The increase in temperature was found to hinder the formation of CaCO3 from Ca2+ and CO32−, thus reducing the efficiency of hydration carbonation. The increase in particle size was not conducive to the leaching of C2S and C3S to the surface of the reaction phase, which in turn reduced the degree of decalcification and polymerization of the C–S–H gel in the carbonation phase. It was concluded that the optimum LFS hydration and carbonation reactions were achieved at 20 °C and with a LFS particle sizes < 38 μm.Ladle furnace slag (LFS) can undergo hydration and carbonation reactions as cement. This article explores the effect of LFS hydration and carbonation reactions on cementitious substances at different temperatures and different LFS particle sizes, determining the effect of these varying conditions on the microstructure and formation mechanism of cementitious substances. The results show that in the early stages, C The mineral phase components of LFS mainly contain calcium metasilicate , dicalcium silicate , tricalcium silicate  and free calcium oxide (f-CaO)2. LFS exhibits poor stability and poor compactness due to its high content of f-CaO. In addition, it exerts a filler effect that can increase the density of cementitious substances, as its mineral phase composition is similar to that of Ordinary Portland cement (OPC) and it contains the same oxides that are present in OPC, while also having the advantages of a high affinity to cement-based materials and highly abundant availability3. The addition of LFS to cement-based materials as a mineral additive can accelerate the hydration reaction and increase the formation of hydration products such as calcium silicate hydrate (C–S–H) gel4. As the most important hydration product of cement slurries, C–S–H gel is an important source of mechanical properties in cementitious materials and considerably influences the carbonation of LFS and the preparation of cement5.Ladle furnace slag (LFS) is an alkaline solid waste material with a high calcium content, that is produced as a by-product of secondary refining processes in the iron and steel industry6. The poor stability and poor compactness of LFS can be mitigated by absorbing CO2 to produce stable calcium carbonate (CaCO3). Xu7 used CO2 to immobilize heavy metals in LFS and enhance its strength. Monkman8 established that the extractable CaO content of carbonated LFS was reduced by about 95% compared with uncarbonated LFS, while its strength is greatly improved when formed into mortar. Therefore, carbonation can not only effectively achieve CO2 storage but also improve the compressive strength of the final product. In the early stage of carbonation, the cementitious substances in LFS (C2S and C3S) undergo hydration reactions to form C–S–H gel. When comparing the changes of C–S–H gel during natural carbonation or accelerated carbonation, Auroy9 concluded that CaCO3 was more conducive to forming crystalline spherical aragonite with a relatively stable structure after accelerated carbonation. Therefore, hydration and carbonation reactions are particularly important in the process of LFS resource utilization.The calcium-based substances in LFS exhibit good carbonation performance with a theoretical carbonation efficiency of 30–60%10 analyzed the controlling factors that affect the carbonation reaction, including particle size, temperature, solid–liquid ratio, stirring speed, and aeration rate and demonstrated that temperature and particle size are the two most important factors. Ashraf11 compared the carbonation process of CS, C2S, and C3S at different temperatures, reporting that the reactivity of CS samples was lower than that of C2S and C3S samples. Previous studies13 have reported that reducing the average particle size of slag can shorten the leaching channels from the metal ion core to the inner surface, thus promoting the utilization of slag and interactions between the slag-water–gas system. Suh14 reported that heating an anhydrous calcium silicate clinker to 200 °C resulted in a gradual increase in Q1 structure (SiO4 tetrahedra connected to a SiO4 tetrahedra) and Q2 structure (SiO4 tetrahedra connected to two SiO4 tetrahedra) peak intensities, according to deconvolution of the recorded 29Si solid-state nuclear magnetic resonance (NMR) spectra. Suh’s results show that the hydration reaction of anhydrous calcium silicate can be accelerated by increasing the reaction temperature within a certain range. The aforementioned studies have generally assessed the effects of hydration and carbonation reactions in isolation on mineral phase composition, with only a few studies having combined both reactions to analyze the change mechanism of the cementitious substances under varying temperature and particle size conditions.Huijgen2S and C3S in LFS and the carbonation reaction of C–S–H gel when applying different temperatures and LFS particle sizes, were explored using solid-state 29Si NMR technology. The samples were characterized by scanning electron microscopy (SEM), thermogravimetry (TG), differential thermogravimetry (DTG), X-ray diffraction (XRD) and Brunner–Emmet–Teller (BET) analysis of nitrogen adsorption–desorption isotherms. Peak Fit software was used to analyze the changes in the cementitious substances during the hydration and carbonation stages and revealed their mechanism of microstructure formation. The present study aimed to understand the effect of different temperatures and particle sizes on cementitious substances subjected to hydration and carbonation reactions, providing theoretical guidance for the application of LFS and LFS-based cement materials.In this study, changes in the hydration reaction of C2S, C3S and CaAl2Si2O8. The solid LFS material was pulverized using a jaw crusher  and then ground with a vibrating disc grinder . Varying particle sizes were obtained by passing the ground sample through 180 μm, 96 μm, 48 μm, and 38 μm pore size sieves before experiments.The LFS used in this study was taken from a steel plant in Xinjiang (China), with the chemical composition and morphology of the material shown in Table 2 ventilation rate of 500 mL/min, with continual stirring at 700 rad/min. Hydration and carbonation reactions were considered to be complete when the mass remained constant. To determine the influence of particle size, slurries with LFS particles sizes of 96–180 μm, 48–96 μm, 38–48 μm and < 38 μm were used at a reaction temperature of 20 ℃. To analyze the influence of temperature, samples with a particle size < 38 μm were reacted at 20 ℃, 40 ℃, 60 °C and 80 °C. At the end of the experiment, the solids in the slurry were separated from the liquid and dried at 105 °C for 8 h to obtain carbonation products for analysis.A three-phase reaction tank was employed to investigate the influence of particle size and temperature on the carbonation of LFS. Slurries of LFS fractions containing each respective particle size range, were prepared with a solid–liquid ratio of 1:5, for use in experiments at a CO15. A scanning electron microscope  was used at an accelerating voltage of 15 kV to analyze the micro-morphology of samples, which were sprayed with gold before the test to stabilize images. Analysis of the microstructure changes during C2S and C3S carbonation was carried out via 29Si NMR analysis , with a 4-mm ZrO2 rotor at a rotation speed of 8 kHz and TMS solution as a 29Si chemical shift calibration reference.X-ray diffraction analysis was performed to identify carbonation products using an X-ray powder diffractometer . For all materials, two samples were analyzed under the following experimental conditions: Cu Kα radiation (λ = 0.154 nm), scanning range of 10°–70° and a scanning rate of 2.0°/min. The mineral phase composition was determined via thermal stability analysis (TG-DTG) using a simultaneous thermogravimetric analyzer  with 10 mg of sample analyzed at a temperature range from ambient temperature to 900 °C at a heating rate of 10 °C/min in a single measurement, as variations in outcome were expected to be small2S, and C3S, with additional amounts of calcium hydroxide Ca(OH)2 and anorthite (CaAl2Si2O8). After the hydration and carbonation reaction processes, the diffraction peaks of the calcium silicate phases decreased or disappeared. Simultaneously, three diffraction peaks appeared at around 2θ = 28.5°, 31.2°, and 61.2°, which were assigned to C–S–H gel19, indicating that the calcium silicate phases formed C–S–H gel during the hydration and carbonation reaction, and LFS has potential pozzolanic activity, Si and Al will also react with Ca(OH)2 to form C–S–H gel. As C2S and C3S are known to undergo hydration reactions20, C2S and C3S present on the surface of LFS particles can undergo hydration to form C–S–H gel in the early stage of the reaction. As the reaction progresses, the C–S–H gel releases Ca2+ to maintain the required alkaline environment21, resulting in decalcification of the C–S–H gel. CO2 gas was continuously passed through the solution to generate H2CO3 of weak acidity, which is extremely unstable and rapidly ionize in solution to generate HCO3− and CO32−. The free Ca2+ in the solution combines with CO32− to form stable CaCO3. C–S–H gel will eventually provide more Ca2+ than the present Ca(OH)2 when cement paste is exposed to high concentrations of CO222, Finally, Ca2+ in CaCO3 mainly originates from the C–S–H gel hydration product and since the presence of moisture is a necessary condition to initiate the carbonation reaction24, hydration will occur prior to the carbonation reaction. The XRD patterns in Fig. 3 in the reaction at different temperatures and different LFS particle sizes, indicating that the main product after carbonation was CaCO3. As CS cannot undergo hydration26, the CS present on the surface of LFS particles does not form C–S–H gel in the early stage of the reaction, but instead directly combines with CO32− in solution to form CaCO3. In addition, the diffraction peaks for CaAl2Si2O8 did not change significantly following the hydration and carbonation reactions, indicating that the anorthite structure was relatively stable and did not participate in the reaction process, which is consistent with the results reported by Ashraf27. With the increase in reaction temperature, the CaCO3 diffraction peaks gradually decreased, which may be due to the faster movement of CO2 molecules and expansion of the materials volume, resulting in an increase in the distance between the molecules of CO2 and H2O, weakening the intermolecular forces and hindering the generation of CO32-28. In addition, the increase in temperature also caused the solubility of CO2 to gradually decrease29, ultimately leading to a reduction in carbonation efficiency. With the decrease in particle size, the diffraction peaks of the calcium silicate phases and Ca(OH)2 gradually decreased, which might be explained by the simultaneous increase in surface area enhancing the contact area between particles and the solution, increasing the rate of reaction occurrence. However, a considerable amount of calcium silicates and Ca(OH)2 did not participate in the hydration and carbonation reactions in the largest LFS particle size, resulting in an incomplete reaction.Figure 30; (2) 300–500 °C, which was attributed to the dehydration of Ca(OH)231; (3) 500–850 °C, which was attributed to the decarbonization of CaCO332. In terms of dry weight, the mass loss over the entire temperature range was referred to as the total mass loss. The mass of CaCO3 can be obtained from the CO2 content generated by CaCO3 decarbonization33. The weight-loss peak appearing in the range of 300–500 °C before the hydration and carbonation reactions may be caused by the formation of Ca(OH)2 due to the combination of calcium-based components of LFS with atmospheric water under natural conditions. Two weight-loss peaks appeared in the range of 100–300 °C and 500–850 °C after the hydration and carbonation reactions, indicating that the calcium based components of LFS underwent hydration and carbonation reactions successively, resulting in C–S–H gel, Ca-deficient C–S–H and CaCO3 being generated.Figure 3 and total mass losses measured by TG analysis at different reaction temperatures or with different LFS particle sizes, showing that the mass of CaCO3 decreased with increasing temperature or LFS particle size. The content of CaCO3, C2S, and C3S was calculated using the reference intensity ratio (RIR) method. Results show that, as the temperature decreased, the C2S and C3S content reduced, indicating that increasing amounts of C2S and C3S participated in the hydration reaction, while the C–S–H gel continuously participated in the carbonation reaction, further promoting the hydration reaction. Therefore, a reaction temperature of 20 °C was more advantageous to the occurrence of the carbonation reaction. With the increase in particle size, the content of C2S and C3S increased, possibly because C2S and C3S in LFS require longer leaching channels to participate in the hydration reaction. Furthermore, this also negatively affected the later carbonation reaction. Therefore, larger particle sizes were not conducive to the subsequent occurrence of the carbonation reaction.Table 3 crystals  with a cubic structure 2. During carbonation, the C–S–H gel is decalcified to form Ca-deficient C–S–H gel and CaCO3. Furthermore, Ca(OH)2 directly reacts with CO32− in solution to form CaCO3. As illustrated in Fig. 3 and Ca-deficient C–S–H gel adhere to the surface of the reaction phase and the entire structure becomes dense, inhibiting the diffusion of Ca2+ into solution, which eventually prevents further progression of the reactions. According to the results shown in Table 2+ ions leached into the solution decreased, further reducing the formation of CaCO3. Therefore, increased temperature or particle size was detrimental to the hydration and carbonation reactions.Figure 29Si NMR chemical shift range of Qn structural units in silicates36, while Fig. 29Si NMR spectra following hydration and carbonation of LFS at different temperatures or when using different LFS particle sizes. In NMR analysis, Q0 represents isolated SiO4 tetrahedra, Q1 often appears at the end group of a straight chain, Q2 mostly appears as a middle group of a straight chain, Q3 has a double-stranded polymer structure or a layered structure and Q4 represents SiO4 tetrahedra connected to four SiO4 tetrahedra, in a three-dimensional network structure37. As shown in Fig. 0, with a few Q1 peaks appearing around -79 ppm, which may be due to the formation of the C–S–H gel by C2S and C3S hydration under natural conditions. As shown in Fig. 2, Q3, and Q4 peaks were present.Table 29Si NMR analysis. No Q3 and Q4 peaks were found to be present before the hydration and carbonation reactions, indicating that it is more difficult to complete C2S and C3S hydration and carbonation reactions under natural conditions. After the hydration and carbonation reactions, the content of Q0 gradually decreased or even disappeared, while the Q2 peak appeared, indicating that at this point C2S and C3S began to participate in the reaction due to the action of the hydration reaction, resulting in the formation of a C–S–H gel with longer chain lengths. Since the Si atom radius is smaller than the Ca atom radius, the bond length of the Si–O bond is shorter than that of the Ca–O bond, resulting in the bond energy of the Si–O bond being greater than that of the Ca–O bond. Furthermore, Si exhibits weak non-metal properties, making it difficult to form ions alone in compounds. Therefore, as the reaction time increased, the Ca–O bonds in the C–S–H gel were gradually broken under the action of the carbonation reaction, while the decalcification of the C–S–H gel results in Ca2+ combining with CO32− in solution to form CaCO3. After decalcification, the C–S–H gel was negatively charged due to the loss of Ca2+. To maintain the charge balance, H+ in solution is adsorbed by the C–S–H gel and combines with the broken Si–O– to form –Si–OH via protonation, which subsequently undergoes a condensation reaction with the adjacent –Si–OH. This condensation increases the mean silicate chain length and forms bridges between neighboring regions, thus pulling them closer together and causing shrinkage. This results in the formation of Q3 and Q4 structures of Ca-deficient C–S–H gel with longer chain lengths and a higher degree of polymerization. CS is composed of chains of SiO4 tetrahedra (Q2), which can directly form CaCO3 during the carbonation process38. Equations (d), the degree of polymerization (Pol) of the C–S–H gel, and the degree of hydration (H) of C2S and C3S, respectively39, as follows:Table quations , 2), an, an29Si 2S and C3S hydration, Ld reflects the degree of separation of Ca2+ in the C–S–H gel and Pol reflects the degree of re-polymerization of the SiO4 tetrahedra after decalcification of the C–S–H gel. According to the results shown in Table d was significantly increased to 13, indicating that more Ca2+ leached into the solution, leading to more carbonation in the rapid carbonation phase. Figure 2, thereby inhibiting the carbonation reaction. The observed trend in Pol was negatively correlated with the LFS particle size, which may be due to the decrease in particle size causing the –Si–OH groups in Ca-deficient C–S–H to connect and form bridges more easily with adjacent Si–OH groups, with this polymerization accelerating the carbonation reaction. Furthermore, the released Ca2+ combines with CO32− in solution to form CaCO3, which leads to a decrease in Ca2+ concentration. According to Kurdowski's theory, a low concentration of Ca will accelerate the hydration reaction of C2S and C3S and generate more C–S–H gel and Ca-deficient C–S–H gel to participate in the carbonation reaction. Therefore, the carbonation reaction promotes the hydration reaction.While H reflects the degree of C2S and C3S to form C–S–H gel. To maintain the required alkaline environment, C–S–H gel undergoes decalcification to form Ca-deficient C–S–H gel and Ca2+, with the Ca2+ and CO32− in solution forming structurally stable CaCO3. The Ca-deficient C–S–H gel undergoes condensation with adjacent –Si–OH groups to maintain charge equilibrium, resulting in a Ca-deficient C–S–H gel structure with a higher degree of polymerization of Q3 and Q4 units. Although increasing the temperature is beneficial to the conversion of Ca2+ in C2S and C3S, it hinders the combination of Ca2+ with CO32− and thus, inhibits the hydration carbonation efficiency. Increasing the particle size was not conducive to the hydration reaction of C2S and C3S, which in turn affects the subsequent carbonation reaction. Therefore, the hydration and carbonation reactions influence and promote each other.According to the obtained XRD, TG-DTG and SEM results, the hydration and carbonation reaction processes start with the hydration of C29Si NMR spectra revealed that the highest degree of hydration of C2S and C3S, the largest C–S–H gel decalcification effect and the highest degree of polymerization were achieved at a temperature of 20 °C, indicating that within the investigated temperature range the hydration carbonation reaction was optimal at this temperature. Moreover, the hydration of C2S and C3S and the polymerization of C–S–H gel decreased gradually with increasing temperature, indicating that the increase in temperature was not favorable for the hydration and carbonation reactions.Deconvolution of the 29Si NMR spectra further indicated that the highest degree of hydration of C2S and C3S and the highest level of decalcification and polymerization of the C–S–H gel were achieved when the LFS particle size was smaller than 38 μm, indicating that the hydration and carbonation reaction was optimal at this particle size. The polymerization degree of the C–S–H gel decreased gradually with increasing particle size, showing that larger particle sizes were not conducive to the hydration and carbonation reactions.Deconvolution of the In future research, the effect of cementitious substances on the properties of LFS-based cement materials will be investigated.


At concentrations suitable for NMR experiments, the slow formation of a supramolecular polymer was observed, followed by precipitation. Single crystals X‐ray structure elucidation confirmed the formation of a polymer with 1 : 1 stoichiometry in the solid state.Inclusion of polymethine cyanine dyes in the cavity of macrocyclic receptors is an effective strategy to alter their absorption and emission behavior in aqueous solution. In this paper, the effect of the host‐guest interaction between cucurbit[8]uril (CB[8]) and a model trimethine indocyanine (Cy3) on dye spectral properties and aggregation in water is investigated. Solution studies, performed by a combination of spectroscopic and calorimetric techniques, indicate that the addition of CB[8] disrupts Cy3 aggregates, leading to the formation of a 1 : 1 host‐guest complex with an association constant of 1.5×10 The different outcome of the dye Cy3 inclusion in CB[8] cavity in the solid state and in water has been elucidated via crystal structure determination and NMR, ITC and fluorescence titrations respectively. The initial 1 : 1 complex formed in water evolves spontaneously toward an insoluble linear polymeric structure. Bearing an additional glycoluril unit Scheme , CB[8] hH‐iodolium iodide (Cy3), with CB[8] prop‐l‐enyl]‐3,3‐dimethyl‐31H NMR spectroscopy in D2O. The high‐resolution MALDI‐TOF spectrum of an aqueous solution of the two compounds in stoichiometric ratio evidenced the presence of the binary complex . Figure 1H NMR spectrum of Cy3 at 0.1 mM and the spectra obtained after addition of CB[8]. A sizable upfield shift was observed for the aromatic protons  upon addition of 0.4 equiv. of CB[8] . Therefore, the largest perturbation was observed for the protons in the aromatic region, thus suggesting an asymmetric binding of the longer Cy3 molecule with respect to the depth of the CB[8] cavity. Indeed, symmetric 1 : 1 binding should expose the aromatic protons outside the macro‐ring. On the other hand, it is notable that the spectrum still shows symmetric signals for the Cy3 molecule. This can be rationalized in terms of fast exchange between complexed and uncomplexed Cy3 molecules. Additionally, when the 1H NMR spectrum was recorded with one equivalent of CB[8]  spectrometry and T=25 °C where the cell contains a 0.1 mM aqueous solution of CB[8], and Cy3 is in the syringe at a concentration of 1.0 mM. The observed exothermic binding curve has a sharp inflection point at a molar ratio of one indicating the formation of a 1 : 1 assembly in aqueous solution. A second and less pronounced binding event is present when 0.5 equivalents of guest are added to the host solution, suggesting the coexistence of a 2 : 1 host‐guest complex. The data fitting of the titration with a “two sequential binding sites” model gave an association constant Ka,1=3.9×107 M−1 with a ΔH1 of −5.1 kcal mol−1 for the first binding of the guest and a Ka,2 of 1.4×105 M−1 plus a Δ2H of −0.7 kcal mol−1 for the interaction with the second host. The corresponding calculated thermodynamic parameters ΔG1 and TΔS1 are −10.4 kcal mol−1 and 5.3 kcal mol−1, respectively, while Δ2G=−5.6 kcal mol−1 and TΔS2=4.9 kcal mol−1, making the two binding processes both enthalpy and entropy driven. These thermodynamic values are in agreement with the displacement of high energy water molecules from the cavity by the guest, which is known to be one of the main driving forces of CB[8] complexation.Isothermal titration calorimetry (ITC) experiments were performed to investigate the binding strength, stoichiometry and thermodynamic parameters for the host‐guest complexation of Cy3 by CB[8]. Figure maxλ along with a decrease in the absorption coefficient. A precise isosbestic point is observed at 545 nm only at low CB[8]/Cy3 ratio, suggesting that at higher CB[8] content the equilibrium is not between two unique species. This spectral shift is in line with a change in the microenvironment of the dye caused by its inclusion in the hydrophobic cavity of CB[8], as already reported in literature for the tetrafluoroborate analogue.The spectroscopic behavior of the dye upon addition of CB[8] was studied in aqueous solution at a concentration of 1 μM to minimize dye self‐aggregation and any effect of inner filter. As shown in Figure −3 ns−1) than in water solution (2.99×10−3 ns−1), consistently with the observed decrease in absorption coefficient upon complexation, while the non‐radiative decay rate stays practically constant (0.287 vs. 0.234 ns−1). Since we know that only one Cy3 molecule is present in the complex, the increase of the radiative decay constant (related to transition dipole moment) for free Cy3 in water suggest the presence of aggregates when CB[8] is not present in water. In particular, the results are consistent with slightly superradiant aggregates, which are disaggregated upon complexation with CB[8].Fluorescence spectra show a small red shift upon addition of increasing equivalents of CB[8], as well as a significant decrease of the intensity (the fluorescence quantum yield amounts to 1 % free Cy3 and to 0.7 % for the 1 : 1 complex). This last observation is in contrast with the idea of an enhanced emission upon complexation in a restricted environment and deserves more investigation. Fluorescence decays were measured, getting an average fluorescence lifetime of 3.48 ns for free Cy3 in water, and of 4.24 ns for a solution containing the supramolecular 1 : 1 complex . Fluorescence intensity and lifetime data unexpectedly lead to a smaller radiative decay rate for Cy3 in the complex  for free Cy3 in water. Cy3 molecules in these aggregates are probably very loosely interacting, as suggested by the small spectral variations with respect to the complex and by the ease of rupture of the aggregates when adding CB[8].To prove this hypothesis, anisotropy measurements were performed for free Cy3 both in water and in a much more viscous solvent . For free Cy3 in water a fluorescence anisotropy value r=0.26 was found, while the measurement in glycerol gave the fundamental anisotropy value r−5 M, we prepared a mother solution and filtered it, to be sure to remove any non‐solubilized compound. The filtered mother solution was then diluted by a factor 8 and by a factor 16. The corresponding spectra , show a decrease of extinction coefficient and of concentration‐normalized emission intensity upon dilution. This strongly supports the formation of aggregates, favored in more concentrated solutions, and the superradiant nature of the aggregates (related to increased transition dipole moment).To unambiguously confirm the presence of Cy3 aggregates in water, we performed absorption and fluorescence measurements on solutions at different concentration. Since Cy3 does not have a good solubility in water already at concentrations of the order of 10Ka estimated by ITC is only an apparent binding constant. We therefore used the fluorescence titration (at much lower Cy3 concentration with respect to the ITC experiment) to obtain a more realistic  Ka. The fluorescence data could be fitted through the Benesi‐Hildebrand equation, and confirmed a 1 : 1 host:guest complex, with a Ka=1.5×106 M‐1 . Interestingly, the major effect of complexation on fluorescence intensity seems to be played by the rupture of aggregates upon the formation of the complex.Under the premise of dye aggregation, the 1 space group. The solid state structure shows that the asymmetric unit contains one CB[8] macrocycle, one trans Cy3 cation with its chloride counterion and a total of 13 co‐crystallized water molecules  were grown in water. Diffraction data from small twinned crystals was collected using synchrotron radiation at 100 K. The crystal belongs to the polar orthorhombic Pna2s Figure a. Therefs Figure b. The ara axes. One aromatic ring of the dye is almost exactly centered in the hydrophobic cavity, with distances of its barycenter from the mean planes of the two CB[8] oxygen portals of 3.16 Å for the entry portal and 2.98 Å for the second portal ‐N‐methylpyridinium6 M−1, obtained via fluorescence titration. The optical spectroscopic investigation suggests the presence of Cy3 aggregates in water which are disrupted by addition of CB[8]. The 1H NMR spectra confirm the complexation process and evidence a slow formation of a supramolecular polymer with subsequent precipitation after a few hours. The X‐ray structure obtained from single crystals confirms the formation of a polymer with 1 : 1 stoichiometry in the solid state. Both aromatic terminals of Cy3 are hosted by two adjacent CB[8] units and each CB[8] hosts the aromatic terminals of two Cy3 guests, thereby forming linear supramolecular polymeric chains. The columnar arrangement of the Cy3⋅CB[8] complex represents an interesting nanotubular framework with incorporated dye molecules.In conclusion, we investigated the interaction of a indocyanine trimethine dye, Cy3, with cucurbit[8]uril, CB[8], by MS, NMR, ITC, UV‐Vis absorption and fluorescence spectroscopy and single crystal X‐ray diffraction. The solution studies indicate the formation of a 1 : 1 complex of Cy3 and CB[8]. The ITC measurements reveal that the complexation is an enthalpically and entropically driven process, with an apparent association constant of 1.5×102092004 contain(s) the supplementary crystallographic data for this paper. These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service.Deposition Number(s) The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.Supporting InformationClick here for additional data file.


Bacillus thuringiensis and are toxic against lepidopterans, reason why the vip3Aa gene has been introduced into cotton and corn to control agricultural pests. Recently, the structure of Vip3 proteins has been determined and consists of a tetramer where each monomer is composed of five structural domains. The transition from protoxin to the trypsin‐activated form involves a major conformational change of the N‐terminal Domain I, which is remodelled into a tetrameric coiled‐coil structure that is thought to insert into the apical membrane of the midgut cells. To better understand the relevance of this major change in Domain I for the insecticidal activity, we have generated several mutants aimed to alter the activity and remodelling capacity of this central region to understand its function. These mutants have been characterized by proteolytic processing, negative staining electron microscopy, and toxicity bioassays against Spodoptera exigua. The results show the crucial role of helix α1 for the insecticidal activity and in restraining the Domain I in the protoxin conformation, the importance of the remodelling of helices α2 and α3, the proteolytic processing that takes place between Domains I and II, and the role of the C‐t Domains IV and V to sustain the conformational change necessary for toxicity.Vip3 proteins are produced by  Spodoptera exigua. The results show the crucial role of helix α1 for the insecticidal activity and for restraining the Domain I in the protoxin conformation, the importance of the remodelling of helices α2 and α3, the proteolytic processing that takes place between Domains I and II, and the role of the C‐t Domains IV and V to sustain the conformational change necessary for toxicity.To better understand the relevance of the major change in Domain I of Vip3 proteins in the transition from protoxin to activated toxin for the insecticidal activity, we have generated several Vip3Aa mutants aimed to alter the activity and remodelling capacity of this central region to understand its function. These mutants have been characterized by proteolytic processing, negative staining electron microscopy, and toxicity bioassays against  Bacillus thuringiensis, and are toxic against a wide range of lepidopteran pests  in combination with the Cry insecticidal proteins to prevent the evolution of insect‐resistant populations  are produced during the vegetative growth phase of the bacterium The Vip3 proteins are secreted as protoxins of ~89 kDa which, once ingested by the larvae, are processed by the midgut proteases into two different fragments of 19–22 and 62–66 kDa that remain attached and conform to the activated protein  and Spodoptera frugiperda Sf21 cultured cells, or for exerting toxicity against the latter , revealing that they consist of a homotetramer in which each subunit is composed of five structural domains  (residues 10–789) was cloned in LIC 1.5, as previously described  containing each of the mutant vectors. Cells were grown in Luria‐Bertani broth to exponential phase (OD600 ~ 0.6), then induced with 0.5 mM isopropyl β‐d‐1‐thiogalactopyranoside (IPTG), incubated at 37°C for 3 h and then centrifuged and stored at −20°C. For purification, thawed cells were resuspended in buffer A  pH 8.0, with 1 mM phenylmethanesulfonyl fluoride and 1 mM Tris (2‐carboxyethyl)phosphine hydrochloride, and sonicated for 5 min. The cell lysate was centrifuged at 25,000 g for 30 min and the clarified supernatant was loaded into a 5 ml Streptrap column . The protein was eluted with 5 mM d‐desthiobiotin dissolved in buffer A pH 8.0. The StrepII‐tag was removed adding 3C PreScission protease  to the Vip3Aa mutants at a 1:1/25 (protein:protease) molar ratio followed by dialysis. The sample was then subjected to two other steps of affinity chromatography with a Streptrap and a GST‐Trap column to separate the non‐tagged protein from any remaining tagged protein and protease. The non‐tagged protein was further purified by gel filtration chromatography using a ProteoSEC 16/60 6–600 HR . Samples containing the purest protein were concentrated to ~5 mg/ml, aliquoted, frozen with liquid nitrogen, and stored at −80°C for further use.The production of Vip3Aa WT and mutants used in this study was described previously  and the supernatant containing the processed proteins was incubated for 10 min at room temperature with 10 mM AEBSF [4‐(2‐aminoethyl)benzenesulfonyl fluoride HCl] serine protease inhibitor to avoid further processing of the proteins. Proteolytic fragments were separated on 12% SDS‐PAGE.The reaction was carried out in a final volume of 20 μl, in buffer A pH 7.5 or in 20 mM carbonate buffer pH 10, depending on the pH conditions required. All samples were incubated for 1.5 h at 37°C for trypsin and α‐chymotrypsin digestions and at 30°C for midgut juice and BBMV digestions. Then, samples were clarified by centrifugation  uranyl acetate as described previously . Control larvae were just fed with the buffer‐dye mixture. Individual midguts of treated larvae were homogenized in 10 μl of 10 mM AEBSF and incubated at room temperature for at least 10 min to stop any further proteolytic processing of the proteins. Samples were subjected to 12% SDS‐PAGE along with a Dual Color Molecular Marker (Bio‐Rad). After SDS‐PAGE, the proteins were transferred to a nitrocellulose membrane  by humid transference in blotting buffer  using the Bio‐Rad system. The lane of the molecular marker was separated from the rest using a clean razor. The two pieces of the membrane were blocked overnight with 5% skimmed milk dissolved in PBST buffer (1× PBS + 0.1% Tween 20). The part of the membrane containing the proteins was incubated for 1 h at room temperature (with shaking) with a primary antibody anti‐Vip3Aa16 raised in the rabbit at 1/5000 antibody dilution in PBST containing 0.5% skimmed milk. After 3 washing steps with PBST (5 min each), the membrane was incubated for 1 h at room temperature with the secondary antibody anti‐rabbit conjugated with HRP peroxidase (Sigma Aldrich) at 1/20.000 antibody dilution in PBST containing 0.5% skimmed milk. The part of the membrane containing the molecular marker was incubated for 1 h at room temperature with Precision Protein StrepTactin‐HRP (Bio‐Rad) at 1/20.000 dilution in PBST. After three washing steps (5 min each) with PBST, the bands were visualized with the Amersham ECL Prime Western Blotting Detection Reagent using Image Quant LAS 4000.Purified WT and Vip3Aa_195‐AVAA‐198 proteins, diluted in buffer A pH 7.5, were mixed with phenol red and sucrose (18 μl of 2 mg/ml protein +2 μl of phenol red +2.5 μl of 1.6 M Sucrose). Then, To better understand the role of the structural features of Domain I in the toxicity, we have engineered several mutants targeting different regions or processes which can be grouped into four categories: (i) mutants directed to affect the N‐t part of the protein, named as Vip3Aa_∆α1, Vip3Aa_41‐KVKK‐44 and Vip3Aa_M34L, (ii) mutants directed to affect the proteolytic processing and conformational change of the activated protein, named as Vip3Aa_195‐AVAA‐198 and Vip3Aa_S‐S, (iii) mutants directed to reduce the internal diameter of the tetrameric coiled‐coil observed in the toxin conformation, named as Vip3Aa_L146F and Vip3Aa_L146M, and (iv) a mutant lacking the C‐t Domains IV and V (Vip3Aa_∆IV‐V). A detailed description of the mutations and their expected effect on the structure and/or properties of the protein, according to the structural data, are summarized in Table S. exigua larvae (SeMJ). As can be observed in Figure SeMJ, only the 65 kDa band was observed  were analysed by negative staining transmission electron microscopy to explore if they had an effect at the structural level to influence the switch from protoxin to toxin conformation . Interestingly, we observed that the mutant Vip3Aa_∆α1 adopted the toxin conformation independently of the incubation with trypsin. This result indicated that the N‐t comprising helix α1 had a relevant role in the stabilization of the helices at Domain I. Indeed, in the protoxin conformation helix α1 interacts with the core of the tetramer formed by Domain II and it is also connected to the loop α1α2 that allows the twist of helix α2 Figure . Thus, iThus, our results indicate that remodelling of Domain I is driven by a fine‐tuned mechanism where not only proteolytic cleavage is necessary but also an appropriate free energy barrier to favour the conformational change.S. exigua. The WT protein showed LC50 and LC90 values of 19.5 (8.6–28) ng/cm2 and 62.9 (47–98) ng/cm2, respectively  ng/cm2, whereas it essentially lost all toxicity at the LC90 level, indicating a different dose–response compared to the WT protein  and 27.7 (17–46) ng/cm2, respectively. The mutant Vip3Aa_195‐AVAA‐198, in which the cleavage site was eliminated, showed toxicity values very similar to that of the WT protein  and the single mutants Q96C and L100C also showed similar toxicity as the WT . The mutants Vip3Aa_L146F and Vip3Aa_L146M, which according to the amino acid substitutions in the atomic models likely count with a reduced internal diameter in the tetrameric coiled‐coil . We mutated the sequence 41‐DTGG‐44, which contains two Gly, for 41‐KVKK‐44, resulting in the reduction of the toxicity of this mutant. Although our mutation was intended to introduce a cleavage site for the release of helix α1, the mutation has demonstrated that stiffness in the loop between helices α1 and α2 is deleterious. Thus, flexibility in this region is important for remodelling and therefore, for the insecticidal activity of the protein.S. exigua (Banyuls et al., A fourth structural feature that unexpectedly has resulted to be critical for remodelling is the C‐t part of the protein, formed by Domains IV and V which are not interacting with Domain I. Their absence in Vip3Aa favoured the protoxin conformation and abrogated toxicity. This is in agreement with previous reports showing a drastic lack of toxicity of C‐t truncated Vip3Aa in bioassays with several lepidopteran pests, including S. exigua. Our results are in contrast to those obtained by Zhang et al. (S. exigua. It is important to note that the mortality values obtained by these authors for all the Vip3Aa proteins tested were much lower than ours, suggesting important differences in the susceptibility of the S. exigua colonies against Vip3Aa proteins.Finally, the 195‐AVAA‐198 cleavage site is also an essential structural feature for remodelling and toxicity. Surprisingly, we have found that alternative proteolytic processing may also take place in vivo, and this explains why the toxicity of this mutant does not differ from that of the WT, at least against g et al. , where tIn conclusion, we have demonstrated here the crucial role of helix α1and the remodelling which takes place in the toxin conformation at Domain I for the insecticidal activity of the Vip3Aa protein. We have proposed several structural features that are relevant for the timing in which Vip3Aa remodelling occurs to drive its biological activity: the role of helix α1, essential for toxicity, holding the Domain I in the protoxin conformation and most probably interacting with the membrane in the toxin conformation, the flexibility of loop α1α2, the remodelling of helices α2 and α3, the proteolytic processing that takes place at the loop α4α5 between Domains I and II and the role of the C‐t Domains IV and V to sustain the conformational change.ML‐B, YB, PC, and JF designed the experiments. ML‐B, FP‐M, RN‐R, and YB performed the experimental work. ML‐B, YB, EA‐P, PC, and JF discussed and interpreted the results. ML‐B, PC, and JF wrote the main manuscript text. ML‐B, EA‐P, and PC prepared the figures. All authors reviewed the manuscript.The authors declare no competing interests.Appendix S1Click here for additional data file.


After stimulation, it diffuses from the plasma membrane where it is produced to the endoplasmic reticulum where its receptors are localized. Based on in vitro measurements, IP3 was long thought to be a global messenger characterized by a diffusion coefficient of ~ 280 μm2s−1. However, in vivo observations revealed that this value does not match with the timing of localized Ca2+ increases induced by the confined release of a non-metabolizable IP3 analog. A theoretical analysis of these data concluded that in intact cells diffusion of IP3 is strongly hindered, leading to a 30-fold reduction of the diffusion coefficient. Here, we performed a new computational analysis of the same observations using a stochastic model of Ca2+ puffs. Our simulations concluded that the value of the effective IP3 diffusion coefficient is close to 100 μm2s−1. Such moderate reduction with respect to in vitro estimations quantitatively agrees with a buffering effect by non-fully bound inactive IP3 receptors. The model also reveals that IP3 spreading is not much affected by the endoplasmic reticulum, which represents an obstacle to the free displacement of molecules, but can be significantly increased in cells displaying elongated, 1-dimensional like geometries.Inositol 1,4,5-trisphosphate (IP In many instances, Ca2+ increases are initiated by the formation of inositol 1,4,5-trisphosphate (IP3) at the plasma membrane2. IP3 diffuses in the cytoplasm and binds its receptors located on the endoplasmic reticulum that contains a large quantity3 of rapidly mobilizable Ca2+. Activity of these receptors is also regulated by cytosolic Ca2+, both positively and negatively4. At the spatial level, IP3 receptors (IP3R) are not homogeneously scattered on the ER membrane, but rather grouped in clusters of ~ 10–20 channels6. Because the number of clusters in cells is limited, coupling between clusters has been much investigated to understand how Ca2+ signaling can be coordinated at the cellular level, giving rise to Ca2+ oscillations and waves9. Most studies have focused on Ca2+-mediated communication between clusters, since it was assumed that all clusters rapidly experience the increase of IP3 that results from the stimulation of the cell. In cytosolic extracts of Xenopus oocytes, apparent diffusion coefficients of Ca2+ and IP3 indeed equal 38 ± 11 μm2s−1 and 283 ± 53 μm2s−1, respectively10. Considering in addition the respective rates of Ca2+ removal from the cytoplasm and IP3 metabolism, these values led Allbritton et al.10 to conclude that Ca2+ mostly acts in restricted domains and that IP3 is a global messenger.Within all cell types, Ca3 acts as a global messenger was however contradicted by indirect in vivo observations11. To evaluate the rate at which IP3 diffuses in an intact cell, the group of Ian Parker used the IP3-evoked liberation of Ca2+ from a cluster of IP3Rs as a detector of the presence of IP3 at the cluster location. Such Ca2+ increases evoked by clusters of IP3Rs are well-known as Ca2+ puffs12. If a non-metabolizable IP3 analog is released at one extremity of an elongated SH-SY5Y cell, the time lapse between the localized IP3 increase and the occurrence of the first Ca2+ puff significantly increases with the distance between the spot of IP3 release and the location of Ca2+ rise. This time lapse, called latency, reflects the time taken by IP3 to diffuse on this distance. Accordingly, when the IP3 analog is uniformly released across the entire cell, latency does not show any systematic variation along the cell length but decreases with increasing IP3 concentration. The fact that latency increases with the distance from the IP3 release spot clearly indicates that IP3 does not act as a global messenger in these conditions. From these observations, Dickinson et al.11 inferred the value of the IP3 diffusion coefficient by resorting to a simplified mathematical expression for the probability of Ca2+ puff occurrence coupled to 1-dimensional (1D) simulations of IP3 diffusion. These calculations predicted an effective IP3 diffusion coefficient lower than 10 μm2s−1, i.e. about 30 times slower than in vitro estimations.The notion that IP3 diffusion within cells as compared to cytosolic extracts was ascribed to the presence of IP3Rs that are not fully bound to IP313. IP3Rs are indeed tetramers that release Ca2+ only when each IP3R monomer is occupied by IP314. Given that IP3 binding on IP3Rs is not cooperative, most of the receptors are partially bound as long as [IP3] remains lower than the KD of IP3 binding, i.e. ~ 100 nM (KD = 119 nM reported by Taylor and Konieczny13) and thus act as a buffer of IP3. Assuming fast binding and unbinding of IP3 to and from its receptor, the resulting effective diffusion coefficient15 is given by:D represents the IP3 diffusion coefficient in the absence of buffers, ST the concentration of IP3R monomers, and KD the equilibrium dissociation constant of IP3 from its receptor. ST is cell dependent, in the range of 80 nM to 2 μM18 with an estimated value of 542 nM in SH-SY5Y cells18. Thus, the buffering effect described by Eq. 3] = KD. In Dickinson et al.11 experiments, [IP3] was in the Ca2+ oscillatory range. Because IP3 concentrations are typically of the order or lower than the KD of the IP3R in the oscillatory range19, one can estimate that the effective diffusion coefficient of IP3 considering IP3 buffering by not fully bound receptors (DI) should be ~ 100 μm2s–1 . These states describe the global behavior of a cluster composed of close-by IP3Rs. The phenomenological model was shown to reproduce experimentally observed statistical properties of Ca2+ puffs31 and to describe the passage from localized puffs to global Ca2+ spikes when clusters are effectively coupled by Ca2+ diffusion9.We performed explicit stochastic simulations of  Ca3, this model is extended to take the dependence of puff occurrence on IP3 concentration into account. Thus, in the Gillespie's simulations, the propensity of transition of the cluster from the closed (C) to the open (O) state now writes:kCO stands for the rate constant that characterizes the passage of the cluster from the closed to the open state, NCa is the number of cytosolic Ca2+ ions and Ω is the extensivity parameter. NIP and KIP represent the number of IP3 molecules and the IP3 dissociation constant of the IP3R (multiplied by Ω), respectively. Although the model does not simulate individual IP3 receptors, we assumed that puff firing probability is related to the probability of one tetrameric IP3R to be fully bound to IP313. As shown below, Eq. 3 concentration reported experimentally32.To evaluate the effective diffusion coefficient of IP3 concentration is described deterministically and two different protocols of photorelease of caged IP3 are simulated. The first one corresponds to a spot photorelease of caged IP3 in a small region of the cell:The evolution of IP3 is liberated at different spots to get an increase in [IP3] that is nearly spatially homogeneous in the whole cell. In this case:In the second one, called “distributed photorelease”, IPDI stands for the effective IP3 diffusion coefficient, 3 release upon laser flash and H for Heaviside function. This function equals 1 if t ≤ tph and 0 otherwise. Considering that 3 is distributed among the ten IP3 releasing sites. Basal IP3 concentration is set to 50 nM33.In Eqs. –6, DI stcase Eq.  because https://github.com/RobertoOrnelasGuevara/ca2-puffs/tree/main. Except for the process describing IP3 dynamics listed here above, the values of parameters are taken from Voorsluijs et al.9 and are listed in Table Supplement 2.A detailed description of the algorithm is provided in the Supplementary Information (Sect. 1), together with a Table listing the propensities used for the stochastic part of the algorithm and the evolution equations used in the deterministic part (Table Supplement 1). Code are available at 3 typically evoke local Ca2+ signals known as Ca2+ puffs34. As the concentration of IP3 increases, Ca2+ puffs become more frequent and transform into Ca2+ waves spreading regeneratively across the cell. These repetitive waves, also known as Ca2+ spikes, are more regular than puffs and are often referred to as Ca2+ oscillations. Their stochastic origin is visible by the linear relation between the variance on the interspike interval and the mean interspike interval itself35. In experiments, Ca2+ waves propagation can be hindered by loading the cells with the slow Ca2+ buffer EGTA, which reduces communication between puff sites36. At high EGTA concentrations, each cluster practically behaves as an independent entity and thus generates Ca2+ puffs on a large range of [IP3].Low concentrations of IP3 concentration, with and without coupling between clusters by Ca2+ diffusion. In all cases, [IP3] is constant in time and space. To model puff dynamics when clusters are uncoupled (corresponding to the presence of EGTA), a single cluster was simulated and the local Ca2+ concentration averaged in 1fL around the cluster was monitored. The resulting dynamics of [Ca2+]  also decrease with [IP3]. These results were used to infer the values of the rate of release of caged IP3 in response to the flash, i.e. the value of parameter θ in Eqs. 11 for the distributed photorelease of IP3 -and replotted in Fig. 3 concentrations that, in the simulations, gave the same mean latencies as those reported experimentally using Fig. θ allowing to reach these spatially uniform concentrations of IP3 when assuming 10 photorelease spots of durations equal to 0.1, 0.2 and 0.5 s respectively, as in the experiments. Good agreement between mean latencies and flash durations was found for θ = 600 μM/s , we reasoned that the discrepancy between the idealized simulations of cells containing one single cluster and experiments may be due to a slight stimulation of puff activity by Ca2+ in the experiments, which is not totally buffered by EGTA. At 5 μM EGTA, the distance on which free Ca2+ ions diffuse before being captured by EGTA is indeed between 2.1 and 3.8 μm (see Sect. 2 of Supplementary Information). As these distances are of the order of the mean distance between clusters in the SH-SY5Y cells, there is some Ca2+ stimulation of puff activity in the distributed photorelease protocol. Because we only model one cluster at a time, this effect does not occur in our simulations. Thus, the value θ = 600 μMs−1 deduced in the previous section overestimates the IP3 release rate and must be seen as an upper limit.We investigated the relation between first puff latency and distance from spot photorelease by performing independent simulations for twelve clusters, each one located at a different distance from the photorelease spot in an ellipse-shaped 2D cell. Such a configuration allows to reproduce the absence of communication between the cluster sites that results from the addition of EGTA. Binding of IP see Eq. . The ellθ (250 μMs−1) representing the rate of IP3 release due to the flash  and decreasing the value of DI. In this case, despite the agreement for mean latencies, there is a systematic and important overestimation of the minimal first puff latencies. Such disagreement cannot be ascribed to a reduced number of experimental observations because a larger number of data is only expected to further decrease the shortest possible latency at a given distance. In contrast, it reveals that the low value of DI is unable to simulate the quite rapid, moderate IP3 increase away from the spot. The larger concentration of IP3 due to the overestimated θ explains why mean latencies agree with observations, as they compensate in average for the slow diffusion. Simulations also predict that with θ = 250 μMs−1 best agreement is obtained with DI = 100 μm2s−1, as compared to smaller or larger values  InsP3 5-phosphatase-transfected cells, a loss of Ca2+ signal occurred in only 1.1% of the mutant InsP3 5-phosphatase-transfected cells41. Such a sensitivity to the location of an IP3 metabolizing enzyme could not be observed if IP3 was indeed such a fast-diffusing molecule that rapidly becomes homogeneously distributed in the whole cell.IP3 was predicted by Dickinson et al.11 on the basis of their observations of puff latencies in SH-SY5Y cells in response to the photorelease of a caged non metabolisable IP3 analog. Although this indirect evaluation of the rate of IP3 spreading is submitted to uncertainties due to the stochastic nature of Ca2+ puffs and to their dependence on Ca2+ concentration that is not fully controlled by EGTA, it has the unique advantage of corresponding to realistic cellular conditions. Based on a simplified description of Ca2+ puff dynamics, these authors proposed that observations can be accounted for if the effective diffusion coefficient of IP3 in these cells is in the 3–10 μm2s−1 range. This conclusion was re-examined in the present study, based on a detailed description of Ca2+ puff dynamics. Our results also point to a lower value of the effective diffusion coefficient of IP3 in intact cells than in cytosolic extracts of Xenopus oocytes but concluded to a decrease by a factor ~ 3 instead of ~ 30. On the basis of their in vitro measurement, Allbritton et al.10 predicted the range of action of IP3 to be ~ 24 μm. This is an estimation of the range on which IP3 can diffuse before being metabolized into another inositol phosphate. Given that this range is proportional to DI value inferred in Dickinson et al.11. Since most mammalian cells have a diameter of ~ 12 μm42, the two values lead to significantly different physiological conclusions. With an effective diffusion coefficient of 100 μm2s−1, IP3 acts as a global messenger in many cases, although this would not be the case in very large cells such as oocytes or some adipocytes.Accordingly, a much lower value of the effective diffusion coefficient of IP3 diffusion in the 1D-like astrocytic process  in three ways. First, only one spatial dimension was considered in the latter study, while we performed 2D or 3D simulations. As illustrated for simulations of IP016 in th3 diffusion coefficient is indeed larger than 10 μm2s−1, we simulated the experimental protocol of distributed photorelease of IP3 used in Dickinson et al.11 to induce a spatially uniform rise in IP3 concentration and described in the methodology section here above. Simulations performed with DI = 10 μm2s−1 and shown in Figure 3 concentration remains spatially inhomogeneous up to at least 10 s after the flash. This is not in agreement with the observation that first puff latencies are independent from the distance from cell end under this protocol. In contrast, when DI = 100 μm2s−1, IP3 rapidly equilibrates in the whole cell . Surprisingly, the presence of the ER, which acts as an obstacle to free diffusion, does not much affect mean effective diffusion. This lack of global effect can be ascribed to the fact that while some paths are slower because of obstruction, others are faster because of the presence of channel-like structures which favor fast diffusion thanks to the absence of dilution effect. It should be noted that we did not consider the tubular-shaped of this network, nor its dynamical evolution into a more fragmented structure44.Simulations predict that IPell Fig. , in a 3Dell Fig.  or in a ell Fig. . However2s−1 for the effective diffusion coefficient of IP3 that emerges from our simulations is in line with the buffering effect exerted by the non-fully bound IP3R tetramers, as proposed previously13. Thus, it is expected to be smaller in cell types with higher expression levels of IP3R. In the same line, effective diffusion is much accelerated upon increasing [IP3] because buffers become saturated. Thus, upon cell stimulation by an agonist that generally leads to a surge in [IP3] followed by a decrease, the properties of diffusion are expected to vary. Even more significant in this respect are the IP3 oscillations that have been observed in several cell types and that arise from the activation by Ca2+ of IP3 synthesis by PLC45 and/or of IP3 metabolism by a 3-kinase47. Further studies are required to assess the consequences of the interplay between the temporal changes in IP3 concentration and its diffusional properties.The value of 100 μmSupplementary Information.


Release of soluble O2 from H2O2 by catalase is promising, but spatiotemporal control of the process is challenging to achieve. Here, we show monitoring and control by optical sensing within a porous carrier of the soluble O2 formed by an immobilized catalase upon feeding of H2O2. The internally released O2 is used to drive the reaction of d‐amino acid oxidase (oxidation of d‐methionine) that is co‐immobilized with the catalase in the same carrier. The H2O2 is supplied in portions at properly timed intervals, or continuously at controlled flow rate, to balance the O2 production and consumption inside the carrier so as to maintain the internal O2 concentration in the range of 100–500 µM. Thus, enzyme inactivation by excess H2O2 is prevented and gas formation from the released O2 is avoided at the same time. The reaction rate of the co‐immobilized enzyme preparation is shown to depend linearly on the internal O2 concentration up to the air‐saturated level. Conversions at a 200 ml scale using varied H2O2 feed rate (0.04–0.18 mmol/min) give the equivalent production rate from d‐methionine (200 mM) and achieve rate enhancement by ∼1.55‐fold compared to the same oxidase reaction under bubble aeration. Collectively, these results show an integrated strategy of biomolecular engineering for tightly controlled supply of O2 substrate from H2O2 into carrier‐immobilized enzymes. By addressing limitations of O2 supply via gas‐liquid transfer, especially at the microscale, this can be generally useful to develop specialized process strategies for O2‐dependent biocatalytic reactions.While O 2 substrate to biotransformations in porous carriers requires tailored solutions beyond bubble aeration. Here, the authors used immobilized catalase (CAT) to produce O2 from H2O2 inside the carrier to generate O2 for reaction of a co‐immobilized D‐amino acid oxidase (DAAO). O2 measurement in liquid and inside particle (based on a co‐immobilized luminescence dye) guided development of a H2O2 feeding strategy for tightly controlled O2 supply. This enabled rate enhancement compared to the same oxidase reaction under bubble aeration.Supply of O CATBordetella pertussis)catalase (from DAAOd‐amino acid oxidase (from Trigonopsis variabilis)Z‐CAT and Z‐DAAObasic2 fused to the N‐terminus of the enzymeCAT and DAAO harboring the cationic binding module Z12‐dependent oxidation reactions have considerable importance in organic synthesis, carried out up to the industrial manufacturing scale  entrained into the bulk liquid  of solid particles used as carriers for enzyme immobilization , there are essentially two options. One is to increase by partial pressure the O2 concentration in the bulk liquid and in that way enhance the O2 transfer rate by pore diffusion  immobilized in the solid carrier  in which defined gas‐liquid contacting can be complicated technically and spatiotemporal control over the available O2 is desired  with high affinity and selectivity. The immobilized Z‐CAT retained up to ∼80% of the specific activity of the soluble enzyme. Second, the ReliSorb SP400 carrier with Z‐CAT immobilized on it was labeled with a Ru(II) luminophore for optical sensing of O2. Time‐resolved measurements from inside the solid carrier were used to monitor the O2 released from H2O2 by the immobilized Z‐CAT. When H2O2 was supplied, a strongly positive O2 concentration gradient between the carrier (high [O2]) and the surrounding bulk liquid (lower [O2]) was developed. In the presence of an O2 consuming reaction in solution (d‐glucose oxidation by glucose oxidase), the gradient of [O2] (Δ[O2]) reached values that exceeded by up to ∼1.5‐fold the air‐saturated equilibrium O2 concentration in solution at atmospheric pressure. Note: provision of O2 substrate in the bulk liquid through gassing necessarily results in an O2 concentration gradient of opposite (negative) sign when the O2 consuming enzymatic reaction happens in the solid carrier. The ratio of the locally available [O2] and the enzyme Km for O2 determines the degree to which the rate of the reaction catalyzed by the immobilized enzyme depends on the O2 concentration  was examined , the degree of use of the available oxidase activity shows effectively linear dependence on the carrier‐internal O2 concentration.The current study builds on advance from Bolivar et al.  in two wd Figure . The DAAd Figure . It is a22.13, was from ABCR GmbH. Chemicals were of analytical grade from Sigma‐Aldrich. ReliSorb SP400 carrier was a kind gift from Resindion. As stated by the supplier, the carrier is porous (pore diameter: 40–50 nm) and spherical (particle diameter: 75–200 µm). Horseradish peroxidase and Aspergillus nigerd‐glucose oxidase  were from Sigma‐Aldrich.Dichloride  ruthenium (II), Ru(dpp)T. variabilis) and Z‐CAT (from B. pertussis) were produced in Escherichia coli BL21 (DE3) as described by Wiesbauer et al.  at 240 nm in a stirred quartz cuvette . O2 was available from air‐saturated substrate solution. One unit of activity is the enzyme amount for 1 µmol/min substrate consumed or product released under the conditions used.Roti®Quant protein assay (Carl Roth) was used with BSA as reference. Activity of Z‐CAT  was determined  by measuring the decrease in absorbance of H2.2E. coli lysate  was diluted into buffer  containing 0.25 M NaCl. The total volume (1 ml) was incubated with 100 mg (dry mass) ReliSorb SP400 for 1 h in an end‐over‐end rotator at ~22°C (room temperature). Solid immobilizate was recovered. The supernatant was used to determine residual soluble protein and activity (measured at air saturation). The immobilizate was washed once with immobilization buffer and stored at 4°C in the same buffer lacking NaCl. Enzymes were co‐immobilized by incubating Z‐DAAO and Z‐CAT together at once or sequentially in varied order. Sequential immobilization involved washing of the carrier two times with buffer (1 ml) before adding the second enzyme.Enzyme in YA, %) was determined from the activity balance in the supernatant according to Equation  in the loading buffer and AL is the volumetric activity in the supernatant remaining after the immobilization. Both activities were measured at air saturation. Control experiments performed in the absence of carrier showed that A0 of both Z‐DAAO and Z‐CAT was stable during the time of the immobilization.The immobilization yield (Equation . A0 is aI) was measured directly using the activity assays described in Section The observable activity at air saturation of the immobilized enzyme preparation (η) compares the observable and theoretical activity of the immobilized enzyme preparation according to Equation (aT is obtained as (A0−AL)/g dry carrier. Note the importance of a stable enzyme to determine aT.The effectiveness factor (Equation . aT is 2.32 concentration in the bulk solution was measured with an ultrahigh speed fiber‐optic oxygen microsensor (PyroScience GmbH) connected to a fiber‐optic oxygen meter  with a working volume of 4 ml . The vial was placed in a water bath (30°C) and magnetic stirring  was used for mixing. Unless otherwise mentioned, the carrier concentration used in the reported experiments was 5.0 mg/ml.Enzymatic reactions were done in an open glass vial (1.2 cm diameter) at atmospheric pressure  was added to buffer‐soaked carrier (~1.0 g), incubated for 60 min and washed with buffer. The Ru(dpp)3 was stably absorbed by the carrier. The general setup for measuring O2 inside the carrier was described in Bolivar et al. . The experiment in Supporting Information: Figure 2] in a solution of 100 mM glucose was brought to its maximum value by bubbling with pure O2 gas. The O2 consumption was then started by adding glucose oxidase to give a final activity of 5 U/ml. The decrease in the bulk [O2] measured with the microsensor was correlated with the phase shift change r et al. . Brieflyr et al. , and con2 sensor was identical to that of the bulk microsensor in the calibrated range of 10 µM to ~1.0 mM (solubility limit of O2 at ambient pressure), as shown in earlier studies , an O2 consumption rate between 0.15 and 50 µM/s was established. The time courses, hence the rates, recorded with the O2 particle sensor were identical to those recorded with the fiber‐optic microsensor. Absence of delay in the response of the particle sensor was thus confirmed for the relevant range of reaction rates.The manufacturer‐stated response time of the bulk microsensor for measurement of aqueous samples was 0.3 s or less. The dynamic response of the particle O2O2 were made from a concentrated stock solution (200 mM) in 50 mM potassium phosphate buffer, pH 8.0. The added volume was adjusted (25–100 µl) to supply the desired amount of H2O2 (5.0–20.5 µmol), resulting in concentration changes between 1.25 and 5.0 mM. The total volume change from several additions (≤10) was ~10% for most experiments. A few initial experiments were performed at higher additions of H2O2 (Δ[H2O2] = +5 mM), here the volume change was ~25%. The reported results are corrected of the volume change.Periodic additions of H2.42] in the bulk solution. Immobilized Z‐DAAO was used and the d‐Met substrate was saturating   2] in thKm (mM) is the apparent Michaelis constant of the immobilized Z‐DAAO and Vmax is the maximum volumetric activity in mmol/(L min). The Vmax results from aI (U/g) times the carrier concentration used (g/L). kO2 is a first‐order rate constant (min−1), formally equivalent to the apparent efficiency of the enzyme preparation (Vmax/Km).2] from the interval was used. Reaction rates dependent on [O2] are thus calculated  using the function Differentiate . The time interval was 20 s and the mean  of 150 µM (±15%).Additional experiment involved O2.6d‐Met by Z‐DAAO. The assay is based on reaction of α‐keto‐acid with 2,4‐dinitrophenylhydrazine  was used for determination of the α‐keto‐acid product formed from d‐Met and α‐keto‐γ‐(methylthio)‐butyric acid were quantitated (UV absorbance at 210 nm) using authentic reference compounds (Sigma‐Aldrich) for calibration. A Shimadzu Prominence HPLC‐UV system (Shimadzu) equipped with a Chromolith® HighResolution RP‐18 column  and a UV detector (210 nm) was used. Water containing tetrabutylammonium phosphate (6 mM) as an ion‐paring reagent was used for elution (40°C) at a flow rate of 2 ml/min.Reaction samples were further analyzed with ion‐pairing HPLC. For this, they were mixed with the same volume of pure acetonitrile to stop the reaction. Solid material was centrifuged off for 30 min. 33.1d‐Met oxidation) were performed with the immobilized enzyme preparations and change in the O2 concentration was monitored in the carrier  and in solution . As shown in Supporting Information: Figure 2] decreased faster than the external [O2], resulting in a temporary gradient  of negative sign. For immobilized preparations involving enzyme loadings of 400 U/g carrier or higher, the Δ[O2] was as large as −120 µM. A fast initial depletion of the internal [O2] to just ∼30% of the corresponding [O2] available in solution at air saturation was observed  of the immobilized Z‐DAAO was only 8.5% (600 U/g), likely explainable by the low supply of O2 from the bulk liquid into the solid carrier. Previous studies of the authors  showed almost complete consumption of the O2 in the carrier and in solution within ∼150 s 3  of the two enzymes in the co‐immobilized catalyst preparation appeared to be lower by ∼50% (Supporting Information: Table η. Labeling with Ru(dpp)3 did not change the η value (Supporting Information: Table N = 6) U Z‐CAT/g and 27  U Z‐DAAO/g.From earlier work on the immobilization of Z‐CAT , with Z‐CAT and Z‐DAAO immobilized in them, gave a suitable response to change in [O2] in the bulk solution. This is shown in Supporting Information: Figure 2] due to the reaction of soluble glucose oxidase was monitored with both the O2 microsensor and the labeled particles. Note: no reaction by the immobilized Z‐CAT and Z‐DAAO took place in this experiment. The time courses of [O2] recorded from signal of sensor and labeled particles were almost superimposable, thus validating the particle‐based method of [O2] measurement. Calibration of the method is shown in Supporting Information: Figure The labeled ReliSorb SP400 particles (2.5 mg Ru(dpp)2] relative to the [O2] in bulk solution. In both enzyme preparations, as shown in Supporting Information: Figure 2] decreased considerably faster than the [O2] in bulk solution. Measurement of the internal [O2] in the co‐immobilized enzyme preparation was validated from these results. Multiple measurements showed excellent reproducibility (≤5% deviation) of the internal and external [O2] time courses when the same batch of co‐immobilized enzyme was used. The largest variation resulted from Z‐CAT activity (≤13%) in different preparations of co‐immobilized enzyme.Comparing the reaction of the co‐immobilized enzyme preparation with the reaction of Z‐DAAO immobilized individually, we observed a similar trend for the time‐dependent change of the internal [O2 consumption rate was linearly dependent on the liquid bulk O2 concentration up to the maximum of ∼250 µM used (air saturation). This implies enzymatic reaction by first‐order kinetics with respect to [O2] in the relevant concentration range. In other words, the Km of the immobilized Z‐DAAO for O2 (which is indeterminate from the results shown) exceeds by far the maximally used [O2]. This emphasizes the importance of efficient supply of O2 to the reaction of the immobilized Z‐DAAO.From progress curve analysis as shown in Figure 3.22] recorded upon the addition of H2O2 to the enzyme co‐immobilizate at a point when the soluble O2 in the system had been largely consumed by the oxidation of d‐Met. Overall, both the internal and the external [O2] increased temporarily, passed through a maximum and decreased later again. The shape of the recorded course for the internal [O2] was strongly dependent on the amount of H2O2 added  into the liquid bulk before the maximum [O2] is reached. In the decline phase of the [O2] time course, the O2 consumption in the particle is due to the reaction of the immobilized Z‐DAAO, while the decline of [O2] in the bulk is caused by the diffusion of O2 from bulk into the particle.Figure d Figure . The intd Figure . In the e Figure , the int2] was dependent on the immobilized Z‐CAT activity, we prepared a second co‐immobilizate in which the immobilized Z‐DAAO activity was the same, but 10,000 U/g instead of 50,000 U/g of the Z‐CAT were immobilized. The maximum [O2] released internally was considerably lower (∼75 µM compared to ∼185 µM) when a smaller Z‐CAT activity was immobilized , an observed decrease in the internal [O2] can arise from physical diffusion to the surrounding bulk in addition to enzymatic consumption inside the carrier. We therefore performed experiments to assess the relative contributions of diffusion and Z‐DAAO reaction to the dynamics of the internal [O2]. After gassing out the O2 in bulk liquid (10 mM d‐Met) with N2, we added H2O2 to exactly comparable preparations of immobilized Z‐CAT that lacked or contained additionally immobilized Z‐DAAO, and recorded the corresponding time courses of internal and external [O2] as shown in Figure 2 was considerably higher in the absence  exactly as intended. Detail of Figure 2] dropped below the external [O2] at longer incubation times (≥12 min), can probably be explained by O2 diffusion from the particle to the surrounding liquid phase and O2 diffusion from the liquid phase to the surrounding air. It can be noted that the O2 in the liquid phase reached a stable concentration.The co‐immobilized enzyme preparation involved a 174‐fold higher activity loading of Z‐CAT than Z‐DAAO. When the internal  served to control the substrate supply from multiple additions of the H2O2.Experiments were performed in small‐scale (4 ml) reactions. The H2O2 was added (Δ[H2O2] = +2.50 mM) in intervals of ∼1 min, resulting in the fast accumulation of internal and external [O2] with each addition. After several additions of H2O2, as observed visually, gas bubbles were formed in the bulk liquid. This went along with a substantial decrease in the signal‐to‐noise ratio for measurements with the bulk microsensor and the intraparticle sensor (at >900 µM [O2]). The deteriorated performance of the intraparticle sensor can probably be explained by gradual removal (flotation) of the labeled particles with the rising gas bubbles. In Figure 2O2 in intervals of 3–4 min consistent with the results of kinetic simulation. Fresh H2O2 was added only when the internal [O2] had decreased to ∼50% of the maximum value from the previous addition. Balance in the formation and consumption of internal O2 could thus be shown experimentally.In Figure 3.42O2 as a nonspecific oxidant of proteins that causes inactivation broadly among enzymes , an overall O2 consumption rate  was calculated for each feeding step . Results showed earlier  from the steepest decline in the internal [O2]  was present over six additions of the H2O2. This result shows that the immobilized Z‐CAT activity could not have been limiting the conversion of the externally supplied H2O2.A decrease in r Figure  suggest ] Figure . Like r3.52O2 in a fully continuous manner. Thus, it should be possible to gain improved control over the [H2O2] that the enzymes are exposed to during the reaction, with consequent benefits on their operational stability. To better manage the volume change from the H2O2 feed than was possible in the small‐scale 4 ml working volume, the whole reaction was scaled up to ∼200 ml. The carrier concentration was increased to 15 mg/ml to improve the signal‐to‐noise ratio (∼2.5‐fold compared to 5 mg/ml) for the measurement of the internal [O2] in the larger vessel.In a next step of development, we considered supply of H2O2 feed was switched on after the O2 concentration inside the particles and in the bulk had approached low values that appeared to be somewhat steady over time. Starting from 0.04 mmol/min, the rate of H2O2 addition was ramped up to 0.16 mmol/min (absolute oxidase rate: ∼0.091 mmol/min) in steps of 0.04 mmol/min every 3 min. The absolute oxidase rate is the activity of the immobilized Z‐DAAO measured at air saturation under the same conditions as in the reactor, but in the absence of additionally supplied H2O2. The final rate of H2O2 addition (~0.16 mmol/min) was therefore close to double the absolute oxidase rate (∼0.091 mmol/min). Slow start of the H2O2 addition served the purpose of preventing the excessive formation of O2 in the initial reaction phase. In its main part (30–200 min), the reaction was performed at a constant rate of 0.16 mmol/min, resulting in constant and similar O2 concentrations (∼130 µM) inside the carrier and in solution. After correction for volume change, the total amount of H2O2 added was ~27 mmol. At the end of the reaction, the H2O2 feed was switched off and the rapid consumption of the O2 showed that the immobilized Z‐DAAO was still active. Comparison of the absolute O2 consumption rates at the start (0.076 mmol/min) and the end of the reaction (0.058 mmol/min) indicated that the Z‐DAAO had retained at least 76% of the initial enzyme activity. Considering that the amount of H2O2 added through continuous feed was ∼10 times that added batchwise in six steps  formed on the externally added H2O2  and determined the corresponding product formation. Samples were analyzed (colorimetric assay) from early reaction times (≤15 min) that largely excluded oxidative decomposition (decarboxylation) of the α‐keto acid promoted by the H2O2  with a slope of around unity (0.95 ± 0.05), as expected for a near‐perfectly atom‐economic usage of the H2O2 supplied. It is noted from Figure 2O2 addition rates, the rate ratio of product formed and H2O2 supplied exceeded the theoretical maximum of 1. Limited supply of O2 from the surrounding air can plausibly explain the effect. To provide a point of reference, we show in Figure 2 supply by bubble aeration. Unfortunately, the air bubbles interfered with continuous online measurements by the internal and external O2 sensors. Lacking suitable monitoring and control in the system used under these conditions, the O2 supply from gas (air or pure O2) was not further pursued. We also showed that the α‐keto acid production rate was linearly dependent on the loading of co‐immobilized enzyme  and the H2O2 feed rate could be adjusted conveniently to match the demand of the increased conversion rate.Provided that the H2 Figure . To demo2], to perform heterogeneously catalyzed O2‐dependent conversions under controlled supply of O2 substrate from H2O2 directly inside the solid catalyst  versus the O2 concentration (≤250 µM) in the liquid bulk. It stands to reason, and we have shown this in related studies of Z‐DAAO immobilization . The highest H2O2 feeding rate yielded a ∼1.5‐fold enhancement of the d‐Met conversion rate compared to the bubble‐aerated reaction. The rate enhancement with respect to the initial rate measured without external O2 supply was ∼2.3‐fold. The internal [O2] of ∼130 µM under continuous supply of H2O2  and ≤ 50 µM for the reaction in air‐saturated buffer  in the same porous carrier in suitable activity ratio. Ideally, the O2 supply is balanced, providing a suitable intraparticle [O2] for a high production rate, while preventing O2 overproduction in the bulk surrounding the particle, to avoid loss of control over the reaction. Continuous online measurement of the internal [O2] enables controlled feeding of H2O2. This ensures a programmable O2 release rate and avoids enzyme inactivation by excess H2O2. Enhanced supply of O2 to the solid enzyme catalyst can be useful to achieve reaction intensification. The approach demonstrated here for DAAO seems applicable broadly to O2‐dependent enzymes. It can facilitate the development of heterogeneous catalysis applications for biocatalytic transformations with O2 as the substrate. It presents advance for a general concept of reaction engineering important in applied biocatalysis, not only with O2‐dependent enzymes but also with cells (Birmingham et al., The co‐immobilization with CAT enables in‐situ supply of OSupporting information.Click here for additional data file.


Expression of human equilibrative nucleoside transporter‐1 (hENT1) is reported to predict survival of gemcitabine (GEM)‐treated patients. However, predictive values of immunohistochemical hENT1 expression may differ according to the antibodies, 10D7G2 and SP120.We aimed to investigate the concordance of immunohistochemical hENT1 expression between the two antibodies and prognosis.The subjects of this study were totally 332 whose formalin‐fixed paraffin‐embedded specimens and/or unstained sections were obtained. The individual H‐scores and four classifications according to the staining intensity were applied for the evaluation of hENT1 expression by 10D7G2 and SP120, respectively.hENT1 mRNA level with H‐score (p = .258). Although the hENT1 mRNA level was significantly different among four classifications using SP120 (p = .011), there was no linear relationship among them. Multivariate analyses showed that adjuvant GEM was a significant predictor of the patients with low hENT1 expression using either 10D7G2  or SP120 . In contrast, agent for adjuvant chemotherapy was not significant predictor for the patients with high hENT1 expression regardless of the kind of antibody.The highest concordance rate (79.8%) was obtained when the cut‐off between high and low hENT1 expression using SP120 was set between moderate and strong. There were no correlations of The present study suggests that the two antibodies for evaluating hENT1 expression are equivalent depending on the cut‐off point and suggests that S‐1 is the first choice of adjuvant chemotherapy for pancreatic cancer with low hENT1 expression, whereas either S‐1 or GEM can be introduced for the pancreatic cancer with high hENT1 expression, no matter which antibody is used. We measured the cycle threshold (Ct) value, which is inversely proportional to the amount of cDNA. Analysis was performed in triplicate for all samples. The expression level of each sample was shown as the value of each target divided by the GAPDH value.Representative hematoxylin and eosin‐stained sections (10 μm‐thick) were reviewed by a pathologist who marked out cancer predominant areas, which were removed by macro‐dissection. RNA was extracted from the removed tumor tissue with the RNeasy FFPE Kit , and cDNA was prepared using a High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor  according to the manufacturer's protocol. Expression of the 2.5U test or Kruskal Wallis test, as appropriate. Categorical variables were compared using the chi‐square test or Fisher's exact test, as appropriate. The cut‐off value of hENT1 mRNA levels was explored using receiver operating characteristic curves for the overall survival and Youden's index. Overall survival rates were estimated using the Kaplan–Meier method and compared by the log‐rank test. The Cox proportional hazards model was used for univariate and multivariate analyses, and treatment arms (GEM or S‐1) and all factors found to be significant predictors of overall survival (p < .10) in univariate analysis were entered into multivariate analysis. All statistical analyses were performed using the SPSS 24.0 software package . p < .05 (two‐tailed) was considered significant.Continuous variables are presented as median and range, and were compared using the Mann–Whitney 33.1The clinical and pathological characteristics of the 332 patients Figure  in the p3.2High), and IHC expression of hENT1 using SP120 were strong in 17, moderate in 31, weak in 31, and absent in 10 patients  was set between moderate and strong (concordance rate: 79.8%). When the cut‐off between SP120Low and SP120High was set between weak and moderate, the concordance rate was 58.4%. The cut‐off set between absent and weak showed a concordance rate as low as 32.6%.Correlation between IHC expression of hENT1 using SP120 and H‐score (high/low) by TMA using 10D7G2 is shown in Table 3.3hENT1 mRNA level and H‐score was investigated in the 84 patients  and hENT1 mRNA level (p = .350).Relationship between s Figure . There whENT1 mRNA level and hENT1 expression with IHC by SP120 were evaluable in 310 patients  in tissues with strong, moderate, weak and absent IHC staining were 24.3 (3.73–228.7), 14.0 (1.02–272.1), 16.2 (1.69–160.3) and 26.2 (2.20–163.6), respectively. Although the hENT1 mRNA expression level was significantly different among patients with four SP120 IHC expressions  of the 48 patients receiving adjuvant chemotherapy with GEM had 10D7G2Multivariate analyses showed that hENT1 expression with 10D7G2 (high vs. low)  and sex   were significant predictor for the survival of the patients who received with GEM as adjuvant chemotherapy agent  was one of significant predictors for the survival of the patients who received S‐1 as adjuvant chemotherapy. However, when the cut‐off was set between absent/weak/moderate and strong, which showed the highest concordance with TMA with 10D7G2 Ab  or SP120 (n = 31)  Table .4The patients' clinical and pathological factors in the present study was not much different from those in the JASPAC 01 study,hENT1 mRNA level and hENT1 expressions using either 10D7G2 or SP120 Ab and suggests that the concordance rate between the two Abs for evaluating hENT1 expression depends on the cut‐off point set among four groups according to IHC staining with SP120 Ab. However, there were several problems in the studies focusing on this issue. First, the platform between the two Abs, IHC and TMA, was different. Second, even among studies that used 10D7G2 Ab, some studies evaluated hENT1 expression with H‐scores,The present study showed no correlations between the High to derive any survival benefit from adjuvant chemotherapy with GEM. In the systemic review about the utility of hENT1 expression to predict sensitivity to GEM, the positive rate of hENT1 expression varied from 39% to 80% (10D7G2).H‐scores are obtained as continuous values, and cut‐off values for high and low expression, to predict sensitivity to GEM, were different among studies. In earlier studies which used the median H‐score as their cut‐off value, median H‐scores 4818 varieLow and SP120High was set between moderate and strong, which showed the highest concordance with TMA with 10D7G2 Ab. However, with cut‐off set between weak and moderate, the positive rate will be compatible to those of previous studies. Therefore, the utility of hENT1 expression in predicting survival will differ depending on the cut‐off value regardless the platform and Abs for its evaluation.On the other hands, almost all studies using SP120 AbWe used the different measurement platforms for hENT1 expression with two Abs (TMA for 10D7G2 and IHC for SP120) in the present study. Expression of hENT1 as assessed by both 10D7G2 and SP120 Abs was matched in 79.8% when the cut‐off was set between moderate and strong, leading to a low positive rate. The concordance rate was higher than those of earlier studies .hENT1 mRNA level was another important issue. In the present study, we analyzed expression levels of both hENT1 using two kind of Ab and hENT1 mRNA level. To our knowledge, no other studies had compared immunohistochemical expression of hENT1 with hENT1 mRNA levels in PC tissues, but Raffenne J et al. showed the correlation between the hENT1 expression evaluated by 10D7G2 and hENT1 mRNA levels during preparing this manuscript.hENT1 mRNA level might include components other than cancer cells because PC with a strongly tendency to have stromal tissues. Considering that there were low association between mRNA level and survival in spite of some relation with IHC and TMA, there might be unknown process from the mRNA level to the protein levelThe inconsistency between immunohistochemical expression of hENT1 (for both 10D7G2 and SP120) and Low and SP120Low groups, which suggests that S‐1 not GEM should be introduced for the patients with low hENT1 expression no matter which Ab is used. In contrast, the kind of adjuvant agent was not significant predictor for survival in both 10D7G2High and SP120High groups, which suggests that either S‐1 or GEM can be introduced for the patients with high hENT1 expression no matter which Ab is used.Considering the actual clinical practice for selecting the adjuvant chemotherapy agent, the present study suggests 10D7G2 Ab is more useful to predict the overall survival than SP120 limited in the GEM‐treated patients. In the subgroup analyses according to hENT1 expression by each 10D7G2 Ab and SP120, introducing GEM as adjuvant agent was significant unfavorable predictor for survival both in 10D7G2High was a significant favorable predictor for the survival in the GEM‐treated patients, and the overall survival rate of the 10D7G2High group was significantly better than that of the 10D7G2Low group; 5‐year overall survival for the GEM‐treated 10D7G2High group was 41.7%, which was similar to the survival rate for patients treated with adjuvant S‐1 in JASPAC 01 study,High group. This result may suggest that GEM, rather than S‐1, is the better choice for adjuvant chemotherapy among patients with 10D7G2High.From another view by the kind of adjuvant agent, 10D7G2Now, there are several options for adjuvant chemotherapy  and the institutional review board of each participating institution. Written informed consent for this study was obtained as required by the Institutional Review Board.Figure S1. Examples of cases with the intensity of immunohistochemistry staining of cytoplasmic human equilibrative nucleoside transporter‐1 (hENT1) expression using the SP 120 antibody. In the scale bar, 100 showed 100 μm.A: no, B: weak, C: moderate, D: strong.Click here for additional data file.Figure S2. A: Kaplan–Meier curves for overall survival of the patients in the present study.B: Kaplan–Meier curves for relapse‐free survival of the patients in the present study.Click here for additional data file.Figure S3. Correlation between human equilibrative nucleoside transporter‐1 (hENT1) expression levels (H‐scores by 10D7G2 antibody [A] and intensity of immunostaining by SP120 antibody [B]) and mRNA expression levels.Click here for additional data file.Figure S4. A: Kaplan–Meier curves for overall survival in the gemcitabine arm, stratified by human equilibrative nucleoside transporter‐1 (hENT1) expression by SP120.B: Kaplan–Meier curves for overall survival in the S‐1 arm, stratified by hENT1 expression by SP120.Click here for additional data file.Figure S5. Receiver operating characteristic curves of hENT1 mRNA levels for predicting 2‐year overall survival (A) and for predicting 3‐year overall survival (B).Click here for additional data file.Figure S6. A: Kaplan–Meier curves for overall survival in the 10D7G2Low group, stratified by adjuvant chemotherapy agent.High group, stratified by adjuvant chemotherapy agent.B: Kaplan–Meier curves for overall survival in the 10D7G2Low group, stratified by adjuvant chemotherapy agent.C: Kaplan–Meier curves for overall survival in the SP120High group, stratified by adjuvant chemotherapy agent.D: Kaplan–Meier curves for overall survival in the SP120Click here for additional data file.Table S1. The clinical and pathological characteristics of the patients whose H‐score by TMA with 10D7G2 Ab were evaluable.Click here for additional data file.