HRTEM confirmed the Cu substitution in the SmCo5 lattice. Non-magnetic Cu was substituted at “2c” site in the SmCo5 crystal lattice and effectively stopped the coupling in its surroundings. This decoupling effect decreased magnetic moment from SmCo5 (12.86 μB) to SmCo4Cu (10.58 μB) and SmCo3Cu2 (7.79 μB) and enhanced anisotropy energy from SmCo5 (10.87 Mega erg/cm3) to SmCo4Cu (14.05 Mega erg/cm3) and SmCo3Cu2 (14.78 Mega erg/cm3). Enhancement of the anisotropy energy increased the coercivity as its values for SmCo5, SmCo4Cu and SmCo3Cu2 were recorded as 4.5, 5.97 and 6.99 kOe respectively. Being six times cheaper as compared to Co, substituted Cu reduced the price of SmCo3Cu2 up to 2%. Extra 15% Co was added which not only enhanced the Mr value but also reduced the 5% of the total cost because of additional weight added to the SmCo3Cu2. Method reported in this work is most energy efficient method on the synthesis of Sm–Co–Cu ternary alloys until now.SmCo With the Ku value larger than 1.19 × 108 erg/cm3 SmCo5 alloy has a very high uni-axial magneto-crystalline anisotropy and the highest Curie temperature among permanent magnetic materials7. Because of high curie temperature they also have special applications, such as high-end engines in motorsports, where magnets are exposed to temperatures above 240 °C. Transition metals substitution for Co into SmCo5 can change the values of coercivity, Mr and Ms11. It was theoretically predicted that by substitution of Cu with Co, coercivity of SmCo5 can be increased on the expense of Mr and Ms values12. Decrease in the Mr is the serious disadvantage that reduces the energy density/BHmax despite of enhancement of the coercivity.Among permanent magnet materials, SmCo5 magnets is their high cost. Sm is the most expensive element among its group and Co is also more expensive than Fe and these make the SmCo5 most expensive class of the permanent magnets. Biggest reason behind high cost of SmCo5 is the low quantity of the Sm and Co in the earth crust as shown in Fig. 13. Hence some ores those are primarily source of the Co (e.g. Carrollite) are also an important source of Cu.Second disadvantage associated with SmCo5. But the price of SmCo5 is critically determined by the Sm due to the high price of Sm (750 USD/kg) as compared to the Co (28 USD/kg). Although Co is six times more expensive as compared to the Cu but Co substitution with Cu (in SmCo3Cu2) decreases the price of the magnet produced, only up to 2%. Another important factor that may reduce the price of the SmCo3Cu2 is use of extra fused Co phase. This extra Co not only enhances the magnetic properties but also increases the overall weight of the produced SmCo3Cu2. SmCo5 consists of almost 33% of Sm by weight, hence 15% addition of Co saved the 5% of the Sm. By considering both the factors (substitution of Co with Cu and addition of extra Co), overall price of SmCo3Cu2 could be reduced up to 6%. Reduced price of the SmCo5 in this work may have a large impact, because global market value of the SmCo5 magnet produced per year is approaching worth 1 billion USD in near future  at 1000 °C. Nishida et al. performed the annealing at more than 800 °C for 160 h16. Gabay et al. annealed the Sm, Co and Cu for 100 h (50 h at 1050 °C then 50 h 350–450 °C)17. Researchers those did not use simple annealing, used expensive methods like sputtering, arc melting, electron beam heating and Induction melting. Even after induction melting at 1430 °C, Suresh et al. later annealed the sample at 900 °C for 4 h for homogenization18. Annealing at such high temperature also leads to the loss of Sm which is evaporated and compensated with the addition of extra Sm. In this work reduction diffusion method is used for the synthesis of the magnetic particles which energy and time efficient as compared to the regular physical methods22.Third problem with the synthesis of Sm–Co–Cu ternary alloys is the high consumption of energy. Te´llez-Blanco et al.5 particles. In this energy efficient process, precursors were annealed at 900 °C for only 2 h. Cu substitution enhanced the coercivity, while reduction in the Mr value caused by Cu substitution is compensated by addition of extra Co. Cost of the SmCo5 is reduced by substitution of Cu with Co. Extra added Co also increased the weight of SmCo5−xCux magnet which further reduced its cost.This study suggests an eco-friendly chemical method for the synthesis of the Cu substituted SmCo3.6H2O, 99% purity), cobalt chloride (CoCl2.62H2O), copper chloride , and potassium chloride  were obtained from Sigma Aldrich. 16 mesh granular Ca (99.5% purity) was obtained from alfa Aesar. These reagents were used without further purification. Milli-Q IQ 7000 water purifying system was used to obtain deionized water.Samarium chloride , CoCl2.6H2O (2.186 g) and CuCl2.2H2O (0.341 g) were dissolved in 100 ml of water. Meanwhile for synthesis of SmCo3Cu2, SmCl3.6H2O (0.870 g), CoCl2.6H2O (1.643 g) and CuCl2.2H2O (0.682 g) was dissolved in 100 ml of water.Experimentally for synthesis of SmCo24. Hence, the product was washed with water again and again to remove CaO completely and washed twice with acetone before being stored in inert conditions. All steps of the experimental procedures are shown in Fig. A 3.5 M NaOH solution was added drop-by-drop to raise the pH of the solution up to 13, so that all of the metal chlorides could be changed to metal hydroxide. The reaction mixture was stirred continuously during the addition of NaOH solution. While maintaining the pH at 13, the solution was stirred for 1 h. Then, products were washed twice with DI water and ethanol, and dried at 80 °C for 1 h. XRD, TEM, and TEM-EDS analysis of SmCoCu hydroxides are provided as Fig 5 and SmCo5−x Cux, samples were dispersed in hexane and the intermediate co-precipitation product samples were dispersed in ethanol. Ni TEM grid was used for TEM and HRTEM analyses. Magnetic properties (demagnetization curves) of SmCo5 and SmCo5−xCux products were measured by Physical Property Measurement System  in the vibrating sample magnetometer mode.Crystal structure analysis was done with X-ray diffractometer (Rigaku MiniFlex) with Cu-Kα source radiation wavelength of 0.15418 nm. EDX, TEM and HRTEM characterizations were done on JEM-2100F by JEOL Ltd. For these characterizations SmCo5 and SmCo5−xCux particles were produced by the experimental process described in the experimental section. XRD patterns for all SmCo5 and SmCo5−xCux particles produced are similar  in XRD pattern  and stay for longer time at 900 °C. More annealing time can lead to the Ostwald ripening and resulted in increased size of particles.SEM images in Fig. 5−xCux particles as shown in EDX mapping image of Fig. 5−xCux alloy particles. TEM-EDS of SmCo5 is provided in 4Cu line. Line mapping confirms the homogeneous distribution of Cu throughout the particle. This also suggests the presence of Cu in the SmCo4Cu crystal, as elemental distribution is very regular. However, density of Sm, Co, and Cu is much higher in the center of the particle. This is because of higher thickness of the particle in the center. Small cobalt particles are attached on SmCo3Cu2, can be detected in TEM-EDS images, Fig. 3Cu2/Co interface is explained in Fig. 25.Sm, Co and Cu are distributed evenly in the SmCo4Cu, and SmCo3Cu2. Other possibility is that these Co particles were fused with SmCo4Cu, and SmCo3Cu2 at high temperature during the reduction diffusion process, which was further proved by HRTEM analysis. Fused Co is identified at [100] facet in Fig. 5 structure which is taken along “a/b” axis showing four Sm atoms connected by rhombus25.There is possibility that Co particles detected in TEM-EDS might be left after the reduction diffusion process and now exist as the free detached, Co particles in SmCo5−xCux/SmCo5. These four Sm atoms are arranged in the “a/b” axis of the crystal lattice. HRTEM analysis confirms the presence of Co and SmCo3Cu2 phases and their fusion in a single particle.Figure 26. Hence Cu reduces and diffuses much faster than Co and all Cu becomes the part of the SmCo5−xCux crystal lattice  as compared to the Co 238.4 kJ/Mol) kJ/Mol265 and SmCo5−xCux have hexagonal close pack crystal structure. Crystal parameters in the hcp were calculated with the help of the following equation as reported by the Sinha et al.29Enhancement in the crystal parameters “a, b, c” and crystal volume was recorded after substitution. SmCo4Cu) and 5% (SmCo3Cu2) as shown in the Fig. In the next step peak with the [111] facet was selected. Then “d” value for the [111] facet was determined and were put in the equation above. With the already known value of “a” parameter, value of “c” parameter was determined. In the hcp crystal “a = b”, hence no calculations for the “b” were performed. Substitution increased the unit cell volume by 3.9% SmCoCu and 5%6 6s2, Sm has 6 unpaired electrons in 4f orbital. Before coupling with Co, electrons in the “f” orbital of Sm hybridize and move to the 5d orbital. Hence actual electronic configuration of the Sm in SmCo5 is [Xe] 5d6 6s2 and it has four unpaired electrons in the valance shell. However, after the hybridization, 5d electrons are in the spin down state30. Valance unpaired electrons in Co are in spin up configuration, hence Sm couples antiferro-magnetically with the Co  of the rigid band of the Cu is located in the “s” orbital which indicates that “3d” orbital is completely filled. It further tells that there is no inter-orbital movement of electrons in between 3d and 4s orbitals. All electrons in 3d orbitals are retained there unpaired therefore, being non-magnetic there is no chance for the Cu to couple with Sm or Co either. In case of Sm or Co, energy level of 3d electrons is similar to the 4s electrons, hence movement of electrons between the 4s and 3d orbital is possible. Fermi level of the Sm and Co is almost in the center of 3d band therefore, 3d band is partially filled . The measured hysteresis curves for the SmCo5 and SmCo5−xCux are shown in Fig. Being non-magnetic after substitution, Cu strongly affects the magnetic properties of SmCo5, SmCo4Cu and SmCo3Cu2 were recorded as 4.5, 5.97 and 6.99 kOe, respectively. The increasing order of coercivity of all three products was determined as SmCo5 < SmCo4Cu < SmCo3Cu2. Simultaneously decreasing order of magnetic moment is SmCo5 > SmCo4Cu > SmCo3Cu2.Reduction in magnetic moment critically affected on the anisotropy energy and coercivity. Coercivity (Hc) values of SmCo5, SmCo4Cu and SmCo3Cu2 were recorded as 10.87, 14.05, and 14.78 M erg/cm3 respectively. Furthermore, it is also evident from the hysteresis loop that squareness ratio was also increased after the Cu substitution. Complete hysteresis loop is given in the 5−xCux is also explained in the Energy density or energy product is intrinsic property and is the amount of energy stored in the lattice because of arrangement of the atoms in the crystal. It was found that Cu substitution enhanced the energy density. Energy densities for SmCo5−xCux is very important. Cu enhances the coercivity at the cost of magnetic moment. Formation of SmCo3Cu2 after the substitution of Cu in SmCo5 can reduce the Mr value up to 35%12 and 32%31. Reduced Mr value will reduce the energy density drastically despite of enhancement of the coercivity. Purpose of keeping the extra Co phase was to maintain the Mr value as well as the cost reduction of the product. Proposed mechanism of reduction of Mr value by coupling between Co and SmCo3Cu2 is explained in Fig. Role of the extra Co phase present in the SmCo5 particles produced by chemical method is quite low as compared to the SmCo5 magnets. But the coercivity reported in our work is still relatively lower as compared to the coercivity reported in other recent chemical methods. One reason behind the lower value of the coercivity is the presence of small Sm2Co17 phase 33. In our work, particles with the similar size (average size 300 nm) were also separated and they exhibited much better magnetic properties. Coercivity of SmCo3Cu2 was recorded as 13.5 kOe. Detail of this experiment and results obtained is described in the Coercivity of the SmCo5−xCux magnetic particles were synthesized by energy efficient, chemical method. Microstructure confirmed the Cu substitution in the SmCo5 lattice and presence of Co phase fused together with SmCo5−xCux. After substitution at “2c” site in the SmCo5 crystal lattice, Cu almost blocked the coupling in the surrounding. The resulted decoupling in the crystal lattice affected the magnetic moment, anisotropy and coercivity. Magnetic moment was reduced as the result of Cu substitution, but coercivity and anisotropy energy were enhanced. The substitution of Co with Cu and extra Co phase decreased the price of SmCo5−xCux, up to 5%.SmCo5 and SmCoSupplementary Information.


In the article titled “Salvianolic Acid B Protects Intervertebral Discs from Oxidative Stress-Induced Degeneration via Activation of the JAK2/STAT3 Signaling Pathway” , there w“Figures 4(a)–4(d) demonstrate that in the H2O2 group, ROS and MDA levels were clearly less, and GSH and SOD2 levels significantly higher than those of the control group” should be corrected to“Figures 4(a)–4(d) demonstrate that in the H2O2 group, ROS and MDA levels were clearly higher and GSH and SOD2 levels significantly lower than those of the control group.”Additionally in The authors confirm that this does not affect the results and conclusions of the article, and the editorial board agrees to the publication of a corrigendum.


Combined Rietveld analysis of neutron and X‐ray powder diffraction data shows a novel ordering of oxygen vacancies not known before for barium cobaltates. A combination of neutron powder diffraction, magnetic measurements, and density functional theory (DFT) studies confirms G‐type antiferromagnetic ordering. From impedance measurements, the electronic conductivity of the order of 10−4 S cm−1 is determined. Remarkably, the bifunctional catalytic activity for oxygen evolution reaction (OER) and oxygen reduction reaction (ORR) is found to be comparable to that of Ba0.5Sr0.5Co0.8Fe0.2Oy3–, confirming that charge‐ordered anion deficient non‐cubic perovskites can be highly efficient catalysts.A new vacancy ordered, anion deficient perovskite modification with composition of BaCoO A new charge ordered oxygen deficient perovskite in barium cobaltate system is reported with bifunctional catalytic activity for oxygen reduction and oxygen evolution comparable to that to that benchmark perovskite catalyst BSCF. ABXy3‐ compounds have received a lot of attention due to their application in the fields of magnetism, fuel cells, solar cells and batteries.ABX3) can be described as a cubic close packed (ccp) arrangement of 3AX layers with B sites occupying 1/4of octahedral voids resulting in a corner sharing octahedral network. The anion sublattice in such a structure is highly flexible and can accommodate a large amount of vacancies  are known and for higher values of y, the cubic modification or ccp‐related vacancy‐ordered variants are known (BaCoO2.22 and BaCoO2),[ccp‐related arrangements can also be stabilized by the uptake of water (BaCoO1.80(OH)0.86).For BaCoOccp‐related modification of barium cobaltate with a composition of BaCoO2.67  prepared by a two‐step heating process together with its electrocatalytic and magnetic properties. The heating steps, times and temperatures were found to be of significant importance for the successful synthesis of the title compound. After the first heating step at 1273 K for 60 h under a flow of argon gas, X‐ray powder diffraction data indicate the formation of a barium cobaltate phase with a diffraction pattern similar to the vacancy‐ordered monoclinic modification of BaFeO2.52.46. In order to determine the topochemical oxidation behavior of this compound and suitable reaction conditions for oxygen uptake, simultaneous thermal analysis  was carried out in oxygen atmosphere, which showed an onset of oxygen uptake at 455 K. This mass increase corresponds to an uptake of ∼0.21 moles of oxygen, which means an average oxidation state of 3.34 for cobalt and a composition corresponding to ∼BaCoO2.67. Iodometric titrations confirmed this composition. At 535 K a second mass increase and oxygen uptake is observed, which would lead to a final composition of BaCoO2.80. The underlying phase changes were investigated by temperature dependent XRD measurements starting from the phase mixture with composition of BaCoO2.46 obtained after the first heating step in oxygen atmosphere. Changes in diffraction pattern are observed starting at 448 K . At 473 K, the X‐ray diffractogram shows the appearance of a single phase of the title compound , whereas the patterns recorded at 573 K and above show the presence of a hexagonal‐type modification , which agrees well with the further oxygen uptake observed in STA. The appearance of this hexagonal phase is in close agreement to what was reported previously by Raveau et al.Herein, we report a new vacancy ordered 2.67, a larger batch of this material was prepared from the as‐determined optimized reaction conditions of the second heating step and X‐ray diffraction as well as neutron diffraction data were recorded and analyzed, see Figure a=10.1718(3) Å, b=5.6035(2) Å, c=6.9248(2) Å and β=91.465(4)°, where the lengths of the axes indicate a 6×2×3supercell of the cubic aristotype structure (we note that ∼3.23(1) wt % of CoO were found as impurity for the larger batch, which indicates close to full homogeneity of the powder). Pawley fits indicated the centrosymmetric monoclinic space group P21/m as the most likely symmetry in agreement with the missing (0 1 0) reflection, which is forbidden and could be observed neither in the X‐ray nor the neutron diffraction data, further indicating the presence of the 21 screw axis . This symmetry had been previously found for the compound BaFeO2.33F0.33[2.67, of the Ba2.67 family was recently prepared, and it appears to be isotypic to both BaFeO2.33F0.33 and BaCoO2.67, which we will report in a separate article. The structural similarity between these compounds is plausible since they contain similar overall anion contents showing that in the setting of a barium‐rich anion‐deficient perovskite lattice, only certain anion compositions can give highly favorable structural stabilization and in agreement with our previous finding that hydrated barium ferrates and cobaltates possess close structural similarity.[2.33F0.33 suggested by Clemens et al.2.33F0.33, the 2a anion site was found to be vacant which, in combination with smaller structural relaxations, gives rise to cobalt being present in three different coordination environments, i. e., tetrahedral, square pyramidal and octahedral . This is in agreement with field and zero field cooled measurements  μB aligned along the c‐axis. The magnetic moment obtained from the refinements suggests an intermediate spin state for Co with ∼1–2 unpaired electrons on average. Density functional theory (DFT) based calculations also found consistently that the G‐type antiferromagnetically ordered state (G‐AFM) is energetically more favorable than the ferromagnetic (FM) state, which nicely supports the experiments above .Field dependent measurements of magnetization (M−H) were recorded at 10, 80, 250 and 320 K 0.862+δ.Electrochemical impedance spectroscopy measurements  were carried out to determine the conductivity of the sample. From the NYQUIST plot a single depressed semicircle was observed which was fitted using a single R‐CPE (constant phase element). The room temperature conductivity of the sample was determined from the intercept on the X‐axis and calculated to be 2.89×102.67 and compared to that of Ba0.5Sr0.5Co0.8Fe0.2Oy3– (BSCF) which is the benchmark perovskite catalyst[0.5Sr0.5Co0.8Fe0.2Oy3‐. The performance towards OER is much better for Ba0.5Sr0.5Co0.8Fe0.2Oy3‐ which might be related to the integration of iron in the system, as it is known for several OER catalysts.The catalytic activity for OER and ORR was measured for the title compound BaCoO2.67 particles showed a much coarser crystallinity . From the ORR region, we see that the potential of 0.7 V vs. RHE (reversible hydrogen electrode) a kinetic current density of 2.3 mA cm−2 is achieved and an onset potential of 0.81 V for BaCoO2.67. On the other hand, BSCF shows a kinetic current density of 2.1 mA cm−2 at 0.7 V with the onset potential of 0.785 V. The positive shift of the onset potential and larger current density therefore indicate a slightly better ORR activity for BaCoO2.67 as compared to BSCF.Once carbon is added, the catalytic activity of the title compound is very similar to that of the BSCF  was calculated to be 0.95 V for both BaCoO2.67 and BSCF which is much lower compared to the BaFex1–CoxOy–δ3–(OH)y seriesIn addition to ORR we also investigated the OER characteristics of both BaCoO2.67 in comparison to BSCF, though no morphological optimization of the compound was performed. According to Suntivich et al.[g orbital filling of the transition metal ion with an average occupation of one electron being desirable. For mixed‐valent BaCoO2.67, all Co ions are located in polyhedra with a local site symmetry of m. Such an average filling of eg states is plausible if Co is present in the intermediate spin state. This would result in the electronic configuration of t2g5eg1 for Co3+ (and high spin eg3t2g2 for Co4+), which could be brought in principle agreement with the local symmetry and thus non‐degeneracy within eg/t2g levels,2g5eg∼1.25 (average Co oxidation state of +2.75 determined from iodometric titrations).2.67 . Although determining the exact spin state of Co in BaCoO2.67 on the different crystallographic sites with octahedral, square pyramidal, or tetragonal coordination is hindered experimentally, the catalytic activity observed here in combination with the magnetic moment observed at room temperature by neutron diffraction is plausible in relation to electron configurations found in similar compounds, with an overall occupation of the higher d‐orbital energy levels by 1.33 electrons for BaCoO2.67. Thus, ordering of vacancies can be considered to be the origin for the resulting ordering of charges and electron configurations, which in turn contribute to achieve a high catalytic activity in BaCoO2.67.In relation to the suggested dependency of the catalytic activity on electron configuration as provided by Suntivich et al.,[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.SupplementaryClick here for additional data file.


In the Methods section, the age range of the youngest category should have appeared as “0-24.” In the key to Figure 2, the top age category should have appeared as “≤24.” This article has been corrected.1In the Research Letter titled “Trends in Drug Overdose Mortality in Ohio During the First 7 Months of the COVID-19 Pandemic,”


It leads to high energy products, in good yield, via a newly identified mechanism, which is likely to apply widely to multiple ionisation by almost all impact processes.Using multi-electron–ion coincidence measurements combined with high level calculations, we show that double ionisation of SO Upon fragmentation, such dications are expected to produce the low energy fragments (A+ + B+) predominantly. All fragmentations tend to produce the lowest energy accessible products, but recent experimental and theoretical studies have shown that state-to-state fragmentations of small neutral molecules may lead to unexpected results. For instance, in some VUV photodissociation of CO2 there is no formation of ground state CO + O species. Instead the O photoproduct is formed in the 1D or the 1S excited states3, whose dipole forbidden emissions are prominent features in the visible spectra of terrestrial atmospheres4. Similarly, the VUV photodissociation of N2 by ns or fs lasers leads to at least one excited atomic nitrogen instead of two 4S ground state nitrogen atoms6. Very recently, Zhou et al. showed that the UV photolysis of H2S produces excited S(1D) atoms (+ H2) instead of ground state S(3P)7. Fragmentation of even quite large singly charged molecular ions can also be state-selective rather than statistical, of which a classical example is the dissociation of C2F6+8. These state specific processes are important to planetary atmospheres, plasmas and photochemistry at the fs and shorter time scales. By contrast, no such unexpected behavior has been reported hitherto for molecular dications.Since their detection almost a century ago by mass spectrometry, doubly charged molecular ions have attracted both experimentalists and theoreticians to investigate their formation, stability and reactivity. Their stability is a balance between Coulombic repulsion and chemical bonding, and small polyatomic dications  SO22+ → O+ + SO+ or (2) SO22+ → O+ + S+ + O or (3) SO22+ → O2+ + S+or (4) SO22+ → O + SO2+. A fifth pathway producing two O+ ions with neutral S is detectable but has extremely low intensity. Dissociative double ionization is brought about not only by direct vertical double ionization, but also by an indirect process shown up clearly in PEPECO studies of this and other small molecules20 by the signatures of distinct electron energies from autoionization of super-excited O, N and other atoms21. The well documented indirect process can be represented for SO2 as: SO2 + hv → SO2+* + e1−; SO2+* → SO+ + O*: O* → O+ + e2−.From existing studies, we know that double ionization of SO22+ ion with fragmentation leading either to charge separation (SO+ + O+) or to the thermodynamically unfavoured charge retaining (SO2+ + O) products of O–SO bond breaking. A vital characteristic of our technique is that both ions and electrons from ionization at a point-like source are collected and detected with high overall efficiency, being uniquely capable to extract complete mass spectra from single and double ionization separately. This is done by requiring the coincident detection of a single photoelectron of appropriate (relatively high) energy in the one case, and coincident detection of an electron pair within the right energy range in the other. As references—single and double ionization mass spectra of SO2 photoionized at 40.8 eV are shown in the SI. These spectra confirm that the most abundant products at this energy are O+ + SO+ and that SO2+ is the only doubly-charged ion formed.We here report multi-particle coincidence measurements using the TOF-PEPEPIPICO technique (cf. SI). This work makes the first direct connection between the energies and states of nascent SO22. The bands in this spectrum due to the direct population of SO22+ states located in the Franck–Condon (F.C.) zone accessed from SO2(X1A1) have been identified in previous work9, and again in the present computations. Whatever the equilibrium geometry of the SO22+ dication, the electronic states are labelled in the C2ν point group since single-photon ionisation occurs from the bent ground neutral SO2 (X 1A1) state. We also mapped the multi-dimensional potentials of the doubly charged ions in the Cs point group (cf. SI for more details). Dissociation following the most abundant ion pair pathway of O+ + SO+ formation sets in at the very onset of double ionization near 34 eV and persists over most of the range of ionization energies. Of the distinct features of the overall spectrum, this charge-separation pathway follows from population of the prominent vibrationally resolved state at 35.3 eV, identified as 11A29, but apparently not from population of the states represented by the bands centered at 36.5 and at 37.5 eV. The onset of the charge retaining pathway forming SO2+ coincides with the peak of the resolved band, and also with the thermodynamic threshold for O + SO2+ at ca. 35.46 eV (Table + + SO+ yield) at all energies above threshold, and specifically by decay from a state or states between 37 and 38 eV, seen as a broad peak in the resolved spectrum and apparently correlated with the population of the 31A1 (41A′ in Cs symmetry) state. Contributions of the triplet states lying in the same energy range cannot be ruled out, in particular the {13A1, 23B1, 23B2} set of states. Of the channels not shown in Fig. + + S+ + O . Channel (3) will be discussed in a separate paper.Figure eV Table . This chfrom SO2XA1 have bcf. Fig. , sets in24 with generally concordant results, but without initial state selection. The most detailed measurements made possible by use of a position–sensitive ion detector23, gave the full KER distributions for 40.8 eV photoionization. Although we can accurately select ranges of ionization energy (IE), the present experimental data are not ideally suited to the extraction of KERs. In contrast to previous experiments where a continuous light source could be used24, in our set-up there has to be a relatively long delay between the photoionisation event and ion extraction (to ensure that even the slowest electrons leave the source region), ions spread out in the source and the TOF peaks get broadened. Nevertheless, we can estimate the mean KEs released from dications formed in limited ionisation energy ranges. To use the available statistics efficiently we have extracted mass spectra from 1 eV wide IE ranges which correspond quite well to the main features in Fig. Insights into the fragmentation mechanisms can be obtained from the kinetic energies released in the fragmentations. The KER magnitudes can be extracted from TOF peak widths and shapes, and for the charge-separating channels this has been done several times before+ + SO+ we find that the mean KER varies slightly, between 4.5 ± 0.2 to 4.8 ± 0.2 eV (cf. Table +(X2Π) + O+(4Su), at least at threshold.For the charge separation reaction forming O2+ the KER is much smaller and produces roughly triangular peak shapes, as is known from the work of Field and Eland24 , where a full list of SO2+ KERs as a function of ionization energy range (Table 2 in Ref.24) is also presented. The KER values given in the present work agree satisfactorily with the more complete values given in the past24. In the 34–40 eV range, this two-body channel SO2+ + O shows two distinct energy distributions: (i) a close to zero KER (0.0–0.2 eV) for 35 < IE < 37 eV range and ~ 0.5 eV for 37 < EI < 40 eV  + O(3P) must be formed at threshold. The rise in KER as a function of IE seems continuous, with no special excursion at the energy of the state(s) near 37.5 eV, which decay specifically to these products.For the reaction(s) producing SO22+ ions more deeply we have performed ab initio computations on the potential energy surfaces (PESs) of the singlet and triplet mainly two-hole (2 h) electronic states located in the whole 33–40 eV range above SO2(X1A1). These computations are done at the CASSCF/MRCI/aug-cc-pV(Q + d)Z level for a wide range of nuclear configurations  + O+(4Su), so intersystem crossings (from singlets) and internal conversions (from higher triplets) are needed to populate the lowest triplet that leads to these products  + SO+(X2Π) via the following mechanisms 1A′ state.Internal conversions within the singlets manifold of states populating the lowest (ii)1A′ to the lowest 3A″ potential. This triplet can be also reached via internal conversion within the triplets.Spin–orbit conversion from this (iii)3A″ leads to the O+(4Su) + SO+(X2Π) products. The spin–orbit integrals presented in Fig. -1, which are large enough for such internal conversions to be rapid.Afterwards, the lowest triplet To identify the SO+ + SO+ continues to be formed over the whole range of ionisation energies up to 40 eV with little change in kinetic energy release means that the products must be created with increasingly large amounts of internal energy. Only a limited amount of energy can be accommodated as vibration in undissociated SO+26, so much of the excess energy must be electronic excitation of the product ions. This is consistent with the observation of the sequential formation of O+ + S+ + O and of optical emission in coincidence with the O+ + SO+ ion pair with a significant quantum yield27, attributed particularly to SO+(A2Π → X2Π). It seems to indicate that internal conversions and intersystem crossings within the SO22+ state manifold, although dominant, are not quite complete.The fact that O2+ + O channel, we refer to Fig. 1A′, 1A″, 3A′ and 3A″ PESs along the SO distance, and to the KERs listed in Table 1A′ and/or the 1A″ MEPs. For 1A′, we find a Morse-like potential that leads to a plateau at intermediate bond extension (ca. 4 Bohr) very close in energy to the SO2+(X1Σ+) + O(3Pg) asymptote. We also found a 1A″ potential with a small potential barrier (of 0.04 eV) before a level at almost exactly the same energy. We suggest that production of ground-state SO2+ may occur by intersystem crossing from these surfaces to one of the triplet surfaces correlated to SO2+(X1Σ+) + O(3Pg), which are expected to be relatively flat at these distances in the presence of charge-induced dipole attraction and the absence of Coulomb repulsion. At such distances other competitive curve crossings, which could lead to eventual SO+ + O+ production are less frequent  and levels of the vibrationally resolved 11A2 state in regions where there are multiple curve crossings. There is also a weak peak at 35.7 eV on the SO2+ yield curve in Fig. 1A2 state at 35.3 eV, decay into this channel rather than to the lowest energy products. It follows that to support the proposed mechanisms, we still need an explanation of how the long O–SO bond extension required can be reached in significant yield, without undergoing prior conversion to lower energy states.For the SO2+ in the range of IE above 37 eV, we measure a KER of ~ 0.5 eV. This value is sufficiently different from the previous case to suggest that a different potential surface is involved. Figure 2+(X1Σ+) + O(1Dg) asymptote where there is no difficulty over the spin. Computations indicate that the SO22+(41A′/31A1) state which leads directly to these products can be populated by direct ionization in the F.C. zone. Figure 22+(41A′/31A1) surface exhibits a potential barrier toward dissociation of ~ 0.4 eV, which is in good agreement with the measured KER at these IEs  towards both optical emission and autoionisation28. The cores of the higher Rydberg states, being doubly ionised, excited and hardly influenced by the attached electron, will start to dissociate in the same way as bare SO22+ ions. Many will follow the most favorable path of O+–SO+ bond extension leading to breakage. A departing O+ may then capture the Rydberg electron on its way out, producing the autoionisating O* states observed. The same or other Rydbergs, some with shorter but still relatively long lifetimes to autoionisation, will undergo significant O–SO bond extension before production of nascent SO22+. The new geometry at the moment of autoionisation can bring the molecular dications into regions of parameter space where curve-crossings and the consequent state conversions are rare, allowing the formation of energetically disfavored products with apparent state selectivity, exactly as is seen in the case of SO2+. At the same time, the molecular autoionisation will contribute to the whole double ionisation spectrum, adding to the direct vertical process. This mechanism should be operative in all molecular double ionisation by photon or electron impact not just in SO2.The clue to an alternative mechanism is given by the observation in double ionisation of autoionising O* over the range of ionisation energy from near threshold right up to the photon energy, as visible in the electron pair coincidence map2+ Figs. –S6 there2. In BrCN2+, ICN2+29 and CF3I2+30 decays no state-specific behavior can be seen. The N2O2+ ion decays radiatively from specific states, but it shows no other state-specific decay behavior. The CS22+ ion both emits fluorescence from two specific excited states and also decays to C+ + S2+ from states in a limited energy range31, and is the closest to SO22+ in respect of this behavior. The electron–electron coincidence map for CS2 at 40.81 eV shows evidence of autoionisation29 akin to that in SO2, further confirming the validity of the proposed mechanism.The present technique is the only existing means of acquiring such detailed information on dicationic decays from the states of ions formed by photoionisation. We note that similar information for the different set of mainly singlet states accessed by the Auger effect could be acquired by Auger-electron–ion coincidence techniques. There are only six other molecules whose fragmentation after double ionisation can be compared with that of SO2. The mechanism almost certainly operates more widely. In the case of SO2, we have identified particular electronic states as probable surfaces upon which the dissociations to high energy products occur. The mechanism is one of indirect ionisation, akin to the usual Auger effect, providing a non-vertical overall pathway to double ionisation of molecules, outside the F.C. zone. In many cases the outcome will be indistinguishable from vertical double ionization in the Franck–Condon region, but in some cases it can permit energetically disfavored pathways to be followed to a much greater extent than would be expected on the basis of free internal energy flow.We have here identified a new non-conventional mechanism involved in the unimolecular decomposition of doubly charged molecular dications which accounts for the observation of energy-disfavored dissociation channels with substantial quantum yield in the decay of doubly ionized SO9. The TOF-PEPEPIPICO apparatus which has been described before27 allows continuous (simultaneous) detection of two electrons with energy measurement and two positive ions with mass/charge measurement. In brief, ionisation occurs where molecules in an effusive jet from a hollow needle intersect wavelength-selected light from a pulsed discharge in He. At the moment of ionisation there is a weak electric field in the source region which accelerates any near-zero energy photoelectrons towards a 2 m distant detector, giving them sufficient velocity to arrive there in less than 10 μs. A continuous, divergent strong magnetic field in the same region guides almost all photoelectrons, irrespective of kinetic energies of less than a few hundred eV, into a solenoidal field which carries them all to the detector. After about 150 ns, when all relevant electrons have left the source region, a strong electric field pulse extracts ions in the opposite direction through a two-field time-of-flight mass spectrometer to a separate detector. The strength of the ion drawout pulse and the associated acceleration field, as set for time-focus conditions, determine the mass resolution, and the time-width of signals for fragment ions formed with more than thermal kinetic energies. The drawout pulse field strength affects the detectability of ion pairs of equal mass-to-charge ratio (m/z) such as O+ + O+ or O2+ + S+ because the ion detector and associated electronics used in these experiments imposed a deadtime of 50 ns.Multi-electron multi-ion coincidence experiments were done at 304 Å (40.8 eV), well above the estimated adiabatic single-photon double ionization onset 33.5 eV2 except equal mass pairs were fully resolved under all conditions, but the 34S isotopic variants were only partially resolved.At the time of these experiments the efficiency was about 30% for electrons and 10% for ions at m/z = 100. Because the final ion energy at the detector was less than 2 keV, the efficiency was somewhat higher for lighter ions. Electron resolution (E/ΔE) was about 20 throughout, while mass resolution ranged from 30 to 100 (FWHM) according to the strength of the drawout pulse; all ions of interest from SO32. In the present study, we mapped the SO22+ six lowest A′ and six lowest A′′ three-dimensional potential energy surfaces (PESs) of singlet and triplet spin multiplicities along the SO distance and the in-plane angle, θ = OSO varying from 40° to 180°. The other SO distance is kept fixed at 2.7 Bohr i.e. its value in SO2(X1A1) at equilibrium.All the ab-initio electronic calculations with different levels of theory were performed using the MOLPRO program suite34 approach followed by the internally contracted multi reference configuration interaction method (MRCI)37. The CASSCF active space is constituted by the whole set of all configurations which is allowing all the possible excitations of all valence electrons in valence orbitals. The electronic states having the same spin multiplicity are computed according to the state-average procedure, as implemented in MOLPRO. For MRCI calculations, all configurations having a coefficient greater than 0.005 in the CI expansion of the CASSCF wave functions were taken into account as a reference. The computations were done in the Cs point group with wave functions leading to more than 7 × 108 uncontracted configurations for the singlet electronic states. For the triplet electronic states, we considered more than 26 × 108 uncontracted configurations. For these calculations, the aug-cc-pV(Q + d)Z and the aug-cc-pVQZ basis sets of Dunning et al. were used to describe the S and O atoms, respectively40. In fact, adding the tight-d functions to describe the sulfur atom improve the accuracy of the results as shown in different works43. Furthermore, to investigate the spin-obit coupling at the crossings between some electronic SO22+ states, the spin orbit integrals where computed using the CASSCF wave functions and the uncontracted spd cc-pVTZ basis set. We used the Breit–Pauli spin–orbit Hamiltonian as implemented in MOLPRO. Indeed, the spin–orbit integrals were evaluated, in Cartesian coordinates, over the CASSCF wavefunctions, where the effective Breit–Pauli SO operator, HSO, as implemented in MOLPRO was used44.The PESs have been generated using the Complete Active Space Self Consistent Field (CASSCF)22+ forming the [SO + O]2+, the partially spin restricted coupled cluster method including perturbative treatment of triple excitation (RCCSD(T))48 was used. For the RCCSD(T) computations we used the aug-cc-pV(5 + d)Z basis set for sulfur and the aug-cc-pV5Z for oxygen.To evaluate the energies of the dissociation limits of SOSupplementary Information.


Zymoseptoria tritici, is one of the most economically important diseases of wheat. Recently, both factors of a gene‐for‐gene interaction between Z. tritici and wheat, the wheat receptor‐like kinase Stb6 and the Z. tritici secreted effector protein AvrStb6, have been identified. Previous analyses revealed a high diversity of AvrStb6 haplotypes present in earlier Z. tritici isolate collections, with up to c.18% of analysed isolates possessing the avirulence isoform of AvrStb6 identical to that originally identified in the reference isolate IPO323. With Stb6 present in many commercial wheat cultivars globally, we aimed to assess potential changes in AvrStb6 genetic diversity and the incidence of haplotypes allowing evasion of Stb6‐mediated resistance in more recent Z. tritici populations. Here we show, using targeted resequencing of AvrStb6, that this gene is universally present in field isolates sampled from major wheat‐growing regions of the world in 2013–2017. However, in contrast to the data from previous AvrStb6 population studies, we report a complete absence of the originally described avirulence isoform of AvrStb6 amongst modern Z. tritici isolates. Moreover, a remarkably small number of haplotypes, each encoding AvrStb6 protein isoforms conditioning virulence on Stb6‐containing wheat, were found to predominate among modern Z. tritici isolates. A single virulence isoform of AvrStb6 was found to be particularly abundant throughout the global population. These findings indicate that, despite the ability of Z. tritici to sexually reproduce on resistant hosts, AvrStb6 avirulence haplotypes tend to be eliminated in subsequent populations.Septoria tritici blotch (STB), caused by the fungus  Zymoseptoria tritici populations revealed a marked decrease in diversity and a global shift to virulence on Stb6 wheat.A snapshot of AvrStb6 isoforms diversity amongst earlier versus more recent natural  Avr factors may be lost through frameshift or nonsense mutations , is one of the most economically important fungal pathogens of wheat, with fungicide control costs exceeding €1 billion per year in Europe alone  Cadenza  in the AvrStb6 protein have been suggested as being critical for the pathogenicity on wheat cultivars carrying Stb6  was found to be the most abundant in several regions of the world examined. This study therefore provides a rare insight into temporal changes in pathogen effector diversity in response to the host‐imposed pressures and highlights the population shifts that can occur when a single corresponding disease resistance gene is deployed extensively.In this study, we resequenced the 22.1AvrStb6 gene were designed and used for PCR amplification of this gene from a collection of 383 Z. tritici isolates sampled from different field‐grown bread wheat (Triticum aestivum) cultivars in 10 countries on five continents. PCR products of expected size were obtained from all analysed samples, indicating AvrStb6 is present in all isolates in the collection. Sequencing of the PCR products revealed a total of 52 AvrStb6 haplotypes  and another well‐studied isolate IPO88004 (H52/I44), which are avirulent or virulent on Stb6‐containing wheat, respectively.Primers flanking the AvrStb6 sequences (361 of 381) were 365 bp in length from the ATG start codon to the TGA stop codon and including two introns, with others ranging in lengths from 362 to 366 bp. Most of the nucleotide polymorphisms were in exons 2 and 3, with exon 1 being more conserved, and particularly within the sequence coding for the N‐terminal signal peptide   containing a nonsense mutation in the exon 1, and H24 and H46  that contained single nucleotide deletions/frameshift mutations located in exon 3 and exon 2, respectively. The majority of recovered ) Figure , and S1.) Figure .2.2By far the most prevalent AvrStb6 isoform in the collection was I02, identified in 280 of 381 isolates (73.5%). This isoform was the most common in almost every geographic region studied, including Australia (96.6%), western Europe (91.8%), the United States (82.7%), and Chile (80%)  in western Europe, South America, and Turkey. All other identified AvrStb6 isoforms were represented by one to eight isolates in the collection. Remarkably, 29 haplotypes and 25 AvrStb6 protein isoforms each were uniquely present in single isolates in the collection, representing well over half of the identified AvrStb6 isoforms Figure .2.3Z. tritici isolates were sourced from smallholder farmers’ wheat fields in Turkey as part of this study. From this sample, 29 AvrStb6 haplotypes corresponding to 26 protein isoforms were identified. This represents a far higher rate of gene and protein sequence diversity than found in any other region studied here, with more than half of all identified AvrStb6 isoforms being found in Turkey and 25 being unique to this region. The network diagram of AvrStb6 haplotypes  and a near‐isogenic line that contains the susceptibility haplotype 3 of Stb6 developed from a cross between Chinese Spring and a susceptible cv. Courtot. The previously characterized avirulent and virulent Z. tritici isolates IPO323  only for the Z. tritici isolate T1‐A2017.24 possessing the AvrStb6 isoform I13 and the control avirulent isolate IPO323.Other than the avirulent reference isolate IPO323 (I01), only one other isolate could potentially be defined as demonstrating reduced virulence on 6 Figure . This is6 Figure . One oth2.5Stb6‐containing wheat cultivars. The cultivar information was available for 254 out of 381 sampled Z. tritici isolates, and it was possible to obtain 20 cultivars—sources of 210 isolates—for further study  Z. tritici isolates in our collection, whereas the other 11/20 analysed cultivars contained nonfunctional susceptibility haplotypes 3 or 7 of Stb6 and they represented sources of 137 (65.2%) isolates  to compare the expression levels of AvrStb6 in the 10 selected Z. tritici isolates (representing 10 different most frequent AvrStb6 isoforms) that were virulent on Stb6 wheat versus an avirulent isolate IPO323 during infection of a fully susceptible wheat cv. Taichung 29 that does not possess Stb6  becoming potentially extinct in the recent Z. tritici populations, and emergence of multiple virulence isoforms with one becoming predominant globally. We hypothesize that these changes in Z. tritici populations may be imposed by a worldwide increase in the deployment of commercial wheat cultivars carrying Stb6  in the host wheat plants sampled   Z. tritiAvrStb6 haplotypes did not identify I02 as the dominant isoform in Argentina and Uruguay, and this isoform was completely absent from the Turkish Z. tritici population. It is possible that the local wheat cultivars used for sampling Z. tritici isolates for this study in these countries may have unique genetic makeups, although it was not possible to test this hypothesis in this study. The Turkish population is of particular interest, given it has a substantially higher AvrStb6 haplotype diversity compared to those in the other wheat‐growing regions studied here. Whilst the exact reasons for this difference are unknown, we hypothesize that the widespread use of local genetically diverse landraces, instead of genetically more similar commercial wheat cultivars, in Turkey is a driver for matching diversification of Z. tritici genomes. The location of Turkey near or at the hypothesized geographic region of the origin of wheat , Ireland (n = 8), France (n = 12), Germany (n = 12), Australia (n = 58), and Turkey (n = 57), and from single fields in Chile (n = 10), Argentina (n = 10), Uruguay (n = 10), and the United States (n = 52). Most of these isolates were collected between 2015 and 2017, with the exception of isolates collected in Australia (2014) and Turkey (2013–2016), and the control isolates IPO323 (avirulent on Stb6 wheat) and IPO88004 (virulent on Stb6 wheat) collected in 1981 and 1988 in the Netherlands and Ethiopia, respectively  glycerol at −80 °C.The 381 4.2Z. tritici isolates as previously described  of the gene using publicly available and own whole‐genome sequencing data for European Z. tritici isolates . Primers avrstb6.f1 and avrstb6.r1 were selected for amplification of AvrStb6 from European isolates, and primers avrstb6.f3 and avrstb6.r1 were used to amplify AvrStb6 from North and South American isolates  of the harvested pycnidiospore suspensions was measured by spectrophotometer. This was converted to spores/ml by comparing against a standard curve produced by measuring OD600 of control suspensions containing defined pycnidiospore numbers as counted using a haemocytometer.STB disease symptoms on each inoculated leaf were visually assessed at 21 days postinoculation (dpi) as previously described  calculated using the 2−ΔΔCt method Click here for additional data file.FIGURE S6 Alignment of the part of the Stb6 coding sequence (exon 1) with the corresponding region in the CRISPR/Cas9‐induced wheat Cadenza ΔStb6 mutantClick here for additional data file.TABLE S1Zymoseptoria tritici isolates used in this studyClick here for additional data file.TABLE S2 Primers used in this studyClick here for additional data file.


Transplantation of stem cells into wounds has become popular in regeneration therapies. As stem cells for transplantation, human dental pulp stem cells (hDPSCs) are known to be pluripotent cells that are relatively easy to collect from the pulp of deciduous or wisdom teeth. The purpose of this study was to investigate whether hDPSCs treated with fibroblast growth factor 7 (FGF7) would contribute to the regeneration of wounded rat submandibular glands (SMGs).in vitro studies, hDPSCs were treated with or without FGF7 and mRNA expression levels were examined at days 3, 7 and 14 using qRT‐PCR. The target genes analyzed were BMI1 as an undifferentiated marker, AQP5 as an acinar cell marker, CK19 as a ductal epithelial cell marker, αSMA as a myoepithelial cell marker and VIMENTIN as a fibroblast marker. In in vivo studies, hDPSCs treated with or without FGF7 for 14 days were mixed with type I collagen gels and were transplanted into wounded rat SMGs. Hematoxylin–Eosin and immunohistochemical staining were performed at days 3 and 7, and the numbers of positive cells were counted. The primary antibodies used were against BMI1, AQP5, αSMA, PanCK and VIMENTIN.In in vitro studies, mRNA levels of BMI1 were decreased and αSMA were increased at days 3, 7 and 14, while AQP5 was increased at day 14 in the FGF7 group. In the in vivo studies, the proliferation of hDPSCs and cell islands was observed at day 7 in the FGF7 group. Few BMI1‐positive cells were observed, while numbers of AQP5‐positive and αSMA‐positive cells were increased at days 3 and 7 in the FGF7 group. Moreover, cell islands were AQP5‐positive.In the These results suggest that FGF7‐treated hDPSCs differentiate into AQP5‐positive and αSMA‐positive cells. Moreover, AQP5‐positive cell aggregations were formed. Fibroblast growth factor 7 (FGF7) is known as a keratinocyte growth factor that is important in epithelial regeneration and differentiation  derived from adult human third molars were purchased from Lonza for use in this study. Those cells are primary cultures that express CD105, CD166, CD29, CD90 and CD73, but are negative for CD133, CD34 and CD45. Those hDPSCs have the phenotypic and functional characteristics of bone marrow‐derived mesenchymal stem cells.2.2The dental pulp stem cell growth medium (DPSC‐GM) was prepared by adding the contents of a DPSC SingleQuots Kit  to DPSC Basal Medium . The DPSC SingleQuots Kit contains human Dental Pulp Stem Cell Growth Supplement, L‐glutamine, ascorbic acid and gentamicin/amphotericin‐B.2.3Recombinant FGF7 powder (10 μg)  was dissolved in 1.0 mL phosphate buffered saline (PBS) to produce the FGF7 stock solution (10 μg/mL). This FGF7 stock solution was diluted 100‐fold in the DPSC‐GM described above to a final concentration of 100 ng/mL.2.42  and were cultured in DPSC‐GM that was changed every 3 days at a temperature of 37°C with 5% carbon dioxide. hDPSCs were detached using Trypsin/EDTA and were passaged. When hDPSCs reached confluence after 2 passages, the medium was changed to DPSC‐GM containing FGF7, and this group was designated as the FGF7 group  and were collected at days 1, 3, 5, 7, 9, 11 and 13 in each group using a cell scraper. The hDPSCs were counted using a Coulter Counter  and the number of cells was plotted.The hDPSCs were seeded in 24 well dishes at a cell density of 5000 cells/cm2.6BMI1: Hs00409825_g1) as an undifferentiated marker, aquaporin 5 (AQP5: Hs00387048_m1) as an acinar cell marker, cytokeratin 19 (CK19: Hs00761767_s1) as a ductal epithelial cell marker, α‐smooth muscle actin (αSMA: Hs00426835_g1) as a myoepithelial cell marker, vimentin (VIMENTIN: Hs00185584_m1) as a fibroblast cell marker and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH: Hs02786624_g1) as an endogenous control  and were reverse transcribed into complementary DNAs (cDNAs) using ReverTra Ace qPCR RT Master Mix with gDNA Remover . qRT‐PCR was performed using TaqMan Gene Expression Assays . The target genes characterized in this study were: BMI1 . Adult male Sprague‐Dawley rats, each weighing approximately 200 g , were used in this study. No animals suffered infection or death during the experimental period.2.8An acid‐soluble type I collagen solution derived from porcine tendons was prepared to a concentration of 3.0 mg/mL at a pH of 3.0  for use in this study. This solution was quickly reconstituted into collagen gels with consistently high strength and transparency required for use.2.9in vivo study. Sheets of hDPSCs were collected using a cell scraper and were separated into single cells by pipetting. The supernatant was removed after centrifugation and a collagen gel solution at a concentration of 100,000 cells/50 μg was added to the remaining hDPSCs. The collagen gel solution containing hDPSCs, which was in a liquid condition, was reconstituted by incubation for 30 min in an incubator at 37°C to produce a gel and was used as a graft material.hDPSCs cultured with or without FGF7 for 14 days were used in the 2.10in vivo experiment was performed according to the method of Kobayashi et al. . Whole rat SMGs were resected and were immersed in 10% neutral buffered formalin for 24 h at room temperature. Specimens were dehydrated in ethanol before being embedded in paraffin. Paraffin sections, 4 μm in thickness, were cut horizontally using a sliding microtome. Paraffin sections were stained with hematoxylin and eosin (H‐E) for light microscopy observations.Three rats in each group were euthanized using an overdose of somnopentyl at days 3 and 7 after the surgery  with an electric pot at 98°C for 45 min. Sections were immersed in methanol containing 0.3% aqueous hydrogen peroxide at room temperature to block endogenous peroxidase activity. The sections were blocked for 60 min with 10% goat serum at room temperature to reduce non‐specific binding. Sections were reacted with primary antibodies overnight at 4°C. The primary antibodies used were: BMI1  as an undifferentiated marker, AQP5  as an acinar cell marker, αSMA  as a myoepithelial cell marker, PanCK  as a ductal epithelial cell marker and VIMENTIN  as a fibroblast marker. The sections were then reacted with MACH 2 Universal HRP Polymer Detection  as a peroxidase‐conjugated secondary antibody for 30 min at room temperature. After that, the sections were stained with DAB, and nuclei were stained with hematoxylin. The sections were observed with a light microscope. The number of cells that stained positive for each antibody at each time period in each group were counted at five locations chosen from randomly drawn rectangles, the size of 350 × 245 μm2.13p value <.05 are considered statistically significant.Quantitative data are presented as means ± standard deviation (SD) and were analyzed using two‐way ANOVA in GraphPad Prism7 . Differences with a 33.1The proliferation of hDPSCs treated with or without FGF7 increased day by day until day 7, after which the cell numbers plateaued in both groups. There was no significant difference in proliferation between the FGF7 group and the control group Figure .3.2BMI1 mRNA was significantly lower in the FGF7 group compared to the control group at days 3, 7 and 14  (Nyman, Lindhe, Karring, & Rylander, in vitro and in vivo. FGF7 is a member of the FGF family, which plays important roles in regulating the proliferation and differentiation of a wide range of cells (Morita & Nogawa, in vitro study (Kera et al., In this study, the effects of hDPSCs treated with FGF7 on wound healing of rat SMGs were evaluated in vitro study, expression levels of BMI1 mRNA decreased over time in the FGF7 group compared to the control group, and this indicates a decrease in the multipotential ability of stem cells. This is consistent with the fact that only a few BMI1‐positive cells were seen immunohistochemically in the in vivo study. Taken together, the results suggest that FGF7 promotes the differentiation but not the proliferation of hDPSCs. VIMENTIN is mainly expressed in mesenchymal cells and is used as a fibroblast marker. The VIMENTIN mRNA expression level increased with time and there was a significant increase in the control group at day 14 compared with the FGF7 group in the in vitro study. Moreover, VIMENTIN‐positive cells were significantly increased in the control group compared with the FGF7 group in the in vivo immunohistochemical study. These results suggest that hDPSCs were differentiated into fibroblasts in the control group, suggesting the possibility of wound healing by scar tissue.BMI1 is known as an undifferentiated marker of cells derived from the neural crest. In our Salivary glands are composed of three kinds of epithelial cells; acinar cells, ductal epithelial cells and myoepithelial cells. In terms of salivary gland regeneration, these three kinds of epithelial cells along with structural characteristics are required to evaluate the regeneration of salivary glands.αSMA mRNA increased significantly in the FGF7 group compared to the control group at all time periods. Furthermore, αSMA‐positive cells were significantly increased in the FGF7 group compared with the control group at all time periods. αSMA is a cytoskeletal protein of smooth muscle cells and is a myoepithelial cell marker. This suggests that FGF7 causes hDPSCs to differentiate into αSMA‐positive cells, which could be myoepithelial cells.The results demonstrated that the expression levels of AQP5 mRNA expression levels increased more in the FGF7 group at day 14 than in the control group in the in vitro study. Furthermore, the number of AQP5‐positive cells increased in the FGF7 group compared with the control group in the in vivo study (Ma et al., In addition, CK19 mRNA in the in vitro study. Furthermore, only a few PanCK‐positive cells were observed in the in vivo immunohistochemical study. CK19 and PanCK are markers of ductal epithelial cells. This suggests that FGF7 does not affect the differentiation of hDPSCs into PanCK‐positive cells, which could be ductal epithelial cells.There was no change in the expression level of in vivo study, aggregations of cells and small cell islands, which are like acini‐like structures, were positive for AQP5 at day 7 in the FGF7 group. The origin of acinar cells are progenitor cells of ductal epithelial cells that differentiate at the embryonic stage. However, in this study, no ductal epithelial cells, which should be negative for CK19 and PanCK, could be found around the AQP5‐positive cell masses. Also, there were no myoepithelial cells with basement membranes around the AQP5‐positive small island masses. Kobayashi et al. (In the i et al.  used sca5These results suggest that hDPSCs treated with FGF7 differentiate into AQP5‐positive and αSMA‐positive cells, and form AQP5‐positive cell aggregates when transplanted into experimentally damaged rat SMGs.The authors declare that there is no conflict of interest.


High glucose promoted expression of AKT3, a direct target gene of miR‐29b, by regulating circHIPK3 that functioned as ceRNA to sponge and down‐regulate miR‐29b. As a potential target gene of miR‐29b, AKT3 plays a crucial role in the pathogenesis of myocardial ischaemia/reperfusion (I/R) injury, and this study aimed to investigate the potential role of high glucose in the outcome of I/R injury. qPCR and luciferase assay were carried out to investigate the relationship between the expression of circHIPK3, miR‐29b and ATK3 mRNA. Immunohistochemistry and TUNEL were performed to analyse the relationship between AKT3 expression and apoptosis of myocardiocytes in vivo. No obvious difference in myocardial functions was observed between I/R and control rats under hyperglycaemia (HG) and normal glucose (NG) conditions, except that the infarct size/area at risk (IS/AR) ratio and the amount of h‐FABP expression were different under HG and NG conditions. The expression of circHIPK3 and ATK3 was significantly elevated in the rats preconditioned by NG, whereas the expression of miR‐29a was remarkably decreased. Meanwhile, the apoptosis of myocardial tissue was reduced in the rats preconditioned by NG. Luciferase assay confirmed that miR‐29a played a repressive role in the expression of circHIPK3 and ATK3. And subsequent study indicated that the over‐expressed AKT3 could rescue the increased cell apoptosis rate induced by the knockdown of circHIPK3. In this study, we demonstrated that high glucose protects cardiomyocytes against I/R associated injury by suppressing apoptosis and high glucose promoted the expression of AKT3 by regulating the expression of circHIPK3/miR‐29b. More importantly, the increased expression of circHIPK3 in HAECs and HUVECs apparently suppressed the growth and survival of endothelial cells while promoting the apoptosis of these cells. In contrary, the silencing of circHIPK3 expression by using specific siRNA sequences further promoted the apoptosis of endothelial cells already induced by the HG condition.By splicing the transcripts of RNAs, a new class of RNAs named circular RNAs (circRNAs) can be produced. As members of non‐coding RNAs (ncRNAs) endogenously present in many types of cells, circRNAs can compete with their target microRNA (miRNA) to alter the expression of miRNA.A recent study showed that circHIPK3 can play a regulatory role by sponging the function of miR‐29b‐3p to decrease the expression levels of several genes such as a‐SMA, COL3A1, as well as COL1A1.It has been shown that HG may alter the expression of circHIPK3, and circHIPK3 may regulate the expression of miR‐29b.22.1In this study, 32 male adult Wistar rats (with an average bodyweight of 250‐300 g) were acquired from our experimental animal centre and then given purified water at ad libitum along with a regular diet. After 7 days of adaptation, the rats were randomly divided into four groups with eight rats in each group, that is (a) control normoglycaemic group (C‐NG group); (b) I/R injury preconditioned normoglycaemic group (PC‐NG group); (c) control hyperglycaemic group (C‐HG group); and (d) I/R injury preconditioned hyperglycaemic group (PC‐HG group).4, 25.0 mmol/L of NaHCO3, 1.18 mmol/L of KH2PO4, 2.5 mmol/L of CaCl2 and 11.1 mmol/L of glucose). In the C‐NG and C‐HG groups, the collected heart tissues were subjected to the occlusion of the left anterior descending (LAD) coronary artery for 30 minutes and subsequent reperfusion of 120 minutes carried out by releasing the clamp used to induce occlusion. In the PC‐HG and PC‐NG groups, the collected heart tissues were exposed to two cycles of ischemic PC, which was carried out by inducing the occlusion of LAD coronary artery for 5 minutes and subsequent reperfusion of another 5 minutes.To collect heart tissues, the rats were first placed under anaesthesia with ip injection 50 mg/kg of sodium pentobarbitone in conjunction with heparin. In the next step, the heart was rapidly removed and dipped into a perfusion buffer equilibrated at 4°C. Then, the heart was cannulated through the aorta and subsequently perfused at a 75 mm Hg perfusion pressure using a KH solution , −dP/dtmax, diastolic pressure in the left ventricle, systolic pressure in the left ventricle, LV developed pressure (LVDP) and index of relaxation were measured by utilizing the Power‐Lab/8SP Chart 7 software (ADInstruments).In this study, the pressure in the left ventricle of each heart was determined by utilizing a balloon filled with water and inserted into the left ventricle. The balloon was inflated to get a 7 mm Hg end‐diastolic pressure before it was linked to the pressure transducer. In addition, the +dAll animal operations in this study were carried out in strict compliance with the Guide for the Care and Use of Laboratory Animals issued by the NIH and Institutional ethical committee has approved the protocol of this study.2.2In this study, the area at risk (AR) size and the infarct size (IS) were determined by utilizing double staining using 2,3,5‐triphenyltetrazolium chloride (TTC) as well as KMnO4 following the protocol described in a previous paper.2.3After the establishment of I/R injury, the effluent from the heart was collected and then randomly divided into six portions that were 20 minutes apart. Two millilitres of the sample taken from each portion of the effluent was dialyzed at 4°C against double distilled water overnight by utilizing a cellulose membrane connected to a dialysis piping. After dialysis, 1 mL of the dialyzed sample was taken, lyophilized into powders, and then dissolved in 100 µL of a sample diluent. In the next step, the solution was analysed by utilizing an ELISA assay kit of rat h‐FABP in accordance with the protocol provided by the kit manufacturer. The measured concentration value of h‐FABP in each sample was then multiplied by utilizing the volume of collected effluent to calculate the content of h‐FABP in the effluent obtained from each heart.2.4The total RNA content in collected tissue and cell samples was isolated first by utilizing a TRIzol reagent (Invitrogen). In the next step, the reverse transcription of isolated RNA to cDNA was carried out by utilizing a cDNA synthesis kit (Exiqon) in accordance with the protocol provided by the kit manufacturer. Then, the real‐time PCR reaction was carried out by utilizing an ExiLENT SYBR Green real‐time PCR kit (Exiqon) in accordance with the protocol provided by the kit manufacturer. The real‐time PCR was carried out on a Bio‐Rad real‐time PCR instrument (Bio‐Rad laboratories). After the real‐time PCR was completed, the relative expression of circHIPK3, miR‐29b and AKT3 mRNA was calculated using the delta‐CT method, and the expression of housekeeping gene U6 and GAPDH was utilized to normalize the results.2.53, 5.37 mmol/L of KCl, 136.89 mmol/L of NaCl, 0.44 mmol/L of KH2PO4, 5 mmol/L of d‐glucose and 0.338 mmol/L of Na2HPO4 (pH 7.4). After the 48 hours induction of hypoxia was completed, the culture media were quickly replaced by utilizing a fresh DMEM. In the normoglycaemic groups, the cells were exposed to a 5.0 mmol/L concentration of glucose for 48 hours. In the hyperglycaemic groups, the cells were exposed to a 15 mmol/L concentration of glucose for 48 hours. For the transfection experiment, the cells were cultured to 70% confluence and then transfected with corresponding vectors using Lipofectamine 3000 (Invitrogen) in accordance with the protocol provided by the manufacturer.H9C2 cells were maintained in a DMEM medium  added with 10% FBS and necessary antibiotics. Then, the cells were randomly divided into four groups, that is (a) Normoxic and normoglycaemic group (NX‐NG group); (b) Reoxidation and normoglycaemic group (HR‐NG group); (c) Normoxic and hyperglycaemic group (NX‐HG group); and (d) Reoxidation and hyperglycaemic group (HR‐HG group). At first, the cells were exposed to a hypoxic condition at 37°C induced by utilizing the D‐Hank solution containing 4.166 mol/L of NaHCOAlso, H9C2 cells were divided into four groups to evaluate the effect of circHIPK3 on the apoptosis in vitro, that is (a) Cells transfected with NC siRNA group (NC siRNA group); (b) Cells transfected with circHIPK3 siRNA group (circHIPK3 siRNA group); (c) Cells co‐transfected with circHIPK3 siRNA and empty pcDNA vector group (circHIPK3 siRNA +pcDNA group); and (d) Cells transfected with circHIPK3 siRNA and pcDNA overexpressing AKT3 (circHIPK3 siRNA+pcDNA‐AKT3 group).2.6Our results of sequence analysis showed that both the 3′ UTR of circHIPK3 and AKT3 contained potential binding sites of miR‐29b. In order to further explore the regulatory relationship between circHIPK3 and miR‐29b, as well as between miR‐29b and AKT3, we established luciferase vectors containing wild‐type and mutant 3′ UTR of circHIPK3 and AKT3 as follows: First, the wild‐type fragments of 3′ UTR of circHIPK3 and AKT3 containing the binding sites of miR‐29b were cloned into separate pcDNA3.1 luciferase reporter gene vectors (Promega) downstream of the luciferase gene to generate wild‐type vectors of circHIPK3 and AKT3, respectively. In the next step, the miR‐29b binding sites in the 3′ UTR of wild‐type circHIPK3 and AKT3 were subjected to site‐directed mutagenesis, respectively, carried out by using a Quick Change II mutagenesis kit (Stratagene) in accordance with the protocol provided by the kit manufacturer. Then, the mutant type fragments of 3′ UTR of circHIPK3 and AKT3 containing the mutated binding sites of miR‐29b were also cloned into different pcDNA3.1 luciferase reporter gene vectors downstream of the luciferase gene to generate mutant vectors of circHIPK3 and AKT3, respectively. Thereafter, H9C2 cells were cultured to 70% confluence and then co‐transfected with mutant or wild‐type vectors of circHIPK3 and AKT3 in conjunction with miR‐29b using Lipofectamine 3000 in accordance with the protocol provided by the manufacturer. At 48 hours post‐transfection, the luciferase reporter assays were carried out using a Dual Luciferase Assay kit (Promega) in accordance with the protocol provided by the kit manufacturer to determine the luciferase activity of transfected cells.2.7Collected tissue samples were fixed by utilizing 4% formaldehyde, rinsed by PBS, permeabilized at room temperature by utilizing 0.1% Triton X‐100, blocked for 15 minutes at room temperature by utilizing 3% BSA, and then incubated with anti‐AKT3 primary antibody (Millipore) and fluorescein isothiocyanate‐tagged secondary antibodies (Thermo Fisher Scientific) in accordance with the protocol provided by the manufacturer. After counter staining at room temperature for 5 minutes with 4′,6‐diamidino‐2‐phenylindoledihydrochloride (DAPI), the relative expression of AKT3 protein in each sample was analysed under an IX71 fluorescence microscope (Olympus).2.8The collected myocardial tissue samples were subjected to TUNEL assay to observe the apoptotic status. The samples were stained using a TUNEL kit (Beyotime Biotechnology) following the protocol provided by the kit manufacturer, and the stained samples were processed following common routine. Finally, the processed samples were observed under a fluorescence microscope, with 10 visual fields being randomly selected to calculate the ratio of positive cells and the apoptotic index.2.9H9C2 cells were processed and fixed in 70% prechilled ethanol following common routine. After rinsed with PBS, the cells were incubated with 10 μL Annexin V‐FITC and 5 μL PI in the dark at room temperature for 15 minutes. Flow cytometry was performed to measure cell apoptosis at 488 mm.2.10P < .05.All experimental results were shown by means ± SE The statistical significance of inter‐group differences was evaluated using one‐way ANOVA. All statistical analyses were carried out using SPSS 21.0 (IBM). The level of statistical significance was set to 33.1P/dt)max, Figure P/dt)max, Figure Control and ischaemia/reperfusion (IR)‐conditioned rats were subjected to hyperglycaemia (HG) and normal glucose (NG) treatments, respectively, before a series of myocardial parameters were measured. No obvious differences were observed in terms of the heart rate  ratio was significantly decreased in the I/R preconditioned rats. However, under the HG condition, the IS/AR ratio was remarkably increased in the I/R preconditioned rats. For the control rats, there was no obvious difference in the IS/AR ratio between NG and HG conditions. However, for the preconditioned rats, the IS/AR ratio was dramatically elevated Figure . For the3.3Real‐time PCR was carried out to analyse the expression of circHIPK3, miR‐29b and AKT3 mRNA under different conditions. The expression of circHIPK3 was the highest in PC rats treated under NG condition and decreased gradually in non‐PC control rats treated under HG condition, followed by control rats treated under NG condition, while the preconditioned rats treated under HG condition showed the lowest expression of circHIPK3 Figure . In cont3.4TUNEL was carried out to evaluate the apoptotic status of myocardial tissues of rats treated under different conditions. The apoptotic level of myocardial tissues was the lowest in PC rats treated under NG condition and the highest in preconditioned rats treated under HG condition Figure .3.5Sequence analysis showed that circHIPK3 Figure  and AKT33.6H9C2 cells were cultured under different conditions before qPCR was performed to analyse the expression of circHIPK3, miR‐29b and ATK3. The expression of circHIPK3 Figure  and ATK33.7H9C2 cells were, respectively, transfected NC siRNA, circHIPK3 siRNA, circHIPK3 siRNA+pcDNA and circHIPK3 siRNA+pcDNA_AKT3. The expression of circHIPK3, miR‐29b and AKT3 mRNA was then evaluated and compared. As shown by the results, the expression of circHIPK3 Figure  was evid4In this study, we established an I/R rat model and treated them under HG and NG conditions. We measured the release of IS/AR, AR/LV and h‐FABP from the heart of these rats. Different patterns of IS/AR, AR/LV and h‐FABP release were observed, revealing that hyperglycaemia up‐regulated the expression of circHIPK3 and ATK3 in these rats while down‐regulating the expression of miR‐29a.As an important circRNA, circHIPK3 expression is reduced under HG conditions due to the increased level of miR‐124, thus down‐regulating the expression levels of several pro‐survival factors such as STAT3 as well as SphK1. On the other hand, the suppression of miR‐124 expression can protect endothelial cells against HG‐induced apoptosis, suggesting that the increase of miR‐124 expression following the down‐regulation of circHIPK3 expression acts as a critical mediator in the injury of endothelial cells induced by HG.In MI, myofibroblasts are derived from myocardial fibroblasts and such differentiation of myocardial fibroblasts can cause excessive accumulation of extracellular matrix.The stimulation induced by a high level of glucose can modulate the progress of cell apoptosis, while the suppression of MIAT expression can reverse HG‐induced apoptosis. In addition, the knockdown of miR‐29b expression can apparently reverse the effect of MIAT in inducing cell apoptosis.As a signal downstream to PI3K, Akt plays an anti‐apoptotic role in PIK3R3/Akt3‐mediated I/R injury.5In conclusion, the findings of this study demonstrated that high glucose protects cardiomyocytes against I/R associated injury by suppressing apoptosis and high glucose promoted the expression of AKT3 by regulating the expression of circHIPK3/miR‐29b.The authors declare that they have no conflicts of interest.Nan Cheng: Conceptualization ; Formal analysis ; Investigation ; Writing–original draft ; Writing–review and editing . Chang Jin: Investigation ; Methodology ; Resources . Ping Jin: Formal analysis ; Investigation ; Visualization . Dan Zhu: Investigation ; Resources ; Validation . Zuoxu Hou: Conceptualization ; Funding acquisition ; Investigation ; Supervision ; Validation ; Writing–original draft ; Writing‐review and editing .