Artificial particle ices are model systems of constrained, interacting particles. They have been introduced theoretically to study ice-manifolds emergent from frustration, along with domain wall and grain boundary dynamics, doping, pinning-depinning, controlled transport of topological defects, avalanches, and memory effects. Recently such particle-based ices have been experimentally realized with vortices in nano-patterned superconductors or gravitationally trapped colloids. Here we demonstrate that, although these ices are generally considered equivalent to magnetic spin ices, they can access a novel spectrum of phenomenologies that are inaccessible to the latter. With experiments, theory and simulations we demonstrate that in mixed coordination geometries, entropy-driven negative monopoles spontaneously appear at a density determined by the vertex-mixture ratio. Unlike its spin-based analogue, the colloidal system displays a “fragile ice” manifold, where local energetics oppose the ice rule, which is instead enforced through conservation of the global topological charge. The fragile colloidal ice, stabilized by topology, can be spontaneously broken by topological charge transfer. Spin ice materials are defined by ice rules, local constraints that lead to frustrated interactions and macroscopic numbers of degenerate configurations. Here the authors show that the ice rule in colloidal ice is emergent, limited to certain geometries, and demonstrate how it can break down under changes of the lattice structure. In the 1930s Giaque and Ashley3 found that the specific entropy of water at very low temperature was not zero, despite the ordered, solid structure of ice. In water ice the oxygen atoms reside at the center of tetrahedra, sharing four hydrogen atoms with four nearest neighboring oxygen atoms. Two hydrogen atoms are covalently bound to each oxygen, and two form hydrogen bonds with neighboring oxygen atoms. In the so-called ice rule introduced by Bernal and Fowler1, this situation is described as having two hydrogen atoms pointing “in”, and two pointing “out” of the tetrahedron. As Linus Pauling explained4, the freedom in choosing such an arrangement on a large lattice leads to a degeneracy that grows exponentially with the number of tetrahedra, generating the residual entropy.The ice rule2Ti2O7 and Dy2Ti2O76. These pyrochlore systems were called “spin ices” because the cations Ho3+ and Dy3+ carry a large magnetic moment directed along the lattice bonds, which can be associated to a classical, binary Ising spin. At low temperature, frustration ensures that two spins point in and two out of each vertex, reproducing the ice rule and preventing the spontaneous magnetization of the material.This idea proved to be not limited to water. The ice rule was recognized to occur in exotic magnets, namely the rare earth titanates such as Ho26 that generalize spin ices and are broadly called artificial spin ices. There, exotic states of matter and emergent dynamics often not found in natural systems can be deliberately designed and externally controlled in artificial nano- and micro-scale materials.The ice rule was eventually exploited to design new artificial frustrated systems based on magnetic nano-islands, confined colloidal particles, or vortices in superconductors27, emergent descriptions30, topological constraints 32, and complex dynamics of magnetic  charges34 can be tailored, nano- or micro-engineered, and characterized at the level of the constitutive degrees of freedom, often providing remarkable vistas of statistical mechanics in action37. Such generality is not surprising since the ice-rule is a powerful topological prescription for conceptualizing the effects of frustration in a broad class of physical systems.In such systems frustration produces complex disordered manifolds where fascinating effects, such as dimensionality reduction38 with nodes of various coordination number z. Assign binary variables on the edges of the graph, in the form of spins directed along the edges and impinging in the nodes. Then we can define a “topological charge” q for each vertex as the difference in the number of spins n pointing toward the vertex and the number of spins z − n pointing away from it, or q = 2n − z. In magnetic spin ices q is proportional to the magnetic charge of the vertex40 leading to a rich phenomenology for magnetic charge currents41, charge ordering43, charge screening33 or dynamical arrest44. In this language, the ice rule corresponds to the minimization of the absolute value of the topological charge |q|. The charge is called “topological” insofar as it depends upon the connectivity of the system, and its definition does not change for continuous deformations of the lattice. It is therefore a topological invariant for the vertex configuration (though it does not completely define the spin configuration45). On vertices of even coordination the minimization of |q| on each vertex implies that q = 0, and when z = 4 we recover the original 2-in/2-out ice rule of water ice. On vertices of coordination z = 3, the minimum occurs for q = ±1, corresponding to 2-in/1-out or 1-in/2-out.To understand the topological nature of the ice rule in the broadest generality, consider a general lattice, even a graph, or network29, mixed coordination28, and the introduction of dislocations;32 indeed it was found that even isolated clusters of magnetic vertices obey the ice rule at low energy46.In the magnetic spin ice-like systems mentioned above, the low energy ensembles all obey the ice rule, which has proven to be extremely robust. The ice rule survives all sorts of weak or strong alterations, including decimationHere we add to the already rich history of the ice rule by introducing a system where the ice rule becomes “fragile”, meaning that it can be easily destabilized by topology. Through a combination of theory, simulations, and experiments, we demonstrate that the colloidal ice falls in a different class of geometrically frustrated ices, or “fragile ices.” There, the ice rule is spontaneously broken in lattices of mixed coordination, leading to a rich and unique set of phenomena, including topological charge transfer and charge screening, that are completely absent in nanoscale magnetic ices or indeed in most ice systems known to us. It is important to understand that we are describing fragility, not a breakdown. As we will see, most of the system still obeys the ice rule, and only specific charges, in the form of negative monopoles, appear. Crucially, these monopoles are not excitations, but instead belong to the low energy state, and their density can be controlled..23. When we decimate our system by removing colloids . We achieve our decimation using a partial, random dimer covering of the edges  in a random fashion, in order to create a lattice of mixed coordination ges Fig. , where rNt the number of traps in the original square lattice, which form a total of Nv = Nt/2 vertices of coordination z = 4 .We introduce some nomenclature that will prove to be useful later. Considering the thermodynamical limit of the system, and neglecting boundary effects, we call = 4 Fig. . We deciξ = 1/4. Note that η → + ∞ when ξ → 1/4−, since z = 3.The maximum possible decimation corresponds to a complete random dimer covering realized when all the vertices are covered by one and only one dimer. Then the number of dimers is half of the number of vertices and therefore a quarter of the number of traps. Thus the maximum decimation corresponds to a removal of 25% of the traps, or z = 3 and z = 4 arranged in order of increasing energy, which also corresponds to increasing topological charge. Note that in computing the vertex energy we adopt a nearest neighbor approximation and consider only the interaction of the particles close to the vertex. Vertices of coordination z = 4 can have even charges q = −4, −2, 0, 2, 4, whereas vertices of coordination z = 3 can have odd charges q = −3, −1, 1, 3. We label the vertices by their topological charge, and call q and coordination z = 4,3, respectively. We define the relative vertex frequencies as Figure We have performed experiments on mixed-coordination lattices at various levels of decimation. We corroborate our experimental results using overdamped Langevin dynamics on larger samples. For both experiments and simulations, at each level of decimation the results are obtained by averaging over ten different, randomly generated lattices obtained via the random dimer algorithm described above.B perpendicular to the plane to induce a tunable, perpendicular dipole moment m ∝ B in each colloid. The resulting interaction between two colloids a distance r apart is repulsive, isotropic, and given by B increases, so does the mutual repulsion, and the colloids, originally disposed randomly, rearrange to a collective low-energy configuration24. Our experimental system extends over a square lattice composed by 11 × 8 vertices corresponding to a total of Nt = 195 traps in the undecimated case. We note that the size of the experiments is limited by two factors: the trapping objective constrains the field of view, and the time required to populate the system must be small enough to keep the suspension electrostatically stable.The experimental system is based on a monolayer of paramagnetic colloids confined above a square lattice of lithographic, microscopic double-wells Fig. . Each grz = 4 sublattice, but very specifically: only q = −2 charges appear spontaneously, while all other vertex type follow the ice rule. At the same time, the ice rule is still obeyed on the z = 3 sublattice, where only charges q = ±1 are present.In Fig. 49.). In Fig. z = 4 vertices only. Furthermore, on a larger scale we find that the disordered charge transfer between z = 4 and z = 3 vertices implies the breakdown of the well known antiferromagnetic order11 of the square ice manifold. This structural transition to disorder has been recently proved theoretically48, but here we experimentally observe it at a decimation of approximately 12% of the traps, about halfway to the maximal decimation of 25%.We corroborate these experimental findings with numerical simulations on larger samples (2500 vertices with periodic boundary conditions) than those used in experiments (which contained only 88 vertices). We employ over-damped, Brownian dynamics precisely parametrized to mimic the experimental setting . Figure q = 1 vertices always exceed q = −1 vertices in number and thus the z = 3 vertices have an overall positive charge. They can therefore adsorb the extra negative charge introduced by the z = 4 vertices without leaving the ice-manifold simply by shifting their relative ratio in favor of vertices of charge q = 1. Moreover, Fig. z = 4 vertices tends to zero), the fraction of vertices of charge q = 1 and q = −1 both tend to the same value of 1/2 as expected in a single coordination, z = 3 lattice.Remarkably we find that the ces Fig.  always oη < 1. There, z = 3 vertices are sparse and surrounded by z = 4 vertices. The density of z = 3 is too small to adsorb all the available charge coming from the z = 4 and therefore the numerical simulations show larger q = +3 charges forming on them. In the experimental data only, we also see very few q = +2 monopoles forming on z = 4 vertices at low decimation. This is likely a consequence of lack of complete equilibration at low decimation, where the system is close to order, and of finite size of the sample, where positive charges can form on the boundaries as explained in the next subsection and in ref.48. Indeed, this type of defects was also present in previous work on non-decimated, ordered square lattice systems23.Small deviations from this picture only happen at QNN, the average charge neighboring a z = 4 vertex. We find that negative q = −2 monopoles are surrounded by a positive average charge that largely exceeds the average charge surrounding non-charged z = 4 vertices. Thus, as monopoles of charge q = −2 spontaneously appear on z = 4 vertices, they are screened by positive charges q = 1 on the surrounding vertices of coordination z = 3. This suggests that charge screening is not unique to magnetic charges that interact via a Coulomb law in magnetic ices45. In fact, charge rearrangement and ordering was also recently observed numerically in the disordered ensemble of kagome colloidal ice49. There, it was shown, it is a consequence of the 1/r3 long range interaction among colloids, the same present in this work. Charge effects driven by charge-charge interactions were also seen in magnetic ice systems: charge ordering within the ice state of kagome artificial spin ice42, and monopole screening by magnetic charges in Shakti ice28. Unlike in Shakti, here charges screen not excitations, but rather monopoles which belong to low energy state.Moving away from the global picture, disorder of the ensemble produces fascinating local effects of spontaneous screening of topological charge. In Fig. 47, along with non-trivial rearrangement of the topological charge being transferred. We reiterate that such a breakdown is not possible in magnetic spin ice systems, where the ice manifold has been shown to be completely robust46.These results unambiguously demonstrate the breakdown of the ice rule in particle based ice as suggested in Ref.E ~ q2 . In colloidal ices, however, the energy of s repulsive colloids in a vertex scales as 47, thus favoring vertices of large, negative charge, in violation of the ice rule. Obviously, the total charge must be zero, so it is not possible for all vertices to be negatively charged. Individual vertices can only push their charge to the boundaries, and the resulting charge accumulation is limited by the size of the edges. Therefore, the density of topological charge in the bulk must scale at least as the reciprocal length of the boundaries, leading to the emergence of the ice rule (zero charge) in the thermodynamic limit. There is thus a collective, non-local reason for the ice rule in colloidal systems, which is quite unlike the local, energy-enforced origin of the ice rule in magnetic systems. Indeed, the latter is observed locally even in small spin ice clusters46.To understand the nature of the ice rule breakdown in colloidal ice, we first need to understand its origin, as the former differs essentially from the magnetic ices. In magnetic spin ice the topological charge minimization that corresponds to the ice rule is enforced by the local energetics, since the energy of frustrated spins meeting at a vertex typically scales quadratically with the vertex charge, or z = 4 vertices now have an internal boundary consisting of z = 3 sub-lattices onto which topological charges can be pushed. Because the global charge must remain zero, the two sub-lattices develop opposite nonzero charges. As a consequence, the ice rule is very selectively broken in the z = 4 sector by the appearance of negative charges q = −2, corresponding to 1-in/3-out vertices. The ice rule still applies to the z = 3 vertices, since the plasma of charges in an odd-coordination spin ice can absorb and screen charges without breaking the ice rule.The boundary size constraint is lifted in our decimated system, since the 47. For simplicity, we can treat vertices as uncorrelated, but constrain the total charge to be zero. Then the thermodynamic ensemble at equilibrium still follows a Boltzmann distribution but in the effective vertex energies ϕ is a Lagrange multiplier enforcing the requirement of zero total charge. Thus, for a lattice of coordination z, the choice ϕ = (z − 1)/4 returns a spin-ice-like effective energetics, given by ϕ that can generate an effective ice-like energetics for vertices of more than one coordination. For z = 4,3, charge conservation imposes ϕ = (3 − 1)/2 = 1/2 and thus the effective energetics maintains the ice rule on z = 3 vertices. On z = 4 vertices, however, it ascribes the same effective energy to the negative (q = −2) monopoles and to the ice rule (q = 0) vertices47. This explains why those are the only vertices seen in our simulations and experiments.We now make these considerations more quantitative48. It was demonstrated theoretically that a decimated particle-based ice is energetically equivalent to a spin ice stuffed with extra, negative topological charges, placed at the two ends of each decimated trap. For a spin system this implies accumulation of positive charge on decimated vertices  and, because the total charge of the occupied traps must be zero, the consequent formation of negative monopoles on undecimated vertices (here z = 4) vertices.Another way to understand the same effect was reported recentlyFrom these considerations one can quantitatively predict the charge transfer and thus the vertex statistics using a very simple entropic argument. In Methods we show how to obtain a very simple entropy density based on the simplifying assumption of uncorrelated vertices. Such entropy depends only on the amount of the charge transferred η < 1 where the system is still largely in an ordered state ; it corresponds to a percolation threshold in the dimer model on which our decimation protocol is based48, a subject currently under numerical study by others50. The formalism reported above and in47, relying on uncorrelated charges, applies to disordered manifolds, and therefore does not predict such a transition.The focus of this work is to prove the ice rule fragility in particle-based ices. We now describe another interesting effect brought about by the topological charge entropy which invites further study: the breakdown of order in the system. Undecimated square ice is antiferromagnetically ordered23. For instance, the first panel of Fig. 19). The charge alternation is thus broken on those pinning sites, generating a net topological charge on the z = 4 sector.Clearly in the low-decimation, ordered phase there no topological charge transfer and no ice-rule fragility is predicted. Instead we see in Fig. 48, the residual entropy of the ground state and the topological charge transfer are associated with the appearance of emergent lines composed solely of negative charges, connecting q = −2 monopoles on the z = 4 vertices that belong to decimated plaquettes. These lines must thread through nearest neighboring decimated plaquettes, and thus they exist only at decimations large enough that the decimated plaquettes percolate. Below that threshold, in the ground state, no such emergent lines exist, no topological charge transfer occurs, and the system remains ordered. The lines can still appear as small energy excitations, where they must include not only q = −2 monopoles, but also ice rule vertices that do not belong to the ground state .This mechanism is better understood by considering how the disorder of the ground state sets in above critical decimation. Following ref.48. In slightly excited states at low decimation, the system forms ordered domains separated by lines pinned to the decimated plaquettes. As the decimation increases, these domains shrink, until at the percolation threshold for the decimated plaquettes, no order is discernible. This mechanism is apparent in the panels of Fig. 19. While the domains of the system are strongly correlated at low decimation, the domain walls are not, explaining why our uncorrelated-charge treatment above can capture the numerical data even at low correlation, and testifying to the solidity of the use of topological charges as degrees of freedom for describing this phenomenon. Indeed it was suggested48 that dynamical arrests of the topological charges, though not necessarily of the colloids, could occur. The associated weak ergodicity breaking might thus make it impractical to observe the predicted structural transition in real systems, an issue that invites further theoretical and experimental investigation.Thus we have the following interesting picture: in the equilibrium ground state below a critical decimation, the system is ordered and obeys the ice rule, while above it the system is disordered and the ice-rule is violatedWe have added a chapter to the long history of the ice rule by demonstrating the fragile nature of particle-based ice. Previously, in magnetic spin ice, the ice rule had proven remarkably robust to the introduction of all types of structural defects, doping, or dislocations. In contrast, in colloidal ice, the ice manifold is of a collective, non-local origin and can be destabilized by topology, leading to the spontaneous formation and accumulation of extensive topological charges which can rearrange and screen.53. Topological states are increasingly studied in classical settings and in soft matter systems56, where they are generally associated with stability produced by topological protection. In this context, our work poses a set of questions regarding whether such topological protections are robust to dilution or are instead fragile.Our work has implications beyond ice rule systems for understanding classical topological phases. For example, the ensemble of ice-rule-obeying configurations, most of which are disordered, is called a Coulomb phase and is an example of a topological phaseFurthermore, from an applied perspective, the possibility of controlling the dynamics and flow properties of topological charges via lattice decimation can inspire the engineering of novel dissipation-free magnetic storage and logic devices at the micro and nano-scale. For example, in domain wall engineering, interfaces of mixed coordination would be charged and possibly semi-permeable to defects, while in driven kinetics, entropically spontaneous charges could be suppressed or enhanced by an ac driving field.4 has played a fundamental role in frustration, inspiring celebrated theoretical models58 and appearing in an increasing number of physical and non-physical systems60. Our findings open a path toward a different phenomenology in geometrically frustrated, ice-rule based systems that is completely absent in traditional spin ices.More fundamentally, geometric frustration is a topic of considerable interest, as it encompasses a large class of physical systems in condensed matter and beyond, including biological systems. The ice-rule24. In brief . The optical tweezers is mounted in a custom inverted optical microscope equipped with a white light illumination LED (MCWHL5 from Thorlabs) and a CCD camera (Basler A311f). The external magnetic field is applied with a custom-made coil oriented perpendicular to the sample cell and connected to a computer controlled power amplifier (KEPCO BOP-20 10M).The samples used in this work were prepared following a process similar to that described in Refs.see Fig. , we use see Fig. . Within 2min−1. The structures are designed using commercial software . Each double well is drawn on the mask as a stadium-shaped transparent region, with a small rectangular opaque spot in the center. The outer region has a length of 21 μm and a width of 7 μm, while the spot covers an area of 3 μm × 2 μm. The Cr mask is then used to etch the microfeatures on a 2.8 μm layer of photoresist AZ-1512HS . The photoresist is deposited on top of a 100 μm thick glass coverslip by spin coating  at 500 rpm for 5 s and afterwards at 1000 rpm for 30 s, both steps with an acceleration of 500 rpm/s. Different thicknesses of the photoresist could be obtained by varying the rotating speed; however, we find that ~3 μm works well to create topographical traps capable of capturing the particles within the double wells for most of the applied fields. After the deposition process, the photoresist is irradiated with UV light passing through the Cr mask for 6 s at a power of 25 mW/cm2 . The light passing through the motifs of the mask uncrosslink only the exposed part of the photoresist. The exposed parts are then eliminated by submerging the film in a AZ726MF developer solution . At this thickness, the size of the spot is too small for the lithographic process, and results in a small hill with a lower height at the center of the islands.For the lithographic fabrication procedure, we write a square lattice of double wells on a mask made by a 5-inch glass wafer and covered with a 500 nm layer of Cr. Direct Write Laser Lithography  was used for this purpose, based on a 405 nm laser diode and working at a speed of 5.7 mmNt = 50 × 50 × 2 = 5000 etched double-well grooves arranged in a square lattice with a lattice constant of 29 μm giving a total of Nv = 2500 vertices. We use periodic boundary conditions in both the x and y directions. The double-well trap consists of two halves of a parabolic well joined by an elongated part. The particle in either parabolic half is tethered to the center of the well with spring constant of 1.212 pN/μm. Along the elongated part of the pinning site, this same tethering force confines the particle perpendicularly, while a repulsive force with a spring constant of km = 0.352 pN/μm repels the particle from the middle of the pinning site, reaching a maximum value of Fm = 1.758 pN in the middle of the pin and vanishing as it reaches the center of either well. These combined substrate forces acting on particle i are written as z direction produces a repulsive particle-particle interaction force Fpp(r) = Ac/r4 with r apart. Here χm is the magnetic susceptibility, V is the particle volume, B is the magnetic field in mT, and all distances are measured in μm. This gives Fpp = 7.231pN for r = 20 μm at B = 50 mT, the maximum field we consider. The dynamics of colloid i are obtained using the following discretized overdamped equation of motion:D = 7000 nm2/s, the mobility μ = 1.729 μm/s/pN and the simulation time step Δt = 1ms. The first term on the right is a thermal force consisting of Langevin kicks of magnitude FT = 2.163 pN corresponding to a temperature of t = 20 °C . Here, N denotes a random number drawn from a normal (Gaussian) distribution with a mean of 0 and a standard deviation of 1. Each trap is initially filled with a single particle placed in a randomly chosen well, or left empty in the case of decimation. We increase B linearly from B = 0 mT to B = 50 mT, consistent with the experimental range. We average the results over 10 simulations performed with different random seeds.We conduct Brownian dynamics simulations of the decimated colloidal ice system comprised of magnetically interacting colloids with a radius of 5.15 μm placed in an array of s colloids and having coordination z = 4,3, respectively. From this, s colloids in each sector. Finally, we write z = 4, z = 3, which are given by We let 47, at equilibrium all the z = 4 vertices are expected to be either obeying the ice rule—and thus of type 2-in (q = 0)—or to break the ice rule as lowest charge monopoles—and thus of type 1-in (q = −2). In contrast, all the z = 3 vertices must obey the ice rule, meaning that they are of type 2-in (q = 1) and 1-in (q = −1). The z = 3 vertices screen the extra charge by changing the relative admixture of ±1 charges. From the conservation of topological charge, we obtain the constraintη ≥ 2.As explained above and demonstrated in Ref.z = 4 vertex η ≤ 2 and η ≥ 2. Indeed, Eq. (η → ∞. Knowledge of charge transfer from Eq. (Since configurations corresponding to partial charge transfer are in general entropically favored, the charge transfer is mostly entropic. Consider a charge transfer between two vertices of different coordination as in Fig. entclass1pt{minimaPeer Review FileDescription of Additional Supplementary FilesSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4Supplementary Movie 5Supplementary Movie 6


Urease are responsible for several pathogenic states in human as well as in animals and its inhibition is utmost urgent. Clinically used drugs are associated with many side effects; recently several researches have shown that flavonoids have good urease inhibition properties. Morin, a natural flavonoid has been investigated for urease inhibition studies which includes designing of library of morin analogues and their in-silico evaluation with the help of Schrodinger’s maestro package of molecular docking software against crystallographic complex of plant enzyme Jack bean urease (PDB ID: 3LA4) followed by synthesis and in vitro evaluation.Anti-H. Pylori activity. In-vitro results revealed that a large number of synthesized compounds were found to possess excellent antioxidant and urease Inhibition properties.Best thirteen derivatives of morin were selected on the basis of their interaction energy and dock score for synthesis and further investigated for in-vitro antioxidant, urease inhibitory and N-(2-chlorophenyl)-N-((4E)-2--3,5,7-trihydroxy-4H-chromen-4-ylidene)thiourea (M2b) and N-(4-bromophenyl)-N-((4E)-2--3,5,7-trihydroxy-4H-chromen-4-ylidene)thiourea (M2i) were found to be most potent urease inhibitor and antioxidant with IC50 value 10.74 ± 0.018, 11.12 ± 0.033 and 7.37 ± 0.024, 7.73 ± 0.015and 7.795 ± 0.003 µM. Derivative M2i exhibited good anti-H. pylori activity having MIC = 500 μg/ml and zone of inhibition 15.00 ± 0.00 mm as compared to standard AHA having MIC = 1000 μg/ml and zone of inhibition 9.00 ± 0.50 mm determined against H. Pylori bacterium  by well diffusion technique. Furthermore, molecular docking study explained the binding pattern of synthesized ligand within active cavity of jack bean protein and drug similarity was explained by ADME studies by quikprop module of molecular docking software.Among the synthesized compounds, The online version of this article (10.1186/s13065-019-0562-2) contains supplementary material, which is available to authorized users. Structure of protein was prepared first of all before docking studies by using pre-process method of Prepwiz module of Schrodinger’s Maestro molecular modeling. Bond orders were assigned, partial charges and hydrogen were added, side chains and loops having missing atoms were fabricated, all waters  were deleted. Structure of proposed Ligands was drawn using Chemdraw Ultra 8.0 (ChemOffice Package) software which was saved as mol. file. Preparation of ligand was done prior to docking by Ligprep module of Schrodinger’s Maestro molecular modeling. Optimization of structure was done by the Gaussian 03 package. Ideal conformation of ligands was acquired by the adding or removing hydrogen bonds, computing partial charges according to the OPLS-2005 force field with 32 stereo isomers, tautomers, and other options like ionization at the physiological pH 7.2 were set as default options. Ligands each conformation was docked within radii of 20 Å into the receptor lattice at the coupling destinations of receptor structures as per their minimum potential energy. Glide/XP by default joined with Induced Fit docking conventions was connected for the forecast of energy of binding and interaction within protein and ligand at the catalytic sites of enzyme. Induced Fit Docking (IFD) methods give the springiness to ligand as well as the residues present at active site all over the docking study where it was rotated in all three angles [GScore) scoring function was obtained as the output for each ligand given in Table In-silico studies were done to study the interaction pattern of newly designed ligands in the catalytic cavity of jack bean urease enzyme. X-ray crystal structure of Jack Bean Urease enzyme having resolution 2.05 Å was downloaded from Protein Data Bank . Readings were recorded in triplicate set in a final volume of 200 μL using thiourea as standard. Each assay was performed at pH 8.2 . Calculation of Inhibition in percentage of synthesized derivatives was done by the formula given as:control is control absorbance; Asample is test absorbanceUrease enzyme inhibition investigation studies for all synthesized compounds were done by the method developed by Weatherburn  named Incontrol is control absorbance; Asample is test absorbanceDPPH method was adopted for measuring the antioxidant nature of the synthesized derivatives. According to this method synthesized compounds were allowed to react for 0.5 h. at 37 °C with stable free radical, 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH). The DPPH was taken in a concentration of 300 µM. Decrease in value of absorption at 515 nm was measured after the period of incubation using a microplate reader using ascorbic acid as a standard. Molecule which can donate a proton to DPPH and cause its reduction act as an antioxidant. Following reduction deep violet coloured DPPH solution changes to yellow depending upon the nature of antioxidant compound, which brings a measurable decrease in value of absorption at 517 nm. The proton or electron donating capacity of the derivative was estimated from the fading of deep purple colored methanolic solution of 1, 1-diphenylpicrylhydrazyl (DPPH). Incubation of mixture was done at room temperature for 30 min and measurements were done at 517 against blank , 29. CalH. pylori bacterium  for their antibacterial activity by using Well diffusion technique. The stock solution of compounds in DMSO (1000 μg/mL) were prepared. Cell suspension was prepared from culture grown on BHI broth. The cell suspensions of all the cultures were adjusted to 1–2 × 105cells/ml. Helicobacter pylori (100 µl) was inoculated by spread plate technique on agar plates (90 mm). Agar wells (5 mm) were made on H. pylori inoculated media and impregnated with 2500 µg of each sample and standard which were incubated @ 35 °C for 24–48 h with 5% CO2 and observed for zone of inhibition around the well. MIC was determined against Helicobacter pylori by micro broth dilution technique as per NCCLS method [Novel synthesized derivatives were investigated against S method –45.Kinetic parameters i.e. Km, Vmax and Michaelis constant were calculated by verifying the concentration effect of substrate on the rate of reaction of urease enzyme in presence as well as in absence of synthesized compounds. Mechanism behavior of derivatives was studied by dissolving 1% of them in dimethyl sulfoxide and inhibition mode studied by Lineweaver–Burk plots was found to be non-competitive as Km was contant but Vmax changed. Urease enzyme kinetic studies of interaction with synthesized was done GraphPad Prism 7 software.Output of statistical analysis has been depicted in mean ± SEM whereas statistical examination of data collected experimentally was done by one-way analysis of variance (ANOVA). Considerable difference revealed by ANOVA p < 0.05 was regarded significant. Evaluation of statistical data was done by Graph Pad Prism 5.0 Version for Windows .50 110.74 ± 0.018, 11.12 ± 0.033 and 12.71 ± 0.027 µM even two folds more active than standard drug thiourea and also displayed good antioxidant behavior with IC50 value 7.37 ± 0.024, 7.73 ± 0.015 and 7.795 ± 0.003 µM. Moreover these are found to be most potent by having excellent dock score 10.977, − 10.273, − 9.225 and binding energy − 59.062, − 45.27, − 61.834 kJ/mol as compared to standard drugs − 3.459, − 3.049 and − 21.156 kJ/mol and − 17.454 kJ/mol of standard thiourea and Acetohydroxamic acid. These top molecules were again further examined for anti-H. Pylori activity and M2i was found to be more potent as compared to standard drug AHA with MIC = 500 μg/ml and zone of inhibition 15.00 ± 0.00 mm as compared to standard having MIC = 1000 μg/ml and zone of inhibition 9.00 ± 0.50 mm. Derivative M2i can be the potential lead compound in future for treatment of pathologies caused by urease as well as against H. Pylori infection.In conclusion, thirteen Morin derivatives have been synthesized successfully via. a simple two step reaction and evaluated for their DPPH- free radical scavenging activity and urease inhibitory activity against jack bean urease. Among the series M2b, M2i and M2a (ICAdditional file 1. Supplementary file for spectral data.


The pedicle screw is a surgical device that has become widely used in spinal fixation and stabilization. Postsurgical complications such as screw loosening due to fatigue loading and screw breakage still need investigations. Clinical parameters such as the screw insertion type and depth, the bone density, and the patient degree of mobility greatly affect the mechanisms of the implant's failure/success.  The current finite element study focused on the prediction of the pedicle screw pullout strength under various conditions such as insertion type, insertion depth, bone quality, and loading mode.  As depicted in this study, the preservation of the pedicle cortex as in the N1 insertion technique greatly enhances the pullout resistance. In addition, the higher the screw-anchoring depth, permitting to gear a maximum number of threads, the better the protection against premature breakouts of pedicle screws.  In agreement with experimental data, the type of insertion in which the first screw thread is placed immediately after the preserved pedicle cortex showed the best pullout resistance for both normal and osteoporotic bone. Spinal surgery has been remarkably improved by using pedicle screws. However, the use of such a successful surgical tool can achieve its objectives only by means of theoretical knowledge and rigorous practice. The critical location of the screws near the main nerve roots makes these elements very delicate in spine surgery causing serious and traumatic postoperative complications to the patient when inserted inaccurately .Although the penetration of bony landmarks is reliable, the demands for multistage instrumentation require adjustments that necessitate an extensive and practical knowledge of spinal morphology . The ratMoreover, several observed cases showed through standard radiographs that the implanted screws can have not only diverse trajectories but variable depths of penetration as well. Due to excessive mechanical stress, some of these screws may have undergone premature failure in vivo. Sterba et al.  have addFrom a biomechanics perspective, a scoliotic spine will exert pullout forces on the pedicle screws that tend to extract them from their anchorage. These pullout efforts vary in terms of amplitude and rate of loading depending upon the patient's mobility. They might be either slow, low in magnitude leading to static-loading conditions, or fast and high in magnitude as in the case of dynamic- or impact-loading conditions. The consequence of these loading conditions on the bone at the vicinity of pedicle screw anchorage is devastating, especially in the early months of stabilization. The need to study the bonding mechanisms between the pedicle screws and vertebra requires a close collaboration between orthopedic surgeons and engineers .The treatment of certain spinal injuries requires a clear vision about the surgical procedure and the hardware shape and materials to be adopted. In practical terms, the surgeon must first identify the access area on the posterior aspect of the vertebra, the screw insertion angles in both sagittal and transverse planes, and finally the shape of the screw to be selected and to which depth it has to be inserted.All these facts and parameters show the importance that pedicle screws can play in spinal surgery and justify, as well, the interest of this research in terms of simulation and modeling. For this reason, the present study focused on the development and use of 2D axisymmetric finite element models to compute the pullout efforts acting on the pedicle screws in spinal surgeries as well as the stress state in the immediate vicinity of the implants for different configurations of screw insertion levels . These pThe stress distribution in the bone was predicted and expected to help understand the mechanical behavior of the screw during the tearing process, under the influence of the abovementioned geometric, physiological, and surgical parameters –15. LughIn order to generate the 2D computer-assisted design (CAD) model, a portion of the vertebra and the screw was isolated at the screw centerline level in order to keep the axis of symmetry of the screw, through which the pullout efforts are transmitted. This permits abridging a complex 3D structure into a simpler 2D axisymmetric model that is accurately representative of an adult lumbar vertebra.To reflect the multiscale aspect of the bone, a partition into three areas was made during the model development see . This coWhile The screws used in orthopedic surgery have very intricate shapes and dimensions . The majThe detailed CAD model for the pedicle screws and surrounding bone was developed using Abaqus/CAE preprocessor. Precautions were taken into account while developing the axisymmetric model, particularly at the junction between the pedicle screw and the vertebra body, to accurately capture the stress state generated by the loading. In order to optimize the FE mesh, the round fillets at the top of the standard crests were eliminated and replaced by sharp edges see . It is wE) and the plastic yield stress (σp) and strain (εp) were deduced from density values based on empirical relations. Also listed in this table are the mechanical properties of the titanium alloy used for the pedicle screws.The mechanical behavior of the human vertebral cortical, subcortical, and trabecular bone structures is described in several studies , 21. To The discretization process of the model passed through critical steps prior to mesh generation. A preliminary investigation was performed to find the optimal mesh parameters for each of the partitions shown in The linear axisymmetric 4-node quadrilateral elements were preferred to their linear triangular counterparts due to their ability in producing regular meshes on both sides of the interacting surfaces. Such a regular and identical mesh at the screw-bone interface for the various models permits a better comparison platform of the computed results. A mesh refinement characterized by a minimal element size of 0.1 mm was implemented in such areas, leading to a total number of nodes and elements for all models of around 28,000 and 27,000, respectively.By their morphological aspect, highly porous vertebrae raise a problem when it comes to computing their contact stresses and load transfer mechanisms while being in contact with an extremely stiffer material, namely, titanium. Special attention was focused on modeling the contact at the screw-bone interface. A free surface-to-surface contact was adopted for such an interaction with nodes on the softer bony area playing the role of slave nodes while their counterpart on the harder screw were set to be master nodes. For all conducted analyses, either in small or large deformation formulations, the friction coefficient between the two surfaces was set to 0.2 .μm shift applied to the RP at a rate of 2 mm/min was found to trigger such behavior and was adopted for the future large deformation analyses. On the opposite, using the default Abaqus standard solver, a 10 μm shift on the RP was selected for small deformation situations to simulate static, low-magnitude loading conditions. An MPC equation was set to account for the material continuity of the vertebral transverse process and ensure the transmission of the cohesive forces. Finally, a fixed boundary condition was set on the outermost boundary while symmetry boundary was administrated to nodes on the model's axis of symmetry  coupling condition. The coupling condition consists of selecting the nodes on the flat area of the screw head to be coupled to a reference point (RP) located on the screw centerline to which a movement is prescribed to reproduce the tensile test. To get closer to the ultimate conditions that supposedly lead to implant failure, large deformation formulation was made active in Abaqus explicit solver, and trial loadings were carried out to establish the amount of load that induces large deformations on the bone structure. A 500 etry see .The failure mechanisms are known to be most active at the bone-screw interface. Several scenarios can schematically predict the mechanisms of bone failure leading to implant pullout. To help elucidate the failure mechanism in bony structures, the von Mises and shear stresses developed due to small deformation and large screw excursions were thoroughly investigated.The most common screw insertion techniques adopted by surgeons in spinal fixation surgeries were modeled in the current study to analyze their respective effects on the stability of pedicle screws when implanted in the vertebral body. Pedicle screws have, in general, standard shapes and sizes; they are fully or partially implanted during surgery, depending on the spine segment to treat, the trajectories of access to the anchorage sites, and the quality of bone . To mimic this situation, five depths of implantation characterized by a number of 2, 5, 8, 11, and 14 engaged threads were modeled for the N1 insertion type. Mechanically, this is equivalent to modifying the contact stress at the bone-implant interface, affecting by the same fact, the overall tension and tearing solicitations imposed on the implant.Since it is one of the most broadly used techniques in surgery, the “N1” insertion type was selected for the analysis against the “N3” type, expected to play a less resistive role in screw pullout situations. μm) clearly shows the high von Mises stress value of about 100 MPa raised in the pedicle subcortical bone for the N1 insertion, in which the first thread presses against the cortical wall impeding the screw-tearing mechanism. Conversely, the deep placement of the screw in the N3 insertion usually breaks the cortical wall leading to a more active pulling mechanism. The von Mises stresses induced in the subcortical bone, for instance, increased by almost five folds when the N1 is preferred to the N3 insertion type.The stress distribution portrayed in the subcortical bone for the last loading step , and type of loading imposed on the pedicle screw . Under small deformation formulation, a maximal resistive effort of about 155 N was computed for the N2 insertion case, as shown in The load-displacement curves computed under large deformations for the four types of screw insertion techniques, for normal and osteoporotic bones, showed that the N1 insertion technique provided the highest pullout effort (~2300 N) in contrast to the N3 and N4 techniques, supporting each at about 1870 N  lost with age. The use of acrylic cement and pedicle screws with special hydroxyl apatite coating may strengthen in that case the anchoring sites and improve the pullout strength of such implants.


Egyptian poultry suffer from frequent respiratory disease outbreaks associated with Infectious Bronchitis Virus (IBV) variant 2 strains (Egy/VarII). Different vaccination programs using imported vaccines have failed to protect the flocks from field challenge. Recent studies confirmed a successful protection using homologous strains as live attenuated vaccines. In this study, a newly developed live attenuated IB-VAR2 vaccine representing the GI-23 Middle East IBV lineage was evaluated in day-old commercial broilers in an IBV-endemic area. A commercial broiler flock was vaccinated with the IB-VAR2 vaccine at day-old age followed by IB-H120 at day 16. The vaccinated flock was monitored on a weekly basis till the slaughter age. The health status and growth performance were monitored, and selected viral pathogen real-time RT-PCR (rRT-PCR) detection was conducted on a weekly basis. Finally, the flock was compared to a nearby farm with only the classical IB-H120 vaccination program. Results showed that the IB-VAR2 vaccine was tolerable in day-old broiler chicks. The IBV virus rRT-PCR detection was limited to the trachea as compared to its nephropathogenic parent virus. Respiratory disease problems and high mortalities were reported in the IB-H120-only vaccinated flock. An exposure to a wild-type Egy/VarII strain was confirmed in both flocks as indicated by partial IBV S1 gene sequence. Even though the IB-VAR2-vaccinated flock performance was better than the flock that received only IB-H120, the IBV ELISA (enzyme-linked immunosorbent assay) and log2 Haemagglutination inhibition (HI) antibody mean titers remained high  until the 28th day of age. The current study demonstrates the safety and effectiveness of IB-VAR2 as a live attenuated vaccine in day-old commercial broilers. Also, the combination of IB-VAR2 and classical IBV vaccines confers a broader protective immune response against IBV in endemic areas. Gammacoronavirus, family Coronaviridae [Infectious bronchitis virus (IBV) is a single-stranded enveloped RNA virus that belongs to the genus aviridae . The viraviridae .® vaccine followed by a classical IB-H120 vaccine administered at 16 days of age were evaluated in day-old chicks under normal field circumstances.In Egypt, Massachusetts D3128, D274, D08880, 793B, variant 1 and 2 strains . Studies® employed is an IBV vaccine containing the variant 2 IBV strain Eg/1212B/2012, Genbank accession no.: JQ839287 . The classical IBV vaccine is a commercial live attenuated H120 strain . All the vaccines were given at the manufacturer’s recommended doses.The ME VAC IB VAR2The study location was selected as being a high-density area for poultry production with recent history of frequent IBV outbreaks. A naturally ventilated commercial broiler chicken farm stocked with 7600 Ross broiler chicks at El-Saff, Giza, Egypt, was selected (Farm 1). Due to limited access to other neighboring farms, another neighboring farm with 7000 Ross broiler chicks was used as a control farm for comparison purposes at the selling age (Farm 2). The main concerns, from a biosecurity point of view, included the lack of optimal distances between farms (the distance between the two farms is 0.35 km), the high frequency of IBV outbreaks at the region, and the lack of shelters and enclosure systems that may allow contact with wild birds.® vaccine at day-old age, then a booster dose using IB-H120 at day 16 in farm 1, while IB-H120 was used at both ages in farm 2; along with the IBV vaccine, other vaccinations were routinely performed in the studied flocks as shown in Thirty serum samples were collected from farm 1 at day-old age to assess maternally derived antibodies (MDA) against IBV, Infectious bursal disease (IBD), and avian influenza of both H9 and H5 subtypes (AI-H9 and AI-H5). The only difference in vaccination programs between farms 1 and 2 was the use of IB-VAR2The flock health status was monitored on a daily basis for any clinical problems and/or any mortality. The final body weights measured at selling age, the total feed intake, and the feed conversion ratio (food conversion rate (FCR) = feed intake/weight gain) were calculated. Mortality was recorded daily and cumulative mortality was obtained. Farm 1 was compared to farm 2 at the selling age in terms of feed total weight gain, conversion ratio, and cumulative mortalities.® IBV Ab and ProFLOK® IBD Ab  for IBV and IBD viruses, respectively, following the protocols recommended by the manufacturer. Haemagglutination inhibition (HI) test was carried out using IBV antigens (IB-H120 and IBV-VAR2) prepared at our laboratory as previously described by the OIE manual [Collected sera were tested using ProFLOKE manual , using rViral RNA was extracted from tracheal swabs using the Patho Gene-spinTM DNA/RNA Extraction Kit  according to the manufacturer’s instructions. The Quant one-step RT-PCR Tiangen Kit  was used to perform single-step rRT-PCR assays using specific oligonucleotide primers and probes for AI-H5 , AI-H9 , IBV 1818, and vp value = 0.05, using SAS software version 9.4 [The statistical analysis is done using a two-tail T-test with sion 9.4 .The vaccinated flocks at farm 1 and 2 remained free of any clinical disease signs. However, in farm 2 (control farm), respiratory symptoms with increased mortalities were observed by 22 days of age. Comparison of the two flocks revealed that the cumulative mortality rate was 9% and 22% in farm 1 and 2, respectively. Also, farm 1’s total sold weight was 12,250 kg with a FCR of 1.7, while farm 2 yielded a total of 8150 kg with a FCR of 2.1 .The mean maternally derived antibody levels (MDA) for IBV at day-old age was 2372 ± 1123 by enzyme-linked immunosorbent assay (ELISA). At 14-Day post vaccination (DPV), the ELISA titers dropped to 590 ± 155. By 12 days after the second IBV vaccination using the IB-H120 vaccine, the ELISA titers were elevated to reach 2253 ± 1175 . Weekly ® on the other vaccines administered to the flock. The lowest responses to the used vaccines were at three and four weeks of age for AI-H5 and AI-H9, respectively, and at five weeks of age for NDV  was evaluated in a prime-boost IBV vaccination program in commercial broiler chickens under field conditions to monitor safety and efficacy. The laboratory evaluation of specific pathogen-free chicks in Biosafety Level-3 (BSL-3) isolators with ciliary activity evaluation and pathological lesions in the respiratory tract and kidneys were conducted in another trial [In this study, a newly developed live attenuated vaccine prepared from an Egy/VarII strain representing the GI-23 Middle East lineage , with increased mortality in farm 2 considering the same biosecurity concerns in both farms.The IB-VAR2The phenomenon of viral interference was previously reported between IBV and live NDV vaccine strains . HoweverThe IBV immune response was monitored by both ELISA and HI test. MDA titers of IBV were very low using the IB-VAR2 antigen as compared to those detected by the IB-M41 classical IBV antigen. These findings are consistent with previous research, indicating that the IBV HI test is strain-specific . The maxNone of the targeted viral pathogens were detected by rRT-PCR at any time point. IBV rRT-PCR detection was mainly limited to the trachea, and no virus was detected in the kidneys by the 14th day of age. The very limited detection of IBV in the kidneys supports the safety of the vaccine as compared to its nephropathogenic wild-type parent virus. The limitation of the fact that the rRT-PCR assay cannot differentiate between vaccine and field IBV strains  is ackno® vaccine (representing the GI-23 Middle East lineage) in day-old commercial broiler chickens in highly endemic areas; also, the ME VAC IB-VAR2® vaccine was better able to protect against the disease in an IBV-endemic study area. The vaccine does not interfere with the birds’ immune response to both live and inactivated vaccines of other viral pathogens. Moreover, it appears that the combination of ME VAC IB-VAR2® and live classical IBV vaccines confers a protective immune response; however, protection studies are needed to confirm the ability of different vaccine combinations to protect chickens from both homologous and heterologous viral challenges.In conclusion, this study indicated the safety and effectiveness of the newly developed live attenuated ME VAC IB-VAR2


IBV is a major avian pathogen and presents a substantial economic burden to the poultry industry. Improved vaccination strategies are urgently needed to curb the global spread of this virus, and the development of suitable vaccine candidates will be aided by an improved understanding of IBV molecular biology. Our high-resolution data have enabled a precise study of transcription and translation in cells infected with both pathogenic and attenuated forms of IBV and expand our understanding of gammacoronaviral gene expression. We demonstrate that gene expression shows considerable intraspecies variation, with single nucleotide polymorphisms being associated with altered production of sgmRNA transcripts, and our RiboSeq data sets enabled us to uncover novel ribosomally occupied ORFs in both strains. The numerous cellular genes and gene networks found to be differentially expressed during virus infection provide insights into the host cell response to IBV infection. Like all coronaviruses, avian infectious bronchitis virus (IBV) possesses a long, single-stranded, positive-sense RNA genome (∼27 kb) and has a complex replication strategy that includes the production of a nested set of subgenomic mRNAs (sgmRNAs). Here, we used whole-transcriptome sequencing (RNASeq) and ribosome profiling (RiboSeq) to delineate gene expression in the IBV M41-CK and Beau-R strains at subcodon resolution. RNASeq facilitated a comparative analysis of viral RNA synthesis and revealed two novel transcription junction sites in the attenuated Beau-R strain, one of which would generate a sgmRNA encoding a ribosomally occupied open reading frame (dORF) located downstream of the nucleocapsid coding region. RiboSeq permitted quantification of the translational efficiency of virus gene expression and identified, for the first time, sites of ribosomal pausing on the genome. Quantification of reads flanking the programmed ribosomal frameshifting (PRF) signal at the genomic RNA ORF1a/ORF1b junction revealed that PRF in IBV is highly efficient (33 to 40%). Triplet phasing of RiboSeq data allowed precise determination of reading frames and revealed the translation of two ORFs (ORF4b and ORF4c on sgmRNA IR), which are widely conserved across IBV isolates. Analysis of differential gene expression in infected primary chick kidney cells indicated that the host cell response to IBV occurs primarily at the level of transcription, with global upregulation of immune-related mRNA transcripts following infection and comparatively modest changes in the translation efficiencies of host genes. Cellular genes and gene networks differentially expressed during virus infection were also identified, giving insights into the host cell response to IBV infection.IMPORTANCE IBV is a major avian pathogen and presents a substantial economic burden to the poultry industry. Improved vaccination strategies are urgently needed to curb the global spread of this virus, and the development of suitable vaccine candidates will be aided by an improved understanding of IBV molecular biology. Our high-resolution data have enabled a precise study of transcription and translation in cells infected with both pathogenic and attenuated forms of IBV and expand our understanding of gammacoronaviral gene expression. We demonstrate that gene expression shows considerable intraspecies variation, with single nucleotide polymorphisms being associated with altered production of sgmRNA transcripts, and our RiboSeq data sets enabled us to uncover novel ribosomally occupied ORFs in both strains. The numerous cellular genes and gene networks found to be differentially expressed during virus infection provide insights into the host cell response to IBV infection. Gammacoronavirus  and a pathogen of domestic fowl (–8–Avian infectious bronchitis virus (IBV) is a member of the genus tic fowl . IBV inftic fowl . The montic fowl . Two lartic fowl , 5, givitic fowl –8. A feaic fowl –, 10. Inc fowl –8–. Subsequin vivo, although recombinant transfer of the protein from the former to the latter does not restore pathogenicity   isolate 17–genicity . The extThe advent of high-throughput sequencing techniques offers a means to monitor viral gene expression at unprecedented resolution –27. Here–(This article was submitted to an online preprint archive .)RiboSeq and RNASeq libraries were prepared from two biological repeats each of Beau-R-infected, M41-CK-infected, and mock-infected cells. Infections were at a high multiplicity (multiplicity of infection [MOI] = ∼3), and cells were processed at 24 h postinfection (p.i.). An average of 1,156,819 RPFs and 1,727,024 RNASeq reads were mapped to viral gRNA in the virus-infected RiboSeq libraries . The RNASeq read coverage in the library derived from the second biological repeat of M41-CK-infected cells was lower than that in the other libraries due to technical losses. However, 106,741 reads were mapped to the forward strand of the viral gRNA in this case, corresponding to a coverage of approximately 3.8-fold, and these reads were generally evenly distributed along the gRNA; hence, the sequencing depth in this replicate was deemed sufficient for further analysis. The vast majority of RPFs mapping to viral and host protein-coding regions were between 27 and 29 nt in length , consistent with the size of the RNA fragment protected by translating eukaryotic ribosomes from digestion by RNase I , 30, 31,P < 0.01 in both cases) . The sequence logos in The density of RNASeq reads mapping to a given sgmRNA represents the cumulative sum of reads derived from the gRNA and those derived from the overlapping portions of other subgenomic transcripts . TherefoGAACAA), in contrast to the TRS-L, which has a U at this position (CUUAACAA) (Table S2). Chimeric reads assigned to this gene were found to contain either a U or a G residue at position 3  and M41-CK , with the latter matching the TRS-L sequence exactly. In this case, we found that Beau-R-derived chimeric reads could contain either a U (denoted 3/E [U3]) or a G (denoted 3/E [G3]), with the G residue being slightly more common , whereas M41-CK-derived reads contained only the U residue . The more abundant of these (0.6% of chimeric reads) mapped to a position immediately downstream of the IBV Beau-R N gene termination codon within the 3′ untranslated region (UTR). Chimeric reads derived from this site contained the sequence CUUAACAU, the last 6 nt of which could have been templated by the genomic (TRS-B) sequence . There was an AUG-initiated downstream ORF (dORF) in Beau-R beginning 2 nt 3′ of this TRS, which comprised 11 codons (nt 27111 to nt 27143). Inspection of our RiboSeq libraries showed that the dORF is ribosomally occupied . Such AUThe second novel chimeric sequence identified in RNASeq libraries maps to a TRS-B (CUUACCAA) within the coding region of the S gene in Beau-R (nt 21242 to 21249). This is consistent with the previous detection of an sgmRNA of appropriate length via Northern blot analysis . While tin vitro PRF assays  . No protTo estimate the translational efficiency (TE) of virus genes, we summed RPFs whose 5′ end mapped in phase between the first nucleotide of the initiation codon and 30 nt 5′ of the termination codon, thereby excluding RPFs derived from ribosomes paused during initiation or termination (the raw ribosome footprint data are provided in Table S3). The TE of each ORF was measured as the quotient of the RPF density and the abundance of the corresponding mRNA, with separate calculations being performed using TRS chimeric reads counts and the decumulated RNASeq densities  was assoInspection of the profiling data sets revealed a number of genomic locations where RPFs accumulated to a much higher level than at neighboring sites, indicative of ribosomal pausing. As such pauses may have biological significance, we first sought to discount those that may have arisen artefactually. The known translation initiation sites in the virus genome generally showed high ribosome occupancy, but as the infected cells were treated with cycloheximide (CHX) prior to lysis to freeze ribosomes onto the mRNA, these pauses are likely to be overrepresented, as ribosomes can accumulate at start codons during the CHX treatment period . FluctuaIt is noteworthy that in our analysis of ribosomal pause sites, we did not see pausing at the AUG of the previously described 11-amino-acid uORF of the genomic mRNA (AUG at nt 131 to 133 ), and in2 fold changes (log2FC) in transcript abundance and TE  for both strains  . Details of the genes found to be differentially expressed  at the level of transcription  or translation , respectively, are provided in Data Sets S1 and S2. Overall, the patterns of change in host cell gene expression in response to infection were broadly similar for Beau-R and M41-CK, with positive interstrain correlations in the log strains , left, wuced TE)  and C. T2FC (Data Set S3), and the top 100 DEGs (or less) within each category were subjected to STRING analysis  were ranked by loganalysis  to identHere, we describe the first high-resolution study of gammacoronaviral gene expression during infection of primary chick kidney cells. Analysis of RNASeq data sets through chimeric read analysis or decumulation allowed us to quantify the relative levels of viral genomic and subgenomic mRNAs and to define the sequence diversity of strain-specific TRS utilization. The predominant sgmRNA in both strains was that encoding the N protein, and between strains, the M transcript was relatively more abundant in M41-CK. In Beau-R, two novel TRSs were identified, one in the viral 3′ UTR immediately downstream of the N gene termination codon and one mapping to a TRS-B within the S gene. In the former, a short ORF (dORF), initiated 2 nt 3′ of the TRS, was present and ribosomally occupied. The potential biological relevance of this ORF remains to be determined; such dORFs are present in most IBV strains and other avian gammacoronaviruses, but it is lacking in M41-CK (as is the TRS-B). A recent report has described the same sgmRNA  as a novel noncoding RNA of IBV . The S gRiboSeq analysis, in conjunction with RNASeq, revealed that the N protein is not more efficiently translated than other structural proteins, despite being a structural component of IBV virions. It is possible that N expression may be regulated by a putative uORF whose initiation codon is located some 50 nt upstream of the N AUG codon .in vitro measurements of IBV frameshifting efficiency carried out using reporter constructs  and the weak initiation context of the ORF4b start codon, it is possible that a proportion of ribosomes might bypass the ORF4b initiation codon and instead translate ORF4c via leaky scanning , althougSaccharomyces cerevisiae and Schizosaccharomyces pombe yeast cells with cycloheximide can lead to an early block in elongation in stressed cells  could potentiate initiation on the uORFs through stacking of scanning ribosomes, and this could be artefactually increased by the cycloheximide pretreatment used during sample preparation. Two of the other pause sites correspond to ribosomes paused postinitiation early in the coding regions of the S and M genes. A biological explanation for this is lacking at present. We are aware that the treatment of ed cells , 50, buted cells . A possiIn general, the pauses that we discerned during translation of the IBV genome were discrete, substantial in terms of read counts, and reproducible. As mentioned above, their origin is uncertain, but it seems unrelated to the identity of the P-site tRNA. Recent studies have shown that P-site prolyl-tRNA is a strong determinant of ribosomal pausing, partly due to the low rate of peptide bond formation with this amino acid , 52. HowOur data indicate that the host response to IBV is mediated primarily at the level of transcription with the upregulation of hundreds of genes, many of which have immune-related functions. Changes in translational efficiency were modest, with more genes showing decreased rather than increased translation in response to IBV infection. Many of the transcriptionally upregulated genes identified reflect the host response to virus infection, as seen previously with IBV infection of chickens , 57 and MK728875.1) has been described previously (AJ311317.1), except for two point mutations, one in nsp16  and one in N . Primary chick kidney (CK) cells were produced from 2- to 3 week-old specific-pathogen-free (SPF) Rhode Island Red chickens (6 cells/ml) were plated in 10-cm dishes and upon reaching 100% confluence (at 2 days postseeding) were washed once with phosphate-buffered saline (PBS) and infected with 9.6 × 106 PFU of Beau-R or M41-CK (MOI = ∼3). After 1 h of incubation at 37°C in 5% CO2, the inoculum was removed and replaced with 10 ml fresh 1× BES . Cells were harvested at 24 h postinfection when clear regions of cytopathic effect (CPE) were visible.The apathogenic molecular clone of IBV, Beau-R, has been described previously . The pateviously . The twochickens . The chi2, 1 mM dithiothreitol, 1% Triton X-100, 100 μg/ml cycloheximide, 25 U/ml Turbo DNase [Life Technologies]) was dripped onto the cells. The cells were scraped extensively to ensure lysis, collected, and triturated with a 26-gauge needle 10 times. The lysates were clarified by centrifugation for 20 min at 13,000 × g at 4°C, and the supernatants were recovered and stored at −80°C.Cycloheximide  was added directly to the growth medium (to 100 μg/ml), and the cells were incubated for 2 min at 37°C before rinsing with 5 ml of ice-cold PBS containing CHX (100 μg/ml). Subsequently, the dishes were incubated on ice and 400 μl of lysis buffer , and reads shorter than 25 nt following adaptor trimming were discarded. Mapping was performed using the Bowtie (v1) program  rRNA, IBV Beau-R (GenBank accession number NC_001451.1) or IBV M41-CK (GenBank accession number DQ834384.1) gRNA, Ensembl host noncoding RNA (ncRNA), NCBI RefSeq host mRNA, and the host genome. The order of mapping was tested to check that virus-derived reads were not lost accidentally due to mismapping to host RNA, or vice versa. When performing analyses of viral and host gene expression, only 28- and 29-nt RiboSeq reads (corresponding to RPFs mapping primarily in phase 0) and only ≥40-nt RNASeq reads were used. A 12-nt offset was applied to the 5′ mapping positions of RPFs, to approximate the P-site position of the ribosome  were derived from reads mapping to RefSeq mRNAs with annotated CDSs at least 450 nt in length and annotated 5′ and 3′ UTRs at least 60 nt in length. When calculating the translation efficiencies of viral genes, only in-phase  RiboSeq reads were counted.Gallus gallus (v5.0) assembly (December 2015) using the STAR aligner  under the accession number E-MTAB-7849.Sequencing data have been deposited in ArrayExpress (


Allergen‐specific immunoglobulin isotype formation associated with immunoglobulin class‐switching during the lactation period is the immunological background for food allergy in infants. We analyzed the serial changes in the production of feeding type‐related egg‐ and milk‐specific immunoglobulin isotypes from birth to 6 months of age with or without eczema in 84 infants.Allergen‐specific immunoglobulin G1 (IgG1), IgG2, IgG3, IgG4, IgA, and IgE levels of hen's egg and bovine milk were measured in cord blood and blood samples from infants at 2, 4, and 6 months of age by the densely carboxylated protein microarray.Formula and mixed feeding were associated with a rapid increase in cow's milk allergen‐specific immunoglobulins and feeding type‐related significant differences in casein‐specific immunoglobulin levels were detected. Breast and mixed feeding were associated with slow but significant increase in ovalbumin‐specific IgG1 and IgE levels, but not other immunoglobulins. We found two different immunoglobulin isotype formation at 6 months of age with low‐ or high‐affinity IgE against ovalbumin. One isotype formation pattern had relatively high ovalbumin‐specific IgG1 levels, detectable IgG2, and low‐affinity IgE, while the other had low ovalbumin‐specific IgG1 levels, undetectable IgG2, and high levels of high‐affinity IgE. The incidence of eczema was significantly higher in the latter pattern (84.6%), compared with the remaining infants (42.2%).Feeding practice‐related allergen sensitization and immunoglobulin isotype formation were identified during the lactation period. The development of eczema during the lactation period could potentially modify the immunoglobulin isotype formation with high levels of high‐affinity IgE. To our knowledge, no such detailed data are available at present, probably due to the methodological limitation of quantitating the instrument of allergen‐specific IgG1, IgG2, IgG3, IgG4, IgE, and IgA by using small amounts of blood from infants. To monitor immunoglobulin isotype production, we developed recently a highly sensitive densely carboxylated protein (DCP) microarray with high antigen immobilization capacity.It has been reported that the presence of food proteins in human breast milk, such as ovalbumin (OVA),The aim of the present study was to monitor the serial changes in the formation of feeding type ‐related allergen‐specific immunoglobulin isotypes during early infancy. We measured allergen sensitization associated with immunoglobulin class‐switching from IgG1 to IgE and/or IgG2 in each infant against OVA, the major hen's egg allergen and casein, the major bovine milk allergen by the DCP microarray. We also searched for any modification in immunoglobulin isotype formation associated with eczema in producing immunoglobulin isotypes with allergen‐specific low‐ or high‐affinity IgE.22.1Among the healthy pregnant women who visited Tokushima Prefecture Naruto Hospital for delivery between November 2013 and October 2014 and agreed to join this study, the present study enrolled 112 of their infants. The applied exclusion criteria were birth before 37 weeks of gestation and complications associated with any chronic or severe disease. Primary‐outcome data were obtained in 84 (75%) of the 112 eligible participants whereas serial blood sampling could not be achieved in the dropout infants (n = 28) for a variety of reasons such as change of address after childbirth and will of no further blood sampling  samples were collected by the obstetricians at birth by needle puncture of the umbilical vein after careful cleaning of the umbilical cord to avoid maternal blood contamination. CB and blood samples were collected from the infants at 2, 4, and 6 months of age, centrifuged at 15002.3r > 0.9‐0.85).A/L) by each allergen‐specific equation. For instance, correlation coefficient of casein‐specific IgE between UniCAP (kUA/L) and DCP microarray (BUe/mL) and its equation are shown in Figure S1. Based on the small volume of the blood sample and wide assay dynamic range, the DCP assay method has been used in several cohort studies.Hen's egg and cow's milk allergen‐specific IgE, IgG1, IgG2 IgG3, IgG4, and IgA levels in blood were measured by the DCP microarray as described in detail previously.2.450 (nM), which represented the concentration of allergen required for 50% inhibition, as described previously.−6 mol/L and then incubated at 25°C for 30 minutes. After incubation, each reaction mixture was applied to an OVA‐immobilized DCP microarray and the competitive binding assay was conducted at 37°C for 60 minutes. After washing three times, the bound antibody was detected and then IC50 was calculated.The OVA affinity of IgE was analyzed using an OVA‐immobilized DCP microarray, which is based on competition binding inhibition of serially diluted soluble OVA, and expressed as IC2.550 values of OVA‐specific IgE and cumulative incidence of eczema between the red circled infants in group A and black circled infants in group B in Figure P < 0.05 was considered significant in all analyses except for the Wilcoxon signed‐rank test with Bonferroni's correction. P < 0.01 (0.05/predetermined number of comparisons) was judged to be significant for the Wilcoxon signed‐rank test with Bonferroni's correction. All statistical analyses were performed using SPSS for Windows (version 22) .Differences in allergen‐specific immunoglobulins among formula milk, breast milk, and mixed‐feeding infants at each time point were analyzed by the Kruskal‐Wallis test. Differences in continuous variables of allergen‐specific immunoglobulins at four different time points , and differences between two time points were analyzed by the Friedman test and the Wilcoxon signed‐rank test with Bonferroni's correction, respectively. We evaluated IC2.6This study was conducted according to the principles in the Declaration of Helsinki. This study was approved by the Ethics Committee of Tokushima Prefecture Naruto Hospital (Permit #1314) on 14th November 2013. Written informed consent was obtained from each mother at the time of enrollment. This study was carried out in accordance with relevant guidelines and regulations.33.1The primary‐outcome data were obtained from 84 healthy infants in the studies on immunoglobulin isotype production in early infancy Figure . Blood s3.2β‐lactoglobulin.β‐lactoglobulin‐specific immunoglobulins (data not shown).Production of allergen‐specific immunoglobulins in blood is highly dependent on the dose of allergens in the ingested milk. Since casein accounts for about 80% of the total cow's milk proteins,In contrast to the relatively high concentration of casein in formula milk, the average breast milk OVA concentration is less than 1 ng/mL,3.3Serial changes in the production of allergen‐specific immunoglobulin isotypes are related to repeated antigen exposure and the results of immunoglobulin class‐switching recombination.OVA‐specific maternal IgG1 in CB almost disappeared at 2 months of age and then OVA‐specific IgG1 and IgE produced by infants increased slowly after 4 months of age Figure D‐F. At 63.450 values  nM, while those of the black circles in group B had low‐affinity IgE with IC50 value of 57.21 (35.34‐68.27) nM. The IC50 values of the two groups were significantly different (P = 0.0047).For measurement of allergen‐specific affinity of IgE by competitive binding inhibition, we used samples with IgE levels higher than 200 BUe/mL to monitor inhibition of binding and at least five different concentrations of allergen were used for competitive binding inhibition. Infants with IgE levels greater than or equal to 200 BUe/mL Figure F were dis Figure A and OVAP = 0.001). Figure P < 0.0001).The cumulative incidence of eczema history was significantly higher in the red‐circles infants in group A  compared with that of the remaining infants . This finding suggests that eczema or inflammation in the skin associated with transcutaneous allergen sensitization is one of the risk factors of this type of immunoglobulin isotype formation with high levels and high antigen‐affinity of IgE in early infancy. This speculation is also supported by the results shown in Figure P < 0.0001). In this regard, it has been reported that IL‐4 plays a key role in class‐switching processes of γ1 → ε and high interleukin 4 (IL‐4) levels in the inflamed skin lesions can stimulate IgE production.The cumulative incidence of eczema was significantly high in the red circles infants (84.6%), compared with that of 42.2% in the remaining infants  and firm diagnosis of food allergy at 1 year of age are needed to confirm our speculations and conclusions. In addition, we found no significant impact on the levels of OVA‐specific immunoglobulins in the feeding types (Figure In conclusion, our longitudinal study identified two different patterns of allergen‐specific immunoglobulin isotype formation with high‐ and low‐affinity IgE in the first 6 months of life of normal breast‐, formula‐ and mixed‐feeding infants with and without eczema.The authors declare that there are no conflict of interests.MI and HK formulated the research hypothesis; MI, WS, MS, YO, KS, and KK conducted the experimental work, analyzed the data, and interpreted the results; KA and MI conducted the statistical analysis; MI, YO, and HK drafted the manuscript; and HS, YO, LY, and SK critically revised the manuscript.Supporting informationClick here for additional data file.


Membrane-associated guanylate kinase (MAGUK) with inverted domain structure-1 (MAGI1) is an adaptor protein that stabilizes epithelial and endothelial cell-cell contacts. The aim of this study was to assess the unknown role of endothelial cell MAGI1 in response to fluid shear stress. We show constitutive expression and co-localization of MAGI1 with vascular endothelial cadherin (VE-cadherin) in endothelial cells at cellular junctions under static and laminar flow conditions. Fluid shear stress increases MAGI1 expression. MAGI1 silencing perturbed flow-dependent responses, specifically, Krüppel-like factor 4 (KLF4) expression, endothelial cell alignment, eNOS phosphorylation and NO production. MAGI1 overexpression had opposite effects and induced phosphorylation of PKA, AMPK, and CAMKII. Pharmacological inhibition of PKA and AMPK prevented MAGI1-mediated eNOS phosphorylation. Consistently, MAGI1 silencing and PKA inhibition suppressed the flow-induced NO production. Endothelial cell-specific transgenic expression of MAGI1 induced PKA and eNOS phosphorylation in vivo and increased NO production ex vivo in isolated endothelial cells. In conclusion, we have identified endothelial cell MAGI1 as a previously unrecognized mediator of fluid shear stress-induced and PKA/AMPK dependent eNOS activation and NO production.Fluid shear stress stimulates endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) production through multiple kinases, including protein kinase A (PKA), AMP-activated protein kinase (AMPK), AKT and Ca N-terminal oxygenase domain with binding sites for heme, l-arginine and tetrahydrobiopterin; a central calmodulin-binding region and a C-terminal reductase domain containing binding sites for nicotinamide adenine dinucleotide phosphate, flavin adenine dinucleotide and mononucleotide /5% sucrose in the drinking water and was changed at least twice per week. Animals were euthanized by CO2 inhalation followed by neck dislocation. Animal experiments were approved by the Cantonal Office in Fribourg (Ruegg_2014_26_FR) and performed according to Swiss regulations and to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes. We used both male and female mice between 6 and 10 weeks of age. The following primers were used for genotyping the mice: VEC_forward: 5′GACGCCTTAGCCATTGAGAT 3′, VEC_reverse: 5′CAGTAG TAG GTGTTTCCCTTTCTT 3′, MAGI1_forward: 5′ TCATTCCTGGGCATGAGTCCT 3′, MAGI1_reverse: 5′GCCAGGGAAGGAAGGATTGT3′.The human MAGI1 cDNA  was inserted into the Lungs from freshly sacrificed mice were cut and digested in 1% Collagenase and 2.5 µg/mL of DNase I, both from Sigma-Aldrich  for 45 min at 37 °C. After passing through 70 µm filters, cells were washed once in PBS with 2 mM EDTA and twice in PBS only. Cells were resuspended in DMEM:F12 supplemented with 2% FBS, 1% penicillin/streptomycin, 20 ng/mL EGF and 10 µg/mL insulin and plated on Collagen I (10 µg/mL) coated plates.Tissue sections were heated in Tris-EDTA buffer to retrieve antigen epitopes, blocked by 10% normal goat serum and Avidin/Biotin blocking reagent  and stained with the following primary antibodies at 4 °C overnight: anti-MAGI1 , P-eNOS  and total eNOS . Sections were incubated with biotinylated secondary antibodies followed by Vectastain ABC Kit  with DAB peroxidase substrate (Sigma-Aldrich). Sections were counterstained with haematoxylin before mounting.t-test or two-way ANOVA, followed by Sidak’s multiple comparison test, where necessary using GraphPad Prism software . A value of p < 0.05 was considered to be significant. *p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001. N, repeated experiments; n, replicates per experiment. Statistical analysis on data expressed as the mean ± SEM was performed on the basis of unpaired 2), MAGI1 localized at cell-cell contacts as continuous staining in co-localization with VE-cadherin (2) in the cone-and-plate BioTechFlow-system (BTF) . T,7. T1,7]1177 phosphorylation and NO production; ii) MAGI1 overexpression in HUVEC and in endothelial cells of transgenic mice, increased PKA phosphorylation, induced eNOS Ser1177 phosphorylation in vivo, in vitro and ex vivo, as well as NO production in vitro and ex vivo. iii) PKA inhibition suppressed eNOS Ser1177 phosphorylation and NO production. iv) MAGI1 also induced AMPK Thr172 phosphorylation, and its inhibition partially reduced MAGI-induced eNOS phosphorylation. Here we report that the scaffolding protein MAGI1 contributes to PKA- and, to a lesser extent, AMPK-dependent eNOS activation and NO production. This conclusion is based on the following observations: i) Fluid shear stress induced MAGI1 expression in HUVEC in three independent models, silencing of MAGI1 expression impinged on eNOS SerA number of MAGI1 physiological functions and involvements in pathologies, including cancer, have been reported, but only little is known on the putative function of MAGI1 in vascular biology. At the cellular level, MAGI1 plays an important role in stabilizing cell-cell contacts in epithelial cells, through tight junctions . MAGI1 rFurther evidence for a potential contribution of MAGI1 to endothelial physiology is corroborated by its involvement in the induction of KLF4 expression in response to flow. There are numerous studies showing that laminar shear stress is inducing KLF4 expression through both transcriptional  and epigIn view of the contribution of KLF4 in mediating responses to flow and its multiple roles in vascular physiology and pathology, it will be essential to unravel how MAGI1 regulates KFL4 expression, and in particular whether KLF4, in turn, may regulate MAGI1 expression. Some of the ongoing experiments are specifically addressing this important question.Considering the importance of NO metabolism in many cardiovascular diseases, in particular atherosclerosis and hypertension ,52, it wThe presented study demonstrates for the first time that the scaffolding protein MAGI1 regulates shear stress-mediated NO production through a PKA/AMPK dependent eNOS phosphorylation in endothelial cells. These results add a previously unrecognized element to the characterization of MAGI1 function in vascular biology and to the complex mechanisms regulating NO production in endothelial cells.