as partt in CNS .β and 1γ of the pyridoxal-5′-phosphate (PLP) dependent aminotransferases [l-KYN. In the ensuing steps this α-amino group is transferred to the coenzyme, converting the PLP to the pyridoxamine phosphate (PMP) form. Regeneration of PLP is achieved by the transfer of the α-amino group to the α-keto acid, leaving the α-keto acid co-substrate with l-KYN, which spontaneously undergoes a ring closure producing KYNA [P43212, at 1.83 Å resolution, which is the highest available for the hKAT-II structure. The high resolution facilitates further study of the active site role including nearby residues and their possible involvement in the catalysis mechanism. Our structure is also valuable for the rational design of inhibitors for the purposes of their consideration as therapeutics for numerous CNS diseases in humans.KAT-II consists of 425 amino acid residues with its catalytic site at the homodimeric interface. It belongs to the subgroup Isferases ,29 with ing KYNA . In thisUsing the Ni-NTA column chromatography method, the recombinantly expressed hKAT-II enzyme was purified, and its activity with the substrate (KYN) and co-substrate (α-ketoglutarate) was determined using HPLC . The actP43212 with unit cell dimensions of a = 102.46 Å, b = 102.46 Å and c = 86.24 Å . The asymmetric unit contains one molecule with an estimated solvent content of 48.1%. The molecular replacement (MR) solution had a log likelihood gain (LLG) of 12999 and a translation function Z-score of 107. The final fully refined structure was achieved with a final R-free and R-work of 19.89% and 17.30%, respectively, in which the agreement between them indicates over-fitting is unlikely. The final model contains all the residues of a total of 425, containing the one modified residue, LLP , which is substituted for Lys263), and 438 solvent molecules. The geometry and stereochemistry of the structure were assessed by MolProbity [cis peptide conformations were determined, at Pro140 and 203. The hKAT-II structure contains 37% α-helix, 15% β-sheet, 12% turn, 35% coil and 1% 310 helix. Unambiguous electron density was generally observed, and the N-terminal arm consisting of an antiparallel β-sheet  . The PLP ligand linked through the aldimine bond to Lys263, required for activity in the PLP-dependent enzyme families, is not always observed as examples exist in the apo form without the PLP, as in the case of the structures reported by Ku et al., 2006 [et al., 2013 [The crystal of our hKAT-II holoenzyme, with the PLP form of the co-factor, diffracted to 1.83 Å. The crystals belonged to the novel tetragonal space group lProbity  and 95.5d Gly64) a. The std Gly64) b. Also td Gly64) . From prd Gly64) a. The oxd Gly64) b. Our hKl., 2006   for 24 h at 15 °C, and later bacterial pellets were harvested by centrifugation and stored at −80 °C. The pET15b vector added a hexa-histidine (6 × His) tag to the N-terminus of the expressed protein which allowed it to be purified using nickel-nitrilotriacetic acid (Ni-NTA) column chromatography. The purified recombinant hKAT-II was concentrated to 16 mg/mL using an Amicon® ultra 4 mL centrifugal filter unit with a membrane size of 30 kDa. The enzyme purity was evaluated by SDS-PAGE analysis [Using the online Genscript tool followed by manual intervention the hKAT-II gene was optimized for heterologous expression in E. coli and the nucleotide sequence was constructed by Genscript Corporation, Piscatway, NJ, USA. The optimized sequence was sub-cloned into a pET15b vector via NdeI/BamHI restriction sites. The expression vector was transformed into Rosetta 2 competent cells, the protein expression induced by adding 0.1 mM isopropyl β-analysis . Enzyme l-kynurenine, 5 mM of α-ketoglutarate, 40 µM PLP and 0.9 µg purified hKAT2 in 10 mM phosphate buffer (pH 7.5) was used to determine the activity of the recombinant protein. The reaction mixture was incubated at 37 °C for 10 min and then an equal volume of 0.8 M formic acid was added to terminate the reaction. After centrifuging at 11,000× g at 4 °C for 10 min the supernatant was stored for the HPLC assay. KYN and KYNA peaks were detected at 330 nm using a C-18 reverse-phase column with water-acetonitrile (93:7) v/v % as the mobile phase.The activity assay was designed based on the previously described method with slight modifications . A 50 µLCrystals of the recombinant human kynurenine aminotransferase II were grown using the vapor diffusion method with hanging drops technique. Protein (1 μL) at a concentration of 7 mg/mL was mixed with an equal volume of a reservoir solution containing 200 mM NaCl, 0.1 M NaCitrate pH 5.6, 24% PEG4K and equilibrated against 1 mL of a reservoir solution at 20 °C  at 100 K using the Blue-Ice software package . Data prIn order to obtain the high-accuracy ensemble structure as a reference model for molecular replacement, the homology modeling technique was used. The homology model of hKAT-II was constructed using YASARA . The priHuman kynurenine aminotransferase II makes the largest contribution to KYNA production in human brains and plays a key role in several CNS disorders. Therefore, it is a biological target of interest that may be important in considerations of how to overcome neurodegenerative diseases. In this paper the native structure of hKAT-II (PLP form) at 1.83 Å has been determined and refined, providing the highest resolution structure reported. This resultant model gives a complete, detailed description of the binding site of hKAT II, which is of considerable practical use, especially in the context of the challenge to design novel therapeutic lead inhibitors targeting this enzyme.


With increasing data, fundamental questions pertaining to allostery are currently receiving more attention from the mechanism of allosteric changes in an individual protein to the entire effect of the changes in the interconnected network in the cell. Thus, the following novel features were added to this updated version: (i) structural mechanisms of more than 1600 allosteric actions were elucidated by a comparison of site structures before and after the binding of an modulator; (ii) 261 allosteric networks were identified to unveil how the allosteric action in a single protein would propagate to affect downstream proteins; (iii) two of the largest human allosteromes, protein kinases and GPCRs, were thoroughly constructed; and (iv) web interface and data organization were completely redesigned for efficient access. In addition, allosteric data have largely expanded in this update. These updates are useful for facilitating the investigation of allosteric mechanisms, dynamic networks and drug discoveries.Allosteric regulation, the most direct and efficient way of regulating protein function, is induced by the binding of a ligand at one site that is topographically distinct from an orthosteric site. Allosteric Database (ASD, available online at  Allosteric regulation fine-tunes most biological processes, including signal transduction, enzyme activity, metabolism and transport –3. AllosDespite the increasing interest in the development of allosteric drugs as a new tactic in drug discovery, the structural mechanisms underlying allosteric drug action represent a key challenge. The hotspots of conformational changes in the allosteric site are of particular importance to understanding the underpinnings underlying allostery, because the efficacy of an allosteric drug is often determined by specific conformational transitions in the allosteric site . IdentifIn drug discovery, protein kinases and G protein-coupled receptors (GPCRs) are two of the largest targets. Due to the remarkable structural conservation of orthosteric sites in the kinome and homologous GPCRs, there has been a long-standing challenge in the development of highly specific kinase and GPCR orthosteric inhibitors or activators –27. FortThe Allosteric Database (ASD) has been developed to provide comprehensive information characterizing allosteric regulation since 2009 ,45, rangAllosteric molecules and features, including structures, sites, pathways, functions, related diseases and external links, were collected using previously described methods ,45. A deHuman α-amylase , more sites (∼113% increase), more pathways (∼17%) and a greater number of related diseases (∼493% increase)  with that after binding (holo structure) , Ras  and PDK1   as a modulator in distinct allosteric sites of 13 proteins and found that a majority of the allosteric sites (80%) were induced by the triphosphate in ATP as a trigger, whereas a smaller number of allosteric sites (20%) were triggered by the adenine in ATP  ,51,52, w7, etc.) . Likewise in ATP . These sapo/holo paired structures for allosteric modulator action in 308 proteins from 107 organisms were constructed using the same protocol described in ATP action ’ is displayed. Conformational transitions in allosteric site upon binding of the corresponding allosteric modulator ‘KRH’ are identified in ‘backbone’ and highlighted in color. The alignment and identified conformational transitions can also be downloaded as a PyMOL session (PSE) file in the page for further analysis. Of the 1688 allosteric apo/holo paired structures, 92.6% and 5.9% allosteric sites showed local motions in backbone and in sidechain, respectively upon the binding of modulators. Remarkably, 20 allosteric proteins can be regulated by the actions of both agonism and antagonism on the same allosteric site, such as glutamate dehydrogenase (bovine)  ,55. The c-9,10-octadecenoamide)’ ’ , ‘Phosphoamide)’  and ‘1-poamide)’ , based oAllosteric sites, which differ from highly conserved orthosteric sites, offer intrinsic opportunities for drug target by providing higher selectivity, fewer side effects and lower toxicity ,66,67. AThe ASD is a comprehensive and integrated allosteric platform that consolidates manually curated chemical, biological and clinical data about all experimentally confirmed allosteric evidences. Building on previous versions, we have significantly expanded the allosteric molecules and features, particularly modulators, sites, interactions and related diseases, which reflect the recent considerable growth of allosteric studies in both pharmaceuticals and medicine. We also introduced three novel datasets for the investigation of allosteric mechanism including (i) allosteric modulator action; (ii) allosteric network and (iii) protein kinase and GPCR allosteromes in this update. The ASD site was completely redesigned, with greatly improved performance of data access, a cleaner appearance and improved usability. In the future, we aim to continue to curate allosteric data from the primary biomedical literature and to refine the quality of the data according to the research progress of allostery. The ASD will also continue to improve database architecture for easy and intuitive use based on the response from community. In addition to our current knowledge, we will focus on introducing allosteric evolution by interspecies navigation and comparison in order to trace to the beginning of allostery. With the increasing number of clinical mutations discovered in patients using next-generation sequencing, the effects and mechanism of the mutations around the allosteric site could provide new insights in syndromes, physiological abnormalities and diseases, and this is another aspect in which we are planning to curate in the future.


Fusarium culmorum was developed based on homology modelling with the crystallized protein of F. sporotrichioides. Eight phenolic molecules, namely ferulic acid 1, apocynin 2, propyl gallate 3, eugenol 4, Me-dehydrozingerone 5, eugenol dimer 6, magnolol 7, and ellagic acid 8, were selected for their ability to inhibit trichothecene production and/or fungal vegetative growth in F. culmorum. The chemical structures of phenols were constructed and partially optimised based on Molecular Mechanics (MM) studies and energy minimisation by Density Functional Theory (DFT). Docking analysis of the phenolic molecules was run on the 3D model of F. culmorum TRI5. Experimental biological activity, molecular descriptors and interacting-structures obtained from computational analysis were compared. Besides the catalytic domain, three privileged sites in the interaction with the inhibitory molecules were identified on the protein surface. The TRI5-ligand interactions highlighted in this study represent a powerful tool to the identification of new Fusarium-targeted molecules with potential as trichothecene inhibitors.A model of the trichodiene synthase (TRI5) of the wheat fungal pathogen and type-B trichothecene producer  Fusarium genus and are among the major causes of crop loss in cereals [Trichothecene mycotoxins are produced by filamentous fungi belonging to the  cereals ,2. Trich cereals , trichot cereals –6.Fusarium diseases, particularly those based on the use of synthetic fungicides, are not always effective and sometimes have determined a selection pressure on fungal populations, hence facilitating the emergence of resistant mutants [Control strategies taken so far against  mutants . In this mutants . Natural mutants .TRI genes [TRI5, which represents the very first step of the trichothecene biosynthesis pathway. In the presence of three magnesium (Mg2+) ions the TRI5 enzyme catalyses the cyclization of farnesyl pyrophosphate to form the bicyclic sesquiterpene trichodiene and the co-product inorganic pyrophosphate (PPi).The mechanisms by which these compounds are able to interfere with the biosynthesis of trichothecenes are not yet completely understood, and numerous hypotheses have been proposed: transcriptional control of RI genes –11, modiRI genes , inhibitRI genes , alleviaRI genes . One of RI genes . TRI5 isFusarium sporotrichioides with PPi bonded to the catalytic site has been isolated and highly defined by X-ray, providing a useful tool to study protein-molecule interactions with high standards [The crystal structure of TRI5 of tandards –20. The in vitro to assess their activity on vegetative growth and trichothecene biosynthesis by F. culmorum. Significant trichothecene inhibitory activity was observed over a range of concentrations comprised between 0.25–1.5 mM and some of the tested compounds proved fungitoxic.In a previous article  a collecF. culmorum model strain FcUK99 [unpublished results).Here we have selected eight representative compounds to test their efficacy on the n FcUK99 , whose fF. sporotrichioides [F. culmorum TRI5 to carry out modelling and docking studies. Docking data were integrated with in vitro activity to identify molecular structures, functional groups and putative amino acids that are most likely involved in the interaction between selected inhibitory molecules and the F. culmorum TRI5 protein.Aiming to provide further insight into the understanding of structure-activity relationship of trichothecene inhibitors and TRI5, we have adopted an approach based on modelling techniques. The crystal structure of TRI5 of chioides  was usedF. culmorum FcUK99  was used in the in vitro experiments. This strain produces predominantly 3-acetyldeoxynivalenol (3-ADON) and, to a lesser extent, deoxynivalenol [et al., 2014 [Type B trichothecene producer wild-type strain of ivalenol . Strain l., 2014 .1, apocynin 2, propyl gallate 3, eugenol 4, Me-dehydrozingerone 5, eugenol dimer 6, magnolol 7, ellagic acid 8  [et al. (2015) [®7 PHARMA; Wacker Chemie Italia, Peschiera Borromeo, Italy) was added to the medium in order to improve solubility of phenolic molecules.All compounds were purchased by Sigma-Aldrich  except for Me-dehydrozingerone . (2013)  and Dett. (2015) , respectet al. (2014) [4 conidia mL-1). The evolution of pH in media amended with the different compounds was recorded at 0, 1, 2, 4, 8 and 14 days after inoculation (The experiments were carried out as described by Pani . (2014) , with so. (2014) . After aculation . After 1-1) obtained from two separate experiments were expressed as percent value of the untreated control. Data were analysed by one-way analysis of variance (ANOVA) with Dunnett’s test, using Minitab Express for Mac version 1.3.0.Data on total trichothecene production . The starting TRI5 protein sequence for F. culmorum was obtained from UniProtKB/Swiss-Prot databases (accession number Q8NIG9) [The TRI5 protein of nkiewicz  (PDB cod Q8NIG9) ,27.F. sporotrichioides were stripped. Hydrogen atoms were added using the ADT module of MGLTools 1.5.7rc1 [Substitution of 33 amino acids from the B chain of 1JFG was carried out with Swiss-Model, an automated comparative protein model server interconnected with Swiss-PdbViewer 4.0.4 program –30. The 1.5.7rc1 .ab initio quantum chemistry program Gaussian 09W by Density Functional Theory (DFT) with method B3LYP/6-311G basis set [−1 mol−1 and T (Temperature) is 298.15 K. Graphical representation of the hypothetical positions derived from the docking calculation was obtained using the software Chimera [Computational modelling experiments were conducted on multiprocessor machines with OS Ubuntu 13.04 and 14.04 or Windows 7.0. Model compounds were constructed with standard bond lengths and angles from the fragment database with MacroModel 5.5 . Minimisasis set . Visual asis set . Bindingasis set ,38. The asis set  for the  Chimera .Lipophilicity (LogP) of ligands was estimated with ChemBioOffice Ultra 13.0 , while dipole moment  was calculated with Gaussian 09W.1, apocynin 2, propyl gallate 3, eugenol 4, Me-dehydrozingerone 5, and eugenol dimer 6) demonstrated their trichothecene inhibitory activity when tested at 0.5 mM, providing a reduction in the total mycotoxin production ranging from 31% to 98%. One compound (magnolol 7) had a fungicidal action, hence complete inhibition of mycelium growth and no trichothecene production were observed. On the contrary, ellagic acid 8 induced a significantly higher (159%) trichothecene biosynthesis compared to the untreated control . In the RCSB protein data bank (http://www.pdb.org) the trichodiene synthase of F. sporotrichioides is represented in a wide array of crystals, including wild type and mutated structures. The 3D structure homology model of F. culmorum was prepared starting from the chain B of the F. sporotrichioides crystal (1JFG), which contains the three magnesium atoms and the pyrophosphate (PPi), both essential for the enzyme activity. The model was refined by substitution of 33 amino acids from the B chain of 1JFG and by comparing the obtained result and the existent homology model sequence of F. culmorum TRI5  was adopted, treating the docking active site as a rigid system and the ligands as flexible, i.e., all non-ring torsions were considered active and free to rotate. In addition to the active site, five further sites showing significant ligand affinities were identified on the TRI5 protein surface. These five sites, overlapping in the F. sporotrichioides and F. culmorum enzymes, are numbered from 1 to 5  occurring between the two enzymes, only four amino acids are located in the privileged binding site 2 , whereas the others are in its vicinity or far away . The difThe scoring results of docking calculations deduced for ligands, as well as the major ligand receptor interactions, are listed in 1); (ii) molecules with charge 0 ; and (iii) dimeric molecules . Docking studies indicate that sites 1, 2, and 4 of F. culmorum Q8NIG9 are privileged sites for ligands inhibiting trichothecene biosynthesis in vitro. Ligands with a long aliphatic chain and dimeric molecules (hydroxylated biphenyls) interact simultaneously with sites 1 and 2 (The ligands can be clustered into three main groups: (i) molecules characterised by a carboxyl group, likely deprotonated at physiological pH, and having a charge of -1 , hence confirming the reliability of the system .1, the sole charged phenol among the eight ligands tested in in vitro, interacts with the catalytic site in the proximity of the Mg2+ ions with significant docking score (Ki 1.99 μM) and with amino acids of three sites located in the surface of TRI5, identified as site 3, 4 and 5  showed a similar behaviour. Forty per cent of docking runs occurred at the lowest calculated energy of -5.86 Kcal/mole and Ki 50.71 μM  to site 2 with a Ki 133.84 μM , binds in 22% of the cases to site 1 of the TRI5 protein . Despite the low inhibition constant Ki (224.72 μM) calculated for site 1, eugenol 4 binds also (50% of docking run sum) to site 2, with two different conformations. Eugenol 4 shows affinity for this site with energies of -5.12 Kcal/mole and -5.07 Kcal/mole, according to its conformations. Eugenol 4 and apocynin 2 interact also with the catalytic domain with negligible docking run, therefore, these interactions were ruled out.Eugenol 5, a molecule that inhibited 92% of the trichothecene production, interacts with site 2 with two distinct conformations of similar energy with a docking run sum of 54%. Moreover, Me-dehydrozingerone 5 also interacts with site 4 with 14% of docking run and significant Ki (51.63 μM) and estimated free energy of binding .Me-dehydrozingerone 6, magnolol 7 and ellagic acid 8 are dimeric phenols belonging to the family of hydroxylated biphenyls. These dimeric molecules are structurally different from the other two groups as they display a more hindered structure due to the presence of two aromatic rings linked by a single C-C bond. Two lactone groups fused in the C-C single bond make the structure of ellagic acid 8 quite planar, whereas eugenol dimer 6 and magnolol 7 may freely rotate along with the C-C single bond. These latter two ligands enter in site 1 with one of their two aromatic rings, positioning the second one perpendicularly to the first one that then interacts with the amino acids located in site 2, in proximity to site 1. This conformation allows magnolol 7 and eugenol dimer 6 to interact simultaneously with the amino acids of site 1 and 2 by each aromatic ring. Magnolol 7 interacts with sites 1 and 2 with two conformations that are quite equal in energy , and with site 4 in 14% of the docking runs. The energy ranges between -6.91 and -5.91 Kcal/mole with significant Ki up to 8.56 μM , are likely to be in the protonated form, as confirmed by the low pH value detected in the medium since the first days of fungal growth.In the tested experimental conditions, all compounds, among which ferulic acid 3, ellagic acid 8, magnolol 7, eugenol 4, and the eugenol dimer 6 bound preferentially to sites 1 and 2 located on the surface of TRI5 and far from the catalytic domain : due to its almost flat structure, ellagic acid 8 does not overlap with sites 1 and 2, exerting a less intimate contact when compared with conformationally flexible magnolol 7 and eugenol dimer 6. These two molecules also bind to site 5 with a significant docking (E.F.E.B.)  . It is k of view ,45,46. T3, which presents an aliphatic side chain, activates interactions with a large number of hydrophobic amino acids that are common to magnolol 7 and eugenol dimer 6 , confirming the high inhibitory activity observed in vitro. To a lesser extent compared to the other sites, Me-dehydrozingerone 5, magnolol 7, eugenol dimer 6 and ellagic acid 8 activate non-covalent interactions with a common set of amino acids located in site 4 .Propyl gallate 2 involve only site 1 with significant docking run values, whereas Me-dehydrozingerone 5 engages sites 4 and 2, the latter with almost equal docking runs in two molecule conformations. We hypothesize that the far less efficient trichothecene inhibition observed in the case of apocynin 2 may be related to its limited number of non-covalent interactions with TRI5, whereas a large number of amino acids are involved in the interaction between TRI5 and Me-dehydrozingerone 5, which activates interactions with the hydrophobic amino acids present in site 2.The energetically “best” poses for apocynin 6–8 establish multiple H-bonds and hydrophobic interactions with the amino acids present in sites 1, 2 and 4  interactions with amino acids of TRI5 . Bipheny 2 and 4 . Cross-bredicted .6–8 and propyl gallate 3 involve mostly apolar and hydrophobic amino acids  should be taken into account ,48. FromAs mentioned before, sites 1, 2 and 4 were identified as privileged sites of no-charged phenols. Quite interestingly, five amino acids  are represented in the range of 80–100% in sites 1 and 2, where most of the ligand-protein docking occurs. These amino acids have neutral and, except for Asp302, non-polar side chains, assuming a lipophilic character of the potentially binding ligand. A prevalence of hydrophobic amino acids also resulted in site 4.3, Me-dehydrozingerone 5, eugenol dimer 6 and magnolol 7 proved the best trichothecene inhibitors, as predicted by docking studies, where these compounds bind to the same sites of TRI5, and further confirmed by the in vitro bioassay. The reduced inhibitory activity observed in vitro for apocynin 2, eugenol 4 and ellagic acid 8 might be related to restricted ligand-protein interaction.To sum up, propyl gallate 7 behaved as a fungicide in vitro when tested at 0.5 mM concentration. However, in the modelling study, ligand concentrations were not taken into account, and magnolol 7 may therefore switch from fungicide to trichothecene inhibitor at very low concentration, as shown previously [7 should be further investigated since its properties are likely to affect other mechanisms of action related to both primary and secondary metabolism [As mentioned, magnolol eviously . The funtabolism .1 is a cinnamic acid and was included in this study due to its well-known inhibitory activity towards trichothecene biosynthesis [1 likely activates non-covalent interactions.Ferulic acid ynthesis ,51. ThisF. culmorum TRI5 with phenolic acids, we compared the docking score of ferulic acid 1 with other cinnamic acids and benzoic acids whose biological activity has been previously tested in vitro [9, 4-hydroxycinnamic acid 10, 2,5-dimethoxycinnamic acid 11 inhibited trichothecene production by F. culmorum strain MCf 21 (syn. INRA 117) in the range of 1.5 and 1.0 mM, whereas no trichothecene nor mycelium growth inhibition were observed for both 3-methoxybenzoic acid 12 and 3,4-dimethoxybenzoic acid 13 at 0.5 mM [1 , mostly binding to polar and partially hydrophobic amino acids.Aiming to further investigate the interaction of in vitro . 3-Hydro.5 mM [1 . Contrar1 and 10 . NeverthF. culmorum TRI5 model. Apart from the catalytic domain, five binding sites were identified on the surface of TRI5 and among them sites 1, 2 and 4 are privileged binding sites for no-charged phenols. For charged phenols, and particularly ferulic acid 1 which was investigated as a prototype of cinnamic acids, sites 3 and 5 are privileged for binding, despite interaction with the catalytic domain should be also considered for more polar cinnamic acids.In the present study, a docking method was applied to explore and validate key interactions between natural and natural-like phenols with the in vitro assays with a collection of phenols, whose lipophilicity and H-bonding capacity were postulated as key factors in the selection of a good trichothecene inhibitor. An important role might be played by the molecular size, which governs the ability of the ligand to interact with different amino acids at the same time.Docking data confirmed results obtained from Hence, our model assumes that a phenolic molecule bearing substituents with high ability to activate protein-ligand interactions at the same time with a large number of amino acids would be an ideal candidate as trichothecene inhibitor.Fusarium TRI5, shall provide an additional tool to discover new molecules with potential as fungicides or as trichothecene inhibitors, and will guide the synthesis of novel Fusarium-targeted compounds by shortening the time of research and by reducing cost. The long-term goal of our project is to develop natural or natural-like phenolic compounds as effective, environmentally-friendly alternatives to synthetic fungicides. Based on in vitro and in silico characterisation, field testing is now being carried out with selected molecules in order to evaluate their efficacy in reducing FHB symptoms in durum wheat as well as trichothecene contamination in harvested grain.It has to be born in mind that the modelling approach provides only an estimation of protein-ligand interaction, as it does not take into consideration a wide range of interfering factors . Nonetheless, the TRI5-ligand interactions highlighted in this study, along with the availability of a crystal structure of the S1 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.