The unusual non-dipolar magnetic fields of ice giant planets, produced by convecting liquid ionic water, are influenced by exotic high-pressure states of H2O—yet the structure of ice in this state is challenging to determine experimentally. Here we present X-ray diffraction evidence of a body-centered cubic (BCC) structured H2O ice at 200 GPa and ~ 5000 K, deemed ice XIX, using the X-ray Free Electron Laser of the Linac Coherent Light Source to probe the structure of the oxygen sub-lattice during dynamic compression. Although several cubic or orthorhombic structures have been predicted to be the stable structure at these conditions, we show this BCC ice phase is stable to multi-Mbar pressures and temperatures near the melt boundary. This suggests variable and increased electrical conductivity to greater depths in ice giant planets that may promote the generation of multipolar magnetic fields.Recent discoveries of water-rich Neptune-like exoplanets require a more detailed understanding of the phase diagram of H During the formation of Uranus and Neptune in the outer region of our solar system, massive amounts of H2O were accreted and are now stored at hundreds of GPa pressures in their interiors1. During the visit of the Voyager II spacecraft, its magnetometer revealed surprising non-axisymmetric, non-dipolar magnetic fields for the ice giants that differed substantially from the strong dipolar fields of Jupiter and Saturn2. Stanley and Bloxham4 performed numerical dynamo simulations using model geometries to explain Uranus’ and Neptune’s anomalous fields—finding their non-dipolar, non-symmetric magnetic fields are generated by a combination of electromagnetic stresses perturbing the convecting ionic fluid which surrounds a layered, stratified interior. Knowing the phases and properties of H2O at the pressure–temperature (P–T) conditions of ice giant interiors on their isentropes is critical for validating dynamo simulations—but they are not well understood.Understanding the phase diagram of H2O ice, protonic conductivity properties are crucial to constrain planetary dynamo processes . The existence of a proton fluid and an oxygen sub-lattice in the superionic phase raises questions about the response of this phase to electromagnetic stress through protonic fluid motion. Theoretical work has suggested a body-centered cubic (BCC)8, face-centered cubic (FCC)11, or orthorhombic  structure of H2O is stable at hundreds of GPa pressures and several thousand Kelvin, with bonding and transport properties consistent with a superionic phase.Convection of electrically conducting fluids generates magnetic fields in planetary interiors. If dissociation of molecules occurs in water-rich planets, then total conductivity is comprised of an electronic and ionic contribution. Ionic conduction is caused by the movement of negatively or positively charged ions and in the case of high-pressure H14 use optical reflectivity, absorption measurements, and X-ray diffraction (XRD) to demonstrate the low electronic conductivity of ice and provide experimental evidence for superionic conduction of water ice in an FCC crystal structure, stable at pressure (P), temperature (T) conditions of ~ 160 GPa and calculated 3000 K. The insulating solid ice phase at comparable pressure and below 2000 K, ice X, is known to have a BCC lattice structure, but does not have superionic properties. In contrast, we find the first XRD evidence for a BCC structure between ~ 100–200 GPa using compression-based solidification of liquid water calculated temperatures up to ~ 5000 K. This BCC crystal structure phase at these P–T conditions represents a new phase of ice: ice XIX. Our constraints on the phase diagram of water ice near the conditions of the isentropes of ice giants, like Neptune and Uranus, have implications for their dynamos generating magnetic fields.More recent work2O. Here, atomic structure measurements of compressed liquid water  were made using transmission in situ XRD with 7.6 keV X-rays from the X-ray Free Electron Laser (XFEL) at the Matter in Extreme Conditions (MEC) end-station of the Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory  , are azimuthally integrated Fig.  as a fun(Å) see “”. A reprd-spacing of 2.063 Å, indicating that the majority of the window volume is uncompressed. The lowest pressure XRD pattern records the Au (111) and (220) peaks shift to smaller d-spacing and broadening – providing a thermally corrected pressure of 47 ± 3 GPa, 1150 K (Ref.18); Fig. 20. Two new XRD features are seen at 1.961(3) Å and 1.397(4) Å corresponding to the BCC ice VII structure, for (110) and (200), respectively, with a density of 2.78 g/cc, as expected in this regime21. Within uncertainty, the densities of Au, diamond, and ice are all consistent with a pressure of 53 ± 5 GPa, also in agreement with the VISAR measurement of 56 ± 4 GPa. Using previously published reverberation-compression-based equations of state (EoS) for ice VII25, we estimate a temperature of 1150 ± 250 K for this density.Diffraction from the (111) peak of the downstream diamond window shows little or no shift from the ambient Upon increasing compression, the Au peaks are no longer resolvable in the XRD due to possible drive light leakage generating thermal expansion peak broadening. Although we clearly see Au peaks in the ambient patterns, above ~ 50 GPa we can no longer resolve the Au. Loss of clarity in the XRD data is likely due to peak broadening to the point that the diffuse scatter intensity distribution across the CSPAD prevents it from being resolved as discrete peaks. Possible reasons for broadening include: (1) reaching temperatures above the melting point of Au at these pressures, and/or (2) thermal expansion due to drive light leakage through the diamond ablator reaching Au layer before the compression process can take place. It has been documented that the drive laser spatial profile can spill over the chamfered drive side of the target mount and damaging neighboring targets. Due to laser light from a preceding shot reaching an adjacent target, the Al flash coating (150 nm) on the drive side of the neighbor target can be damaged. The purpose of an Al flash coating is to prevent drive light from leaking through the ablator. However, if that Al coating was damaged, drive light can reach the Au layer, resulting in premature thermal expansion such that we cannot resolve the peaks.20. The reported pressures are determined from the XRD of the diamond-ablator and are corroborated, within the uncertainty, by the pressure determined from the velocimetry traces. Pressure uncertainty is taken from the goodness-of-fit value for a Gaussian peak profile of the ablator diamond diffraction peak d-spacing, converted to a density uncertainty and used to estimate the and pressure uncertainty with an equation of state. We see the water ice diffraction peak shift from 1.961(3) Å (at 53 GPa) to 1.725(3) Å at the highest pressure. If we continue to assign this feature as a BCC (110) peak, the ice pressure estimates (via EoS from Refs.21) track well with the compressed diamond ablator estimates up to ~ 160 GPa. Beyond this pressure there are discrepancies in the EoS results between quantum molecular dynamics simulations  and previous experiments  for water. At the highest pressure, 205 ± 10 GPa, indexing the new peak as a BCC (110) gives a lattice parameter of a = 2.440 Å, corresponding to a density of 4.12 g/cc. Unfortunately, the (200) peak for the BCC ice structure falls off the detector d-spacing range above ~ 75 GPa.Peak shifts in the compressed diamond ablator are resolvable and, using these diffraction peaks as a pressure calibrant in combination with the velocimetry record, we track pressure increasing to just over ~ 200 GPaP–T stability fields, we can test the viability of the FCC, hexagonally-close packed (HCP), and orthorhombic structures, assuming the geometric constraints of packing efficiency or close packing oxygen in three dimensions . Our procedure was to test assignment of the new peak visible in pressures above ~ 150 GPa to an FCC, HCP, or orthorhombic (Pbcm) structure and then inspect the 2-dimensional CSPAD images for any diffraction intensity located near a predicted (hkl) d-spacing position for that structure. If we assign the FCC (111) peak to the 1.828 Å feature, the corresponding FCC (200) should be at 1.583 Å which should then shift to 1.50 Å with compression. We do not see any XRD signal at these positions on the CSPADs. Similarly, if the HCP structure were assigned at comparable ice densities, the (101) is missing at its predicted 1.843 Å, or 1.678 Å, respectively for ρHCP = 3 and 4 g/cc. Regarding the orthorhombic Pbcm structure—we also check for peaks using a linear extrapolation of lattice parameters, e.g., Ref.12, to ~ 200 GPa to look for (110) and (101) peaks at 1.96 Å and 1.47 Å, respectively. Figure d-spacing vs pressure and corresponding densities at each pressure assuming a BCC or FCC structure, including previous diamond-anvil cell data. Due to the absence of any corresponding FCC, HCP, or Pbcm peaks, each expected to be within the detector d-spacing coverage with predicted relative intensities of these peaks well above the noise floor of the detectors, and a BCC ice density consistent with velocimetry-based pressure estimates and compressed diamond-ablator pressures estimates, we conclude that the ice structure seen in the XRD data is BCC. At similar shot conditions, the formation of this BCC structure is reproducible  have reported randomly-oriented nanocrystalline growth of the high pressure phase as seen in the uniformity of Debye–Scherrer ring intensity and the relative peak intensities matching a randomly orientated powder distribution. However, we see interesting trends in the change in powder XRD texture for both the ice phase and diamond above 100 GPa. The CVD ablator diamond signal remains spotty, showing a similarly sized grain structure as it compresses in the elastic regime up to the ~ 80 GPa HEL. Above this pressure, we see a gradual increase in ring smoothness and peak broadening up to the highest pressure of ~ 200 GPa where the intensity is more uniform over the azimuthal range available. This may indicate that the grain size is likely decreasing and orientations are becoming more random. The ice VII and ice X diffraction are large, broad and spotty up to over 100 GPa. Then as the ice X transitions to ice XIX above ~ 150 GPa we see these larger spots become more diffuse at the edges, perhaps indicating some increase mosaicity and/or crystallites with preferred orientation with respect to the compression direction. In the highest pressure shot there is an apparent concentration of diffraction intensity for the BCC ice (110) peak near the top of the detector. This is consistent with the horizontally polarized XFEL probe (considering the orientation of the CSPAD active areas with respect to the XFEL propagation direction). Additionally, we note heterogeneous growth of ice crystals on target component interfaces may have a needle-like geometry28, such that nanocrystallites develop preferential orientation with respect to the X-ray probe direction. This could also contribute to a concentration of diffraction intensity at this location on the detector.Previous dynamic compression induced disorder-order transitions , however our small multiplier value is indicative of extensive laser energy loss before reaching the target. This could be due to optics/coating damage in the beam path decreasing the delivered intensity reaching the target – perhaps up to 65%.The HYADES Radiation Hydrodynamics codeage Fig. . These s26). Because of the uncertainty in pressure and temperature conditions for this shot we list out two data points for BCC, one for each peak of the doublet giving: 205 GPa, 3300 K and 205 GPa, 5500 K.Diffraction from the highest pressure shot shows a doublet feature for the BCC (110). The hydrodynamic simulation shows a bimodal temperature distribution at the 7 ns probe time, present in the ice in two discrete ice layers: at ~ 3300 K and at 5500 K. This ~ 2000 K temperature difference between the layers would manifest as a density difference of 30–35% in the ice, which is consistent with the peak separation using the SESAME 7154 Gruneisen parameter  at these conditions causing it to absorb visible light , and we do see the loss of reflectance of the 532 nm probe light off the Au as the shock front transits the H2O layer. However, since our velocimetry records are inconclusive regarding a direct transport property measurement, we also consider alternate explanations. For instance, reflectance loss at ~ 2–8 ns ; λ = X-ray wavelength; β = line broadening at full width at half maximum (FWHM) minus instrumental broadening (0.03°); θ = Bragg angle, the BCC (110) ice peak width gives a grain size of 21 ± 2 nm, similar to the findings of Millot et al.14. This small grain size could lead to scattering of the VISAR probe light, which could cause the apparent loss of Au reflectivity. We do note that previous experiments35 and computations  have shown that the P–T conditions achieved here is within the superionic phase stability field. Applying our new XRD data to the phase diagram of high pressure ice confirms the BCC structure previously theorized is stable at these conditions   along the isentrope. However, this pressure assignment, based on diamond XRD could represent the lower bound for the ice pressure, and in fact, be at higher pressure as indicated by hydrocode assessment. Our results are consistent with all but one of the data points reported by Millot et al.14. The reason for this discrepancy is not yet understood, but could be related to issues in diffraction quality signal/noise and indicates the need for more experimental investigations working to resolve real-time diffraction for phase with higher Q-range, velocimetry for pressure, and pyrometry for temperature determination all collected with in situ diagnostics.Direct observation of the crystal structure of H9. This mobility is commonly assessed in molecular dynamics calculations as the hydrogen diffusion rate36. A linear extrapolation of the hydrogen diffusion constant with density to the conditions probed in this study suggests the BCC hydrogen diffusion constant is 40% higher than the FCC diffusion constant9. The values for the diffusion coefficient vary over many orders of magnitude, 1.8e−3 cm2s−1 (Refs.36) to ~ 0.5 cm2s−1 (Ref.9) for pressures and temperatures measured in this study for a BCC structure, depending on the molecular dynamics simulation parameters. The relationship between hydrogen diffusion coefficient (DH) and protonic conductivity (σ) is governed by the Nernst-Einstein equation38: f is non-dimensional geometrical constant taken to be 1, n is the molar concentration per unit cell volume, q is the charge, R is the gas constant, and T is the temperature, and shows a proportional relationship between hydrogen diffusion coefficient and protonic conductivity. For our BCC lattice parameter (2.440 Å), and a DH of 1.8e−3 cm2s−1 (Ref.36) we find a protonic conductivity of 102 (Ω cm)−1, whereas an FCC structure at the same pressure would be ~ 20% lower conductivity. This is in contrast to the liquid outer region of Neptune where ionic conductivity at 25 to 100 GPa, ranges from < 1 to 30–90 (Ω cm)−1, respectively . Confirmation of the extension of the BCC high-pressure stability field in the superionic regime results in higher than predicted protonic conductivity to greater depths in ice giant interiors , as seen here. Our data change the sub-liquid layer from FCC superionic solid ice to BCC superionic solid ice, resulting in a 40% increase in protonic conductivity. The magnetic fields generated in the ionic liquid layer will interact differently with a BCC ice layer than they would an FCC ice layer due to this difference in conductivity. For example, the magnetic fields generated in the ionic fluid layer are time-varying and the skin depth of penetration of magnetic fields into any solid sub-layer depends on the conductivity of that solid material. A higher conductivity solid sub-layer would preferentially repel magnetic fields, limiting their length scales. Although dynamo simulations have been able to produce multipolar magnetic fields without an enhanced conductivity of a solid interior , the smaller length-scales resulting from the enhanced repulsion of a higher conducting solid interior would promote the generation of multipolar magnetic fields—consistent with measurements made by Voyager II for Uranus and Neptune.Ice giant dynamos are generated in a convecting, fluid layer of electrically conductive water, ammonia, and methane. Solid ice layers cannot participate in the dynamo action through fluid motions. However, dynamo complexities arising from layered superionic ices with high but differing proton mobilities will influence the magnetic field properties. Here we have shown that solid ice is stable to over 200 GPa and 4400 K in a BCC structure. Combined with previous measurements that indicate that water ice is superionic in this 12 photons per pulse, were incident over a 50 μm diameter spot on the target package. An X-ray only shot was collected before the drive shot as a reference. The 50 μm XFEL beam spot did not produce any observable x-ray damage to the target. Metal coatings on the diamond ablator served to absorb the drive laser (150 nm Al on upstream side) and act as the reflective layer for velocimetry measurements (75 nm Au on downstream side).Quasi-monochromatic (dE/E = 0.2–0.5%), fully transverse coherent, 7.603(30) keV x-ray pulses of 40 fs duration with an average of ~ 1013 W cm−2  combined with this temporal drive profile were designed to generate the following sequence of events. First, the AB arm is incident on the diamond ablator and begins the ablation process setting up a shock wave in the ablator diamond. The impedance mismatch sets up a reverberating shock in the diamond ablator. Then, a weak shock traverses the water to ~ 25 GPa on the principal Hugoniot as transmitted by the elastic wave in diamond. The diamond plastic wave overtakes the weak elastic wave in the water and is the main compressive wave in the water reaching the diamond VISAR window at ~ 5 ns. This plastic wave sends a reverberation wave back into the water/ice again due to impedance mismatch. At 5 ns, the second laser fires, EF arm, attempting to support continued reverberations.


Allergens are antigens that generate an IgE response (sensitization) in susceptible individuals. The allergenicity of an allergen can be thought of in terms of its ability to sensitize as well as its ability to cross-link IgE/IgE receptor complexes on mast cells and basophils leading to release of preformed and newly formed mediators (effector activity). The identity of the allergens responsible for sensitization may be different from those that elicit an allergic response. Effector activity is determined by (1) the amount of specific IgE (sIgE) and in some circumstances the ratio of sIgE to total IgE, (2) the number of high affinity receptors for IgE (FcεR1) on the cell surface, (3) the affinity of binding of sIgE for its epitope and, in a polyclonal response, the collective avidity, (4) the number and spatial relationships of IgE binding epitopes on the allergen and (5) the presence of IgG that can bind to allergen and either block binding of sIgE and/or activate low affinity IgG receptors that activate intracellular inhibitory pathways. This review will discuss these important immunologic and physical properties that contribute to the effector activity of allergens. The word allergen refers to a specific molecule, usually a protein that generates an IgE response as opposed to a more general term, antigen that is a molecule that induces any immune response . AllergeBefore discussing the effector function of allergens, it is important to note that the allergens within an allergenic source  that are the most potent for sensitization may be different from the allergens that are responsible for elicitation of the allergic response. As an example, sensitization to peanuts may be mediated primarily by Ara h 1, which is a vicilin, also known as a 7S globulin , 18. In Allergenic proteins are limited to a small number of protein families . In someThus, the ability of an allergen to cross-link IgE/FcεR1complexes on mast cells and basophils is determined by (1) the amount of specific IgE (sIgE) and the ratio of sIgE to total IgE (tIgE), (2) the number of FcεR1 molecules on the cell surface, (3) the affinity of binding of sIgE for its epitope and, in a polyclonal response, the collective avidity, (4) the number and spatial relationships of IgE binding epitopes on the allergen and (5) the presence of IgG that can bind to allergen and either block binding of sIgE and/or activate low affinity IgG receptors (FcγR2 in humans and FcγR2b in mice) that activate intracellular inhibitory pathways –36.e.g. ISAC® by ThermoFisher) has allowed simultaneous measurement of IgE binding to a variety of purified or recombinant allergens  or other similar format. Microarray technology . Blanc et al. showed that sera exhibiting a lower ratio sIgE to tIgE required higher concentrations of allergens to trigger RBL SX-38 degranulation, which was not efficiently induced when the ratio was <2% . HoweverD of 10−9 M), remains on the cell surface for several weeks  that activate intracellular inhibitory pathways , 104. SoThe determinants of the effector function of an allergen are complex and are affected by multiple components . These iAll authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.This work was supported AlimH department of INRAE (SH) and by RO1AI165866 (SCD) and R21AI135397 (SCD).Contents are the authors' sole responsibility and do not necessarily represent official NIH views.SCD has received grant support from the National Institutes of Health. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


E = 57.8 kV/m and f = 1 MHz. The dielectric spectrum results show that this inhibition effect of an alternating electric field on ice crystal growth is closely related to the dielectric relaxation process of hydrated ions.In this paper, the inhibition effect of an alternative current (AC) electric field on ice crystallization in 0.9 wt % NaCl aqueous solution was confirmed thermodynamically with characterization. An innovative experimental and analytical method, combining differential scanning calorimeter (DSC) measurement with an externally applied electric field was created by implanting microelectrodes in a sample crucible. It was found that the ice crystallization, including pure ice and salty ice, was obviously inhibited after field cooling with an external AC electric field in a frequency range of 100 k–10 MHz, and the crystallization ratio was related to frequency. Compared with non-field cooling, the crystallization ratio of ice crystals was reduced to less than 20% when  Water–ice phase transition is a common phenomenon in nature, which has never failed to remain an important topic in many research fields. For example, a significant issue is how to avoid the formation of ice crystals in the cryopreservation of biological tissues ,3,4,5,6.In this paper, an innovative experimental and analytical method, combining differential scanning calorimeter (DSC) measurement with an applied electric field was realized by implanting microelectrodes in a sample crucible. The effect of alternative current (AC) electric field on ice crystallization in NaCl aqueous solution was studied with quantitative calculation. The results show that the crystallization ratio of ice crystals is significantly reduced under external AC electric field, varies with frequency, and is related to the relaxation polarization of hydrated ions. This may provide a potential physical approach and theoretical basis for cryopreservation technology.−4 mol/g, and the osmotic pressure was basically equal to that of mammalian blood plasma. Deionized water was also used as a reference.A 0.9 wt % NaCl aqueous solution, which is also called normal saline, was used as the sample. The corresponding molality was 1.552 × 10Vpp = 20 V, E = 57 kV/m, f = 0~10 MHz). The physical and chemical properties of this crucible with microelectrodes remained stable in the whole experimental temperature range and did not affect the performance of other related instruments and the subsequent test results.In order to ensure that the electric field was applied simultaneously during the DSC  cooling processes, a bespoke crucible was specially designed, as shown in E, constant temperature at −60 °C for 5 min, turning off the voltage source, and rising to 10 °C at 3 °C/min. The procedure of the control group, i.e., the non-field cooling group, was the same, except that E = 0 kV/m during cooling. Deionized water was also tested as a reference in this group. All measurements were repeated three times. In order to compare the results more intuitively and avoid the impact of different sample sizes, 4 μL solution was taken for each test using a micropipette. Rewarming DSC curves were selected for quantitative analysis, since supercooling while freezing brings great interference to the quantitative calculation of cooling DSC curves, and differences in crystallization among samples can be directly reflected through rewarming DSC curves.The DSC procedure is illustrated in ε* was measured, and the dielectric loss tangent tanδ was calculated [The dielectric spectra of 0.9 wt % NaCl solution were also investigated. The complex dielectric constant lculated ,26 in thε′ is the relative dielectric constant, ε″ is the dielectric loss factor, and δ is the dielectric loss angle. tanδ refers to the energy consumed by the dielectric in converting electric energy into heat energy per unit volume per unit time, and is an attribute of the dielectric itself. In many cases, tanδ is much more sensitive to the change of medium characteristics.δ was better than 3 × 10−5, and the measurement resolution was less than 10−5. For each measured frequency, the output result of the spectrometer was an average value obtained by multiple automatic measurements.A broadband dielectric spectrometer  was used for dielectric measurement. The measurement accuracy of tanVrms = 1 V.A liquid parallel plate sample cell BDS1308 with two parallel electrodes inside was used, which was sealed to prevent liquid evaporation. The diameter of electrodes was 20 mm. The gap between electrodes was 0.1 mm, adjusted by two silicon dioxide spacers placed in parallel. The stray capacitance of the sample cell was taken into account by open-circuit calibration before measurements. The root-mean-square value of the AC voltage applied to the electrodes was The sample and the sample cell were pre-cooled to 4 °C for 10 min before the start of measurements, to exclude the influence of thermal history on the measurement results. From 4 °C to −6 °C, the dielectric spectrum was measured every 2 °C. The temperature remained constant during the measurement. Each measurement took about 5 min. The temperature control accuracy was ±0.5 °C.2O [2O ,28. DuriCompared with non-field cooling, the positions of the salty ice peaks and pure ice peaks remain steady after field cooling, which indicates that the physical properties of pure water ice and salty ice do not change under an electric field, since the melting point is one of the symbolic physical properties of a material.The area of each peak equals the melting heat egration ,30:(3)ΔQThe integration results are given in tructure ,32.In order to analyze quantitatively, with normalization, the crystallization ratio of pure ice f = 1 MHz. In addition, the changing trend of The calculation results are shown in To further explore the mechanism of the strong dependence of The relative dielectric constant te model  from theectrodes . Howeverδ has an obvious peak near fmax = 1 MHz, consistent with the optimum frequency of the DSC study above. This reveals that the effect of an external AC electric field on the freezing process is related to some kind of relaxation polarization. Since the orientation polarization of water molecules and the induced polarization of hydrated ions appear in the range of 0.1–20 GHz [The dielectric loss tangent tan1–20 GHz ,37 and 11–20 GHz , respectThus, it is inferred that the relaxation polarization of hydrated ions is the main reason for the inhibition of ice crystallization in a solution when freezing under an AC electric field.To complete the transition from liquid phase to solid phase and integrate into the lattice structure of ice, water molecules need to overcome the constraints of kinetic energy and potential energy generated by thermal motion and overcome the solid–liquid interface energy.+ and Cl− ions are hydrated to form bilayer hydrated ions with opposite charge signs, as shown in In NaCl solution, NaUnder the application of an AC electric field, the polarization relaxation of hydrated ions destroys the original charge balance, interferes with the original molecular arrangement structure in the solution, and increases the kinetic energy of water molecules because of the friction between the weakly bound water and the water molecules surrounding the hydrated ions; thus, inhibiting the crystal growth of ice.When the electric field frequency is low enough, the polarization of hydrated ions can keep up with the change of frequency, and the change in direction of the induced dipole is slow. The relatively small disturbance of the solution has a slight influence on the formation and growth process of ice crystals. With an increase of frequency, the polarization gradually lags behind the electric field, resulting in relaxation polarization, the disturbance of the molecular distribution in solution increases, and this has a significant impact on the growth process of ice crystals. With a further increase of frequency, the polarization of hydrated ions cannot keep up with the change of the electric field and can only make small vibrations at the original position; the disturbance is weakened, and the influence on crystallization is also weakened.Therefore, the influence of hydrated ions on the solution phase transition process under an AC electric field is related to frequency and is the most significant near the range of dielectric relaxation. In addition, this corresponding dielectric relaxation is consistent with the theoretical calculations of the frequency range of induced polarization for hydrated ions in previous works ,38, whicf = 1 MHz, where An innovative crucible with microelectrodes was designed, which enabled a synchronous application of an external electric field during DSC measurements. According to quantitative calculations based on DSC rewarming curves, it was found that ice crystallization in 0.9 wt %NaCl aqueous solution can be effectively inhibited by AC electric field cooling in the range of 100 kHz to 10 MHz. Compared with non-field cooling, the crystallization ratio of pure ice DSC Thermal Analysis Method for Action of Applied Electric Field, Publication No.US-2021-0041381-A1;DSC Electrode System Capable of Applying Electric Field, Publication No.US-2021-0037812-A1.Two patents resulting from the work are in the application process and are listed as follows:


Infectious bronchitis virus (IBV) is a major economic problem in commercial chicken farms with acute multiple-system infection, especially in respiratory and urogenital systems. A live-attenuated and killed vaccine is currently immunized to control IBV infection; however, repeated outbreaks occur in both unvaccinated and vaccinated birds due to the choice of inadequate vaccine candidates and continuous emergence of novel infectious bronchitis (IB) variants and failure of vaccination. However, similar clinical signs were shown in different respiratory diseases that are essential to improving the diagnostic assay to detect IBV infections. Various risk factors involved in the failure of IB vaccination, such as various routes of application of vaccination, the interval between vaccinations, and challenge with various possible immunosuppression of birds are reviewed. The review article also highlights and updates factors affecting the diagnosis of IBV disease in the poultry industry with differential diagnosis to find the nature of infections compared with non-IBV diseases. Therefore, it is essential to monitor the common reasons for failed IBV vaccinations with preventive action, and proper diagnostic facilities for identifying the infective stage, leading to earlier control and reduced economic losses from IBV disease. Infectious bronchitis virus (IBV) is an acute and highly contagious respiratory pathogen in chickens. In general, coronaviruses are classified into four groups  according to antigenic cross-reactivity and nucleotide sequence analysis . IBV belIBV infection has major economic impact on the poultry industry due to mutable tissue tropism and the continuous emergence of various IBV serotypes or genotypes in different geographical regions. Because of its degree of severity and high contagiousness, mortality can reach up to 10%–60% over four to six weeks of broiler age during acute infection with a secondary infection ,20. TherThe failure of the vaccine is a consequence of the incapability of birds to produce a satisfactory immune response after vaccination . VariousThe aim of this review article is mainly to discuss advanced current challenges related to proper diagnostic assay with evidence of vaccination failure. We describe various factors influenced by IBV vaccination, various routes of vaccination, postvaccine challenge, possible immunosuppression, and differential diagnosis to explore future research to elucidate the immune-pathogenesis response of multiple types of IBV.Few assays are established for infectious bronchitis (IB) diagnosis for the identification of viruses from current infections or specific antibody responses from post-viral infection. The selection of the best assay for the successful detection of IBV is difficult and confusing due to various factors, as discussed below.The clinical signs of IBV are similar to those in other respiratory infections except for a few unique lesions that are part of strain variation and difficult to differentiate from other respiratory viral infections. Although clinical signs are matched according to the manual, a serology study is not ever matched according to autopsy lesions. The serology titer is occasionally given higher trends, but it cannot match with clinical signs; therefore, IBV confirmation is problematic. Thus, conformation diagnosis is based on the accumulation of data from farm records, including egg quality with production graph, mortality, seroconversion or antibody levels, vaccination report, and blood serology, which are clinical signs that concurrently match symptoms of IBV chickens through PM lesions ,32,33. SVaccination is the main influence for IBV identification, as it requires the infected IBV-vaccinated strain and other virulent field strains. For unvaccinated flocks, the field challenge can simply be detected by RT-PCR, RT-qPCR, or a positive IBV titer after a few weeks. Therefore, there is great difficulty in identifying a field challenge or active infection in vaccinated flocks due to antibody circulation in the bloodstream. Nowadays, vaccine strains can be distinguished from field strains through the differentiating infected from vaccinated animals (DIVA) technique using inactivated oil emulsion vaccine that can be detected by simple neutralization test, enzyme linked immunosorbent assays (ELISA) and indirect immunofluorescence test ,37,38.The selection of diagnostic techniques is important for proper diagnosis related to sample collection and sampling numbers. Advanced ELISA tests can distinguish antibodies owing to vaccination from those from infection. The concentration of antibody titer tends to be abnormally increased after 2–4 weeks post infection; the titer gradually decreased slowly in subsequent weeks . SubsequThe mean titer provides information on the antibody response of tested birds within a flock after vaccination. Mean titers should reach a predictable range with uniform distribution for normal flocks and unintentionally higher titers for diseased flocks. A study suggested that antibody patterns should not be breached over vaccinal immunity because of field infections . Post inOn the basis of general parameters, a percentage coefficient of variation (CV) that is maintained below 40% after inactivated vaccinations and less than 60% in live vaccines suggests good vaccination uniformity. When CV is less than 20%, the level of titer shows good uniformity, but previous exposure of disease or good vaccination is specified . TherefoThe upper respiratory tract (UTI) is the principal site of IBV replication; subsequently, viremia causes viral multiplication to disseminate to other tissue through blood circulation ,45,46. ISerological monitoring can be directly helpful for the assessment of the immune status of birds, viral challenge, and the duration of viral infection . DiffereGenetic traits have great influence on resistance and susceptibility to IBV. Some experimental studies showed the different genetic traits, mortality, viral growth, and variable histopathology found in different breeding lines after IBV immunization . HoweverThe level and duration of vaccination responses rely on many factors that need to be fully considered during vaccination. It is not sufficient to simply describe a specific IBV vaccine that can protect against infection in a particular situation, since regulatory requirements are commonly challenged.According to Dantzer , stress A protection study is not a simple method for application, but several factors were considered during designing such studies . DifferePrior to vaccination, a program needs to be revised according to epidemiological study, the prevalence of a virulent virus, data of sporadic outbreaks, accurate diagnosis, single or combined IBV vaccine, vaccine potency, and monitoring poultry health systems. Immunization is commonly dependent on the challenge of establishing their effectiveness against multiple variants of viruses; a specific time is favorable for the multiplication of viruses, live-attenuated or killed vaccines, and monitoring the titer level or baseline of determination. Essential information details whether bird immunization is protective or a challenge. Therefore, evaluation of the current immunization schedule is not recommended if certain titer limits exist or a virus challenge is circulating in the field . BecauseSample collection is important for the confirmation of diagnosis and identification during active and post-viral infection. In the acute infection of IBV, the majority of birds have a similar typical clinical sign; then, the number of samples can be reduced. At least five birds are enough to achieve higher confidence of the successful detection of infection by molecular assay . In seroThe level of acquired immunity depends on many factors, especially at the time of vaccination or exposure of infection. Vaccines collaborate with the immune system to generate the same immune response as that in natural infection in birds. The humoral immune response has a significant role in the stimulation of certain levels of immune response compared with natural infection. Some researchers reported that vaccination is the process of immune development of the body originating from previous vaccination or earlier infection ,79. The Maternal antibodies can provide passive immunity to chicks from hatching and temporarily protect against pathogens to the young immune system ,87. Age E. coli. Cook et al. [Chicken breeding line is an important factor to genetic resistance to vaccination since variations can be genetically exposed and significantly influenced between inbred chicken lines that are infected with IBV and k et al.  reportedk et al. . Joiner k et al.  applied Vaccine doses are commonly monitored with regard to recommended manufacturer labels and instructions to deliver the correct volume to each chicken . In a goThe timing of the IBV vaccination is given much attention due to the incidence of viruses circulating in certain farms being vaccinated. Broiler chickens are immunized 1 or 2 times against IBV due to their short lifespan . CurrentDiverse types of serotypes or IBV genotypes are globally documented, but different serotypes of the virus are not fully cross-protected. Despite this, novel, more infectious variants have emerged, but the amino acid sequence of the spike protein (S1) and genome are unchanged. Cross-reaction is particularly protected in chickens because of the interaction of multiple vaccinations covered by more than one included serotype ,81. Birdhttps://apps.who.int/iris/handle/10665/69387). A diluent is mixed before application, and temperature should be maintained by a cooler dropper during ocular and drinking live vaccinations. For IBV vaccinations, the process of live vaccination requires no more than 1–2 h of inoculation or drinking time after exposure to the vaccine [The supervision of vaccine handling, transportation, and storage greatly influences the efficacy and quality of vaccines. A live vaccine is sensitive to over 2–8 °C in storage temperature, so it should be continuously monitored to avoid over freezing until hardening ,110,111. vaccine . In drin vaccine . To ensuSigns of postvaccine reactions or challenges are characterized on the basis of local reaction, fever, and delayed reaction, and anaphylaxis is a common lesion or systemic reaction indicating that the chicken is reacting to a satisfactorily vaccination or good immunized response against IBV. These types of adverse postvaccine reactions are commonly found in infectious laryngotracheitis (ILT) rather than in IBV. A reaction is more severe in a few cases, with secondary infections such as conjunctivitis and swelling in the facial region, as shown in The purpose of vaccines is for disease control and prevention against respective pathogens that are correlated with the limits of good biosecurity measures. However, if there is a sudden breakdown of the biosecurity program with a higher load of field challenge, a vaccination program does not help at a satisfactory level. Many management issues, such as congested spaces, uncontrolled temperature and humidity in the shed, poor nutrition, worm infestations, poor uniformity, and other secondary infections that reduce the bird’s immune system, can directly affect vaccination efficacy. Several studies suggested that continuous exposure to 30 ppm , 52 ppm 2, and hydrogen sulfide (H2S), which are continuously produced by chickens through defecation and urination [The operation of ventilation systems is a vital part to regulate sufficient air exchange to meet the air quality in poultry houses to reduce disease susceptibility and stress. Poultry houses’ ventilation systems impact its ammonia levels, harmful concentrations of COrination ,122,123.rination . A good rination ,125.Some immunosuppressive diseases and cyclosporine are the most common agents that directly influence IBV vaccination. Immunosuppressive agents are stimulated in early bird age to reduce the response of IBV vaccination. Thompson et al.  comparedSpray vaccination is currently practiced in modern hatcheries at an early age of birds in farms, where a huge number of chicks require to be simultaneously vaccinated. The live vaccine of IBV is usually used for mass vaccinations in hatcheries or farms by fine/coarse spray via forcing through small spray nozzles, forming aerosolized droplets that chicks can easily inhale to uptake the vaccine ,128,129,The perfect route of IBV vaccination is normally evaluated through the oculonasal route against respiratory pathogens causing respiratory diseases , as showMost live IBV vaccines are applied in drinking water in broiler chickens 1 week following the booster dose if needed, depending on the farm situation. Broiler breeder and layer birds are commonly chosen for live vaccines in drinking at the pullet stage, and subsequently prime-boosted every 6 weeks in the laying stage, while the birds have lower titers or lower protection. In an attempt to mimic field conditions, drinking-water application is a good technique to avoid stress ,141; nevInactivated killed IBV vaccines are applied in the breast or leg muscle using intramuscular (IM) injections, as shown in A gel diluent is another of the latest practices for the application of the IBV vaccine. This technique was first used in the cocci vaccine in poultry as a delivery route of the vaccination process ,145,146.The success of vaccination performance is measured by mean titer response (mean antibody level) and percentages of coefficient of variation (CV)  in vaccinated flocks. The overall standard for CV% uniformity after vaccination is measured as follows: less than 40%, excellent; 40%–60%, good vaccination; and more than 60%, vaccine methods need to be improved . The ratThe VI score is estimated to provide a higher score  in a good vaccination, and a low score with poor vaccination . Depending on the standard parameters of different commercial kits.E. coli, leading to the increased severity of infection [The actual diagnosis of IBV infection is confusing with regard to clinical signs and postmortem lesions due to similar clinical signs produced by several respiratory pathogens, such as Newcastle disease (ND), avian influenza (AI), infectious laryngotracheitis (ILT) and infectious coryza (IC), and mycoplasma (Mg) . Earliernfection ,158. Thenfection . The synnfection ,161. Mornfection .Currently, a live-attenuated and inactivated vaccine is commonly used in IBV vaccination. However, IBV outbreaks in poultry farms usually persist due to a lack of actual serotypes or variants in vaccination programs, and vaccine failure, which is a significant problem in commercial farming. Laboratory diagnosis is a useful tool for the identification of actual serotypes or variants for vaccination programs to select accurate vaccines for the prevention and control of IBV. Moreover, we reviewed the best ways to protect from IBV through the successful vaccinations in every bird to maintain all necessary factors affecting the failure of vaccines. This review outlines this present situation of various vaccine applications, with correct approaches and factors that influence IBV diagnosis with differential diagnosis to assist in the accurate identification of IBV for necessary prophylactic measures to reduce economic losses from IBV outbreaks.


Intrinsically disordered proteins are involved in many biological processes such as signaling, regulation, and recognition. A common strategy to regulate their function is through phosphorylation, as it can induce changes in conformation, dynamics, and interactions with binding partners. Although phosphorylated intrinsically disordered proteins have received increased attention in recent years, a full understanding of the conformational and structural implications of phosphorylation has not yet been achieved. Here, we present all-atom molecular dynamics simulations of five disordered peptides originated from tau, statherin, and β-casein, in both phosphorylated and non-phosphorylated state, to compare changes in global dimensions and structural elements, in an attempt to gain more insight into the controlling factors. The changes are in qualitative agreement with experimental data, and we observe that the net charge is not enough to predict the impact of phosphorylation on the global dimensions. Instead, the distribution of phosphorylated and positively charged residues throughout the sequence has great impact due to the formation of salt bridges. In statherin, a preference for arginine–phosphoserine interaction over arginine–tyrosine accounts for a global expansion, despite a local contraction of the phosphorylated region, which implies that also non-charged residues can influence the effect of phosphorylation. Intrinsically disordered proteins (IDPs) lack tertiary structure under physiological conditions ,2, such Phosphorylation is a reversible type of PTM, especially prevalent among intrinsically disordered regions and proteins ,7,8. TheIn recent years, phosphorylated IDPs have received increased attention ,21,22,23In our most recent work involving all-atom molecular dynamics simulations of phosphorylated disordered peptides, Amber ff99SB-ILDN in combination with the TIP4P-D water model showed promising results in describing the conformational ensemble of short disordered peptides ,31. HereWe observe that, for these peptides, ranging in length from 11 to 43 residues that net charge is not enough to predict the change in global dimensions upon phosphorylation at two to four sites. Instead, salt bridge formation has great impact, depending on the distribution of phosphorylated and positively charged residues throughout the sequence. Furthermore, in statherin, a preference for arginine–phosphoserine interactions over arginine–tyrosine interactions explains the phosphorylation induced changes.Atomistic simulations of five different disordered peptides in both non-phosphorylated and phosphorylated state, shown in g) and end-to-end distance (Ree) in g and Ree presented in g distribution than the Ree distribution, which only changes shape by the disappearance of a shoulder at lower values. This, however, causes the average Ree, presented in ee upon phosphorylation of Tau1 has been detected by fluorescence resonance energy transfer measurements, as reported by Chin et al. [SN15, Tau2, and bCPP all contract upon phosphorylation, as shown from the peak shift towards lower values of the distributions of radius of gyration , as shown in Tau2 and SN15 are both mainly irregular and report an increase of helicity upon phosphorylation, see e groups .10-helix, since the increase of α-helix is only from 1 to 5%. Hence, the simulations are in qualitative agreement with the experiments, but the quantitative results should be treated with caution. In addition, in SN15, the larger part of the helical increase is due to 310-helix, and an increase of α-helix is supported by CD spectroscopy [For Tau2, a phosphorylation-induced increase of α-helical structure from 5 to 40% in region A239–R242 has been reported . In thestroscopy , once agtroscopy , which ig and Ree, salt bridge formation can also contribute to the compaction observed upon phosphorylation. In Tau2, several salt bridges have been established from NMR measurements, specifically pT231–R230, pS237–K240, and pS238–R242 [While helix formation decreases the R238–R242 . pT231–Rg and Ree. The conformation corresponding to the minimum in the most populated basin for the phosphorylated peptide additionally shows a helix in the C-terminal end, which also contributes to a decreased Rg and Ree.Comparing the energy landscapes of non-phosphorylated and phosphorylated Tau2 in In SN15, the salt bridges pS2–K6, pS3–K6, pS3–R9, and pS3–R10 are the most probable and all form with an approximately 25% occurrence. From the change in contact probability displayed in g and Ree substantially. From the energy landscapes in For bCPP, the secondary structure content is dominated by an irregular structure and is highly similar in phosphorylated and non-phosphorylated states, as shown by Based only on the net charge of non-phosphorylated bCPP, it was expected that it would expand upon phosphorylation. Considering only region E13–E21, which contains the four phosphorylation sites, this effect was noticed. The average distance between the C atoms of residue 13 and 21 increases from g at 0 and 150 mM are within error, there is a slight increase in the phosphorylated variant and decrease in the non-phosphorylated variant, see g between the two variants vanishes, considering the associated error. Note, however, that the distributions still have distinctly different shapes, hence we argue that the conformational ensembles are still different. The same trend is observed in the average Ree values, although a difference with respect to phosphorylation state still remains at 150 mM NaCl, see We previously showed that the addition of 150 mM NaCl had negligible effects on the salt bridges and global conformational properties of phosphorylated bCPP . The samUpon phosphorylation of Stath, the three largest changes in secondary structure are a decrease of β-strand structure, an increase of helical structure, and an increase of turns, according to 1–15 agree with those of the fragment (SN15). In the non-phosphorylated variant, the distributions are also rather similar, except for a sharp peak in both the Rg and Ree distributions, which corresponds to a basin in the energy landscape with the conformation shown in 1–15 is caused by β-strand not forming in SN15, due to lacking its partner further on in the sequence. There are also some differences in bends and turns, but the increase of helical propensity is similar. Hence, overall, the first fifteen residues of Stath behave rather similarly in the full peptide and as a standalone fragment, although especially the presence of the rest of the sequence induces β-strand formation. Despite this discrepancy, we can conclude that phosphorylation of Stath causes a contraction of the first fifteen residues, but an expansion of the full peptide, due to disruption of β-sheets.As presented above, SN15, which is the first fifteen residues of Stath, contracts upon phosphorylation, which was explained by the increased helicity and formation of salt bridges. Some of the peptides in this study contracted upon phosphorylation, while others became more expanded. However, the net charge was not enough to predict the effect in these short peptides. Instead, we have identified factors that appeared to be of greater importance, of which the first is the distribution of charged residues, in line with the influence of linear charge distribution on the conformational ensemble of IDPs . Especia10- over α-helix was also observed, while the experimental data only considered α-helix. Hence, the simulations were better at indicating trends than producing exact measurements of secondary structure. Overall, the simulation results were often in qualitative agreement with available experimental data, suggesting that, despite the deficiency related to secondary structure and the reported tendency of the force field to overestimate charge–charge interactions, simulations with this force field can still contribute to an increased understanding of the implications of phosphorylation.Regarding secondary structure, the separation between phosphorylated and positively charged residues was shown to control the helix propensity, and salt bridges additionally induced changes in the amount of bends and turns. Comparison with experimental data on secondary structure for SN15 and Tau2 indicates that the simulations underestimate the structural content. For these peptides, a preference of 3As a final note, this study shows that there are several factors contributing to the outcome of phosphorylation, and that they are of varying importance in different peptides. This shows that phosphorylation indeed is complex; however, it is still possible to obtain a better understanding of these factors individually. Therefore, we have an ongoing project in which the number of phosphorylated residues and their positions are varied in a controlled manner, to investigate the effects of those factors systematically.All-atom molecular dynamics simulations of the systems shown in -1 isothermal compressibility.Initial configurations of the peptides were constructed from the sequence as linear chains using Avogadro 1.2.0 , optimizEnergy minimization was performed by the steepest descent algorithm until the system was converged within the available machine precision. Initiation of five replicates per system with different starting seeds were performed separately in two steps using position restraints on the peptide. The first step was 500 ps of NVT simulation  performed to stabilize the temperature, followed by the second step of 1000 ps of NPT simulation  to stabilize the pressure. Production runs of the five replicates per system were performed in the NPT ensemble, for at least 1 µs per replicate. bCPPp with 150 mM salt was simulated in 10 replicates for 0.7 µs each. The total simulation time per system is stated in g and Ree were calculated using GROMACS 2018.4 and the gmx analyze routine was used to obtain averages and error estimates from block averaging analysis. Distributions were obtained by Gaussian kernel estimation using the SciPy package version 1.5.4 [g and Ree according to:Ron 1.5.4 . The shaSecondary structure was determined using the DSSP program version 2.2.1  with an g and Ree distributions, and energy landscapes, between the replicates, as well as by observing the auto-correlation function and convergence of the block average error estimate of Rg and Ree in the concatenated simulation. These data are available in Convergence and sampling quality were assessed by comparing the R


Convolvulus pluricaulis (CP) in a zebrafish model of cognitive impairment induced by scopolamine (SCOP). CP is a perennial herb with anti-amnesiac and anxiolytic properties. It contains alkaloid, anthocyanin, coumarin, flavonoid, phytosterol and triterpenoid components. Isoxazole (ISOX) was used as a positive control for AChE inhibition. CP-treated 168 hpf larvae showed a similar pattern of AChE inhibition  as that of ISOX-treated larvae. CP was superior to ISOX as evidenced by the retention of avoidance response behavior in adult zebrafish. Molecular docking studies indicated that ISOX binds Ser203 of the catalytic triad on the human AChE. The active components of CP—scopoletin and kaempferol—were bound by His447 of the catalytic triad, the anionic subsite of the catalytic center, and the peripheral anionic site. This suggested the ability of CP to mediate both competitive and non-competitive modes of inhibition. Surprisingly, SCOP showed AChE inhibition in larvae, possibly mediated via the choline-binding sites. CP + SCOP induced a concentration-dependent increase in AChE inhibition and ACh depletion. Abnormal motor responses were observed with ISOX, CP, ISOX + SCOP, and CP + SCOP, indicative of undesirable effects on the peripheral cholinergic system. Our study proposes the examination of CP, SCOP, and CP + SCOP as potential AChE inhibitors for their ability to modulate cognitive deficits.Acetylcholinesterase (AChE) inhibitors increase the retention of acetylcholine (ACh) in synapses. Although they alleviate cognitive deficits in Alzheimer’s disease, their limited benefits warrant investigations of plant extracts with similar properties. We studied the anti-AChE activity of The online version contains supplementary material available at 10.1007/s13659-022-00332-5. Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by memory loss, behavioral changes, and impaired cognition and language . Around Depleted ACh levels could result from the dramatic loss of cholinergic neurons in the basal forebrain, reduced cholinergic innervation to the hippocampus and neocortex, reduced levels of the enzyme choline acetyltransferase (ChAT), and increased activity of the enzyme acetylcholinesterase (AChE) , 5–8. ACCholinesterase inhibitors increase the synaptic residence time of ACh and enhance postsynaptic cholinergic signaling , 18. TheThe structure of human AChE has been characterized using chemical and kinetic studies. The active site of the enzyme contains two subsites. The breakdown of ACh into acetate and choline is catalyzed within the esteratic subsite . This suDanio rerio) is primarily used as a genetic model system for studying developmental and disease processes. They have biochemical and behavioral responses comparable to mammalian systems, making them suitable for drug testing. They exhibit comparable brain macro-organization, cellular morphology, neuromediator systems, and sensitivity to several classes of neurotropic drugs , 4443, 4A passive avoidance response test was used to test the acquisition of avoidance response in adult zebrafish. As expected, SCOP-treated adults failed to acquire the response  Fig. A. ISOX-tWe studied the binding of the two active components of CP—scopoletin, and kaempferol—on human AChE using molecular docking —suggests that it may act as a competitive inhibitor of ACh. This observation is supported by previous studies that demonstrated its AChE inhibitory activity in vitro , 48 and It must be noted that our docking analysis was limited to only two phytoconstituents of CP (scopoletin and kaempferol) that have shown anti-AChE activity in separate studies. A more comprehensive analysis including alkaloid, anthocyanin, triterpenoid and phytosterol components of CP  should bi = 4 × 10–3) [–2 M and mixed type AChE inhibition at a concentration of 0.5 × 10–2 M in synaptosomal fractions isolated from rat brain [An inhibitory activity of 11.68% ± 2.28 was noted in SCOP-treated larvae compared to untreated larvae ; the ACh level was 86.54 ± 4.90 µM  . It was at brain .Using molecular docking, we found that, in the absence of ACh, AChE was bound by SCOP at Gly234 and Thr238 in the peripheral anionic site with a binding energy of 223.762, the former residue being the one that mediates the inhibitory effect of galantamine, an AChE inhibitor . In the A combination of ISOX and SCOP (ISOX + SCOP) increased the inhibitory activity of AChE to 68.45% ± 0.5  compared to either of their inhibitory activities in isolation Fig. . SimilarWe studied the locomotor patterns of larvae treated with ISOX + SCOP and CP + SCOP. Larvae treated with ISOX + SCOP covered increased distances in comparison with ISOX- treated larvae  Fig. . Larvae We studied the binding of ISOX and SCOP on AChE using molecular docking. When AChE was bound by SCOP at Glu313 in the choline-binding pocket, ISOX was bound by the enzyme at the primary and secondary ACh binding sites at a higher energy of − 12.91 to − 12.72. However, in the presence of ACh at Hsd405, SCOP was bound by the enzyme at Glu313 and ISOX at Ser203 with a lower binding energy of − 13.96. An even lower binding energy of − 14.1 was observed with AChE binding two molecules of ACh at Hsd405 and Phe295  as that of the larvae treated with the AChE inhibitor ISOX, which was used as a positive control. Additionally, CP improved the retention of avoidance response in adult zebrafish compared with ISOX. ISOX was found to directly bind Ser203 of the catalytic triad on the human AChE. The active components of CP—scopoletin and kaempferol—were found to bind to His447 of the catalytic triad, the anionic subsite of the catalytic center, and the peripheral anionic site. Unexpectedly, SCOP, which was used in our study to induce cognitive impairment in zebrafish, showed AChE inhibition in 168 hpf larvae, possibly mediated via the anionic subsite of the catalytic center and the peripheral anionic site, as indicated by docking studies with human AChE. Interestingly, CP + SCOP significantly increased AChE inhibition and depleted ACh levels compared with untreated larvae, a pattern that was also observed albeit in a statistically non-significant way in ISOX + SCOP. Abnormal motor responses were observed individually with ISOX and CP, and in their combinations with SCOP, indicative of undesirable effects on the peripheral cholinergic system. Our study proposes the examination of CP, SCOP, and CP + SCOP as potential AChE inhibitors for their ability to modulate cognitive deficits in Alzheimer’s disease.2, 0.33 mM MgSO4, 10–5% Methylene Blue). The developmental stage of the embryos was observed under a microscope [Adult wild-type zebra fish were maintained in tanks as per standard conditions . Spawnincroscope . EmbryosConvolvulus pluricaulis (CP) was prepared by agitating 2 g of macerated root in 100 ml of distilled water at 150 rpm at room temperature for 24 h. The solution was clarified by filtration, frozen overnight at – 20 °C, and lyophilized for 18 h to obtain 18.5 mg of the extract.An aqueous solution of the root of 50 [24 hpf embryos were treated with the plant extract in 96 well plates according to Zebrafish Embryo Toxicity Test (ZFET) Protocol Standards . ZFET al50 .Ellman’s assay is a spectrophotometric method that quantifies AChE activity in terms of μ moles of acetylthiocholine iodide (ATCh-I) hydrolyzed per minute per mg of the plant preparation . A key pThe reagents for Ellman’s assay included 0.1 M phosphate buffer saline (PBS) at pH 7.0, 0.075 M acetylthiocholine iodide (ATCh) as substrate, and 0.01 M DTNB (dithiobis-(2-nitrobenzoic acid)), prepared by dissolving 39.6 mg in 10 ml phosphate buffer (0.1 M) at pH 7.0 and adding 15 mg of sodium bicarbonate. The digestion buffer was prepared by adding 20 mM Tris/HCl at pH 7.0, 5 mM EDTA and 1% Triton X-100. Embryos were euthanized using a mixture of 1 mL of clove oil and 9 mL of absolute ethanol. 1 mL of this solution was then dissolved in 50 mL of tap water. This solution was then transferred to a Petri dish with 20 embryos to be euthanized. 15–25 embryos were suspended in 150–250 µL digestion buffer  and homogenized by pipetting the suspension in and out. The homogenate was centrifuged at 1500 rpm for 15 min. The supernatant diluted in 0.1 M phosphate buffer at pH 7.0 was used as the enzyme (AChE) source. A blank reaction mixture was prepared with the phosphate buffer, substrate, plant extract solutions at specific concentrations, and DTNB reagent. Test reaction mixtures were prepared with the phosphate buffer, substrate, enzyme source, plant extract solutions at specific concentrations, and DTNB reagent. Absorbance was measured at 412 nm.3+ ions result in a red-brown complex, ACh-acetohydroxamic product, which can be detected through colorimetry [2O (in 1:2 ratio), 0.37 M Fe3+ (as ferric nitrate or ferric chloride) in aqueous 0.1 M HCl and a standard aqueous solution of 4 mM. Embryos were euthanized and washed. 15–25 embryos were suspended in 150–250 µL digestion buffer and homogenized by pipetting the suspension in and out. The homogenate was centrifuged at 1500 rpm for 15 min. The supernatant diluted in 0.1 M phosphate buffer at pH 7.0 served as the source for the enzyme (AChE) and the substrate (ACh). The blank reaction mixture was prepared with the phosphate buffer and the enzyme and substrate source. Test reaction mixtures were prepared with the phosphate buffer, the enzyme and substrate source, and the plant extract solution at specific concentrations. The reaction mixture was vigorously mixed with aqueous hydroxylamine hydrochloride and aqueous potassium hydroxide in the ratio of 1:1. The rapid change in pH stops hydrolysis. The resulting mixture was then mixed for 2 min to allow conversion of ACh to acetohydroxamic acid. The pH was then changed to 1.2 by adding conc. HCl/H2O and aqueous ferric nitrate or ferric chloride. Absorbance was measured at 540 nm. The concentration of ACh was calculated using its molar absorption coefficient  = 785 M−1 cm−1).Hydroxylamine reacts in a strongly alkaline medium with the substrate (ACh) forming acetohydroxamic acid and methanol. Acidification of this mixture with HCl and the addition of Feorimetry . The solKarnovsky’s staining method was used for the visualization of localized AChE activity . The mecThe working solution of the stain normally contains 60 mM sodium acetate, 5 mM sodium citrate, 4.7 mM copper (III) sulfate, 0.5 mM potassium ferricyanide, 1.7 mM acetylthiocholine iodide, and distilled water. However, since this composition failed to stain the embryos even after repeated trials and the use of freshly prepared solutions, we had to optimize the staining solution. Copper ions inhibit acetylcholinesterase and cause neurotoxicity in zebrafish . Based oCaenorhabditis elegans, have been optimized in our lab for zebrafish larvae [The settings of wrMTrck, a freely available ImageJ plugin originally developed for examining multiple behavioral parameters in the nematode h larvae . This soAdult zebrafish treated with a test compound was transferred to an experimental chamber. This chamber consisted of a dark compartment and a lit compartment separated by a movable door. The fish was placed in the dark chamber and allowed to acclimatize for 3 min. After this, the door was opened. A stone was dropped in front of the fish 3 s after it crosses the door. The stone served as the shock stimulus in this scenario. Crossing time estimated as the time taken by the fish to cross the door from the moment the door was opened was recorded after every such ‘trial’. Three such trials made up ‘one training session’ for the acquisition of the avoidance response. Two hours after a training session, a ‘test session’ consisting of a single trial was conducted to assess the extent of learned avoidance response retention. The fish was exposed to the test compound in the experimental chamber during both sessions .The crystallographic structure of human AChE was collected from the PDB database (PDB ID: 4PQE) . 4PQE haAdditional file 1: Figure S1. Time spent by adult fishes in each of the three areas of the tank. Adult zebrafish treated with test compound(s) were transferred to an experimental chamber divided equally into three areas and the extent of lateralization in swimming was measured as time spent in each of the three areas (shown as histograms). Areas 1 and 3 are peripheral, whereas area 2 is central. The time spent in the three areas was determined by analyzing 3 min long videos. 2 min was allowed for acclimatization and 1 min for test response. The fish was either untreated or treated with scopolamine (SCOP), isoxazole (ISOX), and ISOX + SCOP. The fish was exposed to 200 µM of SCOP and/or 31.2 mM of ISOX.


Inhibiting α-glucosidase activity is important in controlling postprandial hyperglycemia and, thus, helping to manage type-2 diabetes mellitus (T2DM). In the present study, free polyphenols (FPE) and bound polyphenols (BPE) were extracted from red quinoa and their inhibitory effects on α-glucosidase and postprandial glucose, as well as related mechanisms, were investigated. HPLC-MS analysis showed that the components of FPE and BPE were different. FPE was mainly composed of hydroxybenzoic acid and its derivatives, while BPE was mainly composed of ferulic acid and its derivatives. BPE exhibited stronger DPPH and ABTS antioxidant activities, and had a lower IC50 (10.295 mg/mL) value in inhibiting α-glucosidase activity. The inhibition kinetic mode analysis revealed that FPE and BPE inhibited α-glucosidase in a non-competitive mode and an uncompetitive mode, respectively. Furthermore, compared to FPE, BPE delayed starch digestion more effectively. BPE at 50 mg/kg reduced postprandial glucose increases comparably to acarbose at 20 mg/kg in ICR mice. These results could provide perspectives on the potential of BPE from red quinoa, as a functional food, to inhibit α-glucosidase activity, delay postprandial glucose increases and manage T2DM. According to the World Health Organization, the number of people with diabetes rose from 108 million in 1980 to 422 million in 2014. In 2019, an estimated 1.5 million deaths were directly caused by diabetes . Thus, nPolyphenols, as a major subcategory of phytochemicals, constitute an indispensable part of our daily diet. Polyphenols from various sources, such as tea , berriesChenopodium quinoa Willd.), native to the Andean regions of Chile, Peru and Bolivia, is considered as “golden grain” by local people. The high nutritional value of quinoa, including its ideal protein profile, and rich contents of vitamins and polyphenols, brings many health benefits, such as antioxidant, anti-diabetic and anti-obesogenic activities  refers to the concentration of substrate (pNPG).The inhibition mode of FPE or BPE against α-glucosidase was determined according to the plots of a double reciprocal line  for at least three replicates for each sample. Statistical analyses were performed using the SPSS program, version 17.0 . Data were analyzed by ANOVA and significant differences were set at p < 0.05), which was in consistency with some previous findings that the content of bound polyphenols was largely distributed in cereal grains, such as rice (~62%), wheat (~75%), corn (~85%), etc. [The TPC and TFC of FPE and BPE are shown in %), etc. .The chemical antioxidant activities of FPE and BPE were evaluated by the DPPH and ABTS assays. Both the DPPH and ABTS values of BPE  were significantly higher than those of FPE , which were in positive correlation with the TPC and TFC values. DPPH as a free radical has been widely used to evaluate the reducing molecules. ABTS is also frequently used in the food industry to determine the antioxidant activities of foods . In compThe components of FPE and BPE were further analyzed via HPLC-MS . Table 150 values of FPE and BPE were 13.013 ± 0.196 mg/mL and 10.295 ± 0.223 mg/mL, respectively. The IC50 value of BPE was lower than of FPE, indicating that the inhibitory effect of BPE on α-glucosidase was stronger than that of FPE. Both FPE and BPE exhibited higher α-glucosidase inhibitory activity than polyphenols from some other grains, such as corn and barley [α-glucosidase, located in the brush border of the small intestine, hydrolyzes terminal non-reducing (1→4)-linked α-glucose residues to release a single α-glucose, which leads to an increase in blood glucose levels. The inhibitory effects of FPE and BPE were compared A,B. Bothd barley .Reversible inhibitors can be classified into competitive, non-competitive, uncompetitive (or anti-competitive) and mixed types based on their interactions with the enzyme, which directly affect the values of Km and Vmax. Competitive inhibitors directly compete with the substrate to bind with the active site of the enzyme, while non-competitive inhibitors do not compete with the substrate and bind to the allosteric site of the enzyme instead. Uncompetitive inhibitors bind to the enzyme–substrate complex away from the active site of the enzyme, while mixed inhibitors bind to either the enzyme away from its active site or the enzyme–substrate complex . The inhp < 0.05). In consistency with previous studies, BPE with the highest level of TPC, TFC and various components of polyphenols prevailed over FPE in terms of inhibiting starch digestion.The effects of FPE and BPE on the digestion of rice starch in vitro are shown in p < 0.05). Compared with the AUC of the maltose group, a 25.8% of reduction was observed in the maltose+acarbose group, and a 21.4% of reduction was observed in the maltose+BPE group (50 mg/kg). The suppressive effect of BPE at 50 mg/kg on postprandial hyperglycemia was comparable to that of acarbose at 20 mg/kg, indicating that BPE can be considered as a natural α-glucosidase inhibitor.To further explore the inhibitory effects of FPE and BPE on α-glucosidase in vivo, an oral maltose tolerance test was performed in a normal ICR mice model. Concentrations of 2g/kg of maltose were administered to the mice. In addition, 20 mg/kg of acarbose was used as a positive control. As presented in T2DM has been a rapidly growing health problem in recent years. An estimated 850 billion USD was spent globally on the treatment and health interventions for diabetes in 2017 . This nu50 of BPE < IC50 of FPE).Diets rich in polyphenols have been extensively studied in the past decade in terms of their effects on T2DM. A number of meta-analysis studies revealed that consuming polyphenol-enriched diets played a vital role in the prevention of T2DM, and this was found to be associated with lower risk of T2DM ,27. SomeCompared with free polyphenols, studies on the enzyme-inhibitory activities of bound polyphenols were limited. Zheng et al. used acid/alkaline hydrolysis to extract bound polyphenols from mung bean skin and showed that it was mainly composed of caffeic acid and protocatechuic acid and could inhibit the activity of α-glucosidase . Yan et The composition of polyphenols might contribute to the differences between BPE and FPE regarding their antioxidant and enzyme-inhibitory effects. HPLC-MS analysis showed that hydroxybenzoic acids were the major components of FPE, while ferulic acid, ferulic acid 4-glucoside and quercetin were the major components of BPE , Table 1To further evaluate the outcome of the inhibitory effects of FPE and BPE on α-glucosidase, an in vitro starch digestion assay  and an iIn conclusion, the components, antioxidant activities, α-glucosidase inhibitory activity and mechanism, inhibition in starch digestion and in vivo postprandial glucose of FPE and BPE were investigated and compared in the present study. Alkaline and acid treatment released a large amount of bound polyphenols from red quinoa, and BPE showed higher levels of TPC and TFC than those of FPE. The HPLC-MS analysis revealed that the components between FPE and BPE varied greatly. Mainly composed of ferulic acid and its derivates, BPE showed stronger chemical antioxidant activities and α-glucosidase inhibitory activity. The Lineweaver–Burk model showed that FPE and BPE inhibited α-glucosidase activity in a non-competitive mode and an uncompetitive mode, respectively. By inhibiting α-glucosidase, BPE delayed starch digestion and suppressed postprandial hyperglycemia effectively. Bound polyphenols have been overlooked regarding their activities and application for a long time. Cereals grains consumed as staple foods all over the world contain rich bound polyphenols awaiting further investigation. Given that T2DM is prevailing around the world, exploiting functional components from cereal grains (staple foods) is necessary to manage T2DM. Our results demonstrate that BPE as a functional food has the potential to control postprandial glucose and, thus, is worth further investigation.


Further investigations also revealed that sanguinarine inhibited the collagen-induced human platelet aggregation and granule release. At the same time, it also prevented platelet spreading and adhesion to immobilized fibrinogen. The molecular mechanisms of its antiplatelet activity were found to be as follows: 1. Reduced phosphorylation of the downstream signaling pathways in collagen specific receptor GPVI (Syk-PLCγ2 and PI3K-Akt-GSK3β); 2. Inhibition of collagen-induced increase in the intracellular Ca2+ concentration ([Ca2+]i); 3. Inhibition of integrin αIIbβ3 outside-in signaling via reducing β3 and Src (Tyr-416) phosphorylation. It can be concluded that sanguinarine inhibits collagen-induced platelet activation and reduces thrombus formation. This effect is mediated via inhibiting the phosphorylation of multiple components in the GPVI signaling pathway. Current data also indicate that sanguinarine can be of some clinical value to treat cardiovascular diseases involving an excess of platelet activation.Sanguinarine, a benzophenanthridine alkaloid, has been described to have an antiplatelet activity. However, its antithrombotic effect and the mechanism of platelet inhibition have not thoroughly been explored. The current study found that sanguinarine had an inhibitory effect on thrombus formation. This inhibitory effect was quite evident both in the flow-chamber assays as well as in a murine model of FeCl Most ofWe also found that sanguinarine inhibited human platelet adhesion and spreading on the fibrinogen-coated matrix a,b. Comp3 in mice, but had no influence on tail bleeding time. This suggests that it had no obvious risk of bleeding.The effects of sanguinarine on arterial thrombosis in vivo and in vitro were investigated in this study. Sanguinarine dose-dependently inhibited human platelet aggregation, α-granule and dense granule release, αIIbβ3 activation and intracellular calcium mobilization induced by collagen. It also inhibited platelet spreading on immobilized fibrinogen. This suggests that sanguinarine inhibits collagen-induced platelet activation as well as integrin αIIbβ3 “outside-in” signal transduction. From the molecular mechanism, it was clear that sanguinarine inhibited the phosphorylation of downstream signal molecules of the collagen specific receptor GPVI signal pathway, including Syk-PLCγ2 and PI3K-Akt-GSK3β. Moreover, it also inhibited αIIbβ3-mediated β3-Src signaling . Sanguin3-induced carotid artery thrombosis, in which multiple anticoagulants and antiplatelet drugs are sensitive to i in platelets induced by collagen, and the dose-dependent trend was consistent with its antiplatelet aggregation effect (The increase of intracellular Caignaling ,36. A stpathways . The keyhibition . Our stun effect a,b.Fibrinogen plays a crucial role in cytoskeleton reorganization and platelet spreading after stabilizing thrombosis and growth by binding to the activated integrin αIIbβ3 and triggering the “outside-in” signal ,38. At tHowever, both previous studies  and our Our study indicated that sanguinarine inhibited arterial thrombosis in a dose-dependent manner. Additionally, sanguinarine inhibited platelet aggregation, granule release and spreading. The underlying mechanisms are as follows : 1. Inhi