Laminaria digitata) beads were prepared by homogeneous Ca ionotropic gelation. In addition, glutaraldehyde-crosslinked poly (ethyleneimine) (PEI) was incorporated into algal beads. The three sorbents were characterized by scanning electron microscopy (SEM) coupled with energy dispersive X-ray analysis (EDX): the sorption occurs in the whole mass of the sorbents. Sorption experiments were conducted to evaluate the impact of pH, sorption isotherms, and uptake kinetics. A special attention was paid to the effect of drying (air-drying vs. freeze-drying) on the mass transfer properties. For alginate, freeze drying is required for maintaining the porosity of the hydrogel, while for algal-based sorbents the swelling of the material minimizes the impact of the drying procedure. The maximum sorption capacities observed from experiments were 415, 296 and 218 mg Pb g−1 and 112, 77 and 67 mg Cu g−1 for alginate, algal and algal/PEI beads respectively. Though the sorption capacities of algal-beads decreased slightly , the greener and cheaper one-pot synthesis of algal beads makes this sorbent more competitive for environmental applications. PEI in algal beads decreases the sorption properties in the case of the sorption of metal cations under selected experimental conditions.Alginate and algal-biomass ( For Cu and Pb, the European Union directive on water quality set the permissible levels to 2 mg·Lectively . As a coectively . The othectively . Numerouectively . Algal bectively , crustacectively ,9 repres−1 for Pb(II) and 51–106 mg·g−1 for Cu(II). This higher removal performance is mainly explained by the presence of alginate (anionic polysaccharides) as one of the major components of brown algae [d-mannuronate (M) and α-l-guluronate (G), is another natural polymer extracted from brown algae, naturally containing carboxyl groups in each constituent residue [2 solution to form alginate gel beads (with “egg-box” structure) has been extensively investigated. However, this “external gelation” (or heterogeneous gelation), where beads are obtained by an external Ca(II) source, was found to lead to heterogeneous beads with a more rigid surface layer than center layer due to the delay of Ca(II) diffusion after the formation of the surface layer [By comparing the sorption capacity of non-chemically modified green, red and brown algae on Pb(II) ,13,14,15wn algae , whose hwn algae ,21. Usuawn algae , so theswn algae ,24. Thiswn algae  or the p residue . In addi residue ,28 and l residue . Alginat residue , food te residue  or maghe residue . However residue  due to i residue . The metce layer . In addice layer ,37. The Brown algal species contain approximately 30%–40% alginate , providiPolyethyleneimine (PEI) is well known for its metal chelating characteristic due to the presence of a large number of amine groups  and it iLaminiaria digitata) were used for the synthesis of bead-shaped sorbents: the materials were ionotropically gelled with Ca(II) using the so-called internal gelation method. In addition, glutaraldehyde-crosslinked polyethyleneimine (PEI) was incorporated into algal beads. Though the sorption of copper and lead by alginate beads [−1 in municipal and industrial wastewaters, respectively [Therefore, in this study, alginate and algal biomass . MoreovSEM observation in The presence of other elements such as S in all the sorbents, K in algal-based beads and Al in algal beads were also observed. Sulfur could be associated with fucoidan residues present in the brown algae, from which alginate was extracted, and this could also explain the more intense peak of S in algal-based beads than in pure alginate beads. Light metals such as K are commonly found in untreated biomass while Al can probably be attributed to impurities associated with alginate extraction step . Being sSEM micrographs of the Cu(II)-loaded algal beads shown in 2 bubbles facilitates the formation of very large pores and the gelling is operating in all the directions contrary to the other method that obeys a radial process (shrinking core).The internal ionotropic gelation mechanism is contributing to create more homogeneous porous structure than the conventional external gelation method. In this external gelation a very compact outer skin is formed that may contribute to reduce mass transfer properties. In the case of the internal method: the production of COExperiments were performed in batch under similar experimental conditions with initial pH varying between 1 and 5.5. Experimental conditions  have been selected to avoid precipitation phenomena. Investigating the impact of pH is a key step in the design of the sorption process since this parameter may affect several critical criteria such as the speciation of the metal in the solution , the chemical state of reactive groups on the sorbent (protonation/deprotonation), which, in turn may impact their affinity for target metal ions.q, mg metal g−1) are negligible. This is obviously associated to the protonation of reactive groups at the surface of sorbent beads: the protonation of these reactive groups , which, in turn have weak affinity for metal cations. As the pH increases, the protonation of the reactive groups progressively decreases and the sorption capacity significantly increases. The steep increase in the sorption capacity is essentially observed between pH 2 and 4; above pH 4 the sorption capacity tends to stabilize. The critical changes in sorption efficiency around pH 3.5 can be correlated to the acid-base properties of the encapsulating material . Haug [Ka of carboxylic groups in mannuronic acid units is close to 3.38 while for guluronic acid the carboxylic groups have a slightly higher pKa . Below pH 3.65, carboxyl (COO–) is thus strongly associated with protons rather than with cationic metal ions (Equation (1)) and sorption remains negligible.s). Haug  reportedOver pH 3.38–3.65, the carboxylic groups are less protonated and then gel may form between these groups and Pb(II) or Cu(II) (Equation (2)) by ionotropic gelation , and Pb: the SD is about halved for alginate beads compared to the other sorbents; however, the sorption capacities were not doubled. This is a first indication of the interest of algal-based capsules in terms of rationale use of alginate-based fraction . In the case of algal biomass, the presence of other compounds  makes the modeling and interpretation of pH effects more complex. The amino groups may react with metal ions through different modes depending on the pH, the composition of the solution, and the speciation of the metals : metal anions in acidic solutions may be bound by electrostatic attraction/anion exchange, while metal cations will be preferentially bound to amine groups in near-neutral solutions (through interactions with the free electron doublet on nitrogen). As a consequence, in the case of Pb(II) and Cu(II) that do not form stable anionic complexes in acidic solutions  metal binding is not expected to occur on protonated amine groups and the sorption capacity is not improved due to the presence of these new functional groups. Similar conclusions can be roughly deduced from the sorption behaviors of Algal/PEI beads regarding pH effect. This also explains the fact that the effect of the pH on metal sorption is not significantly changed: sorption capacity increases with the progressive deprotonation of carboxylic groups and amine groups with pH increase.Kas of carboxylic groups in guluronic and mannuronic acid being close to 3.38 and 3.6, respectively, [Moreover, ctively, ), and thThe sorption of Pb(II) and Cu(II) was tested in the presence of calcium nitrate and sodium nitrate. Previous studies have shown that Na(I) ions have a stronger competitive effect on the sorption of divalent cations by alginate than Ca(II) ions . The staThese results contrast with the conclusions raised by Bertagnolli et al.  for the qeq (sorption capacity at equilibrium) as a function of residual metal concentration: qeq = f(Ceq). These curves are characterized by two objective parameters the initial slope of the curve (which is correlated to the affinity of the sorbent for the solute) and the saturation plateau (which is representative of the maximum sorption capacity). The sorption isotherms of Pb(II) and Cu(II) are shown in −1.The sorption isotherms represent the distribution of the solute (here Pb(II) and Cu(II)) at equilibrium between the solid phase (the sorbent) and the liquid phase (the solution), when varying metal concentration. This is represented by the plot of R2 (determination coefficient) and x(10)2 (chi-square). The simulated results and the corresponding parameters are shown in x(10)2  suggest that Sips model can give a better fit than Langmuir and Freundlich models for both metals. The predicted values of qm by Sips model for Pb(II) and Cu(II) were 455 and 138 mg·g−1 for alginate, 338 and 117 mg·g−1 for algal biomass and 230 and 83 mg·g−1 for algal/PEI, respectively. Comparing the predictions from Sips model to the experimental values, Pb(II) could occupy 91 % of the available binding sites in alginate, 87% in algal beads and 69% in algal/PEI beads, while these for Cu(II) are 81%, 66% and 81%, respectively.The experimental data were fitted by the Langmuir, Freundlich and Sips models, respectively. Parameters were evaluated by qm,exp values (in molar units) for Pb(II) are slightly higher (by 13%–19%) than the values for Cu(II) for alginate and algal beads; while for algal/PEI beads the experimental maximum sorption capacity was comparable for Cu(II) and Pb(II) . This result is in agreement with previous work [KH (negative log of hydrolysis constant) than Cu(II)  and 6.7 for Cu(II)). In addition, it is generally observed that higher molecular weight metal ions (Pb = 207.2 g·mol−1) can be more efficiently removed than lighter metal ions (Cu = 63.55 g·mol−1). The maximum sorption capacity observed from experiments  of all the sorbents for Pb(II) and Cu(II) followed the order: alginate > algal > algal/PEI. The satisfactorily performance of alginate in sorption ability may result from the abundant carboxylic group, which provided chemical ligands to form metal-ligand complexes. Obviously, the incorporation of PEI does not improve the sorption of Cu(II) or Pb(II). The qm,exp of algal/PEI beads decreased by 26% for Pb(II) and 12% for Cu(II) when compared to algal beads. This clearly shows that at least in single-metal solutions, the incorporation of PEI does not bring beneficial effects on the sorption of metal cations .Deze et al.  also fouous work , and thrKL and KS  shows a significantly higher affinity of the sorbents for Pb(II) than for Cu(II). Indeed, the KS values for Pb(II) vary according: 174, 153 and 70 L·mmol−1 for alginate beads, algal beads and algal/PEI beads, respectively. In the case of Cu(II) much lower values were obtained: 24, 19 and 25 L·mmol−1 for alginate beads, algal beads and algal/PEI beads, respectively. This means that a certain preference for Pb(II) over Cu(II) could be anticipated for competitive sorption from binary solutions. Complementary experiments are currently performed for investigating the sorption properties of the three sorbents in bi-component solutions. Results demonstrate that the sorption of Pb(II) is less influenced by the presence of Cu(II) than the reciprocal (not shown).According to the Pearson′s theory the hard acids prefer to associate with hard bases and soft acids prefer to associate with soft bases . Actuallw/w). This means that despite a lower amount of alginate in algal biomass the sorption is only increased by 50% for alginate beads while the amount of alginate was multiplied by 3. The presence of other polysaccharides, of other ractive groups (such as proteins), a better dispersion of alginate in the biomass  may contribute to enhance the availability, accessibility or reactivity of functional groups on the polymer chain. Previous studies on silver sorption using chitosan foams reinforced with cellulose fibers [2 at saturation of the material: the dispersion of aminopolysaccharide improved the availability and accessibility of the reactive groups. However, this conclusion should be taken carefully and would probably deserve complementary experimentation since the compositions of alginate and alginate extracted from the L. digitata algal biomass used in this study was inversed . The affinity coefficients (represented by parameters KL and KS in the models of Langmuir and Sips) are of the same order of magnitude for the three sorbents in the case of Cu(II) sorption, while for Pb(II) a significant difference was observed between alginate beads and algal beads on one side and algal/PEI beads on the other side .Comparing the sorption capacities of the three sorbents for both Cu(II) and Pb(II) the materials can be classified according the series: alginate beads > algal beads > algal/PEI beads. It is noteworthy that in the case of alginate and algal beads the principal reactive groups are supposed to be the carboxylic functions of guluronic acid and mannuronic acid , in addition to other polysaccahrides in the case of algal biomass . The alginate beads have sorption capacities that are only 1.4–1.47 times higher than the sorption levels reached with algal beads. The extraction alginate from algal biomass showed a content of about 31%  [Ulva fasciata for Cu(II) [−1 for Pb(II) and Cu(II), respectively with polybenzalimine) resin. Fu et al. [−1 for magnetic poly-acrylic weak acid resin; while Kolodynska et al. [The sorption performance of these materials can be compared to other biosorbents. r Pb(II) , or Ulvar Cu(II) . The conr Cu(II)  reportedu et al.  reports a et al.  highligha et al. , compare2 conditions [Another important criterion is the velocity of metal binding in the design of a sorption process. Uptake kinetics may be controlled by a series of mechanisms involving the proper reaction rate but also several kinds of mechanisms of resistance to mass transfer . Providing a sufficient agitation to the reactor allows reducing the impact of resistance to bulk diffusion and to film diffusion and frequently the uptake profiles are modelled using simple equations that jointly or independently take into account the reaction rate   or the rnditions ). In ordDe) for freeze-dried and air-dried sorbents are summarized in De values were systematically higher for freeze-dried materials than for air-dried materials, regardless of the metal. The freeze-drying process improves the diffusion properties of all the sorbents. Moreover, no obvious trend was found between De for Cu(II) and Pb(II) uptakes. The values of the diffusivity coefficient were found several orders of magnitude lower than the self-diffusivity of Cu(II)  and Pb(II)  in water [The pseudo-second order rate equation and the pseudo-first order rate equation  were tesin water . This coLaminaria digitata) was purchased from Setalg . It was washed, dried at 50 °C overnight and grinded (size fraction: <250 µm). The procedure described by McHugh [L. digitata: . The fractions of mannuronic and guluronic acids (M/G) in alginate and alginate extracted from algal biomass were determined by NMR analysis [Alginate  was supplied by FMC . Brown algal biomass 2 and Pb(NO3)2  dissolved in deionized water. Polyethylenimine  was obtained from Sigma-Aldrich . Calcium chloride (>99.5%) and formic acid (>99%) were purchased from Chem-Lab . Calcium carbonate and glutaraldehyde (50 wt %) were purchased from Sigma-Aldrich. All other reagents used were of analytical grade.The Cu(II) and Pb(II) solutions were prepared using analytical grade Cu under fast agitation. After this step, the mixture was maintained for 24 h at room temperature. Then, it was washed twice with 4 L of demineralized water, filtrated and freeze-dried . Thereafter 2 g of the mixture, grinded and sieved through 200 mesh sieve, were added into the as-prepared algal mixture. The mixture was marked as C.2  and formic acid, CH2O2 . After keeping the gel beads in the CaCl2/CH2O2 solution under agitation for 24 h, the beads were washed twice and maintained in 1 L of demineralized water. Then the solution pH was adjusted to 4 using 0.1 M NaOH. Finally, those beads were rinsed with pure water and then divided into 2 parts. One was air-dried . The other one was freeze-dried .Then the mixture A, B and C were separately distributed dropwise into a solution containing both CaCl−1) of Pb(II) and Cu(II) were prepared by dissolving the exact quantity of Pb(NO3)2 and Cu(NO3)2 in Milli-Q water. Working solutions were obtained by diluting the stock solutions to the desired concentrations. Before the sorption process, the pH of each test solution was adjusted to the required value by using 0.1 M HNO3 or NaOH. The final pH was determined in order to evaluate the potential precipitation phenomena associated with pH variation. Initial and equilibrium metal concentrations were subsequently determined using an inductively coupled plasma atomic emission spectrometer ICP-AES ACTIVA M  after samples were filtered.Stock solutions (1 g·L−1 and contact time of 48 h) and initial pH range of 2–6 for Pb(II)  and 0.38 g·L−1  and contact time of 48 h). The pH of the solution was adjusted using 0.1 M HNO3 or NaOH. The initial and final values of pH were measured using a pH-meter cyber scan pH 6000 .The sorption experiments were conducted within the initial pH range of 1–5.5 for Cu(II) (sorbent dosage of 0.2 g·L3)2 (0–0.2 M) or NaNO3/Ca(NO3)2 (concentration of Na(I)/Ca(II) changed from 0 to 1/0.1 M) with the sorbent dosage of 0.2 and 0.4 g·L−1 for Pb(II) and Cu(II), respectively, pH of 4 and contact time of 48 h. When investigating the effect of Na(I) calcium ions were added to prevent the degradation of the beads; indeed, an excess of Na(I) displaces, by ion-exchange, Ca(II) from ionotropically gelled beads with loss of stability of the material.The influence of Na(I) and Ca(II) was evaluated by sorption experiments in the presence of Ca(NO−1 for Cu(II) and 30 to 300 mg·L−1 for Pb(II). Three sorption isotherm equations were used to analyze the sorption data, which are namely: Langmuir, Freundlich and Sips isotherms and they can be expressed by Equations (3)–(5), respectively.qe (mg·g−1) is the equilibrium amount of metal sorbed per unit weight of adsorbent; Ce (mg·L−1) is the equilibrium concentration of metal in aqueous solution; qm (mg·g−1) is the maximum sorption capacity of the sorbent at saturation of the monolayer; KL (L·mg−1) is the Langmuir equilibrium coefficient; KF (mg·g−1) and Fn are the Freundlich constants, which are indicators of the capacity and intensity of sorption, respectively; KS (L·mg−1) and ns are the constants of Sips model. Values for 1/ns close to zero are generally associated to heterogeneous sorbents, while values closer to 1.0 correspond to sorbent with relatively homogenous binding sites. The parameters of the models were determined using the DataFit software .The sorption isotherms were investigated at pH 4 with metal concentration ranging from 10 to 200 mg·LC0 = 100 mg·L−1; pH0 4; agitation speed, v = 150 rpm; Temperature, T = 25 ± 1 °C). For Cu(II), 0.4 g of sorbent beads were shaken with 1 L of metal solution . Four milliliters of sample were taken out from the solution at desired time intervals over a total period of 2880 min . The intraparticle diffusion coefficient  was determined using the Crank equation (Equation (6)), assuming the solid to be initially free of metal, and the kinetics to be only controlled by intraparticle diffusion resistance:qt and qeq are the metal concentrations in the beads at time t and equilibrium, respectively; r is the radius of the sorbent and qn values are the non-zero roots of the Equation (7):m is the mass of sorbent added into the solution. The Mathematica software  was used to calculate the intraparticle diffusion coefficient, De, and to simulate experimental data .For kinetic studies, air dried and freeze dried sorbents were compared. However, the beads were soaked into water overnight prior to kinetic tests: a small amount of water was dropped onto the beads to moisten and limit the lag phase associated to re-hydration and to limit the flotation of the beads when dropped in the metal ion solution. For Pb(II), 0.2 g of alginate, 0.375 g of algal beads or 0.4 g of algal/PEI beads were agitated with 1 L of metal solution (chi-square test (x2). x2 was calculated as follows:tq is observed value and qc is calculated value.Sorption capacities observed from experiments were compared with those calculated from different models by using L. digitata) were shaped under the form of spherical beads by homogeneous Ca ionotropic gelation. This method, alternative to the conventional external ionotropic gelation method, allows synthesizing stable and homogeneously porous materials with highly porous properties. The method was applied on alginate as the reference material but also for algal biomass . The drying method has a dramatically impact on alginate beads (the collapse of the porous network is irreversible in the case of air-drying compared to freeze-drying) while in the case of algal biomass beads the method of drying has a weaker effect: after rehydration the beads regain a part of their size and porosity  for air-dried materials and completely for freeze-dried materials. The presence of cellulose fibers in the algal biomass is expected to improve this beneficial effect. Then algal biomass offers more flexibility in the management of drying process compared to alginate beads.In this work, alginate and algal biomass (L. digitata) used in this study contains alginate with a high ratio of mannuronic acid to guluronic acid (M/G ratios) [−1 for Pb(II) and 112 mg·g−1 for Cu(II)) are higher than algal beads (296 mg·g−1 for Pb(II) and 77 mg·g−1 for Cu(II)) (L. digitata) encapsulated in alginate beads by adding an extra alginate resource were 87 mg·g−1 for Cu(II) and 372 for Pb(II) under an initial solution pH of 4.5, while for the beads without adding extra alginate in this study, these values were 77 and 296 mg·g−1, respectively under a lower initial pH condition (pH = 4), again revealing that algal beads obtained by one-pot synthesis process not only reduce the preparation cost but also present comparable sorption capacities for Cu(II) and Pb(II). The incorporation of PEI derivatives into algal beads slightly decreased the sorption properties for Cu(II) and Pb(II), meaning that the presence of amine groups provided by the PEI derivatives does not enhance the sorption of these two metal cations. The result of effect of drying process on uptake kinetics show that, in the case of alginate, a strict control of the drying process is required for maintaining the porous characteristics of the hydrogel to get a fast removal of Cu(II) and Pb(II), while in the case of algal-based sorbents the swelling of the material restores the porous characteristics of the original hydrogel and minimizes the impact of the drying process. The composition of the algal biomass (including cellulose-like fibers) is expected to improve these swelling and porous properties.Batch sorption experiments show that algal beads present good sorption performance for both Cu(II) and Pb(II), which could be explained by the fact that the algal biomass ( ratios)  and an i ratios) . Althoug Cu(II)) , it is nIn conclusion, a low-cost and green sorbent, algal biomass beads, was prepared by one pot synthesis process, during which, alginate is simultaneously extracted and used for shaping/conditioning the sorbent as spherical beads. Experimental results demonstrate the high efficiency of this sorbent for Cu(II) and Pb(II) removal. In addition, the swelling process after air-drying is sufficient to maintain its porous characteristic, requiring no strict drying process (freeze-drying). The results of this research have important significance on preparation of algal biomass-based beads by one-pot synthesis process as an efficient heavy metal sorbent. The conditioning of the algal biomass under the form of beads allows using these materials in fixed-bed column while minimizing the risk of clogging and head loss pressure, contrary to the conventional ribbon-like shape of algal biomass.Metal desorption and sorbent recycling were not tested in the present study. However, recent results have shown that slightly acidic solutions  can readily desorb Zn(II), Cu(II) and Cd(II) from metal-loaded Alginate/PEI beads . SimilarFurther studies will investigate the possibility to use this type of material as support for catalytic metals  by binding metal ions such as Pd(II). Post-reduction  will help in designing green supported catalysts. On-going research has shown that these materials can bind small amounts of precious metals while the incorporation of PEI  drastically increases the sorption properties of the composite. The protonated amine groups of PEI are highly reactive for binding tetrachloropalladate anions through anion-exchange/electrostatic attraction. This property gives sense to incorporating PEI in algal beads for the recovery of metal anions in slightly acidic conditions.


However, the magnetism of La0.7Sr0.3MnO3 is strongly modified near interfaces, which was addressed as “dead-layer” phenomenon whose origin is still controversial. Here, both magnetic and structural properties of La0.7Sr0.3MnO3/SrTiO3 heterostructures were investigated, with emphasis on the quantitative analysis of oxygen octahedral rotation (OOR) across interfaces using annular-bright-field imaging. OOR was found to be significantly altered near interface for both La0.7Sr0.3MnO3 and SrTiO3, as linked to the magnetism deterioration. Especially in La0.7Sr0.3MnO3/SrTiO3 superlattices, the almost complete suppression of OOR in 4 unit-cell-thick La0.7Sr0.3MnO3 results in a canted ferromagnetism. Detailed comparisons between strain and OOR relaxation and especially the observation of an unexpected La0.7Sr0.3MnO3 lattice c expansion near interfaces, prove the relevance of OOR for the magnetic properties. These results indicate the capability of tuning the magnetism by engineering OOR at the atomic scale.La Recent studies report that changes of BO6 oxygen octahedra rotation play a decisive role in determining the magnetocrystalline anisotropy213435In LSMO/STO based heterostructures, both LSMO and STO have ABO6 oxygen octahedral rotation and lattice parameters quantitatively. To this end we use state-of-the-art annular bright field (ABF) imaging in a Cs-corrected scanning transmission electron microscope (STEM), in the [1–10] zone axis orientation. We found an unexpected out-of-plane lattice evolution near the interfaces which evidenced that the OOR mismatch, instead of strain, primarily governs the interface structure. An OOR mismatch of ~10° across the LSMO/STO hetero-interface leads to a significant alteration of the rotation angles in both STO and LSMO layers, such that the suppression of OOR in LSMO can be intuitively linked to the deterioration of magnetism. The almost complete suppression of OOR in 4 u.c. thick LSMO within a LSMO/STO superlattice results in a strongly canted magnetism, which discloses the previously reported magnetic-dead-layer phenomenon. These findings suggest an efficient interfacial engineering mechanism of magnetic and electronic properties through OOR coupling at the atomic scale.In this letter, we characterize the structural reconstruction at LSMO/STO interfaces by mapping the BO0.7Sr0.3MnO3 stoichiometric polycrystalline targets onto SrTiO3 (100) substrates. Two representative samples were selected: (1) a triple layer sandwich-like sample (denoted as T2 in the following) consisted of two LSMO layers with thicknesses of 10 and 20 u.c. spaced by a 3 u.c. thick STO interlayer; (2) a LSMO/STO superlattice (denoted by SL15 in the following) consisting of 15 bilayers of 4 u.c. thick LSMO and 6 u.c. thick STO. Summary and comparison of the Curie temperatures for LSMO/STO heterostructures with different LSMO thickness can be found in Epitaxial thin films were fabricated by pulsed laser deposition from LaB/unit cell, in good agreement with the spin-only moment of 3.7 μB/unit cell. In contrast, superlattice sample SL15 does not show a sharp ferromagnetic transition, but a gradual onset of magnetic order approximately around 55 K, see B/unit cell at 10 K and 7 T, see inset to 101213141516The STEM-HAADF images in pc zone axis orientation, where the linear density of the oxygen columns is given by 1/a, the same as the neighboring A/B cation column; a denotes the lattice parameter of the pseudo-cubic perovskite structure, further indicated by the subscript pc. Therefore, the signal from the oxygen columns is weak and masked by the strong contrast from the heavier A/B columns such that it is not easily visible. However, along the <110>pc zone axis the linear density of the oxygen columns increases up to ~1.4/a, whereas the density of the A/B cation columns decreases to ~0.7/a. These atomic density changes boost the enhancement of the relative oxygen/cation contrast; therefore, under the premise of high spatial resolution in a Cs-corrected STEM, the <110>pc zone axis provides a better contrast of the oxygen columns than the <100>pc zone axis. Moreover, considering the characteristics of in-plane oxygen octahedral rotation, such as in the case of a−a−c+ (following Glazer’s notationPnma)pc zone axis introduces a split of the oxygen columns, thus blurring and eclipsing the oxygen atomic propagation, while the in-phase rotation in the [1–10]pc zone axis gives a sharp propagation. Accordingly, the [1–10]pc zone axis was selected in our work for an enhanced contrast of the oxygen and in-phase octahedral rotation in each LSMO monolayer of the heterostructures  with MnO6 tilt of a−a−c+ is fitted into the ABF image. In this zone axis, the in-phase rotation of the oxygen octahedra along the observation direction yielded a sharp oxygen atomic column in the image. A clear zig-zag pattern of the oxygen atoms can be seen along the [110]pc direction which can be used to determine precisely the oxygen octahedral rotation, which is ±10°, close to the value for the bulk structure. The definition of the oxygen octahedral rotation (OOR) angle is sketched in the ABF image with a clockwise-rotation defined as positive.A typical STEM-ABF image of LSMO in the [1–10]1.7–2. Therefore, the pronounced peaks in the HAADF intensity image mark the position of the heavy ions, whereas the oxygen atomic columns are not visible in the HAADF image. According to the HAADF intensity profile, two different components, STO (substrate and middle layer) and LSMO, are clearly distinguished and marked with red and blue shades, respectively. Atomically sharp interfaces were observed for the thin films terminated with the La/SrO plane and an intermixing of Ti and Mn limited to 1 u.c. Average values, over all the atomic rows along the in-plane direction, of the OOR angles and the lattice parameters are shown in the last three panels of A representative STEM-ABF image taken across the substrate and the three sublayers of the trilayer T2 is shown at the top of Whereas the OOR is suppressed on the LSMO side of the interface, it was found to be induced on the STO side, where it follows the oxygen octahedra rotation pattern of LSMO, giving rise to a clearly visible in-plane tilt of 2° within the first 2 u.c. of the substrate and of 3° across the STO interlayer. This induced rotation decays within 4 u.c. in the substrate, indicating a shorter impact length of the interfacial coupling in STO as compared to LSMO. Consequently, near LSMO/STO interfaces the OOR is modified to accommodate the oxygen octahedral rotation mismatch with material dependent propagation lengths of 4 u.c. in STO and 7 u.c. in LSMO.pc direction, i.e. parallel to the LSMO/STO interface, and along the [001]pc growth direction, i.e. perpendicular to the LSMO/STO interface, are shown in the last two panels of c axis of about 0.6% was found at the STO (substrate)-LSMO interface.The corresponding lattice parameters along the in-plane [110]According to our observations, the mechanisms for accommodating lattice mismatch and oxygen octahedra rotation mismatch, although being linked to each other, are different. The magnetic properties are certainly related to both the strain and octahedral rotation state. The magnetic data show a clear difference in the Curie temperatures of 280 K and 320 K of the 10 and 20 u.c. thick LSMO layers with identical transition widths. Since the strain states of the two LSMO layers were mainly identical, we argue that the Curie temperature difference is due to the OOR differences with a metastable plateau state in the 10 u.c. and a fully relaxed bulk-like state in the 20 u.c. layer. In view of the suppressed magnetization in the superlattice, the 60 K transition observed in the trilayer might be related to the interfacial LSMO regions with strongly decreased OOR.We performed the same quantitative atomic analysis for the LSMO-STO superlattice sample SL15. In pc was clamped to the STO substrate lattice constant which is commonly found in superlattice structures. However, the lattice parameter c along the growth direction showed a clear oscillation with the bilayer period. A lattice expansion of around 1.3% was found at all interfaces which is approximately double of that observed in the trilayer sample, indicating a stronger interfacial constraint. In particular, this is clearly visible at the first LSMO layer next to the substrate. The c expansion starts at the last STO unit cell, but also propagates into the first two unit cells of the LSMO layer. Such an expansion of the LMSO c lattice constant is an unusual behavior, since an out-of-plane compression of LSMO is expected for the tensile strain at the STO/LSMO interface. Previous coherent Bragg rod analysis and synchrotron X-ray diffraction have also indirectly reported the occurrence of a c expansion for the first 2–3 unit cells of LSMO thin film growth on STO21c oscillation also results in an inhomogeneous local tetragonality within the LSMO layers. Moreover, the chemical analysis across the interfaces were also shown in the The lattice relaxation of SL15 is shown in the last two panels in Tc is proportional to the electronic bandwidth W1d and the Mn-O-Mn bond angle ϕ: W = cos[ (π-ϕ)/2]/d3.5 45ϕ decreases with the suppression of the octahedral rotations yielding the paradoxical result that the ferromagnetism should actually be enhanced. A change of the eg population due to biaxial strain and concomitant tetragonality was also reported to reduce Tc, with a typical rate of 10% for a tensile strain of 1%eg population change cannot explain the magnetic properties of the superlattice, especially because the STEM analysis showed inhomogeneous tetragonality and a resulting average c/a ratio close to 1.The exact origin of the canted magnetism in LSMO/STO superlattices is difficult to assess. In the manganites, the On the other hand, the magnetic canting seems to be primarily related to the suppression of the OOR that occurs partially nearby the interface of sample T2 or completely in sample SL15. Previous work has reported the occurrence of a spin glass constrained at the interface between LSMO and STO48x2-y2 occupation as expected for tensile strain, very thin film (4 u.c.) has a very small preferential 3z2-r2 occupation3d eg3z2-r2 orbital is preferentially occupied and the role of local strain (that favors respectively 3z2-r2 or x2-y2 for compressible or tensile strain) is a secondary effect5152c/a ratio, can be the origin of a favored eg3z2-r2 population. The microscopic origin of such population change is difficult to elucidate. The OOR suppression can play a role e.g. by altering the electron-phonon coupling leading to Jahn-Teller effect. It is also possible that the heterogeneous c/a might actually average to a slightly compressive situation that favor a slightly eg3z2-r2 population. This will be in agreement with the very weakly positive XLD signal observed for thin LSMO on STORecent work has shown that for LSMO layers, the magnetic easy axis might be strongly altered by the local OOR5152Thus, the evolution of the OOR in LSMO is expected to be the primary mechanism for the thickness-driven magnetism deterioration. The oxygen octahedral mismatch shows impact on both sides of the interface, depending on the elastic properties of the oxygen network; its relaxation length can differ greatly for different materials. In case of the LSMO/STO thin film, relaxation lengths of 4 u.c. for STO and 7 u.c. for LSMO were found. The general dead-layer behavior in LSMO/STO superlattices for LSMO layer thicknesses below 3 u.c., can then be predominantly attributed to a large deviation of the OOR from the bulk value. Therefore, to achieve a high TMR ratio in a LSMO-STO heterostructure, a critical thickness of LSMO of at least 7 u.c. (3 u.c. buffer to each interface) is required for a moderate ferromagnetism, and a critical thickness of STO of at least 5 u.c. (2 u.c. buffer to each interface) for a good insulating barrier layer. These values are in good agreement with previous reports17In summary, the quantitative profiles of the lattice parameters and OOR crossing the coherent interfaces of LSMO/STO heterostructures with relatively thick (larger than 10 u.c.) and ultrathin LSMO layers (4 u.c.) were determined based on STEM-ABF imaging with sub-Å precision. A significant variation of the OOR was found near the interface to accommodate the rotation angle mismatch of 10° between LSMO and STO, which propagates over the LSMO and STO structure for 7 u.c. and 4 u.c., respectively. The suppression of the OOR observed in LSMO was argued to be related to the dramatic thickness-dependent deterioration of the magnetic properties. Especially, in the LSMO/STO superlattice, the almost complete suppression of octahedral rotation in 4 u.c. thick LSMO leads to a strongly canted magnetism, which is in good agreement with other reports and explains the “dead-layer” phenomenon. An unexpected out-of-plane lattice expansion within the LSMO layers near the interfaces further stresses the primary role of OOR mismatch on the interface structural reconstruction. These results indicate the capability of tuning the magnetism by engineering the oxygen octahedral rotation on the atomic-scale with different film thicknesses and adjacent oxygen networks. The strong coupling between the oxygen octahedral rotation and functionalities sheds light on both fundamental research and practical spintronic device design.0.7Sr0.3MnO3 stoichiometric polycrystalline targets onto SrTiO3 (100) substrates. The STO substrates were etched in buffered HF and annealed at 1000 °C for 2 hours in air, in order to obtain substrates with atomically flat terraces and TiO2-termination. Substrate temperature and oxygen partial pressure during film growth were 700 °C and 0.14 mbar O2, respectively. The growth of thin film was in-situ monitored by a reflective high energy electron diffraction (RHEED) system, thus giving precise control on the thickness down to unit cell scale.Epitaxial thin films, trilayers and superlattices were fabricated by pulsed laser deposition  from LaZFC: demagnetizing the sample at 300 K, cooling in nominal zero field down to 5 K, stabilizing the temperature at 5 K, setting the magnetic field to the required value in no-overshoot mode and measuring during warmup.FC: directly after the ZFC run, measuring during cooldown in the same field as in the ZFC measurements.REM: After stabilizing a temperature of 5 K at the end of the FC run, the magnetic field was reduced to zero in no-overshoot mode and the remanent magnetic moment measured during warmup.Magnetization measurements were done with a Quantum Design SQUID magnetometer under varied temperature and magnetic field. The measurements were performed with the following cycle which is typical for the study of spin glasses or high temperature superconductors:pc zone axis of the STO substrate by standard tripod polishing, followed by Ar-ion milling under liquid nitrogen cooling . The microstructure was investigated in a JEOL ARM 200CF scanning transmission electron microscope (STEM) with a probe Cs corrector, utilizing atomically resolved high angle annular dark field imaging , and annular bright field imaging  which enables to directly image oxygen atoms in real space and to map a projection of the oxygen octahedral network. These imaging experiments were performed at an acceleration voltage of 200 kV, with a semi-convergence angle of 21 mrad, showing a typical spatial resolution of 70 pm. In order to diminish the sample drift effect, before starting the image acquisition the electron beam was blocked for at least 5 minutes. The typical scanning step for atom resolution images was 0.06 Å/pixel with a dwell time of 16 μs/pixel, and the scanning direction is perpendicular to the interfaces. The corresponding interfacial chemical distribution was studied by electron energy loss spectroscopy (EELS).Cross-section specimens for electron microscopy study were prepared along the [1–10]How to cite this article: Li, X. et al. Impact of interfacial coupling of oxygen octahedra on ferromagnetic order in La0.7Sr0.3MnO3/SrTiO3 heterostructures. Sci. Rep.7, 40068; doi: 10.1038/srep40068 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


In China, H9N2 subtype avian influenza outbreak is firstly reported in Guangdong province in 1992. Subsequently, the disease spreads into vast majority regions nationwide and has currently become endemic there. Over vicennial genetic evolution, the viral pathogenicity and transmissibility have showed an increasing trend as year goes by, posing serious threat to poultry industry. In addition, H9N2 has demonstrated significance to public health as it could not only directly infect mankind, but also donate partial or even whole cassette of internal genes to generate novel human-lethal reassortants like H5N1, H7N9, H10N8 and H5N6 viruses. In this review, we mainly focused on the epidemiological dynamics, biological characteristics, molecular phylogeny and vaccine strategy of H9N2 subtype avian influenza virus in China to present an overview of the situation of H9N2 in China. Avian influenza (AI) is initially reported in 1878 in Italy to describe the disease resulted in massive poultry death, which was then termed as “Fowl plague” to distinguish from fowl cholera in 1880 . Apart fAvian influenza virus affiliates to the genus of type A influenza virus in the Orthomyxoviridae family, packaged with eight negative-sense and single-strand RNA segments in sequence of PB2, PB1, PA, HA, NP, NA, M and NS according to gene length . Each viIsolation of AI virus in China has been documented since 1970s . During 93-1 (once erroneously identified as H9N3 subtype then), also the earliest published report of AI outbreaks in mainland China [Till 1992, Chen et al. isolated the first H9N2 subtype LPAIV strain AIDnd China . During nd China , 21. A fnd China –24. Howend China , 26. Accnd China . Even toTheoretically, emerging diseases could possibly be effectively controlled by a stamping-out policy before disseminating into vast areas . HoweverH9N2 subtype AI virus is extensively distributed worldwide, generally divided into two major lineages of North-American lineage and Eurasian lineage. Specifically, the Eurasian lineage further blooms into various virus clusters, as represented by A/chicken/Beijing/1/1994(BJ/94-like) or A/duck/Hong Kong/Y280/1997(Y280-like), A/quail/Hong Kong/G1/1997(G1-like), A/duck/Hong Kong/Y439/1997(Y439-like), A/chicken/Shanghai/F/1998(F/98-like) and so on –36. CompTo further systematically understand the evolutionary dynamics of H9N2 subtype AI virus globally, four stem evolutionary clades of h9.1–h9.4 have been designated by Jiang et al. to map the HA gene phylogeny through comparing more than 1000 HA sequences retrieved from GenBank, as referred to the nomenclature of the Asian H5N1 HPAIV defined by the WHO/OIE/FAO H5N1 working group , 38. ParOwing to the segmented nature of AI virus genome, when two or more virus strains concurrently infect a single cell, exchange of gene segments would occur among different virus particles via gene reassortment to generate a series of newborn viral descendants inheriting parental components. Certainly, H9N2 subtype AI virus is also without exception that distinct virus clusters could reassort with each other or with other AI subtypes to produce various genotypes, which is defined on the basis of the combination of each individual gene phylogenies. For instance, virus harbored all the gene constellation from BJ/94-like is designated as genotype A, variant of three polymerase genes and NP gene from F/98-like while the remaining four genes from BJ/94-like is assigned for genotype H. Thus far, H9N2 subtype AI virus in China has evolved into diversified clusters and genotypes (A–W), showing clear spatio-temporal divergence Table  7, 26, , 39, 40.The premier isolates of H9N2 just infected turkeys, rarely encroached on chickens, but have gradually adapted to chickens and acquired pathogenicity after years of evolution , 3. SincIt is acknowledged that the variation mechanism of AI virus mainly includes antigenic drift and genetic shift, with the former featured by point mutation of key amino acids in major immunoprotective proteins whereas the latter resulted from genomic reassortment . As compPresently, H9N2 subtype AI virus has been widely spread in China, and has established stable lineages in commercial chicken flocks with endemicity . DespiteThe majority of commonly used AI vaccines are killed whole-virus vaccines, prepared from formaldehyde inactivation of virus-containing allantoic fluids proliferated via chicken embryos and accompanied with adjuvants, manifesting favorable immune efficacy . DomestiInactivated whole virus vaccine mainly elicits humoral immune response, deficient in inducing effective mucosal and cellular immunity. Furthermore, it also interferes with immunological surveillance and epidemiological investigation of AI virus under the condition of current technology. Therefore, novel DIVA  vaccines against H9N2 come into being, including recombinant live virus vectored vaccine, subunit vaccine, DNA vaccine, VLPs (virus like particles) vaccine and so on. They could supplement certain shortages of traditional vaccines and are popular for AI vaccine development nowadays. Frequently used live virus vectors contain recombinant fowlpox virus, Newcastle disease virus, Marker’s disease virus, etc –76. SubuApart from various kinds of poultry, H9N2 subtype AI viruses could also infect pigs, the long considered mixing vessel for mammalian and avian influenza variants. It is revealed by epidemiological survey that H9N2 viruses were isolated from pigs naturally when transported from southern China to Hong Kong for sale, as early as in 1998 . SubsequWhat’s more noteworthy, H9N2 subtype AI viruses have already acquired the ability to break through species barrier and directly invade human beings without intermediate hosts. The first documentation of human-infecting H9N2 viruses in China traced back to 1998, as described that five H9N2 strains were cultured from laryngopharyngeal mucus of flu-like outpatients and inpatients in southern regions . FurtherAlthough being classified as LPAIV, H9N2 subtype AI virus is extensively distributed in chicken flocks to pose a persistent challenge. In China, traditional raising system of livestock including free-ranging and polyculture, continuously occupies a crucial status yet. It is inevitable for chicken to contact with domestic or wild waterfowl, which harbored large amount of H9N2 viruses. These apparently healthy latent birds could serve as the “Trojan horses” in chicken flocks to cause the circulation of H9N2. Furthermore, the LPMs extending throughout China still played an indispensable role in hosting and disseminating of H9N2 AI virus, as evidenced by significant higher rates of virus isolation than other locations. However, focusing on LPMs management, innovative control measures targeting principally against the emerging avian influenza A(H7N9) virus such as closure of LPMs or other more sustainable but yet effective interventions including washing and cleaning once a day, disinfecting once a week, having rest days once a month and banning live poultry overnight, as well as separating of aquatic and non-aquatic live poultry, would certainly simultaneously reduce the risk of H9N2 contamination at source and deserve high priority in implementation. On account of incessant viral mutation and reassortment, natural variants with increased pathogenicity have been emerging periodically. Even though vaccination remains one of the primary strategies to control H9N2 subtype AI in China, the majority of vaccine recipients are actually still under siege of wild-type variants. Therefore, disease outbreaks would still occur in vaccinated flocks in case of descended protection level or different kinds of immune failure. It is more prior important to establish favorable biosecurity management and take all practicable measures to control infection source, preventing virulent variants from intruding the poultry flocks. On the other hand, the human-infecting events of H9N2 AI virus deserve to be treated scientifically and rationally. Once animal influenza is controlled, should the risk of emerging human pandemic influenza be decreased to minimum level.


Flavivirus, Nairovirus and Phlebovirus of the families Flaviviridae and Bunyaviridae, followed by sequencing and NGS in selected specimens.Ticks are involved with the transmission of several viruses with significant health impact. As incidences of tick-borne viral infections are rising, several novel and divergent tick- associated viruses have recently been documented to exist and circulate worldwide. This study was performed as a cross-sectional screening for all major tick-borne viruses in several regions in Turkey. Next generation sequencing (NGS) was employed for virus genome characterization. Ticks were collected at 43 locations in 14 provinces across the Aegean, Thrace, Mediterranean, Black Sea, central, southern and eastern regions of Anatolia during 2014–2016. Following morphological identification, ticks were pooled and analysed via generic nucleic acid amplification of the viruses belonging to the genera A total of 814 specimens, comprising 13 tick species, were collected and evaluated in 187 pools. Nairovirus and phlebovirus assays were positive in 6 (3.2%) and 48 (25.6%) pools. All nairovirus sequences were closely-related to the Crimean-Congo hemorrhagic fever virus (CCHFV) strain AP92 and formed a phylogenetically distinct cluster among related strains. Major portions of the CCHFV genomic segments were obtained via NGS. Phlebovirus sequencing revealed several tick-associated virus clades, including previously-characterized Antigone, Lesvos, KarMa and Bole tick viruses, as well as a novel clade. A wider host range for tick-associated virus strains has been observed. NGS provided near-complete sequences of the L genomic segments of Antigone and KarMa clades, as well as Antigone partial S segment. Co- infections of CCHFV and KarMa or novel phlebovirus clades were detected in 2.1% of the specimens.Widespread circulation of various tick-associated phlebovirus clades were documented for the first time in Anatolia. Genomes of CCHFV AP92 strains were identified in previously unexplored locations. NGS provided the most detailed genomic characterization of the Antigone and KarMa viruses to date. The epidemiological and health-related consequences must be elucidated.The online version of this article (doi:10.1186/s13071-017-2279-1) contains supplementary material, which is available to authorized users. Ticks are closely-associated with the epidemiology of several microbial pathogens with significant human and animal health impact . They haFlaviviridae or Bunyaviridae, such as notable human pathogens tick-borne encephalitis virus (TBEV), Crimean-Congo hemorrhagic fever virus (CCHFV), Kyasanur forest disease virus, Powassan virus, Alkhurma virus, as well as livestock pathogens African swine fever virus, Nairobi sheep disease virus, and louping ill virus [Nairovirus and Phlebovirus [Ixodes, Haemaphysalis, Hyalomma, Amblyomma, Dermacentor, Rhipicephalus and Boophilus [Many tick-borne viral pathogens, causing severe morbidity and mortality, are classified in the families ll virus –7. Sharill virus . Viral gll virus . Bunyaviebovirus . All bunebovirus . The lenebovirus . The vecoophilus .Haemaphysalis longicornis ticks were suggested as the primary transmission vector [Amblyomma americanum ticks, has been characterized in human cases of febrile disease in the USA [Current evidence suggests a global increase in the incidence of tick-borne diseases . This isn vector , 14. Ano the USA , 16. SFT the USA , 18. The the USA , Africa  the USA , 21, Asi the USA , 23, Aus the USA  and Euro the USA –27. ThesLocated in Asia Minor and Eastern Thrace region of the Balkan Peninsula, Turkey covers a transboundary temperate climate zone between Asia and Europe. Turkey provides suitable habitats for several tick species due to the variety of ecological and climatic conditions observed throughout Anatolia . SeveralBos taurus), sheep (Ovis aries), goats (Capra aegagrus hircus) and dogs (Canis familiaris) were collected at animal shelters. The specimens were kept alive in separate vials, transferred to the laboratory and identified morphologically to the species level using appropriate taxonomic keys [Tick sampling was carried out at 43 locations in Edirne , Aydin and Mugla , Ankara and Cankiri , Sivas and Van (eastern Anatolia), Bayburt and Gumushane , Antalya and Mersin , Diyarbakir, Mardin and Siirt (south-eastern Anatolia) provinces; from April to October during three consecutive years 2014–2016) . All specimens were subsequently subjected to complementary DNA synthesis with random hexamers, using the RevertAid First Strand cDNA Synthesis Kit , performed according to the manufacturers’ guidelines.Individual and pooled ticks with up to 5 individuals were subjected to homogenization using the SpeedMill PLUS homogenizator  and subsequently, total RNA extraction via BlackPREP tick DNA/RNA kit  according to the manufacturer’s instructions. Tick pools with more than 5 individuals were homogenized by vortexing with tungsten carbide or stainless steel beads  in 500–700 μl of Eagle’s minimal essential medium, supplemented with 5% foetal bovine serum and 1% L-glutamine. They were clarified by centrifugation at 1430× Nairovirus, Flavivirus and Phlebovirus of the families Flaviviridae and Bunyaviridae, were employed for the screening of individual or pooled ticks. Generic Nairovirus screening was carried out via a previously published single round PCR protocol with primers targeting the central motif A of the viral polymerase (on the genomic segment L) [Nairovirus, including CCHFV and Nairobi sheep disease virus. Previously quantitated human plasma from CCHFV-infected individuals were employed for assay optimization [A spectrum of assays for the detection of viruses belonging to the genera gment L) . The assmization .http://www.european-virus-archive.com) for tick-borne strains.A nested PCR assay with degenerated primers targeting the NS5 conserved region was used for the flavivirus screening in ticks. The assay provides a sensitive amplification of all major tick and mosquito-borne pathogenic flaviviruses including West Nile virus (WNV), dengue virus, yellow fever virus, TBEV, Murray Valley encephalitis virus, Saint Louis encephalitis virus and Usutu virus . Assay oPhlebovirus screening was performed using two PCR assays optimized for the amplification of tick-borne phleboviruses . The binAmplified products of the screening assays were visualized in a ChemiDoc XRS+ imaging system  via ethidium bromide staining after electrophoresis in 1.3–1.7% agarose gels. Extreme care was taken to prevent carry-over contamination, and extraction, pre- and post-PCR steps were performed in spatially-separated areas.Detectable PCR products of the screening assays were cleaned up using PureLink PCR Purification Kit  and characterized via sequencing in an ABI PRISM 3500xL Dx Genetic Analyzer (Thermo Fisher Scientific), using forward-reverse primers of the relevant assay and BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific). Obtained sequences were handled using CLC Main Workbench v7.8.1 , Bioedit v.7.0.9.0 and MEGA v.6.06 softwares , 42.https://blast.ncbi.nlm.nih.gov/Blast.cgi) [https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and MOTIF Search (http://www.genome.jp/tools/motif/) in the PFAM database [BLASTn, BLASTn optimized for highly similar sequences (MEGABLAST) and BLASTp algorithms were employed for nucleotide similarity searches in the public databases, implemented within the National Center for Biotechnology Information website (ast.cgi) . Nucleotast.cgi) . Pairwisdatabase , 46.Aliquoted RNA of the selected tick specimens were reverse transcribed using random hexamers into double-stranded cDNA via SuperScript IV Reverse Transcriptase  and NEBNext mRNA Second Strand Synthesis Module . Agencourt AMPure XP Reagent  and Agilent 2100 Bioanalyzer  were used for the cleanup, yield and size distribution determination.Fragmentation, adaptor ligation and amplification steps were performed via the NexteraXT DNA Library Preparation Kit , as indicated by the manufacturer. The sequencing run was performed in one lane of the Illumina HiSeq 1500 (Illumina Inc.) in the high output mode.The raw sequencing data was de-multiplexed and extracted in fastq format. Trimmomatic and Bowtie2 v2.2.9 softwares were employed for trimming for quality and length, removal of adaptor and background mapping , 48. AcqRiphicephalus sanguineus complex , followed by Haemaphysalis parva , Riphicephalus bursa  and others .A total of 814 specimens, comprising 13 tick species, were collected  R. sanguineus complex (2/6) and Hyalomma marginatum (1/6) specimens. Generic tick phlebovirus PCR revealed positive results in 48 pools, collected from 6 provinces in eastern, south-eastern and central Anatolia, Aegean and Mediterranean regions (Table R. sanguineus complex (31/48), R. bursa (8/48), H. marginatum (4/48), Hyalomma aegyptium (3/48), Hyalomma excavatum (1/48) and Haemaphysalis parva (1/48) specimens. In 4 pools with R. bursa (3/4) R. sanguineus complex (1/4) specimens, nairovirus and phlebovirus co-infections were identified, with both assays providing positive results  were positive in the screening assays, originating from central Anatolian (Cankiri: 1 pool), eastern Anatolian (Van: 12 pools), Black Sea (Bayburt: 1 pool), south-eastern Anatolian , Mediterranean (Mersin: 28 pools) and Aegean (Mugla: 5 pools) provinces . The sequences aligned to the 6705–7168 nucleotides on the L segment of the AP92 genome (GenBank accession number: DQ211612) and displayed 2.3–2.5% diversity from this strain. In the maximum likelihood analysis, the sequences formed a separate monophyletic cluster with the AP92 strain, distinct from other CCHFV clades, supported by high bootstrap values  [In specimens MG22 and MG31, sequences of 6539 nucleotides were characterized, that represents the near-complete L segment of the Antigone clade. The putative coding regions were located identically, which encompassed the nucleotides 36–6488 on the sequence that encoded a 2161 amino acid product. Though non-coding regions of 35 and 51 nucleotides could be obtained via NGS, the sequences are considered near-complete since the phlebovirus-specific terminal motifs could be identified partially and no additional experiment was performed to ensure the availability of the complete L segment. In specimens MG36 and MG49, 6374 and 6363 nucleotide-long stretches within the coding region could be obtained, which corresponded to the 2124 and 2121 amino acids of the viral RNA-dependent RNA-polymerase. The maximum likelihood analysis based on the putative amino acid sequences revealed a well-supported separation of the Antigone and KarMa clades, sharing common ancestors with Bole and Lihan tick viruses  from the Antigone phlebovirus S genomic segment could be characterized via direct NGS in specimen MG22 Table . BLASTn H. marginatum specimens collected from Bayburt province of the Black Sea region was evaluated via direct NGS, following generic nairovirus PCR reactivity (Table One specimen (MG48) that comprised ty Table . Sequencty Table . The strThe obtained S segment sequence has further been aligned and analysed with previously- characterized AP92-like partial sequences from Turkey as well as several Balkan countries.Pairwise comparison of a 426-nucleotide partial sequence revealed 98.3–99.5% similarity with sequences previously-identified in ticks (GenBank: FJ392601– FJ392603) and humans (GenBank: EU057975 and FJ392604) in Thrace region of Turkey. These sequences from Turkey and AP92-related sequences characterized from Albania, Bulgaria, Iran, Greece and Kosovo formed distinct clusters in the maximum likelihood analysis  of the specimens with detectable viral nucleic acids Table . The KarThe NGS performed on selected specimens with screening reactivity have resulted in the characterization of L and S segment sequences of the Antigone and KarMa clade phleboviruses, for which only limited data have been previously available. The near-complete L segment sequences have enabled a more robust analysis of the genetic distances among partially- characterized tick-borne phleboviruses, confirming the separation of these particular clades Fig. . FurtherAn important issue that remains to be elucidated is whether these identified phleboviruses are pathogenic for humans or animals or not. Among the recently-described tick-borne phleboviruses, SFTSV and Heartland virus are documented human pathogens and SFTSV is a highly-pathogenic agent causing hemorrhagic fever with a case fatality rate as high as 50% –15, 64. We have documented the presence of multiple and divergent tick-associated phleboviruses throughout Anatolia, with four previously-identified as well as a novel clade. A wider host range for particular tick phlebovirus clades and a larger dissemination zone for the CCHFV AP92-like strains have been revealed. NGS has enabled the comprehensive genomic characterization of two phlebovirus clades and the CCHFV AP92-like strain in circulation.Additional file 1: Table S1.Distribution of the tick specimens according to species, sex and collection province. (XLS 31 kb)Additional file 2: Table S2.Pairwise comparison of the nucleotide and putative amino acid sequences of the L segment of tick phleboviruses identified in the study  with related viruses. Similarity rates are given in percent. (XLS 28 kb)Additional file 3: Figure S1.The maximum likelihood analysis of the partial putative nucleocapsid protein (152 amino acids) of phleboviruses. The sequence characterized in this study is given in bold and indicated with the GenBank accession number, pool code and a black circle. Global virus strains are indicated by GenBank accession numbers, virus and strain/isolate names. Bootstrap values higher than 60 are shown. (PDF 422 kb)Additional file 4: Table S3.Pairwise comparison of the nucleotide and putative amino acid sequences of the S segment of tick phleboviruses identified in the study (MG22) with related viruses. Similarity rates are given in percent. (XLS 25 kb)Additional file 5: Table S4.Pairwise comparison of the nucleotide and putative amino acid sequences of the L segment of the Crimean-Congo hemorrhagic fever virus (CCHFV) identified in the study (MG48) with distinct CCHFV strains. Similarity rates are given in percent. (XLS 26 kb)Additional file 6: Table S5.Pairwise comparison of the nucleotide and putative amino acid sequences of the M segment of the Crimean-Congo hemorrhagic fever virus (CCHFV) identified in the study (MG48) with distinct CCHFV strains. Similarity rates are given in percent. (XLS 26 kb)Additional file 7: Table S6.Pairwise comparison of the nucleotide and putative amino acid sequences of the S segment of the Crimean-Congo hemorrhagic fever virus (CCHFV) identified in the study (MG48) with distinct CCHFV strains. Similarity rates are given in percent. (XLS 26 kb)


The population is aging rapidly in many developed countries. Such countries need to respond to the growing demand and expanding costs of healthcare (HC) for the elderly. Therefore, it is important to investigate the factors correlating such HC costs. In Japan, HC is composed of two sections, namely medical care (MC) and long-term care (LTC). While many studies have examined MC and LTC costs on their own, few studies have conducted comprehensive investigations of HC costs. The aim of this study is to examine the risk factors that influence HC costs for the elderly who enroll in the LTC insurance system in Japan.The inclusion criteria in the present study are as follows: being 65 years of age, or older; certified eligibility for, and use of services offered by the LTC insurance system at home or in an institutional setting in December 2009; and being covered by the National Health Insurance (NHI) system. MC and LTC insurance data were obtained from claim records for the elderly in July and December of 2007, 2008, and 2009 . Panel data, per subject, were constructed using MC and LTC claim records. The sample included 810 subjects and 4029 observations.We estimated a regression equation with a censored dependent variable using a Tobit model. Significant associations between MC or LTC costs and interaction terms  were investigated. MC costs significantly decreased and LTC costs significantly increased among subjects living alone during winter. Income level was also a positive determinant of MC costs, while eligibility level was a positive determinant of LTC costs.We recommend that the health policy for the elderly focus more on seasonal effects, household composition, and income level, as well as on eligibility level. According to the Organization for Economic Cooperation and Development (OECD), many developed countries will experience an aging society as the baby boom generation ages. Consequently, the demand for long-term care (LTC) is expected to rise sharply over the coming three decades . Such coJapan, in particular, is at the forefront of countries experiencing an aging society. The healthcare (HC) policy in Japan includes both medical care (MC) and LTC insurance systems. MC insurance is necessary for medical treatment due to illness, injury, preparation for hospitalization, surgery, outpatient visits, and medication treatment. LTC insurance applies when a person requires nursing care. The MC insurance service covers hospitalization, rehabilitation, medication, medical treatment, surgery, and visiting nursing services. The LTC insurance service covers in-home care and community-based care, such as residential facilities and facility care. If an insured person is over the age of 65, he/she is eligible for the MC insurance service and the LTC insurance service. If the insured person needs acute medical care, he/she will be admitted to a hospital and will receive medical treatment. After being discharged, the person can use in-home care services, including housekeeping and nursing care, on a daily basis, and can receive rehabilitation services in order to maintain his/her physical activity ability, which does not fall under acute treatment rehabilitation. These are services provided by LTC insurance. However, the two insurance services are separate and provided independently. Since 1961, Japan has provided a universal National Health Insurance (NHI) system for MC to all its citizens. The LTC insurance system, a mandatory social system supporting LTC for the elderly, was established in 2000 . HC costA behavioral model proposed by Andersen , 6 and AMany studies have reported that seasons have a significant impact on health. For example, studies have reported that institutionalization rates peak in winter , as doesA longitudinal design was applied in the analysis of both MC and LTC insurance data, and a cross-sectional design was used for the questionnaire survey. The study was conducted in city A, Hokkaido, Japan. The name of City A cannot be disclosed because the city administration and the ethics committee restricted sharing the city name in order to protect participant privacy. City A is located near the center of the northern island of Hokkaido and has an inland climate. The average annual temperature is 5.5°C, with a mean annual rainfall of 960 mm and snowfall of 884 cm (data from 1980 to 2010) . The incIn the Japanese LTC insurance system, eligibility is classified into seven levels, depending on an individual’s current cognitive, physical, and mental status. The seven levels are as follows: Support Levels 1 and 2, in which individuals require partial assistance in daily living and can access preventative care services in the LTC insurance system ; and Care Needs Levels 1–5. The latter group ranges from those in Level 1, classified as needing partial care for daily life, to Level 5, in which individuals are unable to conduct daily activities without extensive assistance. In order to determine the eligibility level, a trained local government official visits the home and/or institution. Then, he/she uses a questionnaire, created by the Ministry of Health, Labour, and Welfare, to evaluate the individual’s current physical and mental status. Next, a local expert committee, consisting of medical and care professionals, reviews and evaluates the results and assigns each person to an appropriate level [MC insurance data were obtained from claim records of the NHI and/or MC insurance systems for the elderly aged 65 years or older in July and December of 2007, 2008, and 2009 . In order to preserve the anonymity of the participants, the staff of city A deleted all their personal information after handing it over to the researchers. MC claim records include hospital services, outpatient services, pharmacist services, and home medical care, as well as treatments provided by judo-orthopedists, masseuses, chiropractors, and acupuncturists. LTC insurance data from claim records were also obtained for the same period. LTC claim records include in-home care services, community-based services, and facility care services. In this study, the sum of hospital service fees, outpatient service fees, and pharmacist service fees is used as MC costs. The sum of in-home care services, community-based care costs, and facility care services fee is used as LTC costs. Because neither of the sets of claim records have any data on household composition, to obtain such information (on population characteristics), we formulated our own cross-sectional questionnaire. We then distributed this questionnaire to care staff who visited homes or who visited people in institutions in 2009 (n = 871). Our questionnaire includes questions on the number of people covered by LTC insurance and on household composition. We obtained permission from all LTC service companies in city A to conduct the questionnaire survey. Then, we delivered the questionnaire to the LTC service companies, which included eight in-home care companies, three group home companies, and four facility companies. For people living in institutions, staff members were instructed to report on the household component using data on participants prior to them entering the institution. For subjects who lived at home, home care staff members were instructed to gather information at the time of their visit. Then, each company returned their questionnaires to us. Panel data, per subject, were created from the claim records for both MC and LTC. The MC claim records include information on age, gender, illness, prescribed medication (including cost), and monthly hospital and/or outpatient care costs. LTC insurance records contain information on age, gender, eligibility level, types of services needed, income level, and the monthly costs of care in the home or in a facility.In Japan, medical fees are decided based on the report of the Central Social Insurance Medical Council. In principle, revisions are made once every two years. The medical fee system is a points system. MC costs are calculated as the value of medical fees multiplied by 10 yen. In the revision of 2008, an overall review resulted in no increase in the fees for specific medical services. LTC fees are determined by the Minister of Health, Labour and Welfare after conferring with the Social Security Council. LTC fees are revised every three years and are also based on a points system. LTC costs are calculated as the value of LTC fees multiplied by 10 yen. In the revision of 2009, all fees for LTC insurance services increased in order to increase the salaries of LTC staff. The total remuneration for MC costs was reduced by 0.82%. The LTC insurance system was further revised in 2009, and the total remuneration for LTC costs increased by 3%. Thus, in order to account for the effect of the changes in MC and LTC insurance reimbursements, we multiplied MC costs for 2007 by 0.9918 (1–0.82%) and LTC costs for 2007 and 2008 by 1.03 (1 + 3%).For LTC insurance, income levels are classified from Level 1 to Level 6. People who receive welfare or a welfare pension and who are not required to pay local resident taxes are assigned to Level 1 or Level 2. People who are members of households that are exempt from municipal taxation are assigned to Level 3. People exempt from municipal taxation are assigned to Level 4, and those who pay municipal taxes, but have a total annual income of less than 2,500,000 yen are assigned to Level 5. Lastly, people who pay municipal taxes and whose total annual income is 2,500,000 yen or more are assigned to Level 6 . All datVariables potentially associated with HC costs and services were selected on the basis of prior research –7, 19–21Data were analyzed using the t-test, chi-square test, Wilcoxon signed-rank test, and median test to compare population characteristics and HC costs at the different survey points. The distribution of MC was censored at zero for a large portion of the sample. To examine household composition and seasonal effects, we estimated a regression equation with a censored dependent variable using a Tobit model . In the The study was approved by the Ethics Committee of the University of Tokyo (approved number 1790). This is a joint study between city A and the researchers, and is intended for use in municipal administration. For the study, we obtained comprehensive agreement from residents, including a notice posted in the office of city A. This notice included information about our study’s aim, the right to refuse participation in the research, the right to withdraw consent, and how we protect personal information. Furthermore, the research purpose was also discussed when staff members explained the questionnaire to participants.City A has a population of 31,000. In Japan, local cities, towns, and villages with populations under 50,000 comprise over two-thirds of all municipalities. In 2009, the elderly aged over 65 years accounted for 25.0% of the total population of city A, which was higher than the national average of 22.7%. However, the per capita MC and LTC expenditures for the elderly were similar to the national levels.Of the 871 cross-sectional questionnaires we distributed, 810 were completed, giving a response rate of 93.0%. Of the 61 that were excluded from the analysis, 44 did not return the questionnaire and 17 did not fill in all required items. Tables Tables This study examines the effects of household composition and season on HC costs among the elderly enrolled in the LTC insurance system in city A, Japan. When considering factors that influence HC costs paid by the MC and LTC insurance systems, it is necessary to collect a variety of data from both claim records in a single panel for each person. In general, it is difficult to connect claim records for MC and LTC in Japan, which is why few studies have analyzed data from both. However, city A cooperated with our research and allowed us to connect the data from claim records for MC and LTC insurances. Thus, we were able to collect various data in a single panel for each subject from both sets of records, which were supplemented by the information from our cross-sectional questionnaires. Using a panel approach, we were able to analyze the impacts of both MC and LTC costs. Our results presented three major conclusions.First, the household composition variable alone did not have an effect, but the interaction term between household composition and season was negatively associated with MC costs. Seasonal changes affect the mortality risk and the level of MC expenditure of older people . HoweverSecond, LTC cost was strongly associated with the interaction term between household and season. Our results emphasize that living alone is risk factor for nursing home admission . Those wThird, income level and eligibility level were significantly associated with HC costs. The urban elderly with higher income or their spouses tend to pay for services by themselves or their spouses . It is pThe present study has several limitations. First, the analysis of factors correlating HC costs only consider a single city in Hokkaido and use data for two months in a year. This creates a selection bias, which is why other studies have found a different trend in the expenditure of rural and urban areas . AccordiDespite these limitations, our results are meaningful and contribute to the identification of factors that influence HC costs in Japan. We strongly recommend that health policies for the elderly focus on the effects of household composition, season, income level, and LTC eligibility.This study found that, in addition to income level and eligibility level, household composition and season correlate HC services for the elderly enrolled in the LTC insurance system. There may be a substitutionary effect between MC and LTC among those who live alone and during winter, as suggested by the statistically significant negative and positive coefficient estimates of the Tobit model. MC costs were not correlated by the household variable alone, but were affected by the interaction term of the household and seasonal variables. MC costs decreased significantly. On the other hand, LTC costs were not correlated by the seasonal variable alone, but they increased significantly in the case of the interaction variable. Most elderly persons who use LTC services incur both MC costs and LTC costs. Lastly, the effects of the environment and population characteristics on MC costs and LTC costs differ. In considering health policies for the elderly, it is very important to connect data on MC and LTC insurance, creating one set of panel data.


We collected venous and capillary serum samples from 21 Zika virus‒infected patients on multiple days after symptom onset and found RNA load was higher and median duration of virus detection significantly longer in capillary than in venous blood. These findings raise questions about the role of the capillary compartment in virus transmission dynamics. Flaviviridae and genus Flavivirus, is transmitted to humans by mosquito bites but can also be contracted through sexual and vertical transmission  of serum and urine samples provided by patients during the first few days after symptom onset. We obtained serum samples from the venous and cutaneous capillary blood (collected from the fingertip) sequentially 1‒18 days after the onset of symptoms. The median age of the population was 40 (range 28–63) years and the sex ratio  was 1.6. We observed no co-morbidities, and all participants were found to be free of dengue and chikungunya virus infections by methods previously described  and performed Zika virus RNA amplification using the RealStar Zika Virus RT-PCR Kit 1.0  according to the manufacturer’s instructions. We performed Zika virus RNA quantification using a reference strain provided by the European Zika Virus Archive (SKU no. 001N-01648) and estimated the Zika virus RNA load as logr = 0.54, p<0.0001) but were significantly higher in the capillary samples , except for 3 patients  of 21 patients  and longer in venous blood for 2  of 21 patients (nos. 2 and 21)   Table.These data raise questions about the consequences of longer persistence and higher loads of Zika virus RNA in the cutaneous capillary blood compartment, although we did not test Zika virus replication capacity. The higher load and longer detection of Zika virus RNA in this compartment might be attributable to Zika virus replication in permissive cells of the skin ; capillaries; or both (This study comparing the kinetics of Zika virus RNA load between the venous and capillary compartments highlights the need to further investigate the infectivity and pathophysiology of the virus located in the often neglected capillary compartment. These findings provide new information on this biologic compartment, which plays a key role in vectorborne transmission and transmission dynamics. Moreover, these observations, if validated with more patients and extended to other vectorborne infections, will be vital for preventing and controlling the transmission of Zika virus and other arboviruses.Kinetics of Zika virus load in venous and capillary blood samples from 21 patients, French Guiana, 2016.


Toward the end of 2015, China relaxed its more than three-decade-old one-child policy to address the problems of a rapidly aging population, now allowing all couples to have two children  [As of August 3, 2016, more than 1.5 million people in 68 countries and territories have been affected by Zika virus, including 51 countries and territories with a first reported outbreak since2015 .Aedes aegypti and A. albopictus; however, mounting evidence shows that the virus may be transmitted through Culexpipiens and A. triseriatus, sexual intercourse, blood transfusion, laboratory exposure, and from mother to child [declared Zika virus to be a Public Health Emergency of International Concern [The primary transmission vectors of Zika virus are to child ,5,6. On  Concern , and man Concern .Aedes mosquitoes; therefore, Zika virus may attain the same geographical spread as dengue virus [A. aegypti and A. albopictus)   and a suAn outbreak of Zika virus is most likely to occur in the southern and southwestern parts of China  and in large cities with a large inbound population during the mosquito season. These regions have a very high population density, with tens of millions of women in their reproductive age. Therefore, if a Zika virus outbreak coincides with the baby boom, serious consequences, including increases in birth defects and delayed pregnancy, may occur. This may lead to a baby boom that is smaller than anticipated and a heavy societal burden in the long run, contrary to the original intention of the universal two-child policy.A Zika virus outbreak in China is possible, and public health preparedness is urgently needed. The government could apply lessons learned from controlling dengue to prevent an outbreak of Zika virus. Proactive vector control measures should be taken, and medical supplies should be prepared. Inspection and quarantine policies at entry ports should be strengthened. Moreover, Chinese scientists should participate in international efforts to accelerate vaccine development. A large-scale epidemiology survey should be conducted to identify potential local cases of Zika virus infection. Mass media should be used to educate the public about Zika virus disease prevention, including protection against mosquito bites and safe sex as the main measures to avoid infection.Fortunately, the Chinese government is aware of this situation, and many measures have been implemented in response to the possible challenges posed by Zika virus. A national scheme on the prevention and control of Zika virus disease and a national scheme on the diagnosis and treatment of Zika virus disease has been worked out in China in February 2016 , 16. Als